Eneurology is now here, it is a biannual publication that welcomes original publications, review articles, case records in the field of neurology, psychiatry, neuroradiology, neuropathology, and neurosurgery. You can access it at: http://eneurology.yassermetwally.com
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The author: Professor Yasser Metwally
INTRODUCTION
April 17, 2009 — The primary therapeutic goal of embryonic stem cell (ESC) research is cell replacement therapy. During the last decade, great strides have been made in developing in vitro protocols for differentiating human ESCs into neuroepithelial progenitors. More recent progress has been made in further directing them into becoming cells with specialized regional and neurotransmitter identities, such as midbrain dopaminergic and spinal motor neurons. Along with directed differentiation, other current efforts are aimed at efficient enrichment, avoidance of immune rejection, demonstration of functional integration, genetic modification to regulate neurotransmitter and factor release, directed axon growth, in vivo cell tracking, and measures to ensure safety. This review will focus on the potential of ESCs as a source of transplantable cells for use in cell replacement therapy.
Cell replacement therapy is an exciting research area that offers potential treatment for several developmental, traumatic, and degenerative neurological diseases for which there is currently no cure. The requirement of the replaced cell can be as straightforward as synthesis and release of a protein, to something as complex as the establishment of lengthy projections and integration with host circuitry. A universal cell replacement strategy is unlikely to work.
Cells ultimately used for transplantation can be isolated from the donor organism at different lineage stages. If obtained later in development when their fate is restricted, they can be purified and sorted prior to transplantation. Alternatively, cells can be isolated at an early stage, expanded, specified, and differentiated prior to transplantation. The second option allows expansion and directed differentiation. Ideally, what is needed is an unlimited supply of expandable, stable cells with enormous potential that can be manipulated to give rise to specific cell types.
Significant strides have been made in designing cells with broad potential that can be directed in vitro down a given path to eventually become a specific phenotype. These cells have shown promise for brain repair in preclinical models of disease.[63] Major barriers include immune rejection, the potential of tumor growth, and incomplete understanding of the steps needed to manipulate human cells due to an incomplete understanding of the normal steps of human development.
When designing a cell to use for replacement therapy, it is important to consider the lineage stage and commitment of the grafted cell so that its actions can be predicted after transplantation. Figure 1 demonstrates the predicted relationship between lineage position and cell behaviors following transplantation. In general, as a cell progresses from an ESC to a mature neuron, its capacity for cell division, migration, and differentiation gradually decreases, while the neurotransmitter phenotype predictability and safety increase.
Figure 1. Relationship between cell behaviors and lineage position of cell. As a cell progresses from an ESC to a neuron, production efficiency (in vitro expansion capability) and ability to self renew, proliferate, migrate, and differentiate progressively decreases, while neuronal phenotype predictability and safety of the transplant increases. ES = embryonic stem; N = neuron; NE = neuroepithelial; NP = neural progenitor. *Adapted from original: Guillaume DJ, Zhang SC: Neuronal replacement by transplantation, in Bottenstein J (ed): Neural Stem Cells: Development and Transplantation. Norwell, MA: Kluwer Academic Publishers, 2003, p 309. With kind permission of Springer Science and Business Media. (Click on figure to enlarge)
Each human cell is isolated at a particular stage of development and will possess either broad or restricted potential. If stem cell technology is to become a practical therapy, there would ideally be a readily available stock of starting cells available that can be tailored for specific therapies. The ideal source of starting cells could be expanded to large numbers, stored for extended periods, is effective in cellular replacement, can be manipulated or tailored to fit the specific condition being treated, tolerant to immune rejection, and safe. In the case of neuronal replacement, the cell must possess the capacity to functionally integrate with existing circuitry. Presently, no cell meets all of these criteria. Many cells have been investigated in animal studies and clinical trials. Table 1 lists potential donor cells, their sources, and the advantages and disadvantages of their use in cellular replacement therapy.
Table 1. (Click on table to enlarge)
Primary neural cells are fairly mature cells that are committed to a particular phenotype corresponding to their site of origin. An example would be the human fetal mesencephalic tissue used in clinical trials for PD.[4] Advantages include predictable phenotype and low risk of tumor formation. The disadvantage is that these cells have a limited capacity to expand, and grafts must be prepared from several stage-specific embryos. If these cells were found to be effective in cellular replacement for neurological diseases, problems would arise due to their limited supply. The practical and ethical constraints that accompany the use of primary fetal brain tissue in cell transplantation are too numerous to be covered here.[13]
Adult NSCs can be isolated from the brains of adult animals and humans and expanded in culture.[15,28,31] These cells can generate neurons and glia in vitro and after transplantation.[18,41,45] Stem cells generated from adult neural and nonneural tissues are attractive because they avoid many ethical and immunological issues. They may prove effective for conditions that require synthesis and release of certain enzymes and factors. However, adult NSCs may not be optimal for neuronal replacement because they tend to become primarily interneurons when placed in neurogenic regions and astrocytes if placed in nonneurogenic regions.[45] The full potential of these cells has yet to be realized.
Neural stem/progenitor cells are isolated from embryonic CNS tissue and can be expanded in culture for prolonged periods using genetic or epigenetic approaches. Expanded cells have the capacity to differentiate into neurons, oligodendrocytes, or astrocytes.[20,46] These cells are often confined to a specific regional identity at the time of isolation because they are frequently isolated after rostrocaudal and dorsoventral specification. In this regard, they offer a good source for the cell replacement requirement in many general conditions, but may be limited as a source of specific neuronal replacements. Still, neural stem/progenitor cells isolated within a certain window of development appear to retain the ability to give rise to cells of other lineages. For example, coculture of NSCs isolated from the midbrain and hindbrain with ventral forebrain tissue induces the expression of ventral forebrain markers.[19] Neural precursor cells isolated from the hippocampal dentate gyrus and grafted into the rostral migratory stream migrate to the olfactory bulb and become tyrosine hydroxylase-positive neurons, a type of dopaminergic neuron not normally found in the hippocampus.[45]
Neural stem/progenitor cells can be immortalized with oncogenes. For instance, the clonal line C17.2, created by overexpression of v-myc in cerebellar granular cells, is capable of differentiating into a variety of neurons and glia in a site-specific manner.[42] These cells differentiate into myelin-producing oligodendrocytes when placed into the dysmyelinated shiverer mouse brain,[58] but appropriately become neurons or glia when grafted into areas of injured brain or spinal cord,[55] suggesting that the fate of these cells is influenced by their environment. Because neural stem/ progenitor cells have relatively restricted differentiation potential compared to cells of lower lineage stages, they appear to have a lower risk of tumor formation.
Embryonic stem cells possess several features that make them ideally suited for neuronal cell therapy. These cells, derived from the inner cell mass of a blastocyst,[11,51,52] can be expanded in vitro for years while retaining the capacity to differentiate into any of the specialized cell types that make up an organism. Accordingly, they provide a useful model for understanding early mammalian embryonic development and also offer a source for generating specialized cells such as specific neurons and glia for therapeutic uses.[44,63-66]
Neuroepithelial cells are specified from naive ESCs at approximately Day 7 in mice and near the end of the third week of gestation in humans. At this time, the CNS appears as a slipper-shaped plate of thickened ectoderm known as the neural plate. This plate is located in the mid-dorsal region in front of the primitive pit.[3,17,66] These neuroepithelial or neural stem/progenitor cells generate neurons, astrocytes, and oligodendrocytes in a coordinated temporal and spatial fashion. These cells have positional identity, and their fate is influenced by local environmental cues from neighboring cells. In a reciprocal fashion, these cells influence their surrounding environment. Regionalization of these neuroepithelial cells is attained with morphogen gradients such as Shh, bone morphogenetic proteins, FGFs, retinoic acid, and Wnt proteins that control dorsal-ventral and rostral-caudal fate. When stem cells are isolated during various stages of development, their programmed fate will vary depending on the time and location of their isolation.
With this knowledge, naive neuroepithelial cells such as those generated in vitro from ESCs can be directed to a specific neuronal fate that is dictated by the presence of specific morphogens. For instance, ESC-derived neuroepithelial cells can be made to differentiate predominantly into dopaminergic neurons in the presence of FGF-8 and Shh (which confer midbrain dopamine neuronal identity), while the same cells can be made to generate spinal cord motor neurons in response to caudalizing signals such as retinoic acid. Cells will respond to these directive cues during a critical period, and if this period is passed, they tend to follow their intrinsic program of development.[65]
The potential benefit of ESC therapy was first noted in mouse studies. Mouse ESCs could be differentiated into neuroepithelial[54] and more specialized neuronal and glial subtypes such as midbrain dopamine neurons,[2,23,25] motor neurons,[56] and oligodendrocytes.[7] Moreover, these cells were used successfully in disease models. For example, mouse ESC-derived neurons and oligodendrocytes were shown to reverse the locomotive deficit of parkinsonian rats[2,25] and produce myelin in dysmyelinated or injured rat spinal cords.[7] This work in rodents led to experiments evaluating the potential of human ESCs in similar animal experiments. Human ESCs were initially isolated and shown to possess the potential to produce all cell tissue types of the body.[35,36,51,53] Techniques were developed to efficiently differentiate them in vitro into NPCs[10,34,67] and neuronal and glial subtypes.[27,29,33,57] This source of synchronized neuroectodermal cells provides a starting source that can produce any neural cell as well as offering an in vitro model for scrutinizing the mechanisms of neural induction and cell lineage specification in early human development.
Neural cells differentiated in vitro from hESCs exhibit broad cellular heterogeneity. The key in neuronal transplantation will be obtaining functionally effective cells that are restricted enough to "do the right thing" and resist doing harm, yet unrestricted enough that they can adapt to and integrate with the host environment in which they are placed. The neural differentiation process of hESC-derived NPCs appears to follow the intrinsic programmed temporal course of human neuronal and glial generation, while the ultimate neural subtype fate is largely influenced by the brain region in which the grafted cells reside.[1,12,14,16,50,60] For instance, grafted cells residing in neurogenic brain regions continue to divide, while those in nonneurogenic regions cease cell division.[16] Grafted cells dwelling in gray matter mostly become neurons, whereas those in the white matter differentiate exclusively into glial cells. No matter when the cell was isolated (embryonic or neural progenitor stage), neuronal subtype differentiation is also influenced by its final brain location, suggesting an environmental influence on neuronal differentiation.[12,14,16,47,50]
One primary objective in neuronal replacement is functional integration of the transplanted cell with the host’s endogenous neuronal circuitry, leading to improved function. Recent strides have been made in achieving these goals in preclinical animal studies. There is evidence that hESC-derived NPCs can mature, form synapses, and functionally integrate following transplantation. After injection into the brain of neonatal SCID (severe combined immunodeficiency) mice, synaptic proteins, expressed by differentiated human neurons derived from hESCs, can be demonstrated by confocal immunohistochemistry and immunoelectron microscopy,[16] suggesting potential functional communication between the transplanted cells or between endogenous and exogenous cells. Gage and colleagues[28] have shown that, after transplantation into the embryonic mouse brain, hESC-derived neural progenitors differentiate into mature neurons and functionally integrate into the host brain as demonstrated electrophysiologically. Recently, hESC-derived dopaminergic neurons have been shown to reverse the functional deficit in parkinsonian rats.[59]
Together, these findings support the notion that hESC-derived NPCs produced in vitro have the potential to functionally integrate with host neuronal circuits and restore functional neurological deficits. The site-specific migration and differentiation of hESC-derived neuroepithelial cells in the neonatal and adult brain environment presents an opportunity for directed functional differentiation prior to and/or after transplantation.[34,67]
Directing cell differentiation down a given path requires an attempt to recapitulate the temporal and spatial environment that the cell would normally experience in becoming that final cell type. This means exposing cells to proper concentrations and sequences of factors. To achieve this goal would require a complete understanding of development of the nervous system. Human ESCs, while offering a potential source for cell replacement therapy, are at present proving to be more beneficial in adding to our understanding of human neurodevelopment.
One disease priority in cell replacement research has been PD. Electrophysiologically active dopamine neurons have been generated from hESCs through strategic and systematic application of FGF-8 and Shh.[33,57] Importantly, these cells contribute to locomotive functional recovery in the 6-hydroxydopamine lesioned rat striatum 5 months after transplantation with improvement correlating to the dopamine neurons present in the graft.[37,59] Underscoring the importance of temporal exposure to a given morphogen is the observation that when cells are treated prior to expression of Sox1, they generate dopaminergic neurons with characteristics of midbrain projection dopamine neurons (large cell bodies, complex processes, and coexpression of En1) while treatment after Sox1 expression results in production of dopaminergic neurons that lack En1 expression. Dopaminergic neurons can also be differentiated by coculture of ESC-derived cells with stromal cells such as PA6,[8,24,32,48,62] MS,[33,39] and HepGII.[40]
Amyotrophic lateral sclerosis is a disease associated with the loss of motor neurons. Motor neurons can be specified from hESC-derived neuroectodermal cells in a manner similar to that for PD. Prior to, but not after, Sox1 expression, the neuroectodermal cells can be efficiently posteriorized by retinoic acid and, in the presence of Shh, can differentiate into spinal motor neurons that express HB9, HoxC8, choline acetyltransferase, and vesicular acetylcholine transporter, and are electrophysiologically active.[27] Transplantation of these cells into the developing chick embryo results in robust engraftment, maintenance of motor neuron phenotype, and long-distance axonal projections into peripheral host tissues. Transplantation into the spinal cords of adult rats yielded grafts with a large number of human motor neurons and outgrowth of choline acetyltransferase-positive fibers.[26]
Other cells produced from hESCs include serotonin neurons,[38] γ-aminobutyric adic neurons,[21,67] glutaminergic neurons,[10,67] and myelinating oligodendrocytes.[29] Current efforts are directed towards developing reproducible protocols for directing cells to the appropriate phenotypes.
Embryonic stem cells derived from in vitro fertilized embryos differ genetically from the patient’s own cells and thus could be rejected. One common method for overcoming immunological rejection after transplantation is immunosuppression. Long-term immunosuppression, however, is associated with multiple problems, including opportunistic infection. An alternative strategy involves generating ESCs that are genetically identical to the patients’ own cells.[22,63,65]
Somatic cell transfer allows trans-acting factors present in the mammalian oocyte to reprogram fully differentiated somatic cells to pluripotent stem cells that exhibit the essential characteristics of ESCs. Takahashi et al.[49] induced pluripotent stem cells from mouse fibroblasts by retroviral introduction of Oct3/4, Sox2, c-Myc and Klf4, and these iPSCs were similar to ESCs in morphological characteristics, capacity for proliferation, and tendency for teratoma formation. Okita and colleagues[30] were able to generate similar mouse iPSCs with increased ESC-like gene expression and DNA methylation patterns by selecting for Nanog expression. Successful reprogramming of differentiated human somatic cells into a pluripotent state may allow creation of patient- and disease-specific stem cells.
Recently, Rhesus macaque blastocysts were produced from adult skin fibroblasts using a modified somatic cell nuclear transfer approach, with successful isolation of 2 ESC lines. Deoxyribonucleic acid analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ESC morphological characteristics, expressed key stem-cell markers, were transcriptionally similar to control ESCs, and differentiated into multiple cell types in vitro and in vivo.[9]
In a recent paper, Yu and colleagues[61] were able to reprogram human somatic cell nuclei to pluripotent stem cells that exhibit the essential characteristics of ESCs using Oct4, Sox2, Nanog and Lin28. These human iPSCs have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize hESCs, and maintain the potential to differentiate into all three germ layers. These results, taken together, demonstrate proof-of-concept for so-called "therapeutic cloning." In the future, iPSCs may be produced without the need for viral transduction, and instead via nongenetic approaches.
A serious problem with the use of ESC-derived neural precursors for cellular replacement is the known tendency of undifferentiated ESCs to form tumors. Tumor formation by a transplanted cell population derived from hESCs is thought to be due to the presence of undifferentiated ESCs in the transplanted graft and the inherent ability of ESCs to produce all cell types in the body.[5] Brederlau and colleagues[6] reported "severe teratoma formation" in rats grafted with hESCs predifferentiated in vitro for 16 days. There have been other reports of cell overgrowth and tumor formation in rats receiving grafts derived from hESCs within a short period (8-13 weeks).[37,43] However, in similar investigations by the senior author (S. C. Z.), no evidence of tumor formation was found, as evidenced by lack of rosette structures within the graft and scant Ki 67 expression even 5 months posttransplantation.[59] We believe that the lack of tumor formation in these studies is secondary to the absence of contaminating ESCs in the original tissue graft. In other words, all grafted cells were committed to neural lineage, with no potential to form tissues of other germ layers. This laboratory uses a unique differentiation protocol that essentially eliminates undifferentiated cells.[67] Also, in these studies the hESCs are differentiated in vitro for a total of 7 weeks, a much longer time period than in other reports. Still, concerns over tumor formation are valid, and much work needs to be done before hESC-derived precursor cells will be considered safe for clinical studies.
To eliminate the possibility of tumor formation from ESC-derived progenitor cells, steps would need to be taken to ensure that grafts are free of undifferentiated ESCs. In other words, all grafted cells would be of a lineage stage similar to the NSCs transplanted in other described clinical studies. Strategies to achieve this include positively sorting out the target cells using cell surface markers or by using cell type specific transcription factors through homologous recombination or by use of promotors.[56,68] Alternatively, pluripotent stem cells could be removed using stem cell surface molecules.
In addition, human ESC-derived neuroepithelial cells are at a primitive stage of development. These cells tend to undergo many more cell cycles than fetal-derived NSCs. Therefore, human ESC-derived neural transplants are often associated with an overgrowth of neuroepithelia[37,43] but not with teratoma formation. To avoid such overgrowth, the number of primitive neuroepithelial cells used must be minimized. This may be achieved by expanding the neuropeithelia before transplant, breaking the neuroepithelial rosettes (which keep cells in a primitive stage), sorting the dividing population out, or eliminating the dividing cells with pharmacological agents.
Problems in addition to tumor formation and immune rejection that must be addressed include promotion of migration and dispersement of cells after transplantation, directed long-distance axonal projection, and controlled transmitter release.
Growth cone migration and synaptogenesis are part of a complex 2-way developmental process that cannot be recreated in the mature adult brain environment. Even with establishment of synaptic connections, replaced neurons may not secrete neurotransmitters in a controlled or regulated fashion due to faulty afferent inputs. Efforts are directed toward regulation of neurotransmitter release with means such as genetic manipulation. Another potential problem is the lack of dispersement and/or migration of cells after engraftment. Many of the described diffuse neurological conditions would ideally require dispersement of cells to cover virtually all regions within the brain. No cell has been shown to accomplish this well in mature animal models that are near human size. Human ESC-derived NPCs have perhaps the greatest capacity for dispersement and appear to preferentially migrate throughout the white matter.[16] Efforts to promote cell dispersement are aimed at manipulation of the transplanted cell and/or its environment.
Great strides have been made in the isolation, purification, and directed differentiation of hESCs. Neural progenitor cells derived from hESCs and produced in vitro show promise in animal models with the potential for functional integration and behavioral improvement. Investigations using ESCs have greatly improved our understanding of human nervous system development, brain tumor genesis, and pharmaceutical screening. The use of hESC-derived neural cells in patients is a distant goal. Much work still needs to be done in producing a purified source, promoting dispersement and integration, and ensuring safety.
The author: Professor Yasser Metwally
INTRODUCTION
April 17, 2009 — What is the current status of transplantation or implantation of neural or fetal tissue or stem cells in the treatment of Parkinson’s disease? What are the leading centers for such research and treatment?
The NIH has funded 2 studies of neurotransplantation in Parkinson’s disease (PD), one at Columbia University/University of Colorado (completed) and the other at Mt. Sinai in New York (near completion). In addition, a group in Tampa, Florida, and another in Sweden have been reporting individual cases. All these studies have used aborted human embryonic tissue. The preliminary findings indicate that:
Investigators from the Columbia-Colorado study presented their findings at the 1999 annual meeting of the AAN in Toronto, Ontario, Canada. They reported that the clinical benefit of neurotransplantation in patients with PD was modest and only occurred in patients younger than 50 years, that PET scans showed an increase in dopamine uptake in all patients, and that some patients who were taken off all drugs experienced dyskinesias (runaway dyskinesias) as a side effect.
Thus, we can draw several conclusions about neurotransplantation for PD:
The author: Professor Yasser Metwally
INTRODUCTION
April 16, 2009 — The reported evidence of neurodegeneration in multiple sclerosis (MS) may explain the lack of efficacy of the currently used immunomodulating modalities and the irreversible axonal damage, which results in accumulating disability. To date, efforts for neuroprotective treatments have not been successful in clinical studies in other CNS diseases. Therefore, for MS, the use of stem cells may provide a logical solution, since these cells can migrate locally into the areas of white-matter lesions (plaques) and have the potential to support local neurogenesis and rebuilding of the affected myelin. This is achieved both by support of the resident CNS stem cell repertoire and by differentiation of the transplanted cells into neurons and myelin-producing cells (oligodendrocytes). Stem cells were also shown to possess immunomodulating properties, inducing systemic and local suppression of the myelin-targeting autoimmune lymphocytes. Several types of stem cells (embryonic and adult) have been described and extensively studied in animal models of CNS diseases and the various models of MS (experimental autoimmune encephalomyelitis [EAE]). In this review, we summarize the experience with the use of different types of stem cells in CNS disease models, focusing on the models of EAE and describe the advantages and disadvantages of each stem cell type for future clinical applications in MS.
Multiple sclerosis (MS) is a chronic inflammatory multifocal demyelinating disease of the CNS that affects predominantly young adults. MS is the main cause of chronic neurological disability in this age group. While its pathogenesis is still obscure, and multiple (genetic, environmental and infectious) factors seem to be involved in it, it is widely accepted that the final pathogenetic pathway is that of an autoimmune attack against myelin components. Additional mechanisms have been lately undercovered, including damage of the axons in the CNS and a degenerative process, which is probably the result of inflammation, causing accumulating and irreversible damage with time.[1] Naturally, treatment approaches for MS focus on targeting the immune system, either in a nonspecific way (systemic immunosuppression with cytotoxic agents) or through immunomodulation (to specifically downregulate the myelin-reactive autoimmune lymphocytes or to enhance the regulatory immune networks) in order to control the inflammatory process, which, as mentioned, causes demyelination.[1] Unfortunately, currently existing treatments for MS (both the immunosuppressive ones and the immunomodulating, i.e., glatiramer acetate and IFN-ß) are only partially effective, likely owing to the limited ability of the prescribed medications to exert a significant in situ immunomodulation in the areas of lesions in the CNS, paralleled by a deficiency in growth-factor production and insufficient numbers or mobilization of the resident CNS stem cells.[2,3]
Therefore, it is obvious that, in order to improve treatment outcome in MS, innovative approaches are required for immune regulation rather than nonselective immunosuppression, as well as therapeutic interventions that may offer effective in situ immunomodulation and neuroprotection.
Extensive studies have provided strong evidence for neurodegeneration in MS, including: the finding of amyloid precursor protein accumulation in neurons;[4] a reduction in N-acetylaspartate/creatine ratio in magnetic resonance spectroscopy, which correlates well with the degree of disability,[5] the finding of axonal ovoids/transected axons at the edge and the core of active lesions[6] and of oxidative damage in mitochondrial DNA and impaired activity of mitochondrial enzyme complexes;[7] the reduction in axonal density in normal-appearing white matter (NAWM) early in MS; and a more prominent reduction of axonal density in spinal cord NAWM in progressive MS patients.[8,9]
A logical treatment approach to enhance neuroprotective mechanisms and to induce neuroregeneration in MS is with stem-cell transplantation.[2,3] Stem cells are a diverse group of multipotent cells. In general, these cells are relatively undifferentiated and unspecialized, and can give rise to the differentiated and specialized cells of the body. All stem cells exert two characteristic features: the capacity for self renewal and preserving a pool of undifferentiated stem cells; and the potential to produce various differentiated cell types. There are different kinds of stem cells that can be isolated from embryonic and adult tissues. Embryonic stem cells (ESCs) are cells derived from the inner cell mass of embryos[10,11] at the blastocyte stage (5-9 days after fertilization). The only source for human stem cells is from embryos obtained from in vitro fertilization. Adult stem cells represent a more differentiated cell population than ESCs, and can be isolated from various tissues, including muscle,[12] adipose tissue,[13] CNS (neural stem cells [NSCs])[14,15] and bone marrow (mesenchymal stromal cells [MSCs]).[16,17] A distinct population of non-tissue-specific multipotent adult stem cells (MAPCs) have also been isolated from the bone marrow, muscles and CNS.[18] All of the previously described stem cells carry a potential for tissue repair. Theoretically, the use of ESCs and adult NSCs might represent the optimal source for cell-replacement therapies in CNS disorders such as MS.
In the current review, the various types of stem cells, which were mainly studied in animal models, will be reviewed as a potential therapeutic approach for MS. The main and common mechanisms of action of all stem cells include induction of neuroregeneration and remyelination through the activation of resident stem cells, or production of new CNS cell lineage progenitors, paralleled by local and systemic immunomodulating effects.
Experience With Various Types of Stem Cells in Animal Models
Embryonic stem cells
As mentioned above, ESCs derived from the inner cell mast of the blastocyte are capable of giving rise to cells from all three germ layers. ESCs were shown to differentiate in vitro into several cell types of the body including neuronal stem cells. The actual fate of their differentiation is determined by growth factors, chemical agents and neurotrophic factors, such as EGF, FGF, brain-derived neurotrophic factor (BDNF) and retinoic acid.[19-23] ESCs express distinct surface markers, such as Oct-4, Sox-2 and SSEA-1/2/3/4. Several studies have demonstrated the ability of these cells to differentiate into myelin-producing cells (oligodendrocytes)[24-32] and neurons,[22,33] therefore becoming good candidates for neuroregeneration and remyelination in diseases such as MS. In a recent study, it was found that neurospheres derived from ESCs obtained from ILRIL6 chimeras (soluble IL-6 receptor fused to IL-6) exhibit an enhanced differentiation into oligodendrocytes with more branches and peripheral accumulation of myelin-basic protein in myelin membranes.[34] Transplantation of differentiated oligodendroglial progenitors derived from ESCs into the shiverer mouse model of dysmyelination resulted in integration, differentiation into oligodendrocytes and compact myelin formation.[27] When transplanted in rodent models of induced demyelination, ESCs were able to differentiate into glial cells and re-ensheath demyelinated axons in vivo.[30,31]
ESCs were tested as a therapeutic approach in various neurological disease models such as Parkinson’s disease (PD),[35] stroke[36] and muscle dystrophy,[37] as well as in models of organ failure, such as liver failure[38] and cardiac infarctions.[39]
PD is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ESCs are currently the most promising donor cell source for cell-replacement therapy in PD. Successful production of DA neurons from ESCs has been shown in animal models of PD.[40-47] In a study by Kim et al., it was demonstrated that a highly enriched population of midbrain NSCs can be derived from mouse ESCs.[40] The DA neurons generated by these stem cells displayed electrophysiological and behavioral properties expected of midbrain neurons. Takagi et al. generated neurospheres composed of neural progenitors from monkey ESCs, which are capable of producing large numbers of DA neurons.[35] They demonstrated that FGF20, which is preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ESC-derived neurospheres. The effect of transplantation of DA neurons generated from monkey ESCs into 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, a primate model for PD, revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.
ESCs were also tested in models of spinal cord injury.[28,30,48-52] In a study by Mcdonald et al., neural differentiated mouse ESCs were transplanted into a rat spinal cord 9 days after traumatic injury.[30] The transplant-derived cells survived and differentiated into astrocytes, oligodendrocytes and neurons, and migrated as far as 8 mm away from the lesion edge. Clinically, ESC-transplanted animals showed an accelerated recovery of the injured spinal cord.
One of the earliest applications of ESC transplantation was in rat models of stroke. Since 1992, studies have demonstrated graft survival and even evidence of functional connection with the host brain.[53-58] These early reports determined parameters for future work in stroke, but ultimately had limited efficacy and did not progress to clinical trials. A variety of cell types have been tested for restoration of brain function after stroke, mostly in rodent models. ESCs were used in a recent study by Buhnemann et al..[36] An induction of focal cerebral ischemia by transient middle cerebral artery occlusion in rats was used as a model of stroke. Grafted ESCs were transplanted and were shown to survive within the infarct core for up to 12 weeks after transplantation and to differentiate with a high yield into mature glial cells and neurons of diverse neurotransmitter subtypes. The transplanted cells demonstrate characteristics of electrophysiologically functional neurons with voltage-gated sodium currents that enabled these cells to fire action potentials. Additionally, during the first 7 weeks after transplantation spontaneous excitatory postsynaptic currents were observed in graft-derived cells, indicating synaptic input.[36]
Although the above results were encouraging, researchers have underlined that ESCs could be a ‘double-edged sword’, since they may cause the formation of a non-homologous implant and teratomas within the organ of transplantation.[31,59,60] Immune rejection by the host immune system has been considered to be one of the greatest hurdles for cellular transplantation of ESCs. However, recent data have indicated that ESCs may exhibit immune-privileged properties,[60,61] and may escape rejection through immunotolerance. It was found that the injection of ESCs into immunocompetent mice did not induce an immune response against them. Undifferentiated and differentiated ESCs did not stimulate the proliferation of alloreactive primary human T cells and inhibited third-party allogeneic dendritic cell-mediated T-cell proliferation.
Adult Stem Cells
Adult stem cells are undifferentiated cells that can be found in various body tissues and have the ability to divide, migrate and regenerate damaged tissues. These cells are also known as somatic stem cells, and can be isolated from both fetuses and adults. They can be harvested from adipose tissue,[13] bone-marrow (hematopoietic stem cells [HSCs],[17,62,63] MSCs[16,64-66] and MAPCs[18,32,67,68]), mammary tissue,[69-71] CNS (NSCs and neurospheres),[22] olfactory bulb[72] and others. Several studies suggest that these ault stem cells may have neuroregenerative, and in some cases (especially in neuronal and bone marrow-derived stem cells) immunomodulatory, potential.[2,73-76]
Adult neural stem cells. Adult NSCs or CNS NSCs are cells that are cultured as sparse adherent cells or as aggregates of floating cells termed neurospheres in serum-free medium on a nonadherent surface in the presence of EGF and FGF2.[77,78] It is accepted that in adult mammalian brains there are two sites of cell division in which NSCs have been isolated for in vitro use: the subventricular zone and the subgranular zone of the dentate gyrus of the hippocampal formation.[79-84] NSCs express markers such as Nestin polysialylated form of the neural cell adhesion molecule and Sox2. These cells, as their name implies, can give rise to neural, astrocytic and oligodendrocytic precursors that can, in turn, differentiate into neurons, astrocytes and oligendrocytes.[22] Based on their ability for neural and glial cell generation, NSCs were tested in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). In a study by Pluchino et al., it was shown that adult NSCs cultured and injected into EAE mice, intravenously or intracerebroventricularly, could migrate into the demyelinating CNS area and differentiate into mature brain cells.[85] It was noticed in this study that oligodendrocyte progenitors were especially increased in the areas of the lesions in the CNS. Clinically, EAE symptoms were strongly downregulated in the transplanted animals. In additional studies by Ben-Hur et al.[86] and Einstein et al.[87] from our laboratory, it was found that transplanted neural precursor neuroshperes (intra-cerebroventricularly) migrated into the inflamed white matter in EAE, attenuated the severity of clinical signs and reduced brain inflammation. In these studies, it was also shown that when neural precursors were administered intravenously, EAE was suppressed by a peripheral immunosuppressive effect, which inhibited T-cell activation and proliferation in the lymph nodes.[2,87] Moreover, it was demonstrated that the transplanted neural precursor cells could downregulate the inflammatory brain process in situ, as indicated by the reduction in the number of perivascular infiltrates and of brain CD3+ T cells, a reduction in the expression of ICAM-1 and lymphocyte function-associated antigen-1 in the brain, and an increase in the number and proportion of regulatory T cells in the CNS.[87] The latter may indicate an active immunoregulation induced by the neural progenitor cells (NPCs).
Regarding the migration of NSCs to the inflammation area, Belmadani et al. reported that using hippocampal slice cultures, grafted NPCs migrate toward areas of neuroinflammation and that chemokines are a major regulator of this process.[88] Migration of NPCs was tested following injection of a cocktail of proinflammatory agents (including the cytokines TNF-a and IFN-?, the bacterial toxin lipopolysaccharide and the HIV-1 coat protein glycoprotein 120, or a ß-amyloid-expressing adenovirus) into the area of the fimbria and subsequent transplantion of enhanced green fluorescent protein (EGFP)-labeled NPCs into the dentate gyrus of cultured hippocampal slices. In slices injected with this proinflammatory cocktail, EGFP-NPCs migrated towards the site of the injection in contrast to nonstimulated slices. It was demonstrated that the inflammatory stimuli increased the synthesis of chemokines and cytokines in the hippocampal slices. When EGFP-NPCs obtained from chemokine receptor CCR2 knockout mice were transplanted, they exhibited a weak migration ability towards the sites of inflammation. Similarly, wild-type EGFP-NPCs exhibited weak migration when transplanted into slices prepared from MCP-1 knockout mice. These data indicate that ‘danger’ signals secreted by cells at the sites of neuroinflammation attract neural progenitors and suggest that chemokines such as MCP-1 play an important role in the process of migration.
In PD, which is characterized by an extensive loss of DA neurons in the substantia nigra pars compacta and their terminals in the striatum, NSCs may provide an alternative tissue source that does not share the same ethical concerns as ESCs[89-93] and can give rise to progenitor cells capable of extended self-renewal and able to generate neurons and glial cells. Several studies have demonstrated the ability of NSCs, not only to migrate into the affected brain areas, but also to induce the production of DA neurons, resulting in improvement of behavioral deficits in animal models of PD.[89-93]
Spinal cord injuries are frequently responsible for permanent neurological disability leading to irreversible paralysis of the extremities. Unfortunately, effective therapies to restore spinal cord function are not yet available. Depending on their features, NSCs may provide a promising approach for spinal cord reconstruction.[52,94-99] Pallini et al. transplanted NSCs derived from mouse embryos in mice where an extended dorsal funiculotomy had been performed at the T8-T9 level.[97] At intervals from 4 to 12 weeks after grafting, motor behavior was assessed using an open-field locomotor scale and footprint analysis. It was found that by the 12-week time point, mice engrafted with NSCs significantly improved both their locomotor score on an open-field test and their base of support on footprint analysis. Ogawa et al. showed that, following in vitro expansion and transplantation of the cells at the appropriate time point, NSCs derived from rat fetal spinal cord could divide and differentiate into neurons in vivo and integrate into the host tissue in the injured spinal cord.[98]
Despite these promising results, NSCs are still not considered as the perfect stem cell population for cell-replacing therapy and are associated with significant drawbacks. First, it is difficult to culture neurospheres from regions of the adult brain that do not normally undergo self-renewal.[100] Second, although NSCs can be propagated for extended periods of time and differentiated into both neuronal and glial cells, recent studies suggest that this behavior is induced by the culture conditions of the progenitor cells and might be restricted to a limited number of replication cycles in vivo.[101] Furthermore, neurosphere-derived cells do not necessarily behave as stem cells when transplanted back into the brain.[102] Additional concerns include the potential for immune rejection of NSCs, the danger of tumor development in the host brain and various ethical aspects related to the donor tissue origin.
Hematopoietic stem cells. HSCs are the main stem cells of the bone marrow. These cells typically express the surface marker phenotypes of CD34+, CD133+, CD45+ and CD38-. HSCs are the precursor cells that give rise to all types of blood cells, including T cells, B cells, natural killer (NK) cells, macrophages, red blood cells, granulocytes and other monocytes.[17,62,63,103]
Several studies have shown the ability of HSCs to transdifferentiate into CNS cells including neurons, astrocytes and oligodendrocytes.[104-107] Hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT) is widely used in hematological malignancies. During the last few years HSCT and BMT have been tested in animal models of autoimmunity[108-114] and subsequently applied in human diseases such as MS.[115-119] The rationale for this application has been provided by studies from our group,[108-112] which have shown that high-dose cyclophosphamide for the elimination of immunocompetent lymphocytes, followed by syngeneic BMT rescue, could suppress chronic EAE and induce tolerance to the immunizing antigens. Initial clinical trials with HSCT in MS have shown promising results.[115-119] However, it is important to emphasize that this treatment approach is not intented to evaluate the transdifferentiation potential of HSCs; the main treatment modality in the HSCT protocol is the cytotoxic regimen, which ablates and resets the immune system and the use of HSCs is only to ‘rescue’ patients and induce reconstitution of the immune system.[119,120]
The apparent problem with using HSCT or BMT in autoimmunity is the need for strong (lethal) immunosuppresion, which is associated with significant morbidity and mortality. The use of lower doses of immunosuppression is not only less efficient, but may actually provoke relapses of EAE.[121-123] In addition, despite the significant effects of such protocols in suppressing the inflammatory activity of MS, as evidenced by MRI studies,[116-118] the clinical efficacy still was not equally impressive, especially in the progressed stages of MS. HSCT suppressed the inflammatory lesions of MS as shown on MRI and induced clinical stabilization in more than 60% of patients,[116-119] but it did not prevent the progression of brain atrophy.[124,125]
Moreover, the procedure-related mortality (solely caused by the cytotoxic conditioning and the associated immunodeficiency state) in these studies exceeded 5%, which seems to be unacceptable for the majority of MS patients. A randomized controlled clinical trial with HSCT versus immunosuppressive modalities in MS is underway.[116]
Mesenchymal stem cells MSCs are another important member of the bone marrow stem cell repertoire. These cells are described as nonhematopoietic stromal cells and their classic role is to support the process of hematopoiesis and HSC engraftement and to give rise to cells of mesodermal origin, such as osteoblasts, adipocytes and chondrocytes.[16,64-66] These cells do not have a specific surface marker profile, but it is widely accepted that they are negative for CD34, CD45 and CD14, and positive for CD29, CD73, CD90, CD105 and CD166. Recent studies have depicted some new roles of MSCs: the ability to transdifferentiate cells of endodermal and ectodermal origin,[126-128] including possible neural transdifferentiation[126,129,130] and immunomodulating properties.[73,75,131-151] It was shown that MSCs under different culture manipulations, whether by chemical induction (e.g., using dimethylsulfoxide, butylated hydroxy anisole and retinoic acid) or by the use of growth factors (e.g., BDNF, GDNF, FGF and EGF), can give rise to neural-, glial- and astrocytic-like cells in vitro.[126,129,130] Besides, MSC subpopulations may express a variety of neuroregulatory molecules and promote neuronal cell survival and neuritogenesis.[133] In a study by Crigler et al., it was shown that a subpopulation of MSCs coexpress neurotrophins (e.g., BDNF and NGF) and other potent neuroregulatory molecules, which contribute to MSC-induced effects on neuronal cell survival and nerve regeneration.[133] MSCs were additionally found to possess significant immunomodulating properties; they were found to suppress in vitro T- and B-cell functions and NK cells.[134-139] The exact mechanism for this MSC-mediated immunosuppression is not fully understood. Two mechanisms were suggested to explain the immunomodulatory effects: soluble factors and cell-to-cell contact-dependent mechanisms.[131,134,135,140-148] The soluble factors, TGF-ß1,[149] IFN-?,[145,150] indoleamine 2,3-dioxygenase (IDO)[146] and prostaglandin E2[135,140] have been suggested to be involved in this process. In a study by Krampera et al., it was shown that MSCs suppress the proliferation of both CD4+ and CD8+ T lymphocytes, as well as of NK cells, whereas they do not have an effect on the proliferation of B lymphocytes.[145] The suppressive activity of MSCs was not contact-dependent and required the presence of IFN-?. In the presence of IFN-?, activated B cells became susceptible to the suppressive activity of MSCs. The downregulating effect of IFN-? was related to its ability to stimulate the production of IDO activity by MSCs, which in turn inhibited the proliferation of activated T or NK cells. Cell-to-cell contact mechanisms appear to be less likely involved in the immunomodulatory actions of MSCs.[143,145,151]
These immunomodulating features of MSCs led investigators to evaluate the effect of MSC transplantation in the EAE model of MS.[152-154] In a study by Zappia et al., it was demonstrated that intravenous injection of MSCs in C57BL/6J mice immunized with the peptide 35-55 of myelin oligodendrocyte protein (MOG) significantly dowregulated the clinical severity of EAE, subsequently decreasing CNS inflammation and demyelination.[152] The findings were explained by the induction of T-cell anergy, which occurred at the level of lymphoid organs where MSCs seemed to engraft. In an additional study by Zhang et al., it was shown that intravenous administration of MSCs could suppress proteolipid protein (PLP)-induced EAE in SJL mice;[153] MSCs migrated into the CNS where they promoted BDNF production and induced proliferation of a limited number of oligodendrocyte progenitors. In a recent study by Gerdoni et al., SJL mice, in which EAE was induced with PLP, were injected intravenously with MSCs.[154] The treated mice showed a significantly milder disease and fewer relapses compared with control mice, with a decreased number of inflammatory infiltrates and reduced demyelination, and axonal loss in the CNS. No evidence of green fluorescent protein-labeled neural cells was found inside the brain parenchyma, thus not supporting the hypothesis of MSC transdifferentiation. In vivo, PLP-specific T-cell response and antibody titers were significantly lower in MSC-treated mice. When adoptively transferred, encephalitogenic T cells activated against PLP in the presence of MSCs induced a milder disease compared with that induced by untreated encephalitogenic T cells.[152-154] These cells showed decreased production of IFN-? and TNF-a and did not proliferate on antigen recall, and thus were considered anergic.
Along with these important immunomodulatory features, there are also recent results indicating MSC plasticity and differentiation potential. This might contribute to remyelination and myelin recovery in demyelinating disorders. In rats with an induced focal demyelinated lesion of the spinal cord, intravenous or brain injection of acutely isolated MSCs resulted in remyelination.[155] In a study by Inoue et al., separated bone marrow mononuclear cells were transplanted either intravenously or focally into rats with a demyelinated lesion of the spinal cord.[155] Both injection routes were shown to be effective in inducing remyelination.
We have also tested the therapeutic potential of bone marrow-derived MSCs in the chronic progressive model of EAE induced in C57BL/6 mice with the MOG 35-55 peptide. This model is more relevant for human MS, since it has substantial clinicopathological similarities (including a chronic course and an extensive axonal damage) with the human disease.
To test the neurodifferentiation potential of MSC purified from whole bone marrow, cells (passage 2-3) were cultured with a cocktail of growth factors, containing FGF and BDNF. MSCs cultured under these conditions showed morphological changes towards cells with thin and long processes resembling neural- and glial-like cells. In each differentiation set, there were positive immunostaining for the neuronal-lineage cell markers Nestin (neural marker), tubulin ß-III (neural marker), GFAP (astrocyte marker) and O4 (oligodendrocyte marker) [156]. When injected in mice with chronic EAE induced by the MOG 35-55 peptide, MSCs showed a strong migratory potential to the white-matter lesions, which was in correlation with the degree of inflammation. The clinical course of EAE was significantly ameliorated in animals treated with purified MSCs, following both intracerebroventricular and intravenous administration. In histopathological evaluation of the treated EAE mice, a reduction in lymphocytic infiltrations was observed with a significant preservation of the axons, which was more profound in the intracerebroventricular injection protocol.[156]
Purified MSCs showed immunomodulatory properties and downregulated myelin-sensitized lymphocytes (obtained from EAE mice) in the presence of the MOG peptide or the mitogen ConA.
Clinical Experience With MSCs
The use of bone marrow-derived MSCs provides several advantages over conventional neuronal, embryonic and hematopoietic stem cells:
They can be obtained from the adult bone marrow
They can be easily cultured and expanded in large numbers
They can be injected autologously without the need of immunosuppressive means to prevent rejection
They are less prone to genetic abnormalities during multiple in vitro passages, possessing a low risk for induction of malignancies as compared with other types of stem cells
However, the future use of MSCs derived from the patient themselves should be carried out with caution in diseases where genetic factors play a significant pathogenetic role (e.g., in Alzheimer’s disease), since these stem cells may be affected by the same genetic dysregulation.
The clinical use of purified MSCs or other bone marrow- derived stem cells (reviewed by Giordano et al. [157]) has revealed the feasibility and safety of this method of application of stem cell therapy and indicated some evidence of efficacy in various medical conditions, as described later.
Myocardial Infarction & Cardiac Failure
A pivotal trial in acute myocardial infarction (MI) was carried out by Chen et al..[158] A total of 69 patients within 12 h of onset of infarction were randomized to receive MSC transplantation (n = 34) or saline treatment (n = 35) after percutaneous coronary intervention (PCI). Wall movement velocity over the infarcted area increased significantly in cell therapy-treated patients, but not in the control group. Also, left ventricular ejection was higher in the cell therapy group compared with controls.[158] Another randomized trial (Bone Marrow Transfer to Enhance ST-Elevation Infarct Regeneration [BOOST]) tested the effectiveness of intracoronary injection of autologous bone marrow cells in acute MI.[159] The investigators enrolled 60 patients following PCI, who were randomly assigned to receive either classical postinfarction treatment or bone marrow cell therapy. Bone marrow nucleated cells were injected 4-8 days post-PCI (approximately 24 × 108 cells/kg) into the infarcted artery by a balloon catheter. Compared with the control group, patients in the cell therapy group had increased left-ventricular ejection fraction (LVEF) and systolic wall motion 6 months after transplantation.[159]
Katritsis et al. injected intracoronary autologous bone marrow-derived mononuclear cells (containing culture-expanded MSCs along with endothelial progenitors) in 11 patients with MI, following angioplasty and stent implantation.[160] Patients with both recent and old anteroseptal MI were enrolled in this study. In five of 11 patients in the transplantation group, there was improvement of myocardial contractility in one or more previously nonviable myocardial segments. All of the aforementioned results, along with similar studies,[161] demonstrate that cell therapy with bone marrow cells is feasible, safe and may contribute to regeneration of myocardial tissue following infarction.
A study performed by Perin et al. evaluated the hypothesis that transplants of bone marrow mononuclear cells in patients with end-stage ischemic heart disease may promote neovascularization and prevent impairment of heart functionality.[161] A total of 2 months after treatment, they observed a significant reduction in total reversible defect and improvement in global left ventricular function within the treatment group and between this and the control group. The 4-month follow-up revealed an improvement in ejection fraction and a reduction in end-systolic volume in the treated patients[161]. The same research team further evaluated the effectiveness of their cell therapy protocol by evaluating patients with severe ischemia 6 and 12 months after transendocardial injection of autologous bone marrow cells. They showed that total reversible defect, detected by single photon emission computed tomographic perfusion scanning, was reduced in the cell therapy group compared with controls. Moreover, at 12 months, exercise capacity was significantly improved in cell therapy-treated patients.[162]
In contrast to the mentioned studies, other studies showed bone marrow cells to be less effective.[163-165] In the study by Janssens et al.,[163] Ficoll-separated bone marrow cells were injected after acute MI. During the 4 months of follow-up, no significant differences in overall LVEF were found, although the infarct size was reduced. Lunde et al. did not find any improvement on LVEF in groups treated with mononuclear BMC (the Autologous Stem Cell Transplantation in Acute Myocardial Infarction [ASTAMI] trial).[164] Similarly, in a recent study by Meyer et al., it was found that a single dose of intracoronary BMCs did not provide long-term benefit on left ventricular systolic function after a MI;[165] however, an acceleration of LVEF recovery is suggested after bone marrow cell treatment. These controversies between the studies might be explained by the technical issues concerning the purification, characterization and infusion methods of BMC.[166,167]
Osteogenesis Imperfecta
Based on the classical properties of MSCs, a logical approach for treatment of osteogenesis imperfecta (OI) is by cell therapy with MSCs. Preclinical experiments carried out on animal models showed that transplanted MSCs migrated and became incorporated into the bone and muscle of recipient animals,[168] demonstrating the potential of allogenic bone marrow (whole bone marrow cells) transplantation for children in severe OI. Three children with OI were intravenously infused with unmanipulated bone marrow cells (5.7-7.5 × 108 cells/kg) from HLA-identical or single antigen-mismatched siblings after they received ablative conditioning therapy.[169] Engraftment was associated with improvement in bone histology evaluated at 216 days post transplantation. There was also an increase in the total body mineral content tested by dual energy x-ray absorptiometry.[164] In an extension study, the same authors enrolled seven children with OI, five of whom underwent cell therapy treatment and two were in the control group.[170] The study revealed growth acceleration for the children in the cell therapy group 6 months after the transplantation, in contrast to retarded growth for age-matched controls.
Hurler Syndrome/Leukodystrophies
To improve the efficacy of cell transplantation for Hurler syndrome and metachromatic leukodystrophy, Koc et al. infused allogenic MSCs into patients suffering from such diseases. The authors hypothesized that after implantation, MSCs could migrate to bones, cartilages, peripheral nervous system and CNS, and repair these tissues.[171] A total of 2-10 × 106 cells/kg were infused intravenously. No infusion-related toxicity was observed. In four patients with metachromatic leukodystrophy, a significant improvement in nerve conduction velocities could be detected. However, there was no improvement of the mental and physical status.
Lazarus and Koc et al. tested the hypothesis that MSC infusions could improve recovery of cancer patients receiving myeloablative therapy.[172-176] In a Phase I/II clinical trial, they enrolled 32 patients with locally advanced or metastatic breast cancer who were eligible for high-dose chemotherapy and peripheral blood progenitor cell (PBPC) transplantation. Upon enrollment, 35 days before chemotherapy and PBPC transplants, bone marrow aspirates were collected from patients and MSC cultures were prepared. All patients were discharged from the hospital and only one patient died within 100 days of the transplant. The authors concluded that MSC infusion at the time of PBPC transplantation is feasible and safe and the prompt hematopoietic recovery suggests that MSC treatment may have a positive impact on recovery of patients after high-dose chemotherapy.
Other Ongoing Trials
Several other clinical trials with MSCs are ongoing worldwide. At the National Cancer Institute (OH, USA), a Phase I trial is currently studying the side effects and best dose of donor MSCs in patients with acute or chronic graft-versus-host disease (GVHD) following a donor stem cell transplant. In a similar Phase I/II trial at the Christian Medical College (India), the role of MSCs in the treatment of GVHD is being evaluated. At the St Jude Children’s Research Hospital (TN, USA) a pilot study for MSCs as a therapy for OI is recruiting patients. At the Shaheed Beheshti Medical University (Iran), a Phase I/II study is ongoing for treatment of patients with end-stage liver disease with autologous MSCs.
Explorative Clinical Trials With MSCs in MS & Amyotrophic Lateral Sclerosis
Our data and similar publications that have evaluated the immunological properties and the therapeutic potential of bone marrow-derived MSCs in animal models, together with the accumulating data from the aforementioned clinical studies, showing the safety and efficacy of MSC injection in various conditions,[177] seem to justify a small explorative trial with MSCs in MS and amyotrophic lateral sclerosis (ALS). We have suggested a treatment protocol involving the intrathecal and intravenous administration of autologous MSCs taken from the patients’ bone marrow. The protocol was approved by the Helsinki Committee of our hospital and is underway. Initial experience in seven patients with ALS and spinal trauma treated with our protocol confirmed the safety of the procedure (no significant side effects in 1-year follow-up) and showed some indications of clinical efficacy.[178] An additional Phase I/II trial with intravenous administration of MSCs in MS is underway in Cambridge, UK. A recent study from Italy with seven ALS patients in whom the MSCs were injected in the thoracic spine has also indicated the feasibility and efficacy of the use of MSCs in neurodegeneration.[177]
Expert Commentary
In summary, the use of stem cells may open a new era in the management of MS and other neurodegenerative and neuroimmunological disorders. In the case of MS, the potential of stem cells to migrate into the affected inflammatory CNS areas seems to be the key element for their possible efficacy by inducing local immunomodulation (which is usually inefficient by other means, i.e., systemic administration of immunomodulating drugs) and neuroprotection, accelerating and enhancing the remyelinating mechanisms through induction of growth factor production and activation of the resident stem cell repertoire. The possibility of transdifferentiation into myelin-producing cells or neurons theoretically exists and is supported by several lines of evidence demonstrating that the transplanted cells express markers of neurons and oligodendrocytes in animal models. Others have suggested that fusion mechanisms may be involved and not real transdifferentiation. If this is the case, fusion mechanims may also be of benefit, inducing the rejuvenation of partially damaged CNS cells. Adult stem cells appear to be more appropriate for human use, since they are associated with less danger and ethical problems. Specifically, bone marrow-derived MSCs offer the best compromise and ease of use. Systemic administration may induce strong peripheral immunomodulating effects, but administration directly into the cerebrospinal fluid may offer additional advantages. The only way to have more answers to this exciting treatment approach is by Phase I/II controlled clinical trials.
Five-year View
There is definitely enough preclinical data to justify a clinical trial, epecially with bone marrow-derived MSCs in patients with MS and other neurodegenerative diseases, such as ALS, Alzheimer’s disease, PD and CNS injury. A clinical trial at the Hadassah hospital (Israel) in MS and ALS and spinal injury patients has already began. During the next 5 years results from a controlled study (at least in MS) should be available.
Key Issues
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References
Steinman L. Multiple sclerosis: a two-stage disease. Nat. Immunol. 2(9), 762-764 (2001).
Einstein O, Grigoriadis N, Mizrachi-Kol R et al. Transplanted neural precursor cells reduce brain inflammation to attenuate chronic experimental autoimmune encephalomyelitis. Exp. Neurol. 198(2), 275-284 (2006).
Einstein O, Ben-Menachem-Tzidon O, Mizrachi-Kol R et al. Survival of neural precursor cells in growth factor-poor environment: implications for transplantation in chronic disease. Glia 53(4), 449-455 (2006).
Gehrmann J, Banati RB, Cuzner ML, Kreutzberg GW, Newcombe J. Amyloid precursor protein (APP) expression in multiple sclerosis lesions. Glia 15(2), 141-151 (1995).
Foong J, Rozewicz L, Davie CA et al. Correlates of executive function in multiple sclerosis: the use of magnetic resonance spectroscopy as an index of focal pathology. J. Neuropsychiatry Clin. Neurosci. 11(1), 45-50 (1999).
Trapp BD, Peterson J, Ransohoff RM et al. Axonal transection in the lesions of multiple sclerosis. N. Engl. J. Med. 338(5), 278-285 (1998).
Vladimirova O, O’Connor J, Cahill A et al. Oxidative damage to DNA in plaques of MS brains. Mult. Scler. 4(5), 413-418 (1998).
Lovas G, Szilagyi N, Majtenyi K, Palkovits M, Komoly S. Axonal changes in chronic demyelinated cervical spinal cord plaques. Brain 123(Pt 2), 308-317 (2000).
Evangelou N, Esiri MM, Smith S, Palace J, Matthews PM. Quantitative pathological evidence for axonal loss in normal appearing white matter in multiple sclerosis. Ann. Neurol. 47(3), 391-395 (2000).
Heins N, Englund MC, Sjoblom C et al. Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells 22(3), 367-376 (2004).
Conley BJ, Young JC, Trounson AO, Mollard R. Derivation, propagation and differentiation of human embryonic stem cells. Int. J. Biochem. Cell Biol. 36(4), 555-567 (2004).
Alessandri G, Pagano S, Bez A et al. Isolation and culture of human muscle-derived stem cells able to differentiate into myogenic and neurogenic cell lineages. Lancet 364(9448), 1872-1883 (2004).
Gimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 5(5), 362-369 (2003).
Burnstein RM, Foltynie T, He X et al. Differentiation and migration of long term expanded human neural progenitors in a partial lesion model of Parkinson’s disease. Int. J. Biochem. Cell Biol. 36(4), 702-713 (2004).
Bottai D, Fiocco R, Gelain F et al. Neural stem cells in the adult nervous system. J. Hematother. Stem Cell Res. 12(6), 655-670 (2003).
Lennon DP, Caplan AI. Isolation of human marrow-derived mesenchymal stem cells. Exp. Hematol. 34(11), 1604-1605 (2006).
Bernstein ID, Andrews RG, Rowley S. Isolation of human hematopoietic stem cells. Blood Cells 20(1), 15-23; discussion 24 (1994).
Jiang Y, Vaessen B, Lenvik T et al. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp. Hematol. 30(8), 896-904 (2002).
Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI. Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168(2), 342-357 (1995).
Strubing C, Ahnert-Hilger G, Shan J et al. Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons. Mech. Dev. 53(2), 275-287 (1995).
Fraichard A, Chassande O, Bilbaut G et al. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell Sci. 108(Pt 10), 3181-3188 (1995).
Reubinoff BE, Itsykson P, Turetsky T et al. Neural progenitors from human embryonic stem cells. Nat. Biotechnol. 19(12), 1134-1140 (2001).
Benzing C, Segschneider M, Leinhaas A, Itskovitz-Eldor J, Brustle O. Neural conversion of human embryonic stem cell colonies in the presence of fibroblast growth factor-2. Neuroreport 17(16), 1675-1681 (2006).
Kang SM, Cho MS, Seo H et al. Efficient induction of oligodendrocytes from human embryonic stem cells. Stem Cells 25(2), 419-424 (2007).
Billon N, Jolicoeur C, Raff M. Generation and characterization of oligodendrocytes from lineage-selectable embryonic stem cells in vitro. Methods Mol. Biol. 330, 15-32 (2006).
Mueller D, Shamblott MJ, Fox HE, Gearhart JD, Martin LJ. Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J. Neurosci. Res. 82(5), 592-608 (2005).
Nistor GI, Totoiu MO, Haque N, Carpenter MK, Keirstead HS. Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation. Glia 49(3), 385-396 (2005).
Liu S, Qu Y, Stewart TJ et al. Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc. Natl Acad. Sci. USA 97(11), 6126-6131 (2000).
Srivastava AS, Shenouda S, Mishra R, Carrier E. Transplanted embryonic stem cells successfully survive, proliferate, and migrate to damaged regions of the mouse brain. Stem Cells 24(7), 1689-1694 (2006).
McDonald JW, Liu XZ, Qu Y et al. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat. Med. 5(12), 1410-1412 (1999).
Brustle O, Jones KN, Learish RD et al. Embryonic stem cell-derived glial precursors: a source of myelinating transplants. Science 285(5428), 754-756 (1999).
Zeng L, Rahrmann E, Hu Q et al. Multipotent adult progenitor cells from swine bone marrow. Stem Cells 24(11), 2355-2366 (2006).
Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19(12), 1129-1133 (2001).
Zhang PL, Izrael M, Ainbinder E et al. Increased myelinating capacity of embryonic stem cell derived oligodendrocyte precursors after treatment by interleukin-6/soluble interleukin-6 receptor fusion protein. Mol. Cell. Neurosci. 31(3), 387-398 (2006).
Takagi Y, Takahashi J, Saiki H et al. Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model. J. Clin. Invest. 115(1), 102-109 (2005).
Buhnemann C, Scholz A, Bernreuther C et al. Neuronal differentiation of transplanted embryonic stem cell-derived precursors in stroke lesions of adult rats. Brain 129(Pt 12), 3238-3248 (2006).
Bhagavati S, Xu W. Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice. Biochem. Biophys. Res. Commun. 333(2), 644-649 (2005).
Kuai XL, Cong XQ, Du ZW, Bian YH, Xiao SD. Treatment of surgically induced acute liver failure by transplantation of HNF4-overexpressing embryonic stem cells. Chin. J. Dig. Dis. 7(2), 109-116 (2006).
Min JY, Yang Y, Sullivan MF et al. Long-term improvement of cardiac function in rats after infarction by transplantation of embryonic stem cells. J. Thorac. Cardiovasc. Surg. 125(2), 361-369 (2003).
Kim JH, Auerbach JM, Rodriguez-Gomez JA et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418(6893), 50-56 (2002).
Carpenter M, Rao MS, Freed W, Zeng X. Derivation and characterization of neuronal precursors and dopaminergic neurons from human embryonic stem cells in vitro. Methods Mol. Biol. 331, 153-167 (2006).
Nishimura F, Toriumi H, Ishizaka S, Sakaki T, Yoshikawa M. Use of differentiating embryonic stem cells in the Parkinsonian mouse model. Methods Mol. Biol. 329, 485-493 (2006).
Brederlau A, Correia AS, Anisimov SV et al. Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson’s disease: effect of in vitro differentiation on graft survival and teratoma formation. Stem Cells 24(6), 1433-1440 (2006).
Cho YH, Kim DS, Kim PG et al. Dopamine neurons derived from embryonic stem cells efficiently induce behavioral recovery in a Parkinsonian rat model. Biochem. Biophys. Res. Commun. 341(1), 6-12 (2006).
Yoshizaki T, Inaji M, Kouike H et al. Isolation and transplantation of dopaminergic neurons generated from mouse embryonic stem cells. Neurosci. Lett. 363(1), 33-37 (2004).
Bjorklund LM, Sanchez-Pernaute R, Chung S et al. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc. Natl Acad. Sci. USA 99(4), 2344-2349 (2002).
Nakao N, Yokote H, Nakai K, Itakura T. Promotion of survival and regeneration of nigral dopamine neurons in a rat model of Parkinson’s disease after implantation of embryonal carcinoma-derived neurons genetically engineered to produce glial cell line-derived neurotrophic factor. J. Neurosurg. 92(4), 659-670 (2000).
Zhang SC. Embryonic stem cells for neural replacement therapy: prospects and challenges. J. Hematother. Stem Cell Res. 12(6), 625-634 (2003)
Senior K. Embryonic stem cells used to remyelinate injured rat spinal cord neurons. Lancet 355(9218), 1890 (2000).
Hendricks WA, Pak ES, Owensby JP et al. Predifferentiated embryonic stem cells prevent chronic pain behaviors and restore sensory function following spinal cord injury in mice. Mol. Med. 12(1-3), 34-46 (2006).
Lerou PH, Daley GQ. Therapeutic potential of embryonic stem cells. Blood Rev. 19(6), 321-331 (2005).
Kimura H, Yoshikawa M, Matsuda R et al. Transplantation of embryonic stem cell-derived neural stem cells for spinal cord injury in adult mice. Neurol. Res. 27(8), 812-819 (2005).
Grabowski M, Brundin P, Johansson BB. Fetal neocortical grafts implanted in adult hypertensive rats with cortical infarcts following a middle cerebral artery occlusion: ingrowth of afferent fibers from the host brain. Exp. Neurol. 116(2), 105-121 (1992).
Grabowski M, Christofferson RH, Brundin P, Johansson BB. Vascularization of fetal neocortical grafts implanted in brain infarcts in spontaneously hypertensive rats. Neuroscience 51(3), 673-682 (1992).
Chopko BW, Voneida TJ. Fetal rat cerebellar fragment transplantation into adult rat forebrain lesion cavities. J. Neural Transplant. Plast. 3(1), 63-69 (1992).
Fiandaca MS, Gash DM. New insights and technologies in brain grafting. Clin. Neurosurg. 39, 482-508 (1992).
Jiao S, Wolff JA. Long-term survival of autologous muscle grafts in rat brain. Neurosci. Lett. 137(2), 207-210 (1992).
Wirth ED 3rd, Theele DP, Mareci TH et al. In vivo magnetic resonance imaging of fetal cat neural tissue transplants in the adult cat spinal cord. J. Neurosurg. 76(2), 261-274 (1992).
Deacon T, Dinsmore J, Costantini LC, Ratliff J, Isacson O. Blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation. Exp. Neurol. 149(1), 28-41 (1998).
Yanai J, Doetchman T, Laufer N et al. Embryonic cultures but not embryos transplanted to the mouse’s brain grow rapidly without immunosuppression. Int. J. Neurosci. 81(1-2), 21-26 (1995).
Drukker M, Katchman H, Katz G et al. Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells 24(2), 221-229 (2006).
Wognum AW, Eaves AC, Thomas TE. Identification and isolation of hematopoietic stem cells. Arch. Med. Res. 34(6), 461-475 (2003).
Huss R. Isolation of primary and immortalized CD34-hematopoietic and mesenchymal stem cells from various sources. Stem Cells 18(1), 1-9 (2000).
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 284(5411), 143-147 (1999).
Lennon DP, Caplan AI. Isolation of rat marrow-derived mesenchymal stem cells. Exp. Hematol. 34(11), 1606-1607 (2006).
Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276(5309), 71-74 (1997).
Verfaillie CM. Multipotent adult progenitor cells: an update. Novartis Found. Symp. 265, 55-61; discussion 61-55, 92-57 (2005).
Raedt R, Pinxteren J, Van Dycke A et al. Differentiation assays of bone marrow-derived multipotent adult progenitor cell (MAPC)-like cells towards neural cells cannot depend on morphology and a limited set of neural markers. Exp. Neurol. 203(2), 542-554 (2007).
Woodward WA, Chen MS, Behbod F, Rosen JM. On mammary stem cells. J. Cell Sci. 118(Pt 16), 3585-3594 (2005).
Clarke RB. Isolation and characterization of human mammary stem cells. Cell Prolif. 38(6), 375-386 (2005).
Capuco AV, Ellis S. Bovine mammary progenitor cells: current concepts and future directions. J. Mammary Gland Biol. Neoplasia 10(1), 5-15 (2005).
Roisen FJ, Klueber KM, Lu CL et al. Adult human olfactory stem cells. Brain Res. 890(1), 11-22 (2001).
Frank MH, Sayegh MH. Immunomodulatory functions of mesenchymal stem cells. Lancet 363(9419), 1411-1412 (2004).
Yu JJ, Sun X, Yuan X et al. Immunomodulatory neural stem cells for brain tumour therapy. Expert Opin. Biol. Ther. 6(12), 1255-1262 (2006).
Krampera M, Pasini A, Pizzolo G et al. Regenerative and immunomodulatory potential of mesenchymal stem cells. Curr. Opin. Pharmacol. 6(4), 435-441 (2006).
Puissant B, Barreau C, Bourin P et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells. Br. J. Haematol. 129(1), 118-129 (2005).
Hsu YC, Lee DC, Chiu IM. Neural stem cells, neural progenitors, and neurotrophic factors. Cell Transplant. 16(2), 133-150 (2007).
Campos LS. Neurospheres: insights into neural stem cell biology. J. Neurosci. Res. 78(6), 761-769 (2004).
Cameron HA, Gould E. Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. Neuroscience 61(2), 203-209 (1994).
Eriksson PS, Perfilieva E, Bjork-Eriksson T et al. Neurogenesis in the adult human hippocampus. Nat. Med. 4(11), 1313-1317 (1998).
Gould E, Reeves AJ, Fallah M et al. Hippocampal neurogenesis in adult Old World primates. Proc. Natl Acad. Sci. USA 96(9), 5263-5267 (1999).
Kornack DR, Rakic P. Continuation of neurogenesis in the hippocampus of the adult macaque monkey. Proc. Natl Acad. Sci. USA 96(10), 5768-5773 (1999).
Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97(6), 703-716 (1999).
Alvarez-Buylla A, Herrera DG, Wichterle H. The subventricular zone: source of neuronal precursors for brain repair. Prog. Brain Res. 127, 1-11 (2000).
Pluchino S, Quattrini A, Brambilla E et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 422(6933), 688-694 (2003).
Ben-Hur T, Einstein O, Mizrachi-Kol R et al. Transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis. Glia 41(1), 73-80 (2003).
Einstein O, Fainstein N, Vaknin I et al. Neural precursors attenuate autoimmune encephalomyelitis by peripheral immunosuppression. Ann. Neurol. 61(3), 209-218 (2007).
Belmadani A, Tran PB, Ren D, Miller RJ. Chemokines regulate the migration of neural progenitors to sites of neuroinflammation. J. Neurosci. 26(12), 3182-3191 (2006).
Yasuhara T, Matsukawa N, Hara K et al. Transplantation of human neural stem cells exerts neuroprotection in a rat model of Parkinson’s disease. J. Neurosci. 26(48), 12497-12511 (2006).
Harrower TP, Tyers P, Hooks Y, Barker RA. Long-term survival and integration of porcine expanded neural precursor cell grafts in a rat model of Parkinson’s disease. Exp. Neurol. 197(1), 56-69 (2006).
Ben-Hur T, Idelson M, Khaner H et al. Transplantation of human embryonic stem cell-derived neural progenitors improves behavioral deficit in Parkinsonian rats. Stem Cells 22(7), 1246-1255 (2004).
Snyder BJ, Olanow CW. Stem cell treatment for Parkinson’s disease: an update for 2005. Curr. Opin. Neurol. 18(4), 376-385 (2005).
Lindvall O. Parkinson disease. Stem cell transplantation. Lancet 358(Suppl.), S48 (2001).
Teng YD, Lavik EB, Qu X et al. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc. Natl Acad. Sci. USA 99(5), 3024-3029 (2002).
Iwanami A, Kaneko S, Nakamura M et al. Transplantation of human neural stem cells for spinal cord injury in primates. J. Neurosci. Res. 80(2), 182-190 (2005).
Okano H, Ogawa Y, Nakamura M et al. Transplantation of neural stem cells into the spinal cord after injury. Semin. Cell Dev. Biol. 14(3), 191-198 (2003).
Pallini R, Vitiani LR, Bez A et al. Homologous transplantation of neural stem cells to the injured spinal cord of mice. Neurosurgery 57(5), 1014-1025; discussion 1014-1025 (2005).
Ogawa Y, Sawamoto K, Miyata T et al. Transplantation of in vitro expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats. J. Neurosci. Res. 69(6), 925-933 (2002).
Cummings BJ, Uchida N, Tamaki SJ, Anderson AJ. Human neural stem cell differentiation following transplantation into spinal cord injured mice: association with recovery of locomotor function. Neurol. Res. 28(5), 474-481 (2006).
Shihabuddin LS, Horner PJ, Ray J, Gage FH. Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J. Neurosci. 20(23), 8727-8735 (2000).
Doetsch F, Verdugo JM, Caille I et al. Lack of the cell-cycle inhibitor p27Kip1 results in selective increase of transit-amplifying cells for adult neurogenesis. J. Neurosci. 22(6), 2255-2264 (2002).
Marshall GP 2nd, Laywell ED, Zheng T, Steindler DA, Scott EW. In vitro-derived ‘neural stem cells’ function as neural progenitors without the capacity for self-renewal. Stem Cells 24(3), 731-738 (2006).
Gallacher L, Murdoch B, Wu DM et al. Isolation and characterization of human CD34-Lin- and CD34+Lin- hematopoietic stem cells using cell surface markers AC133 and CD7. Blood 95(9), 2813-2820 (2000).
Brazelton TR, Rossi FM, Keshet GI, Blau HM. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 290(5497), 1775-1779 (2000).
Eglitis MA, Mezey E. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc. Natl Acad. Sci. USA 94(8), 4080-4085 (1997).
Locatelli F, Corti S, Donadoni C et al. Neuronal differentiation of murine bone marrow Thy-1- and Sca-1-positive cells. J. Hematother. Stem Cell Res. 12(6), 727-734 (2003).
Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 290(5497), 1779-1782 (2000).
Karussis DM, Vourka-Karussis U, Mizrachi-Koll R, Abramsky O. Acute/relapsing experimental autoimmune encephalomyelitis: induction of long lasting, antigen-specific tolerance by syngeneic bone marrow transplantation. Mult. Scler. 5(1), 17-21 (1999).
Karussis DM, Slavin S, Ben-Nun A et al. Chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE): treatment and induction of tolerance, with high dose cyclophosphamide followed by syngeneic bone marrow transplantation. J. Neuroimmunol. 39(3), 201-210 (1992).
Karussis DM, Slavin S, Lehmann D et al. Prevention of experimental autoimmune encephalomyelitis and induction of tolerance with acute immunosuppression followed by syngeneic bone marrow transplantation. J. Immunol. 148(6), 1693-1698 (1992).
Karussis DM, Vourka-Karussis U, Lehmann D et al. Immunomodulation of autoimmunity in MRL/lpr mice with syngeneic bone marrow transplantation (SBMT). Clin. Exp. Immunol. 100(1), 111-117 (1995).
Karussis DM, Vourka-Karussis U, Lehmann D et al. Prevention and reversal of adoptively transferred, chronic relapsing experimental autoimmune encephalomyelitis with a single high dose cytoreductive treatment followed by syngeneic bone marrow transplantation. J. Clin. Invest. 92(2), 765-772 (1993).
van Gelder M, van Bekkum DW. Effective treatment of relapsing experimental autoimmune encephalomyelitis with pseudoautologous bone marrow transplantation. Bone Marrow Transplant. 18(6), 1029-1034 (1996).
van Gelder M, van Bekkum DW. Treatment of relapsing experimental autoimmune encephalomyelitis in rats with allogeneic bone marrow transplantation from a resistant strain. Bone Marrow Transplant. 16(3), 343-351 (1995).
Nash RA, Bowen JD, McSweeney PA et al. High-dose immunosuppressive therapy and autologous peripheral blood stem cell transplantation for severe multiple sclerosis. Blood 102(7), 2364-2372 (2003).
Saccardi R, Kozak T, Bocelli-Tyndall C et al. Autologous stem cell transplantation for progressive multiple sclerosis: update of the European Group for Blood and Marrow Transplantation autoimmune diseases working party database. Mult. Scler. 12(6), 814-823 (2006).
Mancardi GL, Saccardi R, Filippi M et al. Autologous hematopoietic stem cell transplantation suppresses Gd-enhanced MRI activity in MS. Neurology 57(1), 62-68 (2001).
Fassas A, Passweg JR, Anagnostopoulos A et al. Hematopoietic stem cell transplantation for multiple sclerosis. A retrospective multicenter study. J. Neurol. 249(8), 10881097 (2002).
Muraro PA, Douek DC. Renewing the T cell repertoire to arrest autoimmune aggression. Trends Immunol. 27(2), 61-67 (2006).
Muraro PA, Douek DC, Packer A et al. Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients. J. Exp. Med. 201(5), 805-816 (2005).
Minagawa H, Takenaka A, Itoyama Y, Mori R. Experimental allergic encephalomyelitis in the Lewis rat. A model of predictable relapse by cyclophosphamide. J. Neurol. Sci. 78(2), 225-235 (1987).
Miyazaki C, Nakamura T, Kaneko K, Mori R, Shibasaki H. Reinduction of experimental allergic encephalomyelitis in convalescent Lewis rats with cyclophosphamide. J. Neurol. Sci. 67(3), 277-284 (1985).
Polman CH, Matthaei I, de Groot CJ et al. Low-dose cyclosporin A induces relapsing remitting experimental allergic encephalomyelitis in the Lewis rat. J. Neuroimmunol. 17(3), 209-216 (1988).
Chen JT, Collins DL, Atkins HL et al. Brain atrophy after immunoablation and stem cell transplantation in multiple sclerosis. Neurology 66(12), 1935-1937 (2006).
Inglese M, Mancardi GL, Pagani E et al. Brain tissue loss occurs after suppression of enhancement in patients with multiple sclerosis treated with autologous haematopoietic stem cell transplantation. J. Neurol. Neurosurg. Psychiatry 75(4), 643-644 (2004).
Bossolasco P, Cova L, Calzarossa C et al. Neuro-glial differentiation of human bone marrow stem cells in vitro. Exp. Neurol. 193(2), 312-325 (2005).
Oswald J, Boxberger S, Jorgensen B et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 22(3), 377-384 (2004).
Talens-Visconti R, Bonora A, Jover R et al. Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells. World J. Gastroenterol. 12(36), 5834-5845 (2006).
Sanchez-Ramos J, Song S, Cardozo-Pelaez F et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp. Neurol. 164(2), 247-256 (2000).
Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61(4), 364-370 (2000).
Krampera M, Glennie S, Dyson J et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 101(9), 3722-3729 (2003).
Uccelli A, Moretta L, Pistoia V. Immunoregulatory function of mesenchymal stem cells. Eur. J. Immunol. 36(10), 2566-2573 (2006).
Crigler L, Robey RC, Asawachaicharn A, Gaupp D, Phinney DG. Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis. Exp. Neurol. 198(1), 54-64 (2006).
Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L. Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 107(4), 1484-1490 (2006).
Sotiropoulou PA, Perez SA, Gritzapis AD, Baxevanis CN, Papamichail M. Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells 24(1), 74-85 (2006).
Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand. J. Immunol. 57(1), 11-20 (2003).
Deng W, Han Q, Liao L et al. Effects of allogeneic bone marrow-derived mesenchymal stem cells on T and B lymphocytes from BXSB mice. DNA Cell Biol. 24(7), 458-463 (2005).
Beyth S, Borovsky Z, Mevorach D et al. Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness. Blood 105(5), 2214-2219 (2005).
Augello A, Tasso R, Negrini SM et al. Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway. Eur. J. Immunol. 35(5), 1482-1490 (2005).
Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105(4), 1815-1822 (2005).
Bartholomew A, Sturgeon C, Siatskas M et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp. Hematol. 30(1), 42-48 (2002).
Corcione A, Benvenuto F, Ferretti E et al. Human mesenchymal stem cells modulate B-cell functions. Blood 107(1), 367-372 (2006).
Glennie S, Soeiro I, Dyson PJ, Lam EW, Dazzi F. Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood 105(7), 2821-2827 (2005).
Jiang XX, Zhang Y, Liu B et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 105(10), 4120-4126 (2005).
Krampera M, Cosmi L, Angeli R et al. Role for interferon-? in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells 24(2), 386-398 (2006).
Meisel R, Zibert A, Laryea M et al. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. Blood 103(12), 4619-4621 (2004).
Rasmusson I, Ringden O, Sundberg B, Le Blanc K. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation 76(8), 1208-1213 (2003).
Zhang W, Ge W, Li C et al. Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells. Stem Cells Dev. 13(3), 263-271 (2004).
Di Nicola M, Carlo-Stella C, Magni M et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99(10), 3838-3843 (2002).
Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp. Hematol. 31(10), 890-896 (2003).
Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan EC. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75(3), 389-397 (2003).
Zappia E, Casazza S, Pedemonte E et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 106(5), 1755-1761 (2005).
Zhang J, Li Y, Chen J et al. Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice. Exp. Neurol. 195(1), 16-26 (2005).
Gerdoni E, Gallo B, Casazza S et al. Mesenchymal stem cells effectively modulate pathogenic immune response in experimental autoimmune encephalomyelitis. Ann. Neurol. 61(3), 219-227 (2007).
Inoue M, Honmou O, Oka S et al. Comparative analysis of remyelinating potential of focal and intravenous administration of autologous bone marrow cells into the rat demyelinated spinal cord. Glia 44(2), 111-118 (2003).
Karussis DM, Grigoriadis N, Ben-Hur T et al. Mesenchymal bone marrow stem cells, migrate into CNS lesions in experimental autoimmune encephalomyelitis, differentiate into neuronal and glial line and downregulate chronic EAE. Neurology 64(6), A407 (2005).
Giordano A, Galderisi U, Marino IR. From the laboratory bench to the patient’s bedside: an update on clinical trials with mesenchymal stem cells. J. Cell. Physiol. 211(1), 27-35 (2007).
Chen SL, Fang WW, Ye F et al. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am. J. Cardiol. 94(1), 92-95 (2004).
Wollert KC, Meyer GP, Lotz J et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 364(9429), 141-148 (2004).
Katritsis DG, Sotiropoulou PA, Karvouni E et al. Transcoronary transplantation of autologous mesenchymal stem cells and endothelial progenitors into infarcted human myocardium. Catheter Cardiovasc. Interv. 65(3), 321-329 (2005).
Perin EC, Dohmann HF, Borojevic R et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 107(18), 2294-2302 (2003).
Perin EC, Dohmann HF, Borojevic R et al. Improved exercise capacity and ischemia 6 and 12 months after transendocardial injection of autologous bone marrow mononuclear cells for ischemic cardiomyopathy. Circulation 110(11 Suppl. 1), II213-II218 (2004).
Janssens S, Dubois C, Bogaert J et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. Lancet 367(9505), 113-121 (2006).
Lunde K, Solheim S, Aakhus S et al. Autologous stem cell transplantation in acute myocardial infarction: The ASTAMI randomized controlled trial. Intracoronary transplantation of autologous mononuclear bone marrow cells, study design and safety aspects. Scand. Cardiovasc. J. 39(3), 150-158 (2005).
Meyer GP, Wollert KC, Lotz J et al. Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months’ follow-up data from the randomized, controlled BOOST (BOne marrOw transfer to enhance ST-elevation infarct regeneration) trial. Circulation 113(10), 1287-1294 (2006).
Rosenzweig A. Cardiac cell therapy – mixed results from mixed cells. N. Engl. J. Med. 355(12), 1274-1277 (2006).
Welt FG, Losordo DW. Cell therapy for acute myocardial infarction: curb your enthusiasm? Circulation 113(10), 1272-1274 (2006).
Ferrari G, Cusella-De Angelis G, Coletta M et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279(5356), 1528-1530 (1998).
Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat. Med. 5(3), 309-313 (1999).
Horwitz EM, Prockop DJ, Gordon PL et al. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 97(5), 1227-1231 (2001).
Koc ON, Day J, Nieder M et al. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant. 30(4), 215-222 (2002).
Koc ON, Gerson SL, Cooper BW et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J. Clin. Oncol. 18(2), 307-316 (2000).
Lazarus HM. Bone marrow transplantation in low-grade non-Hodgkin’s lymphoma. Leuk. Lymphoma 17(3-4), 199-210 (1995).
Lazarus HM, Haynesworth SE, Gerson SL, Rosenthal NS, Caplan AI. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant. 16(4), 557-564 (1995).
Lazarus HM, Koc ON, Devine SM et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol. Blood Marrow Transplant. 11(5), 389-398 (2005).
Lazarus HM, Winton EF, Williams SF et al. Phase I multicenter trial of interleukin 6 therapy after autologous bone marrow transplantation in advanced breast cancer. Bone Marrow Transplant. 15(6), 935-942 (1995).
Mazzini L, Mareschi K, Ferrero I et al. Autologous mesenchymal stem cells: clinical applications in amyotrophic lateral sclerosis. Neurol. Res. 28(5), 523-526 (2006).
Karussis D, Kassis I, Basan GS, Slavin S. Immunomodulation and neuroprotection with mesenchymal bone marrow stem cells (MSCs): a porposed treatment for multiple sclerosis and other neuroimmunological/neurodegenerative diseases. J. Neurol. Sci. (2007) (Epub ahead of print).
The author: Professor Yasser Metwally
INTRODUCTION
April 16, 2009 — In this post the author intend to demonstrate the need for supplementing conventional repair of the injured nerve with alternative therapies, namely transplantation of stem or progenitor cells. Although peripheral nerves do exhibit the potential to regenerate axons and reinnervate the end organ, outcome following severe nerve injury, even after repair, remains relatively poor. This is likely because of the extensive injury zone that prevents axon outgrowth. Even if outgrowth does occur, a relatively slow growth rate of regeneration results in prolonged denervation of the distal nerve. Whereas denervated Schwann cells (SCs) are key players in the early regenerative success of peripheral nerves, protracted loss of axonal contact renders Schwann cells unreceptive for axonal regeneration. Given that denervated Schwann cells appear to become effete, one logical approach is to support the distal denervated nerve environment by replacing host cells with those derived exogenously. A number of different sources of stem/precursor cells are being explored for their potential application in the scenario of peripheral nerve injury. The most promising candidate, transplant cells are derived from easily accessible sources such as the skin, bone marrow, or adipose tissue, all of which have demonstrated the capacity to differentiate into Schwann cell–like cells. Although recent studies have shown that stem cells can act as promising and beneficial adjuncts to nerve repair, considerable optimization of these therapies will be required for their potential to be realized in a clinical setting. The authors investigate the relevance of the delivery method (both the number and differentiation state of cells) on experimental outcomes, and seek to clarify whether stem cells must survive and differentiate in the injured nerve to convey a therapeutic effect. As our laboratory uses skin-derived precursor cells (SKPCs) in various nerve injury paradigms, we relate our findings on cell fate to other published studies to demonstrate the need to quantify stem cell survival and differentiation for future studies.
Injuries of the peripheral nerves are common and debilitating, affecting 2.8% of trauma patients[44] and resulting in considerable long-term disability.[27] The assumption has been that peripheral nerve injuries recover, given the observation of spontaneous axonal regeneration following insult. While this capacity for regeneration is higher than that of the central nervous system, complete recovery is fairly infrequent, misdirected, or associated with debilitating neuropathic pain.[57] In fact, satisfactory results only tend to occur following relatively minor injuries, such as neurapraxia or axonotmesis.[21] Nerve transection is associated with notoriously poor outgrowth compared with other injuries, particularly when the distance between injury and target is long.
Poor outcome from peripheral nerve injury is especially evident when repair is performed after a temporal delay,[31,51,56] occurring frequently in clinical practice. Due to the nature of most nerve injuries where the nerve is left in physical continuity, the propensity for spontaneous recovery is not immediately known.[37] As such, surgical repair is significantly delayed in a great number of cases. Even patients undergoing immediate nerve repair are subject to a lengthy denervation of the distal nerve as a result of the low rate of regeneration (~ 1 mm/day in humans[58]) and the long regeneration distances required to reach the end organ.
Elongation of regenerating axons is initially supported by resident Schwann cells that undergo a phenotypic change from myelinating to growth supportive following initial denervation.[38,52] This switch in Schwann cell phenotype is associated with up-regulation of several growth associated genes including neurotrophic factors, p75 NTR, GFAP, GAP-43, netrin-1, and key transcription factors.[14,38,52] Proliferating Schwann cells are a rich source of neurotrophins, cell adhesion molecules, and cytokines that support axonal regeneration and recruit further cells into the injury site.[14,63,68] Unless axonal contact is reestablished in a timely fashion, however, this growth supportive environment is not maintained.[49,62,72] Denervated Schwann cells progressively lose their ability to express regeneration-assisting genes[32,74] and in effect become "turned off."[14] As the capacity of the denervated distal nerve to support axonal regeneration is highly dependent on proliferating Schwann cells within the basal lamina tubes[7] that guide elongating axons to their denervated target,[50] this loss of vitality and functionality in distal Schwann cells directly translates to poor muscle reinnervation outcomes.[15]
Although placement of interposed autologous nerve grafts offers a cell-rich material through which axons can regenerate, their use is not ideal because of donor site morbidity, lack of donor tissue availability, and nonspecific regeneration.[5,43] Recent advances in tissue engineering have introduced synthetic nerve guide conduits that are capable of bridging small defects in peripheral nerves (up to ~ 3 cm in humans), but their relatively inert microenvironment reduces their value for larger or more chronic injuries.[3,71] It appears that combined approaches with cells[29] or trophic factors[28] within synthetic tubes may extend their functionality. Indeed, delivery of Schwann cells in a variety of repair paradigms has been successful in promoting regeneration and remyelination of the injured spinal cord[48,61] and peripheral nerve.[16] However, human Schwann cells must be derived from invasive nerve biopsies in sufficient numbers for regeneration and are only available after a lengthy expansion time in vitro.[17] Therefore, several groups have turned their attention to identifying more accessible sources of Schwann cell–like cells for transplant therapies.
Emphasis has been placed on exploring stem or progenitor cells that are easily accessible, rapidly expandable in culture, capable of survival and integration within the host tissue, and amenable to stable transfection and expression of exogenous genes.[4] Embryonic neural stem cells or cell lines have been used to repair nerve injuries with demonstration of regenerative success[2,18,42] but suffer the drawback of being somewhat difficult to obtain. On the other hand, adult stem cells have the advantage of being available from relatively noninvasive, autologous harvest methods, and are likely the most promising choice for the majority of clinical nerve injuries. Bone marrow stromal cells have attracted the attention of several groups interested in cellular strategies to supplement nerve repair.[10,12,20,26,54,64,70,75] These mesenchymal stem cells are harvested from the long bones, and when placed in culture medium containing the appropriate cytokine cocktail,[26] transdifferentiate into an adherent Schwann cell–like phenotype expressing S100 protein, GFAP, and p75.[12,64] They have been used with artificial conduits and acellular grafts, where they have contributed to improved electrophysiological, morphometric, and/or behavioral recovery outcomes versus vehicle controls. Although their potential to produce functional myelin in vivo has been questioned,[69] others have shown that these BMSC-derived Schwann cells are at very least capable of myelinating cultured PC12 cells in vitro,[26] further highlighting their therapeutic potential.
More recently, even less invasive sources of stem cells have been discovered. Adipose tissue has been identified as a niche for a multipotent stem cell with a comparable phenotypic profile to the bone marrow stromal cells, and it appears to differentiate into a myelinating Schwann cell phenotype in vitro given the appropriate medium formulation known to promote transdifferentiation of BMSCs.[30,73] Further studies will be required to assess whether they can also translate this advantage to the injured peripheral nerve.
The skin and its associated structures pose another easily accessible source of stem cells. A large population of neural crest stem cells has been found in the bulge area of hair and whisker follicles that can differentiate into neurons, smooth muscle cells, Schwann cells, and melanocytes.[55] Cells isolated from the vibrissal follicle bulge area have been used to repair a gap created in rodent peripheral nerve, where they differentiate into Schwann cell–like cells and improve recovery.[1] Similarly, when stem cells derived from skin were transplanted into artificial nerve guidance tubes bridging a 16-mm gap in rodent sciatic nerve, there was promising improvement in behavioral, electrophysiological, and morphometric parameters measured over vehicle control.[34] It should be noted that cells in this study were used naive and only a small proportion differentiated into Schwann cells in the in vivo environment. The skin dermis contains neural crest–related precursor cells (termed SKPCs) that can differentiate into neural crest cell types in vitro when supplied the appropriate cues, including those with characteristics of peripheral neurons and Schwann cells.[13,36,66,67] The SKPCs respond to neuregulins in vitro to generate Schwann cells, highlighting their potential to serve as transplantable cells for nerve injury models (where neuregulins are liberated from cells within the nerve).[9,33] The SKPCs that are Schwann cell–like in their apparent differentiation (SKPC–Schwann cell), survive and associate with axons within both normal mouse sciatic nerve and distal to crush, where they express a myelinating phenotype.[38] Indeed, SKPCs appear to generate functional Schwann cells as they myelinate both sensory neurons in dorsal root ganglion cocultures in vitro and dysmyelinating shiverer mouse[42] nerve axons in vivo.[38]
Although often not reported, the number of cells delivered to nerve injuries in animal models varies considerably between studies. While some have used as few as 4 × 103 cells,[2] others have transplanted 2 × 107 cells,[20] but there has often been little explanation for the selection of cell numbers in these studies. It is admittedly difficult to compare the number of cells delivered in widely different repair paradigms, but it is fair to state that there are likely an ideal number of cells that should be determined for each cell type or repair scenario. Just as too few cells may not translate to a therapeutic effect, delivery of too many cells may also have detrimental results. This was exemplified beautifully by a study using transplanted Schwann cells delivered in 10-mm nerve gap.[40] When authors used a concentration of 20 × 106 cells/ml, there was no appreciable increase in axonal regeneration distance. Increasing the concentration to 80 × 106 cells/ml proved ideal for regeneration, whereas further increases resulted in slightly poorer regeneration. This same logic likely applies to stem cell transplantation, as they must compete for space and available resources with the cellular milieu of the regenerating nerve. Therefore, optimization strategies should take the number of delivered cells into account. Similarly, the way in which cells are delivered to the injury site has varied between studies, ranging from direct microinjection,[36] suspension within artificial tubes,[10,42,54] and seeding within devitalized muscle or nerve grafts.[25] Although the choice of stem cell delivery method may depend on the type and extent of nerve injury in question, it may be optimized by providing transplanted cells an environment that will favor their survival and integration, such as within structured fibers[12] or biomatrices.[8]
Part of the appeal of using precursor or stem cells for supplementing peripheral nerve repair is their capacity for self-renewal, such that it is possible to deliver large numbers of dividing cells to the injury site.[65] By delivering stem cells into the injured nerve in a naive state, this proliferative capacity is maintained, and it is expected that cells will be prompted by the microenvironment to differentiate into the required cell type.[22] In vitro studies have demonstrated that neural stem/progenitor cells in coculture with cells from the nervous system will take on a phenotype similar to their partner tissue’s origin: dorsal root ganglion cultures will induce a peripheral neuron/Schwann cell/smooth muscle phenotype, and a cerebellar feeder layer will induce differentiation into CNS neurons.[6] Nevertheless, incidence of differentiation from naive precursor cells within the peripheral nerve is rather low in many cases.[10,11,18,46] Choosing to predifferentiate stem cells toward a desired phenotype prior to delivery into the repair site may be an effective strategy to ensure a more precise and complete therapeutic effect. It may be that cells at later developmental stages (vs embryonic stem cells, for example) possess more mature intrinsic molecular programs to direct them to their target destination.[53]
Because it is well known that mature Schwann cells survive denervation events by secreting autocrine factors such as insulin-like growth factor, neurotrophin-3, and plant-derived growth factor–BB,[23] might an appropriately differentiated stem cell also possess similar machinery for self-preservation and thus be an ideal candidate for supplementing the injured peripheral nerve? We have found that SKPCs, when injected as naive sphere-forming cells, do differentiate into GFAP-positive Schwann cells in response to cues found in the local environment of the injured peripheral nerve (Fig. 1). However, long-term survival and maintenance of Schwann cell markers is greatly improved by predifferentiating the cells to a Schwann cell phenotype prior to delivery.[36] On the other hand, others have reported that allowing stem cells to differentiate before delivery accelerates posttransplant cell death, perhaps owing to increased expression of major histocompatibility complex antigens or reduced proliferation rates.[59] In addition to survival of stem cells, their effect on surrounding tissues may be modified based on their level of differentiation prior to transplantation. For example, when naive adult neural stem cells were injected into a lesioned spinal cord, the resulting aberrant sprouting resulted in profound allodynia. If gliogenesis in these cells was suppressed by prior treatment with neurogenin-2, there was an overall greater functional improvement.[19] One of the potentially negative consequences of stem cell therapy in any system is the tumorigenic capability of multipotent precursors. Indeed, when the regenerative potential of C17.2 neural stem cells was assessed in 3 different rat sciatic nerve injury models, there was a high incidence of tumor formation by the transplanted cells.[18,24]
Figure 1. Fluorescent micrographs of a longitudinal section of sciatic nerve. The SKPCs survive and differentiate within the injured peripheral nerve. Naive GFP-labeled SKPCs (green, A) injected into the transected sciatic nerve demonstrate viability after 8 weeks and integrate along host Schwann cells columns labeled by GFAP (red). The occasional double-labeled cells (yellow) on the merged image (B) are likely SKPCs expressing GFAP, suggesting their in vivo differentiation toward a Schwann cell phenotype. Original magnification ×40 (A); ×400 (B). (Click on figure to magnify)
Whether due to technical challenges or oversight, it is an unfortunate reality that survival of stem cells delivered to nerve injury sites is reported only infrequently. When quantified, precursor cells have shown between 0.5 and 38% survival, depending on evaluation time point and cell type.[18,34,36,54] In our laboratory, we have also seen differences in survival based on nerve injury model and differentiation state of the cells at transplantation (unpublished observations). For example, when we delivered naive SKPCs into an acutely injured nerve, survival after 2 weeks was ~ 10.5%, whereas when delivered into a nerve that had been previously chronically denervated, the number of detected SKPCs decreased to 5.8%. Seeing that 78% of the surviving stem cells in the chronic model had differentiated into GFAP-positive Schwann cells, we next used predifferentiated (Schwann cell–like) SKPCs and found that we could increase survival to ≥ 8%. Without quantification of survival in stem cell transplantation experiments, it is difficult to determine whether they are being retained long enough and in enough numbers to confer a sufficient benefit to regeneration. The danger of exogenous cell therapy is of course cell death caused by immune system attack.[59] Although some authors have reported considerable phagocytosis of transplanted stem cells,[54,59] this may be due to species/strain mismatching of donor and recipient, as many others have not observed this trend. In fact, we have observed a highly interesting pattern of surviving transplanted SKPCs that are spatially separate from phagocytic ED-1-positive macrophages (Fig. 2). The question of survival is mechanistically interesting, as improvement in regeneration outcomes has been also been observed in the absence of detection of transplanted cells.[46]
Figure 2. Confocal image. The SKPC–Schwann cells are not immediately cleared by host immune system. Eight weeks following injection into an initially decellularized (by repetitive freeze-thawing) nerve graft bridging a 12-mm defect created in the rodent sciatic nerve, SKPCSchwann cells (red), and ED-1-positive macrophages (green) are spatially segregated within the longitudinal extent of the nerve graft. The finding that there is very little colabeling of SKPC–Schwann cells with ED-1 positive macrophages suggests that transplanted cells are not phagocytosed in any large quantity within the host nerve. Original magnification ×400. (Click on figure to magnify)
If a minimum survival time of stem cells is indeed required to observe a therapeutic effect, strategies should be devised to increase the amount of time cells remain in grafted regions. Survival and effectiveness of transplanted cells can be improved by ex vivo genetic manipulation or concomitant delivery of protective agents or trophic factors. Pan and colleagues[45] found that administration of granulocyte–colony stimulating factor to animals receiving transplants of amniotic fluid mesenchymal stem cells not only improved survival of transplanted cells but also augmented nerve regeneration over that of a primarily cell-based approach. Additionally, differences in the material in which stem cells are delivered have demonstrated varying capacities to support long-term cell survival.[8,47] Finally, immunosuppressive regimens, especially in the light of interspecific transplants may protect stem cells from premature clearance from the nerve injury site.[47]
As with survival, differentiation of stem cells within the injury site has demonstrated a mixed correlation to therapeutic effect. Some studies have demonstrated a need for differentiation to glial phenotype to observe adequate regeneration of neural tissue, and others have shown improvement with little to no differentiation of stem cells at the assessment end point.[60] Furthermore, the glial differentiation of transplanted stem cells within the injured peripheral nerve has tended to vary between studies, even within the same cell type. Keilhoff and colleagues[26] could not detect Schwann cell differentiation of transplanted marrow stromal cells if delivered in a naive state, whereas Zhang et al.[75] observed at least limited expression of S100, p75, and GFAP markers in similarly obtained cells. In this case, the difference in repair paradigms (devitalized muscle graft versus crush injured nerve) may explain the disparity in the ability of these cells to differentiate, outlining the need for careful consideration of the method of delivering stem cells to the injury site. In the cases in which adequate regeneration and improvement of outcomes occurs without Schwann cell differentiation of transplanted precursors, it may be that the cells are supporting axonal growth by additional mechanisms such as the production of cytokines or harnessing the inflammatory response.[42] Although C17.2 neural stem cells show little differentiation into a Schwann cell phenotype in the chronically denervated peripheral nerve, their secretion of various matrix metalloproteinases, capable of breaking down growth-inhibiting chondroitin sulfate proteoglycans, likely underlies their ability to elicit superior regeneration.[18] Similarly, unpublished observations from our laboratory have shown that impure cultures of SKPCs at an early stage of Schwann cell differentiation secrete detectable levels of a number of neurotrophins despite lacking typical Schwann cell morphology or histological markers. Therefore the following question remains: must stem cells fully adopt a stereotypical Schwann cell phenotype to be successful adjuncts to nerve repair? Careful examination of ultimate cell fate with correlation to functional outcome is strongly recommended for future precursor transplant studies and will be required to fully answer this question for each cell type and repair strategy. It may be that, at least for some precursor cell types, there is a minimum level of differentiation to S100β/MBP/GFAP-positive Schwann cells that is required for acceptable regeneration outcomes. If this is the case, effectiveness of precursor transplantation could be improved using technology that exists to directly alter the regenerative microenvironment by continuous delivery of neuregulins, forskolin, or other differentiation-promoting factors.[35]
Given the evidence presented above, it is apparent that studies exploring stem cell transplantation for peripheral nerve repair should give careful thought on strategies to track the fate of transplanted cells over time. There is often little importance placed on prelabeling cells prior to delivery into the injured nerve, and as such authors cannot comment on the mechanism of any advantage conferred by cell therapy. Others have used labeling techniques that are not sufficiently robust or long-lasting to be detected at the study end points.[10] Chemical markers such as bisbenzimide and PKH26 have been used to label Schwann cells delivered to peripheral nerve injuries, but their usefulness is limited to the short term and may in fact affect the viability and phenotype of transplanted cells.[39] Genetic labeling with either lacZ or fluorescent proteins such as GFP is increasingly popular and appears to be a relatively long-lasting method that is not deleterious to transduced cells.[12,40] We have used the lipophilic carbocyanine derivative CellTracker CM-DiI (Molecular Probes) to reliably label SKPCs within a variety of nerve injury models with no dilution or loss of signal for ≥ 10 weeks following transplantation (Fig. 3). These dyes have the advantage of being technically simple to use, rapid, and resistant to leakage to nearby cells. Emerging technologies such as quantum dots offer an exciting alternative to traditional cell labeling methods. These nanoparticles are available in a wide range of photostable colors and are resistant to chemical and metabolic degradation, making them ideal for use in long-term fate tracking of transplanted stem cells.[41]
Figure 3. Confocal image. The CM-DiI is reliable method for long-term cell fate tracking within the peripheral nerve. The SKPC–Schwann cells prelabeled for 20 minutes in 2 μM CM-DiI retain bright labeling (red) for ≥ 8 weeks following transplantation into an acellular nerve graft bridging a gap in the rodent sciatic nerve, allowing for quantification without using additional dye techniques. Original magnification ×200. (Click on figure to magnify)
Animal studies have demonstrated that transplantation of stem and precursor cells has the potential to serve as an adjunct therapy to common practices of surgical nerve repair. Although the application of cell-based strategies in a clinical setting is promising, optimization of cell delivery and careful investigation of the fate of transplanted cells is required to guarantee the safety and maximum efficacy of these therapies. As discussed in this review, it will be important to determine the ideal number and method of cell delivery, and elucidate the extent of transplant cell survival and differentiation that is required to elicit a therapeutic effect. Future studies should place emphasis on using reliable labeling methods to track the long-term fate of transplanted cell. Finally, while many cell types have been investigated for their potential use in cell replacement therapy, few studies have directly compared the utility of different stem cells in augmenting peripheral nerve repair. We believe that cells that are easily isolated from autologous sources such as the skin and that can survive and differentiate to a glial phenotype within the milieu of the injured nerve provide the most promise.
References
The author: Professor Yasser Metwally
INTRODUCTION
April 15, 2009 — Stereotactic radiosurgery (SRS) with the Gamma Knife and linear accelerator has revolutionized neurosurgery over the past 20 years. The most common indications for radiosurgery today are tumors and arteriovenous malformations of the brain. Functional indications such as treatment of movement disorders or intractable pain only contribute a small percentage of treated patients. Although SRS is the only noninvasive form of treatment for functional disorders, it also has some limitations: neurophysiological confirmation of the target structure is not possible, and one therefore must rely exclusively on anatomical targeting. Furthermore, lesion sizes may vary, and shielding adjacent radiosensitive neural structures may be difficult or impossible.
The most common indication for functional SRS is the treatment of trigeminal neuralgia. Radiosurgical treatment for epilepsy and certain psychiatric illnesses is performed in several centers as part of strict research protocols, and radiosurgical pallidotomy or medial thalamotomy is no longer recommended due to the high risk of complications. Radiosurgical ventrolateral thalamotomy for the treatment of tremor in patients with Parkinson disease or multiple sclerosis, as well as in the treatment of essential tremor, may be indicated for a select group of patients with advanced age, significant medical conditions that preclude treatment with open surgery, or patients who must receive anticoagulation therapy. A promising new application of SRS is high-dose radiosurgery delivered to the pituitary stalk. This treatment has already been successfully performed in several centers around the world to treat severe pain in patients with end-stage cancer.
WHEN Lars Leksell presented SRS in 1951,[33] his original intention was to use the technique for the treatment of functional disorders such as PD, psychiatric conditions, and chronic pain syndromes. He developed the first Gamma Knife in 1967 and originally used slit-shaped collimators to make discoid-shaped lesions in the brain with stereotactic guidance and high precision using focused γ-radiation in a noninvasive fashion. The second Gamma Knife model, produced in 1975, had circular collimators as we know them today—producing spherical lesions—and in 1988 the Gamma Knife took on the design and source geometry that has more or less remained unchanged since. With the reports by Solberg et al. in 1998,[65] linear accelerators were introduced as tools for making lesions with small collimator apertures for the treatment of functional disorders, first in an animal model[7] and later in humans.[63]
The purpose of the present study is to provide an overview of the current literature on SRS in the treatment of movement disorders, epilepsy, chronic pain syndromes (including TN), and psychiatric disorders. A search of the online database of the National Library of Medicine was conducted using the following search term combinations: radiosurgery OR Gamma Knife; tremor; Parkinson disease; thalamotomy; pallidotomy; pain; trigeminal neuralgia; epilepsy; and psychiatric disease. We selected articles pertinent to this report and added historical papers. Publications were categorized into 4 groups: movement disorders, pain, epilepsy, and psychiatric disease. Our experience and that of authors from numerous treatment centers is discussed in a critical manner. We have also included personal experiences and unpublished data when appropriate.
Radiosurgical treatment of tremor has been performed successfully for more than 25 years by directing a 4-mm-collimator shot into the posterior ventrolateral thalamus close to the internal capsule (Fig. 1). The authors of several studies have reported success rates in tremor control in patients with PD, essential tremor, and tremor related to multiple sclerosis or other causes[13,17,41,42,43,51,68,69,72] that are comparable to those achieved using other methods. Complications are usually rare, mild, and temporary, although unusual complications[62] and severe, permanent complications[47] have also been reported. The most common applications of SRS in the treatment of movement disorders are summarized in Table 1 .
Table 1. Parameters of Radiosurgery for Movement Disorders (Click on table to enlarge)*
Figure 1. Stereotactic MR Images, Axial (A and B) and coronal (C) Views, Inversion-recovery Sequence, as Used for Planning of a Radiosurgical Thalamotomy. After Identifying the AC-PC Plane, a Single 4-mm-collimator Shot is Placed Over the Target Area in the Ventrolateral Thalamus. The 20% (Outer Circle) and 80% (Inner Circle) Isodose Lines are Plotted. AC = Anterior Commissure; PC = Posterior Commissure. (Click on figure to enlarge)
The first reports of using GKS in the treatment of tremor were published in the early 1990s, when Rand et al.[51] in the United States, Lindquist and colleagues[35] in Europe, and Ohye et al.[44] in Japan described their experiences. The positive findings of these groups have since been duplicated at numerous treatment centers around the world.[7,45,68,72] The most comprehensive series was published by Young and associates,[69] who reported on long-term follow-up of up to 8 years in 102 patients with tremor related to PD, 52 patients with essential tremor, and 4 patients with tremor as a result of stroke, encephalitis, or head injury. Treatment was performed with the Gamma Knife using a 4-mm collimator and a maximum radiation dose of 120-160 Gy. Of special note is the fact that blinded pre- and postoperative rating was performed by a team of independent evaluators skilled in evaluating movement disorders. The severity of PD was scored using a validated rating scale (Unified PD Rating Scale) to assess tremor and rigidity. In 88% of patients with parkinsonian tremor and 88% of patients with essential tremor, there was long-term relief of symptoms after GKS, which was statistically significantly improved over baseline status. In 50% of patients with other tremors there was improvement, and complications were noted in 3 of 158 patients (1.9%; 1 patient with transient and 2 with mild permanent complications).
Sato and associates[59] addressed the question of optimal target selection by comparing the radiosurgical target with the target used for stereotactic radiofrequency coagulation. Because the center of the radiosurgical treatment plan is positioned in such a way that the internal capsule laterally and the ventralis-caudalis sensory nucleus of the thalamus posteriorly do not suffer radiation damage, this radiosurgical target is typically located 1-2 mm more medial and anterior than the "real" target. In their study, Sato et al.[59] describe 4 patients who underwent radiosurgical treatment planning followed by radiofrequency lesioning of the Vim for the treatment of tremor. Electrophysiological recording demonstrated that the tremor-synchronous cells with rhythmic discharge typically found in the Vim region were indeed present within the radiosurgical target. The authors concluded that current planning strategies for Vim radiosurgical thalamotomy were adequate. The issue of extent of necrosis after radiosurgical creation of a lesion was addressed by the same authors in a different publication. Specifically, the area of the blood-brain barrier disruption, seen on contrast-enhanced MR images as a ring of enhancement, was the target of their research. Two patients with tremor in whom previous GKS had failed underwent open stereotactic thalamotomy with neurophysiological recording. The authors found that the area corresponding to the ring enhancement seen on MR images was active neural tissue with viable tremor cells and not, as may be assumed, necrotic tissue.
Radiosurgical pallidotomy has been tried in several institutions but abandoned due to an unacceptable complication rate in most cases. Our own results in 4 patients[14] indicate that the success rate is significantly lower than would be expected with other forms of treatment, such as deep brain stimulation therapy. A postmortem analysis in 1 of our patients with PD who died 30 months after GKS pallidotomy, presumably of unrelated causes,[15] also raises concerns over optimal lesion location, potential injury to the optic tract, and the risk of postradiosurgical vascular changes possibly resulting in a basal ganglia stroke. The authors of other studies[47] also strongly advise against the generalized use of radiosurgery for the treatment of PD.
Radiosurgical pallidotomy was first reported on by Rand et al.,[51] who used the technique in 8 patients, 4 of whom had significant relief of contralateral rigidity. There were no reports of significant side effects. A less encouraging result was obtained by Friedman et al.,[14] who reported on 4 patients with PD treated with unilateral pallidotomy using a maximum dose of 180 Gy. Only 1 patient had reduced dyskinesia afterwards but also experienced temporary dementia and psychosis. The authors questioned the feasibility of performing radiosurgical pallidotomies.
A more positive experience is reported by Young and coworkers[72] who treated 2 groups of patients: 29 patients with radiosurgical pallidotomy and 22 with radiofrequency lesioning in the posteroventral pallidum internum. After a mean follow-up period of 20.6 months, more than 80% of patients in both groups had significant improvement in dyskinesia, and two thirds of patients had improvements in bradykinesia or rigidity. In 27.7% of patients in the radiofrequency lesioning group there was transient postoperative confusion. Only 1 patient in the GKS group (3.4%) had a complication in the form of a homonymous hemianopsia 9 months after treatment.
Duma[9] cautions that radiosurgical pallidotomy may be associated with an unacceptably high risk of complications. In his experience, the combination of poor outcome and high risk have led him to abandon this radiosurgical modality altogether. This concern is shared by Okun and colleagues[47] who reported on a study of 8 patients (who underwent 5 thalamotomies and 3 pallidotomies) with significant complications as a result of radiosurgical lesioning, including hemiparesis, hemianopsia, and pseudobulbar laughter. The authors questioned whether the overall risks of radiosurgical lesioning for PD had been underestimated and strongly recommended that this modality should be offered only with great reservation for the treatment of PD.
For other movement disorders, the worldwide experience in treatment with SRS is limited. An initial case report from 1995[29] in which a patient with hemidystonia was treated with a radiosurgical pallidotomy was followed by the report of 2 cases,[48] and in an additional report in 2002[46] of patients treated with a lesion to the anterior ventrolateral thalamus. All patients were reported to have significant improvements, observable 2-3 months after treatment. A homonymous hemianopsia developed in the patient undergoing radiosurgical pallidotomy; treatment-related side effects in the other patients were not reported. Successful radiosurgical treatment of dystonia has been reported[29,48] with the posteroventral pallidum or the ventrolateral thalamus as the target, and there has also been a report of successful radiosurgical subthalamotomy[23] with long-term follow-up.
The most common applications of SRS for the treatment of pain are summarized in Table 2 .
Table 2. Radiosurgery for Pain Disorders* (Click on table to enlarge)
By far the most common indication for functional SRS is TN. Extensive wordwide research has been conducted regarding underlying pathophysiological mechanisms of pain relief,[26] clinical outcomes,[5,27,28,32,55,58,63] and comparative technologies.[37] Because of the nonexistent procedure-related mortality rate, the low risk of significant complications, and its ability to provide pain control for tic doloreux, SRS for TN has certainly become one of the most important options when recommending treatment modalities to patients. Other forms of pain treatment with SRS have been described mostly in anecdotal form and are much less researched. We continue to rely mostly on clinical experience of some surgical centers when recommending SRS for certain chronic pain disorders. When it comes to treatment of pain related to a malignant growth, there is increasing evidence that SRS applied to the pituitary stalk region is an effective approach for patients with pain from metastatic cancer.[21] The same approach has also been tried in patients with poststroke thalamic pain syndrome; however, a much lower rate of success and a higher rate of complications was found.[20]
Radiosurgical treatment for TN has been subject to extensive research. Both GKS and linear accelerator radiosurgery have been successfully applied to treat tic doloreux refractory to medication after failed attempts at MVD, and ablative procedures such as radiofrequency retrogasserian rhizotomy or glycerol injection.[5,27,28,55,58,63]
Leksell began treating patients with TN in 1953, although his first report of 2 patients was not available until 1971.[32] Both patients were reported to be pain free 1 and 5 months after radiosurgery to the trigeminal nerve. Hakanson examined these 2 patients 20 years after treatment, and they remained pain free (personal communication). The estimated target doses were 16.5-22 Gy. Several years later, Lindquist et al.[35] and Rand et al.[52] reported on their patients with tic doloreux treated with radiosurgery to the Gasserian ganglion. Because the results were inconsistent, the authors concluded that other targets should be considered. The first multicenter study using GKS at the TREZ near the pons was initiated by the University of Pittsburgh and published in 1996.[27] Five centers participated in this study, and 50 patients were enrolled, 32 of whom had undergone previous surgery for facial pain. Patients were treated with low-dose (60-65 Gy) or high-dose (70-90 Gy) radiosurgery. After a follow-up period of 11-36 months, 29 patients (58%) reported complete relief of pain, 18 (36%) had obtained good pain control (50-90% relief), and 3 (6%) experienced treatment failure. The median time to pain relief was 1 month. The authors also noted that pain relief was significantly more likely with higher doses of radiation (70-90 Gy).
In another prospective study[55] radiosurgical treatment of the TREZ using 70-90 Gy, 83 of 100 patients were pain free either with or without medication. A quality-of-life assessment in these patients indicated a statistically significant improvement in all tested areas. The only side effects were mild hypesthesia or paresthesia in the trigeminal area (6% of patients).
A single-center single-physician study compared GKS against MVD surgery.[5] The author reports on 24 patients who underwent surgery and 61 patients who were treated with 75 Gy to the TREZ. After 18 months’ follow-up, complete pain relief was seen in 68% of patients in the group that received MVD, and 24% of patients in the GKS group. Pain relief of 90% or more was reported in 78% of patients who underwent MVD surgery and in 48% of patients who received GKS. There were no significant complications seen in either group. The author concludes that both methods provide good pain control, but that MVD is more likely than GKS to provide complete relief of pain.
Linear accelerators have also been used successfully for the treatment of tic doloreux.[58,63] In the original study from 1995,[28] using either 5-mm or 7.5-mm collimators and applying similar doses to the TREZ (70-90 Gy) as described with GKS, the authors achieved sustained pain relief of 88% of patients, with a mean follow-up period of 23 months. The only reported complication was postprocedure numbness in 25%. Although there is no study comparing linear accelerator SRS to GKS in terms of outcomes or side effects, Ma et al.[37] attempted model dose falloff and error tolerances when planning radiosurgical procedures for TN. The authors found no significant differences between the 2 modalities, provided that a high number of arcs is used (≥ 7), and a small interarc error is guaranteed; however, they caution about increased treatment time for multiarc treatment plans.
The question of whether the TREZ undergoes any kind of permanent change as a result of radiosurgery is addressed in a publication by Shetter and coauthors.[61] They report on 6 patients who were treated with radiosurgery with 80-135 Gy in 1 or 2 sessions. Patients had persistent or recurrent facial pain and elected to undergo MVD surgery. The authors indicate that there were no abnormal findings around the nerve entry zone that were attributable to previous radiosurgery and therefore conclude that previous radiosurgery does not preclude patients from MVD in case of pain recurrence.
Stieber et al.[67] describe the only reported—and successful—treatment of glossopharyngeal neuralgia in a patient who refused MVD surgery. A maximum dose of 80 Gy was delivered to the nerve at the entry into the osseous canal of the jugular foramen using a single 4-mm-collimator shot. The patient experienced gradual pain relief starting 6 weeks after treatment and was pain free without medication 3 months after treatment. She had partial recurrence of pain 6 months after treatment but did not require further medical or surgical therapy. No complications were reported.
Pollock and Kondziolka[50] describe the successful application of a single 8-mm-collimator shot to the sphenopalatine ganglion in a patient with sphenopalatine neuralgia. A maximum dose of 80 Gy was delivered, and the patient initially improved but then had recurrence of her pain that required a second treatment with the same treatment parameters. Six months after the second treatment, she was pain free and her vasomotor symptoms (nasal discharge, injected eye) also improved.
After Steiner[66] and Leksell[31] reported initial successes in patients with chronic pain by making radiosurgical lesions in the thalamus, it took almost 2 decades before the treatment was investigated on a larger scale. Encouraged by some positive results,[70] Young[68] treated 41 patients with chronic pain related to structural spinal disorders or spinal cord injury, postherpetic neuralgia, stroke, and thalamic pain syndrome, or anesthesia dolorosa of the face. The treatment target was the medial thalamus, including the intralaminar nuclei, the lateral portions of the medial-dorsal nucleus, the centromedian nucleus, and the parafascicular nucleus.[71] The 4-mm collimator was used to deliver 140-180 Gy in 1, 2, or 3 isocenters. Although two thirds of patients reported a reduction in their pain of 50% or more, the treatment was associated with significant side effects, including 1 death (personal communication). In another report,[16] these unpredictable lesion sizes were studied and found to be typically seen when more than 1 isocenter was applied or when doses of more than 160 Gy were used.
Radiosurgical treatment of the pituitary stalk for post-stroke thalamic pain syndrome was recently described.[20] In this study, 24 patients underwent GKS of the pituitary gland and stalk with a dose of 140-180 Gy. In 71% of patients, significant pain relief started as soon as 48 hours after treatment. However, after a follow-up period of 12-48 months, only 21% of patients had lasting pain relief. Ten patients (42%) also had reported side effects, which were usually related to hormone imbalance.
After Steiner[66] and Leksell[31] reported initial successes in patients with chronic pain by making radiosurgical lesions in the thalamus, it took almost 2 decades before the treatment was investigated on a larger scale. Encouraged by some positive results,[70] Young[68] treated 41 patients with chronic pain related to structural spinal disorders or spinal cord injury, postherpetic neuralgia, stroke, and thalamic pain syndrome, or anesthesia dolorosa of the face. The treatment target was the medial thalamus, including the intralaminar nuclei, the lateral portions of the medial-dorsal nucleus, the centromedian nucleus, and the parafascicular nucleus.[71] The 4-mm collimator was used to deliver 140-180 Gy in 1, 2, or 3 isocenters. Although two thirds of patients reported a reduction in their pain of 50% or more, the treatment was associated with significant side effects, including 1 death (personal communication). In another report,[16] these unpredictable lesion sizes were studied and found to be typically seen when more than 1 isocenter was applied or when doses of more than 160 Gy were used.
Radiosurgical treatment of the pituitary stalk for post-stroke thalamic pain syndrome was recently described.[20] In this study, 24 patients underwent GKS of the pituitary gland and stalk with a dose of 140-180 Gy. In 71% of patients, significant pain relief started as soon as 48 hours after treatment. However, after a follow-up period of 12-48 months, only 21% of patients had lasting pain relief. Ten patients (42%) also had reported side effects, which were usually related to hormone imbalance.
Leksell used radiosurgery early on to treat pain related to malignant tumors with the medial thalamus as the target.[31,66] Since his initial reports, there have been few studies on using the same technique for cancer-related pain. Frighetto et al.[18] in 2004 described their experience of using a linear accelerator with a 5-mm collimator to create a lesion in the medial thalamus of 3 patients, 1 of whom had metastatic cancer. All patients, including the patient with cancer, who died 2 weeks after treatment, had substantial pain relief and were able to reduce their medications.
Backlund and coworkers[2] used Gamma Knife hypophy-sectomy in the treatment of 8 patients suffering from severe pain from advanced-stage breast cancer and achieved excellent pain relief in all patients. A somewhat different approach—originally reported by Lisák and Vladyka[36] —is described in a multicenter prospective protocol[21] of pituitary gland and stalk ablation with GKS in patients with pain related to bone metastases. One of the inclusion criteria was that the pain had to be responsive to morphine. The authors reported on 9 patients who were treated with either a single 8-mm-collimator shot or a 2-isocenter delivery with the 4-mm collimator. A maximum dose of 160 Gy was delivered, and the radiation exposure of the optic structures was limited to 8 Gy or less. After a follow-up of 1-24 months, all 9 patients experienced significant pain relief without any reported side effects.
In 1998, Ford et al.[12] published the first report of successful treatment of cluster headaches by GKS. Of 6 patients treated with a 70-Gy maximum dose to the TREZ, 4 had excellent pain relief for a follow-up period of 8-14 months. No side effects were reported. A few years later, a prospective study was begun, which was just recently completed and published.[8] Of the 10 patients treated in this study, only 3 had complete or almost complete relief of symptoms. Due to the high incidence of side effects (9 of 10 patients) with trigeminal paresthesia, hypesthesia, or deafferentation pain, the authors do not recommend treatment of cluster headaches with trigeminal radiosurgery. McClelland et al.[38] confirmed the negative findings. Targeting the sphenopalatine ganglion instead of the TREZ could be a promising solution and is being investigated.[30]
The most common applications of SRS for the treatment of epilepsy are summarized in Table 3 .
Table 3. Radiosurgery for Epilepsy (Click on table to enlarge)
Mesial temporal lobe epilepsy and its treatment with SRS is one of the areas in radiosurgery with extensive evidentiary support. After first reports in humans[3,4] a French group[54] conducted basic laboratory tests to gain further insight into the mechanisms underlying radiosurgical anti-epileptogenesis. Rats received doses of 100 Gy to the striatum, resulting in a differential effect on different enzymes (glutamate decarboxylase and choline acetyltransferase) and secondarily between excitatory amino acids and non-excitatory amino acids, particularly γ-aminobutyric acid. Another animal study[40] examined a kainic-acid rat model of epilepsy and the effect of varying doses of radiation. The authors were able to establish a clear relationship between increasing radiosurgical dose and decreasing seizure frequency measured by electroencephalography. A dose in the 40-60 Gy range was found sufficient to provide good seizure control without causing brain tissue necrosis and was therefore deemed appropriate for human use.
In 1985, Barcia-Salorio and colleagues[4] reported on 6 patients treated with a cobalt unit using a 10-mm collimator. The epileptic foci in these patients received an estimated dose of 10 Gy. In 1994, the same group reported a long-term analysis using doses between 10-20 Gy.[3] Five of 11 patients had complete cessation of seizures and another 5 were improved starting 3-12 months after treatment. Further experience was then reported from Sweden by Hellstrand et al.,[22] with more advanced techniques of localizing the epileptic focus, including magnetoencephalography and the use of GKS. In 1993, Régis and associates[56] performed the first selective radiosurgical amygdalohip-pocampotomies for mesial temporal epilepsy. The 2 patients in their study both received a dose of 25 Gy to the 50% isodose and were seizure free within 1 year. Postoperative MR imaging studies in these patients revealed contrast enhancement corresponding to the 50% isodose. The same researchers then initiated a multicenter study, which was conducted at 3 European centers.[57] Twenty patients underwent radiosurgery with 24-25 Gy delivered to the mesial temporal lobe for intractable mesial temporal lobe epilepsy. After a follow-up period of 2 years, 65% of these patients were seizure free, however, it took about 12 months postsurgery for the seizure frequency to begin to drop significantly. The authors also commented on the fact that all of their patients experienced a transient increase in seizures, mostly auras, before the number of seizures started to diminish. Nine patients (45%) were reported to have visual field defects, which, according to the authors, compares favorably with the results of microsurgical selective amygdalohippocampectomy or anterior temporal lobectomy, in which > 70% of patients have postoperative visual field deficits. Other transient complications included headache, nausea, vomiting, depression, and dizziness.
In 1998, Arita et al.[1] reported the first case of a patient with a hypothalamic hamartoma suffering from gelastic and tonic-clonic seizures to be treated with SRS. The patient became seizure free with a follow up of almost 2 years. The reported MRI findings indicate a complete disappearance of the hypothalamic changes. As a result of this report, a multicenter retrospective study was conducted including 10 patients in 7 international centers.[53] All patients reported improvement of their seizures, with 4 being seizure-free after a follow-up period of 12-71 months. The authors advocate a dose to the margin of the lesion of 17 Gy. These findings have since been confirmed using linear accelerator radiosurgery.[60]
Another fairly novel way of using radiosurgery in the treatment of epilepsy was first described in 1999 by Pendl et al.,[49] who described the treatment of 2 patients with longstanding Lennox-Gastaut syndrome and 1 patient with tonic-clonic and absence seizures with a corpus callosotomy of the anterior third of the corpus callosum. After a mean follow-up period of 38 months, all patients had improvement in their seizures. Encouraged by these results, the same group later published a report on 8 patients undergoing either anterior callosotomy (6 patients) or posterior callosotomy (2 patients) after previous surgical callosotomy.[11] The patients were followed up for a minimum of 1 year (range 1-12 years). Drop-attack seizures were abolished in 3 patients, and 3 more patients had a 40-60% improvement of drop attacks. Generalized tonic-clonic seizures were abolished in 2 patients, and 2 other patients had a 50-60% improvement. Other seizure types did not respond as well to treatment, with a 20-70% reduction seen in 3 patients. There were only minor transient side effects in 2 patients, and mild changes on MR imaging were noted in only 25% of patients. The favorable results have since been confirmed and repeated with the use of a linear accelerator[6,64] and have also been reported in children.[10]
The first radiosurgical treatments of psychiatric disease were performed by Leksell in 1953 with a 300-kV x-ray device. Seven patients with OCD were treated by placing radiosurgical lesions into the anterior limb of the internal capsule.[34] After 7 years’ follow-up, 5 patients reported long-term benefits.[35] Further treatments were performed at the Karolinska Institute in Stockholm starting in 1988, first by using the 8-mm collimator and later—because of excessive lesion sizes—with the 4-mm collimator.[25] Long-term follow-up 17 years after treatment of OCD with bilateral radiosurgical anterior capsulotomy at maximum doses of 120-180 Gy has been reported.[24] In 27 of 28 targets, the volume of necrosis was measured on MR images to be 100 mm3 or less, and a radiation dose of at least 110 Gy reliably produced a lesioning effect.
Our institution has been involved in the neurosurgical treatment for psychiatric disease for the past 2 decades. Radiosurgery as a possible tool to treat OCD was introduced in the 1990s, encouraged by some positive reports from the Karolinska Institute.[24,35,39] Because there are significant ethical concerns and organizational issues associated with the treatment of such patients,[19] we have offered this treatment exclusively to an extremely treatment-refractory group of patients who were selected by a team of psychiatrists as part of a strict research protocol. At our own institution we continue to provide radiosurgical treatment for OCD to a small group of very treatment-resistant patients as part of an ongoing open-label research study. Our current treatment protocol calls for 2-isocenter shots with the 4-mm collimator to each side with a maximum dose of 180 Gy per shot (2 lesions on each side are created). With this treatment method we have achieved long-term and significant reduction of OCD symptoms in roughly two thirds of our patients. Preliminary findings are encouraging but longer follow-up is needed.
There is sufficient evidence in the literature to support the use of radiosurgery in the treatment of certain functional disorders. Stereotactic radiosurgery has become a standard neurosurgical tool for the treatment of TN because of favorable results and very limited risk of complications. Radiosurgical treatment of some other pain syndromes is promising with certain techniques (SRS to the pituitary stalk), while other techniques (such as medial thalamotomy) are no longer recommended. Certain types of epilepsy appear to respond well to SRS with acceptable risks. The supportive evidence on the treatment of hypothalamic hamartomas with SRS continues to accumulate. Although some procedures for the treatment of movement disorders are associated with high risks (radiosurgical pallidotomy, for example) it is feasible to offer radiosurgical thalamotomy to patients with tremor, especially in situations where open stereotactic approaches for lesioning or deep brain stimulation therapy are not possible or are too risky. The widespread use of radiosurgery in the treatment of psychiatric disorders is strongly discouraged unless performed within the confines of a research protocol and accompanied by the strong support of a dedicated psychiatry team.
The author: Professor Yasser Metwally
INTRODUCTION
April 9, 2009 — We were queuing for crullers and coffee after a grueling ward round when our eyes were drawn to the choreiform movements of the woman ahead of us. Despite our feeble attempts not to stare, our eyes were drawn to her excruciatingly slow attempts to insert a straw into a glass, extricate cash from her purse, and, balancing her tray precariously, wend her way to a nearby table. Against our better natures, none of us made any real attempt to assist, lest we cause offence or embarrassment. She sat down, adjusted her perfectly positioned spectacles, and began to eat. Later, comfortably ensconced in the doctors’ lounge, we began to discuss the phenomenology of her movement disorder, even postulating that adjusting her spectacles was a voluntary maneuver to mask her involuntary choreiform movements.
In medicine, the art of making a spot diagnosis is much lauded; indeed, William Osler, doyen of clinical medicine, declared, “There is no more difficult art to acquire than the art of observation.”[1] In 1817, James Parkinson wrote a monograph on his eponymous disease after examining Londoners whose stooped posture and shuffling gait caught his attention.[2] Many of his observations hold true to this day, bearing testament to his prescience and clinical acumen. Legend has it that Christian Billroth would start the academic year by sticking his finger into a foul-tasting liquid while informing his students of the two qualities necessary for becoming a doctor: namely, freedom from nausea and the power of observation. He would then enjoinder his students to copy his actions and would smile at their stoic discomfiture, telling them that they had passed the first test but not the second, for he had dipped the index finger and licked the middle finger.
Neurology is unique among the medical specialties in that much of the clinical examination can be appreciated visually and taught by use of video recordings.[3,4] Since 2003, we have conducted a ‘neurological localization course’, during which participants are taught correct clinical examination techniques with the help of patients.[5] Trainees are often impressed by the wealth of clinical information that can be gleaned by observation alone; for example, how the externally rotated, slightly plantar-flexed attitude of the lower limb of a supine patient can hint at the possibility of an underlying footdrop, or how muscle atrophy, diabetic dermopathy and trophic changes can not only provide clues to an underlying peripheral neuropathy, but can even indicate the level of the stocking paresthesia.
Several weeks after our encounter with the woman with choreiform movements, we were enjoying another post-rounds breakfast-cum-discussion when our attention was drawn to a colleague whose subtle neck and facial movements were accompanied by grunting noises while eating—phenomena indicative of complex motor tics, rather than the more facile explanation that he was really enjoying his morning porridge. When he had left, the medical student attached to our team asked the obvious question: with the evidence staring us in the face, why did no one inform him of the diagnosis and proffer appropriate treatment? Having acknowledged the proverbial ‘elephant in the room’, we launched into an animated discussion about a physician’s duty of care, asking whether the ethical imperative to treat exists only in a medical emergency or after the establishment of a formal doctor–patient relationship.
Few would argue that doctors have a moral and legal obligation to render assistance in the event of a medical emergency.[6] A formal doctor–patient relationship likewise provides a doctor with the moral and legal imperative to practice ‘good medicine’. Hence, a neurologist seeing a patient for diabetic polyneuropathy would not hesitate to enquire about symptoms of hyperthyroidism when the patient has a noticeable goiter, despite its apparent irrelevance to the case. Indeed, the same doctor would be thought negligent if he were to ignore or fail to notice a goiter in a patient with myasthenia gravis, in view of the known associations between these two conditions.
Was our group remiss because we did not inform the stranger with choreiform movements or our colleague with tics of their diagnoses, simply to avoid embarrassment? It might be argued that to offer an unsolicited medical opinion to a stranger reeks of bad taste, akin to canvassing patients or ambulance chasing. Some might comfort themselves that failing to inform a woman with a goiter and ophthalmopathy or a man with bilateral ptosis of their probable diagnoses of Graves disease and myasthenia gravis, respectively, is not an egregious or harmful omission. Yet the potential consequences of such inaction could be the development of atrial fibrillation resulting in an embolic stroke, or myasthenic crisis with respiratory failure. As Einstein perceptively pointed out, “The world is a dangerous place, not because of those who do evil, but because of those who look on and do nothing.” We had hoped that, being in a hospital, the woman with choreiform movements had already consulted (or was on her way to consulting) a doctor. It was, however, just as probable that she was merely visiting a patient, and that, like us, other doctors had steered clear of broaching the subject.
Could we have helped the woman? The answer is an emphatic yes. We could have ascertained the cause of her chorea, performed a clinical examination for a cardiac murmur to exclude rheumatic fever and Sydenham chorea, examined her eyes under a slit lamp for Kayser–Fleischer rings, treated any underlying conditions such as hyperglycemia or thyrotoxicosis, recommended neuroimaging, and instituted genetic testing if Huntington disease was suspected. If she had Parkinson disease and unrecognized levodopa-induced dyskinesias, we could have recommended adjustment of her medications or the addition of amantadine, sedatives or nootropics to ameliorate the chorea.[7] Likewise, dopamine depletors or atypical antipsychotics could lessen disabling choreiform movements,[8] thereby increasing her quality of life. It is possible, however, that unsolicited advice could prove unwelcome. In the case of a diagnosis of Huntington disease, for example, the patient would be forced to face the prospect of an incurable and heritable disease that could render her unemployed and without medical insurance.
What, then, is our duty of care to the ‘man in the street’? Are physicians morally and legally obliged to render a medical opinion at all times? More importantly, could we be held liable, in the absence of a formal doctor–patient relationship, for failing to point out to a would-be patient that they might have medical conditions requiring attention? Although we are morally obligated to render assistance in a life-or-death situation, even being protected under the ‘Good Samaritan Act’,[9] there is no legal liability should a physician ‘do nothing’ in an emergency involving a stranger, as long as the physician “is not under a pre-existing duty and has not created a risk of harm to the stranger”.[10] The absence of an emergency situation does not, however, preclude longer-term deleterious consequences, as illustrated by the scenarios described above. For this reason, the issue of our duty of care is worthy of further debate and discussion.
References
Elder NC et al. (2006) The art of observation: impact of a family medicine and art museum partnership on student education. Fam Med 38: 393–398
Parkinson J (1817) An Essay on the Shaking Palsy. London: printed by Whittingham and Rowland for Sherwood, Neely and Jones
Lim EC and Seet RC (2008) Demystifying neurology: preventing ‘neurophobia’ among medical students. Nat Clin Pract Neurol 4: 462-463
Lim EC and Seet RC: Using an online neurological localisation game. Med Educ [doi:10.1111/ j.1365-2923.2008.03188.x]
Lim EC et al. (2006) Neurological localisation course for postgraduate candidates. Med Educ 40: 487-488
Walker AF (2002) The legal duty of physicians and hospitals to provide emergency care. CMAJ 166: 465-469
Lim E (2005) A walk through the management of Parkinson’s disease. Ann Acad Med Singapore 34: 188-195
Cardoso F et al. (2006) Seminar on choreas. Lancet Neurol 5: 589-602
Vernaglia LW (1999) The Good Samaritan physician. Med Health 82: 304-306
Zhang Y (2005) Responding to an emergency in and out of the office. Mo Med 102: 424-428
Metwally, MYM: Textbook of neuroimaging, A CD-ROM publication, (Metwally, MYM editor) WEB-CD agency for electronic publication, version 10.2a April 2009 [Click to have a look at the home page]
The author: Professor Yasser Metwally
INTRODUCTION
April 1, 2009 — Considerable interest has been shown in the potential anti-inflammatory effects of polyunsaturated fatty acids (PUFAs) in multiple sclerosis (MS) and other autoimmune inflammatory disorders. Studies suggest a modest association between consumption of low levels of unsaturated fat and an increased incidence of MS. Moreover, in vitro and in vivo studies have demonstrated that omega-3 and omega-6 PUFA supplementation can reduce immune-cell activation via a number of complex pathways. Noncontrolled and controlled clinical trials of PUFA supplementation in patients with MS have, however, provided mixed results. These studies had important limitations in design and selection of outcome measures, and these factors might partially explain the inconsistent results. We propose that the potential role of PUFAs as disease-modifying, anti-inflammatory treatments for MS should be revisited in proof-of-concept trials that use accepted MRI outcome measures.
The currently approved immunomodulatory therapies for multiple sclerosis (MS) show modest efficacy and can produce a wide range of adverse effects. Interest has, therefore, been increasing in complementary and alternative approaches for the treatment of MS. As disability and impairment accrue, patients with MS often seek unconventional treatments in the hope of halting the disease process.[1] One popular unconventional approach is dietary supplementation with polyunsaturated fatty acids (PUFAs). In view of the reputed anti-inflammatory properties of PUFAs, numerous reviews have cited these agents as potential treatments for immune-mediated disorders, including rheumatoid arthritis and MS.[2,3,4,5,6,7] In a recent survey, 37% of 1,573 patients with MS revealed that they had used omega-3 unsaturated fatty acids at some point in their lives.[8]
In this post, we briefly discuss the nomenclature of PUFAs, as well as describing their sources and derivatives. We then describe epidemiologic studies that have indicated a link between dietary saturated and polyunsaturated fat intake and the risk and severity of MS. These results have contributed to an increased interest in the biological effects of PUFAs in inflammation. We then discuss the effects of PUFAs in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Finally, we review the noncontrolled and controlled clinical trials that have assessed the effect of PUFA administration on disability and relapses in MS. We conclude by discussing the implications of all these findings for further clinical research into the effects of PUFAs in MS.
Polyunsaturated Fatty Acids: An Overview
Polyunsaturated fatty acids (PUFAs) are defined as fatty acids that possess more than two carbon–carbon double bonds. Given that they also contain 18 or more carbon atoms, they are often referred to as long-chain PUFAs. The term omega or n refers to the terminal methyl end of the fatty acid. The numerical designation, such as omega-3 or n-3, refers to the location of the last carbon–carbon double bond from the omega end. For the purposes of this Review, we will use the omega designation to distinguish the two classes of fatty acids, and the term PUFAs to refer to either omega-3 or omega-6 fatty acids.
Omega-3 and omega-6 PUFAs and their derivatives have important roles in metabolic, immunologic, coagulation and inflammatory processes. Omega-3 PUFAs are primarily derived from fish oils, whereas omega-6 PUFAs are obtained from plant sources, including sunflower, safflower, corn, wheat germ and soybean oils (Figure 1). Omega-3 PUFAs, such as a-linolenic acid, are converted, through several reactions, to eicosapentanoic acid (EPA). However, EPA can also be directly obtained through the diet from fish oils. EPA can be converted to docohexanoic acid (DHA), and also to eicosanoids, such as prostaglandins, thromboxanes and leukotrienes, which are essential for inflammatory signaling. DHA is converted to eicosanoids, and, ultimately, to anti-inflammatory mediators such as resolvins and protectins.
 |
Figure 1. Omega-6 and omega-3 PUFAs and their respective sources and metabolic derivatives. After consumption, the PUFAs are metabolized via several pathways (not shown) to active compounds that mediate inflammation and products that promote resolution of inflammation. Possible effects on inflammation are listed in the box at the bottom. Abbreviations: IFN-Y, interferon Y; IL-2, interleukin 2; NFkB, nuclear factor kappa B; PGE2, prostaglandin E2; PPARY, peroxisome proliferator-activated receptor Y; PUFAs, polyunsaturated fatty acids; TGFß, transforming growth factor ß; TNF, tumor necrosis factor. (Click to enlarge figure)
Omega-6 PUFAs are more abundant in the diet than omega-3 PUFAs. Omega-6 PUFAs are converted to other metabolites through various enzymatic processes, and eventually to the key intermediate arachidonic acid, which is subsequently converted to eicosanoids. Omega-3 PUFAs tend to be more potent anti-inflammatory agents than omega-6 PUFAs, but the effects of omega-6 PUFAs might predominate owing to the abundance of these compounds in the diet.[9]
Studies Exploring Diet and Multiple Sclerosis Risk
Epidemiologic studies have reported that MS is particularly common in geographic regions with high levels of saturated fat consumption. However, these findings were derived mainly from ecological studies, which are highly susceptible to confounding factors.[10] Analysis of data pooled from 36 industrialized nations showed that saturated fatty acids, animal fat and animal minus fish fat were all independent predictors of MS mortality (P <0.001 in women and P <0.01 in men). Furthermore, the ratios of dietary polyunsaturated to saturated fatty acids and of monounsaturated plus polyunsaturated to saturated fatty acids showed independent and significant negative correlations with the risk of developing MS (P <0.01–P <0.001 in women, and P <0.05–P <0.01 in men).[11] A multivariate analysis of published data from 48 states in the US found positive correlations between the risk of MS and the sales of meat and dairy products. By contrast, an inverse correlation was evident between MS risk and the sales of vegetable and fish products.[12]
Numerous case–control studies on diet and MS have been conducted over the past 60 years. However, the results of these studies were inconsistent, and did not support an overall relationship between fat intake and MS.[13,14,15,16,17]The inconsistencies probably stem from the retrospective nature of the studies, which renders them vulnerable to bias. Another potential weakness of such studies is their susceptibility to confounding factors.[10] A recent case-control study in Norway found an inverse association between fish intake and risk of developing MS.[18] The investigators believe that vitamin D, which is highly concentrated in cod-derived products, might have confounded the results of previous observational studies. Vitamin D has an important role in the immune system, and high levels have been associated with a reduced risk of developing MS.[19,20,21]
Despite the results from case-control and ecological studies, a large prospective study that analyzed data from two large cohorts (150,000 women, including 195 incident cases of MS) in the Nurses’ Health Studies I and II failed to find any evidence that an increased risk of MS was associated with high intake of saturated fats or a low intake of polyunsaturated fats.[22] A borderline inverse association was noted between linolenic acid intake and risk of MS, but this link was nonsignificant. This longitudinal study design is much less susceptible to recall or selection bias than are case-control and ecological studies.[10]
Several small studies have demonstrated a reduction in PUFA content in serum, cerebral white matter, erythrocytes and lymphocytes in patients with MS compared with controls.[23,24,25,26] However, these observations do not help to clarify the exact nature of the relationship between PUFA intake and MS, as no data were provided on the dietary habits and clinical characteristics of the study participants.
Effects of Polyunsaturated Fatty Acids on Inflammation
The relationship between dietary fat intake and the risk of MS remains unclear, but evidence is growing that omega-6 and omega-3 PUFAs have anti-inflammatory effects. This evidence derives from the results of several in vitro, in vivo and ex vivo studies that are likely to be relevant to MS. The anti-inflammatory effects of omega-3 and omega-6 PUFAs might include competitive inhibition of arachidonic acid, the metabolites of which are involved in promoting inflammation.[27,28,29] In addition, the production of anti-inflammatory prostaglandins E1 and E2, which are derived from the omega-6 PUFA dihomo-Y-linolenic acid, can inhibit the production of proinflammatory cytokines such as interleukin (IL)-2 and interferon Y (IFN-Y).[30,31,32] In vitro, T-cell proliferation can be reduced by supplementation with either omega-6 or omega-3 PUFAs.[33] Both omega-6 and omega-3 PUFAs can modify lymphocyte function through a reduction in the levels of the proinflammatory cytokines IL-1ß, tumor necrosis factor (TNF) and IL-1.[34,35,36] Omega-3 PUFAs might also inhibit the migratory activity of leukocytes—an essential part of the inflammatory response—via alteration of cytoskeletal components.[37]
Recently identified molecules derived from PUFAs have roles in the resolution of inflammation. These molecules include lipoxins, which are derived from arachidonic acid and can promote resolution of inflammation by reducing neutrophil activity and stimulating the uptake of apoptotic polymorphonuclear leukocytes.[38] Resolvins and protectins are two other types of lipid mediator that are derived from omega-3 PUFAs, EPA and DHA, via lipooxygenase-mediated mechanisms.[39,40,41] D-series resolvins (those derived from DHA) are attractive candidates for the control of inflammation in neural tissues, given that these tissues are a source of endogenous DHA. Effects of D-resolvins include inhibition of neutrophil activity and, potentially, modulation of inflammation.[39,42] Protectins are also derived from DHA, and protectin D1 in particular possesses anti-inflammatory properties that include reduction of TNF expression and IFN-Y production, and promotion of T-cell apoptosis.[39,43]
Increasing attention is being paid to the roles of PUFAs as ligands for peroxisome proliferator-activated receptors (PPARs). PPARs are ligand-activated nuclear transcription factors that have various subtypes. PPARs regulate genes that are involved in lipid metabolism and lipid storage, and have important roles in the anti-inflammatory response.[44,45] PUFAs and their respective derivatives can serve as PPAR agonists.[45,46,47] Human T lymphocytes express the PPARY isoform, and omega-3 PUFAs and their eicosanoid derivatives are thought to possess stronger PPARY-agonist properties than omega-6 PUFAs and their derivatives.[48] Oral administration of PPARY agonists can not only ameliorate existing inflammation in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, but can also prevent inflammation from occurring in such models.[49,50,51] These findings suggest a potential role for PPARY agonists in the treatment of MS.[52]
Omega-3 PUFAs have also recently been shown to inhibit the expression of nuclear factor kB (NFkB), a transcription factor that is crucial for the production of inflammatory cytokines, chemokines and adhesion molecules with pivotal roles in MS.[53,54,55,56]
In addition, omega-3 PUFAs can, in vivo, promote the production of molecules involved in myelinogenesis.[57] When EPA and DHA were injected intraventricularly into rat brains, increased expression of gene transcripts for proteolipid protein, mylein basic protein and myelin oligodendrocyte was observed. The investigators showed that EPA adminstration caused a more pronounced effect than administration of DHA.
PUFAs might also alter the production of certain matrix metalloproteinases (MMPs) that have been implicated in disruption of the blood–brain barrier. MMP activity seems to facilitate the migration of leukocytes into the CNS. Dietary supplementation with conjugated linoleic acid and DHA in rats reduced serum levels of MMP2 and MMP9.[58] Various doses of omega-3 PUFAs (DHA and EPA) alone, or of a mixture of DHA, EPA, linoleic acid, arachidonic acid and saturated fatty acids, can lower MMP9 levels when added to lipopolysaccharide-stimulated rat microglial cell cultures.[59]
Trials of Polyunsaturated Fatty Acids in Multiple Sclerosis
Trials in Animal Models
Administration of PUFAs can reduce the clinical severity of EAE, an animal model of MS. Linoleic acid supplementation has been shown to reduce the severity of EAE in guinea pigs when given before EAE induction.[60,61] Another study examined several different dosages of Y-linolenic acid (GLA) and linoleic acid in Lewis rat models of EAE.[62] Oral GLA supplementation at a dose of 500 mg/kg body weight abolished the clinical signs of EAE, an observation that was confirmed histologically. However, oral supplementation of GLA at doses of 200 mg/kg or 1000 mg/kg only delayed the onset of EAE. Linoleic acid doses in the range of 500–1000 mg/kg reduced the severity of clinical EAE in a dose-dependent fashion.[62] Harbige et al. also demonstrated that GLA supplementation could reduce the severity of both acute EAE and the relapsing phases of chronic, relapsing EAE.[63] Further analysis revealed an increase in production of transforming growth factor ß-1 and prostaglandin E2, both of which are associated with a reduction in the inflammatory response in EAE models.[30,32,63,64]
Clinical Trials
Noncontrolled Observations. Over 50 years ago, Swank hypothesized that dietary fat intake contributed to disability progression in MS.[65,66,67,68,69] In what was essentially an open-label observational study, Swank prescribed a low-fat diet to patients with relapsing–remitting MS, in which fats derived from dairy and animal sources were gradually eliminated, and replaced with both omega-3 and omega-6 fatty acids derived from fish and vegetable sources. The patients (n = 47) showed reductions in the frequency and severity of exacerbations—usually after the first year of observation had elapsed—and continued to demonstrate improvements in relapse rate and severity after 5.5 years.[65,66] Those who adhered most closely to the prescribed diet seemed to derive the greatest benefits from the intervention, and no adverse events were reported. The patients were monitored over the next 15 years, and maintained lower levels of disease activity and disability than would be predicted by natural history studies—this was true especially of those who began to follow the diet early in their disease course.[67] In 2000, 15 patients who had been on the diet for 50 years were re-evaluated, and 13 of these individuals were described as ambulatory and fully able to perform activities of daily living.[69]
In another noncontrolled trial, patients with MS (n = 12) who were treated with a combination of EPA and DHA omega-3 PUFAs for 4 months showed minimal change in disability as a group. However, the subset with relapsing–remitting disease (n = 5) showed a decrease in their mean Expanded Disability Status Scale (EDSS) score from 3.30 to 2.70, whereas those with progressive disease (n = 7) showed an increase in mean EDSS score from 6.42 to 7.07.[70] In a similar study, 16 patients with relapsing–remitting MS were treated with fish oil containing DHA and EPA for 2 years.[71] The investigators also provided dietary advice to reduce daily saturated fat intake and increase weekly fish consumption. At the end of 2 years, a significant reduction in mean annual relapse rate and mean EDSS score (P <0.01) was evident when compared with baseline assessments. Serum concentrations of omega-3 fatty acids had also increased significantly (P <0.001).
Controlled Trials. In an attempt to provide a proper assessment of the efficacy of PUFA supplementation in MS, multiple controlled studies have been performed, some of which date back to the 1970s. These studies, however, generally produced inconclusive results.[72] The results of the controlled trials performed to date are summarized in Table 1 .
Table 1. Clinical Trials that Assessed the Efficacy of Polyunsaturated Fatty Acid Supplementation in MS
|
Reference |
Interventions |
Trial design |
Results |
Comments |
| Millar et al. (1973)[73] | Linoleic acid (17.2 g per day) emulsion versus oleic acid (7.6 g per day) emulsion | Double-blind, parallel-group study, 24 months duration, involving 87 patients at two centers with DSS scores from 0–6 | Significant improvement in relapse severity and nonsignificant trend towards lower annualized relapse rates in the linoleic acid group; no differences in disability between the two groups | Disability, as measured by a modified DSS and ability to perform activities of daily living, worsened in both groups; high dropout rate from one site |
| Bates et al. (1977)[76] | Four treatment arms: linoleic acid (0.36 g per day) + GLA (3.42 g per day) capsules versus oleic acid (4.8 ml per day) capsules (placebo), and linoleic acid (11.5 g per day) spread versus oleic acid (4 g per day) spread (placebo) | Double-blind, parallel-group study, 24 months duration, involving 152 patients with chronic, progressive MS | No significant differences in disability (measured on the DSS), relapse rates or relapse severity score among the four groups | 12% early dropouts |
| Bates et al. (1978)[77] | Four treatment arms: linoleic acid (0.34 g per day) + GLA (2.92 g per day) capsules versus oleic acid (4.0 g per day) capsules (placebo), and linoleic acid (23 g per day) spread versus oleic acid (16 g per day) spread (placebo) | Double-blind, parallel-group study, 24 months duration, involving 116 patients with relapsing MS | Linoleic acid plus linolenic acid group had briefer and less-severe relapses compared with placebo group, but accumulated more disability than placebo group | Methods for determining relapse severity and duration were not described |
| Paty et al. (1978)[79] | Linoleic acid 17g per day versus oleic acid 21 g per day (placebo) | Double-blind, parallel-group study, 30 months duration, involving 96 patients with relapsing and progressive MS | No differences in disability, rates or severity of relapse, or timed functional tests between the two groups; significant increase in serum concentrations of linoleic acid in the active arm | Meaningful treatment effect was probably obscured by the enrollment of patients with relapsing MS and with progressive MS |
| Bates et al. (1989)[82] | Fish oil (mixture of EPA 1.71 g per day and DHA 1.14 g per day) capsules versus oleic acid (7.2 g per day) capsules (placebo) | Double-blind, parallel-group study, 24 months duration, involving 312 patients with relapsing MS | Fish-oil group showed a nonsignificant trend toward less disability progression | Both groups were instructed to restrict saturated fat intake and increase omega-6 fat intake, which might have obscured any meaningful treatment effect |
| Weinstock-Guttman et al. (2005)[83] | Fish oil (EPA 1.98 g per day and DHA 1.32 g per day) capsules versus oleic acid (1.0 g per day) capsules (placebo); disease-modifying therapies allowed | Double-blind, parallel-group study, 12 months duration, involving 31 patients with relapsing MS | No differences seen in relapse rates between the two groups; fish oil group had improvements in quality-of-life measures | Both groups were instructed to restrict saturated fat and daily caloric intake, which might have obscured any meaningful treatment effect |
| Harbige et al. (2007)[84] | High-dose GLA (14 g per day) versus low-dose GLA (5 g per day) versus placebo (polyethylene glycol) | Double-blind, parallel-group study, 18 months duration, involving 36 patients with active MS | High-dose GLA group had significantly reduced relapse rates and disability progression (measured on the Expanded DSS) compared with low-dose GLA and placebo groups | Interpretation limited by relatively short study duration, number of patients might be insufficient for adequate power; methods, inclusion criteria and enrollment criteria were not defined clearly |
Abbreviations: DHA, docohexanoic acid; DSS, Disability Status Scale; EPA, eicosapentanoic acid; GLA, γ-linolenic acid; MS, multiple sclerosis.
In a double-blind study by Millar et al., 75 patients with MS were randomly assigned to receive linoleic acid (administered in the form of sunflower seed oil) or oleic acid (an omega-9 PUFA with no known immunomodulatory effects, administered in the form of olive oil) as a control. The authors monitored the effects of this treatment for 2 years.[73] A nonsignificant trend towards lower annualized relapse rates and a significant reduction in relapse severity (P <0.01) were identified in the linoleic acid group compared with the control group. Relapse severity was measured on the basis of a semiquantitative assessment of the type and duration of specific symptoms. Disability status was scored by a modified Kurtzke Disability Status Scale (DSS) and by patients’ ability to perform activities of daily living.[73,74,75] Disability in both groups worsened to similar degrees, and no overall differences were observed between the groups. The investigators reported contrasting trends between the study sites, but did not elaborate any further on this point.[73]
In a 2-year study, Bates et al. randomly allocated 152 patients with chronic, progressive MS to one of four groups.[76] One group received a combination of linoleic acid and GLA in the form of Naudicelle® oil capsules (Naudicelle Ltd, Nantwich, Cheshire, UK) and a second received placebo (oleic acid capsules). Two additional groups received spreads containing linoleic acid or oleic acid. No significant differences were observed in disability (measured by the DSS), relapse rates, or the relapse severity score as used in Millar et al.’s study.[73] In a separate trial, Bates et al. randomly assigned 116 patients with relapsing MS to one of the four treatment arms.[77] The linoleic acid–GLA group had shorter, less-severe relapses than did the oleic acid group, although the methods used to obtain this outcome were not fully described. The linoleic acid–GLA acid group showed more progression of disability than did the oleic acid group (P <0.05). Linoleic acid spread alone was significantly associated with attacks that were shorter in duration, and was also associated with lower attack severity scores.
Paty et al. randomly assigned 96 patients with relapsing or progressive MS to linoleic acid or oleic acid for 30 months of double-blind treatment. This study monitored the effects of this treatment on disability (DSS, Kurtzke functional scales), relapse-related measures, and results of timed functional studies (7 timed tests of upper extremity function conducted by an occupational therapist, and 46 timed tests of upper and lower extremity functions carried out by a physical therapist).[74,78,79,80] Compared with placebo, linoleic acid did not affect disability, relapse rates, relapse severity, or the outcomes of functional tests, despite a marked increase in the serum concentration of linoleic acid in the treated group.[79,80]
The inconsistent results of these studies prompted Dworkin et al. to reanalyze pooled data from three of the four sites (Belfast, Newcastle upon Tyne and Western Ontario, but not London) that enrolled patients in these studies.[73,77,79] A total of 87 patients treated with linoleic acid and 85 controls were examined, and assessments from the first 30 months after treatment initiation were included in the analysis.[81] Dworkin and colleagues hypothesized that the benefits of linoleic acid supplementation would be most pronounced in patients with minimal disability or a short disease duration at baseline. This hypothesis was tested by comparing patients who, at trial entry, had lower (0–2) versus higher (3–6) disability scores, and different durations of disease (0–5 years, 6–10 years or =11 years). The results showed a benefit of linoleic acid with respect to disability progression in patients with minimal or no disability at trial entry; the DSS score showed mean changes of 0.12 in the linoleic acid group and 0.81 in the control group (P = 0.05, one-tailed Mann–Whitney U test). In addition, treatment with linoleic acid reduced the severity and duration of relapses at all levels of disability and disease duration. Considered together, the results of these three trials and the analysis of the pooled data provide support for a beneficial effect of linoleic acid, or the combination of linoleic acid plus linolenic acid, in reducing the severity of relapses and the rate of disease progression in patients with MS.
In a separate trial, Bates et al. randomly assigned 312 patients with relapsing MS to receive either a mixture of omega-3 fatty acids (18% EPA, 12% DHA) derived from fish oil or to placebo (primarily oleic acid) for 2 years.[82] Both groups were also instructed to restrict saturated fat intake and to increase the intake of omega-6 PUFAs in their diets. The fish-oil group demonstrated a trend towards less disability progression (on the basis of DSS scores) compared with placebo (P = 0.07). In a prespecified subgroup analysis, no difference was evident in disability changes between the fish oil and placebo groups for patients with DSS scores =2 or with MS of =5 years’ duration. Furthermore, no significant difference was evident between the fish oil and placebo groups with regard to the frequency, duration or severity of relapses.
Weinstock-Guttman et al. randomly assigned 31 patients with MS to omega-3 PUFAs from fish oil or to placebo.[83] All patients were also instructed to maintain a low intake of saturated fat. The group taking omega-3 PUFAs showed improvements in quality-of-life measures (SF-36®[Medical Outcomes Trust, Inc., Waltham, MA] Physical Component Scale and the Mental Health Inventory) compared with the placebo group after 6 months of treatment, but not after 12 months. No differences in relapse rates were observed between the two groups.
In 2008, Harbige et al. described the results of a randomized, double-blind, placebo-controlled trial to determine the effects of two different dosages of BGC20-884, a GLA-rich oil derived from borage oil, in patients with active MS.[84] A total of 28 patients were followed over 18 months. High-dose GLA treatment (14 g per day; 11 patients) significantly reduced relapse rates (P < 0.05) when compared with placebo (10 patients) and low-dose GLA (5 g per day; 7 patients). The high-dose GLA treatment group also showed reduced disability progression, as measured with the EDSS. However, the authors did not provide detailed information regarding trial design, statistical methods, and the characteristics of the patients.
Limitations of the Trials Performed to Date
The inconsistent results from the clinical trials performed to date are disappointing, but we believe that these trials possess several notable limitations. First, we consider the question of appropriate clinical trial design. The observational and noncontrolled trials mentioned above were extremely prone to selection bias. Furthermore, noncontrolled studies give no indication as to how individuals with MS would have fared had the disease been allowed to take its natural course. A matched control cohort can also help eliminate problems caused by regression to the mean, which is common in MS clinical trials.[85] In patients with MS who have a high level of disease activity, such as frequent relapses, these parameters will tend to return to the average when followed longitudinally. One recent study of 44 patients with relapsing–remitting MS reported that regression to the mean can reduce the relapse rate by as much as 40%.[86] In extension studies such as those performed by Swank et al., individuals who persisted with PUFA supplementation might have had a mild course of disease.[69] Conversely, individuals who were lost to follow up might have had a persistent course of MS that was refractory to diet modification.
Randomized, placebo-controlled trials are often preferable for the demonstration of efficacy in clinical trial research, as they have the design least susceptible to bias. However, these trials have limitations of their own. The randomized, controlled trials that have assessed the efficacy of various PUFAs typically contained small sample sizes, which reduced their power to detect small effects and adverse events. Some studies included both patients with relapsing MS and patients with chronic, progressive disease. Given that these two groups have very different patterns of disease (patients with progressive MS experience relapses to a lesser extent), the outcome measures that were used in these trials might not detect a significant benefit. Indeed, the choice of different and often inadequately validated outcome measures for the various trials might have limited their ability to detect meaningful differences overall. Some of the rating scales used in the early controlled trials, such as those used to measure relapse severity, had not been validated and were not widely employed at the time. Another common limitation lies in the fact that disability measures in MS, such as the DSS and EDSS, are often insensitive to changes that occur over a short time scale. Disability often accrues gradually, and 1–2-year studies are generally insufficient to provide any meaningful data on a treatment-related effect of PUFAs. Another issue is the ability to achieve appropriate statistical power to detect a true difference effect between treatment and control groups. Power is increased with large sample sizes or long study durations. Thus, studies with small numbers of patients conducted over a 1–2-year period are likely to be underpowered.
Another limitation in the controlled trials relates to the choice of an appropriate placebo. In most of the controlled trials mentioned above, olive oil was used as a placebo in the belief that oleic acid is relatively inert.[73,76,77,80,82,83] However, some evidence exists that components in olive oil inhibit NF-kB activation.[87] Furthermore, oleanic acid, which is also found in olive oil, can induce the release of prostacyclin, which can in turn activate PPARY.[88,89] These observations suggest that olive oil might not be an ideal placebo for PUFA trials, a factor that might partially explain the apparent lack of efficacy of PUFAs in the aforementioned trials.
Finally, relative uncertainty exists in relation to the optimal dosing of omega-3 and omega-6 PUFAs. The trials described above assessed various formulations, dosages and delivery vehicles for PUFAs, which hinders the attempt to draw any meaningful conclusions about safety and tolerability.[72] The optimal balance between omega-6 and omega-3 intake is also unknown. Both types of PUFAs competitively use the same set of enzymes, and the omega-6:omega-3 ratio in cell membranes can affect cell fluidity, which could in turn have a marked effect on numerous cellular functions.[48] Furthermore, omega-3 and omega-6 PUFAs might have proinflammatory properties under certain conditions.[90,91]
Implications for Future Research
The recent identification of mechanisms that indicate that omega-6 and omega-3 PUFAs might have beneficial effects on immune function should be explored with conventional proof-of-concept studies that use MRI end points. In particular, the incidence of gadolinium-enhancing brain lesions has emerged as a particularly sensitive measure of anti-inflammatory potential in preliminary proof-of-concept trials.[92]
Studies that examine such MRI activity have several inherent advantages: reduction of intrapatient variability, minimization of the number of potentially uninformative MRI scans, an adequate statistical power despite a relatively low number of patients, and a short study duration.[93,94] As a consequence, additional resources could be allocated for dose-ranging studies and controlled phase III trials that employ definitive clinical end points, in an attempt to demonstrate therapeutic effects of omega-3 or omega-6 PUFAs in MS.
Conclusions
There are some indications that PUFA intake has an inverse relationship with MS risk, but the controlled studies performed to date have not produced definitive results with regard to the potential benefits of PUFA supplementation in patients with MS. To address such potential benefits, well-designed, relatively short proof-of-concept trials with MRI end points are needed. Any successful result should then be followed by substantially larger and longer-term confirmatory trials with clinical end points.
Key Points
Epidemiological studies demonstrate an association between saturated fat intake and the incidence of multiple sclerosis (MS)
In vivo studies demonstrate that polyunsaturated fatty acids (PUFAs) can exert anti-inflammatory effects through multiple, complex mechanisms
Controlled and noncontrolled trials have produced mixed results regarding the efficacy of PUFAs in MS; however, these trials have several limitations that could partially explain the lack of a treatment effect
Despite the lack of definitive evidence that PUFAs can be beneficial in MS, the anti-inflammatory potential of these agents is intriguing
The potential role of PUFAs as a treatment for MS should be further explored in proof-of-concept studies that use MRI-based outcome measures
Abbreviations: DHA, docohexanoic acid; DSS, Disability Status Scale; EPA, eicosapentanoic acid; GLA, Y-linolenic acid; MS, multiple sclerosis.
References
Sastre-Garriga J et al. (2003) Unconventional therapy in multiple sclerosis. Mult Scler 9: 320-322
Rothman D et al. (1995) Botanical lipids: effects on inflammation, immune responses, and rheumatoid arthritis. Semin Arthritis Rheum 25: 87-96
Kankaanpaa P et al. (1999) Dietary fatty acids and allergy. Ann Med 31: 282-287
Mayer M (1999) Essential fatty acids and related molecular and cellular mechanisms in multiple sclerosis: new looks at old concepts. Folia Biol (Praha) 45: 133-141
Zamaria N (2004) Alteration of polyunsaturated fatty acid status and metabolism in health and disease. Reprod Nutr Dev 44: 273-282
Stewart TM and Bowling AC (2005) Polyunsaturated fatty acid supplementation in MS. Int MS J 12: 88-93
van Meeteren ME et al. (2005) Antioxidants and polyunsaturated fatty acids in multiple sclerosis. Eur J Clin Nutr 59: 1347-1361
Schwarz S et al. (2008) Complementary and alternative medicine for multiple sclerosis. Mult Scler 14: 1113-1119
Galli C and Marangoni F (1997) Recent advances in the biology of n-6 fatty acids. Nutrition 13: 978-985
Willett WC (1998) Overview of nutritional epidemiology. In Nutritional Epidemiology, 3-17 (Ed Willett WC) New York: Oxford University Press
Esparza ML et al. (1995) Nutrition, latitude, and multiple sclerosis mortality: an ecologic study. Am J Epidemiol 142: 733-737
Lauer K (1994) The risk of multiple sclerosis in the U.S.A. in relation to sociogeographic features: a factor-analytic study. J Clin Epidemiol 47: 43-48
Westlund KB and Kurland LT (1953) Studies on multiple sclerosis in Winnepeg, Manitoba, and New Orleans, Louisiana. I. prevalence; comparison between the patient groups in Winnipeg and New Orleans. Am J Hyg 57: 380-396
Cendrowski W et al. (1969) Epidemiological study of multiple sclerosis in western Poland. Eur Neurol 2: 90-108
Tola MR et al. (1994) Dietary habits and multiple sclerosis. a retrospective study in Ferrara, Italy. Acta Neurol (Napoli) 16: 189-197
Gusev E et al. (1996) Environmental risk factors in MS: a case-control study in Moscow. Acta Neurol Scand 94: 386-394
Ghadirian P et al. (1998) Nutritional factors in the aetiology of multiple sclerosis: a case-control study in Montreal, Canada. Int J Epidemiol 27: 845-852
Kampman MT et al. (2007) Outdoor activities and diet in childhood and adolescence relate to MS risk above the Arctic Circle. J Neurol 254: 471-477
Hayes CE et al. (2003) The immunological functions of the vitamin D endocrine system. Cell Mol Biol (Noisy-le-Grand) 49: 277-300
Munger KL et al. (2004) Vitamin D intake and incidence of multiple sclerosis. Neurology 62: 60-65
Munger KL et al. (2006) Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis. JAMA 296: 2832-2838
Zhang SM et al. (2000) Dietary fat in relation to risk of multiple sclerosis among two large cohorts of women. Am J Epidemiol 152: 1056-1064
Koch M et al. (2006) Erythrocyte membrane fatty acids in benign and progressive forms of multiple sclerosis. J Neurol Sci 244: 123-126
Gul S et al. (1970) Fatty acid composition of phospholipids from platelets and erythrocytes in multiple sclerosis. J Neurol Neurosurg Psychiatry 33: 506-510
Fisher M et al. (1987) Linoleic acid levels in white blood cells, platelets, and serum of multiple sclerosis patients. Acta Neurol Scand 76: 241-245
Wilson R and Tocher DR (1991) Lipid and fatty acid composition is altered in plaque tissue from multiple sclerosis brain compared with normal brain white matter. Lipids 26: 9-15
Callegari PE and Zurier RB (1991) Botanical lipids: potential role in modulation of immunologic responses and inflammatory reactions. Rheum Dis Clin North Am 17: 415-425
Gil A (2002) Polyunsaturated fatty acids and inflammatory diseases. Biomed Pharmacother 56: 388-396
Namazi MR (2004) The beneficial and detrimental effects of linoleic acid on autoimmune disorders. Autoimmunity 37: 73-75
Mertin J et al. (1984) Prostaglandins and cell-mediated immunity. The role of prostaglandin E1 in the induction of host-versus-graft and graft-versus-host reactions in mice. Transplantation 37: 396-402
Mertin J et al. (1985) Nutrition and immunity: the immunoregulatory effect of n-6 essential fatty acids is mediated through prostaglandin E. Int Arch Allergy Appl Immunol 77: 390-395
Santoli D and Zurier RB (1989) Prostaglandin E precursor fatty acids inhibit human IL-2 production by a prostaglandin E-independent mechanism. J Immunol 143: 1303-1309
Rossetti RG et al. (1997) Oral administration of unsaturated fatty acids: effects on human peripheral blood T lymphocyte proliferation. J Leukoc Biol 62: 438-443
Endres S et al. (1989) The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells. N Engl J Med 320: 265-271
Gallai V et al. (1995) Cytokine secretion and eicosanoid production in the peripheral blood mononuclear cells of MS patients undergoing dietary supplementation with n-3 polyunsaturated fatty acids. J Neuroimmunol 56: 143-153
DeLuca P et al. (1999) Effects of gammalinolenic acid on interleukin-1 beta and tumor necrosis factor-alpha secretion by stimulated human peripheral blood monocytes: studies in vitro and in vivo. J Investig Med 47: 246-250
Ferrante A et al. (1994) Neutrophil migration inhibitory properties of polyunsaturated fatty acids. The role of fatty acid structure, metabolism, and possible second messenger systems. J Clin Invest 93: 1063-1070
Yacoubian S and Serhan CN (2007) New endogenous anti-inflammatory and proresolving lipid mediators: implications for rheumatic diseases. Nat Clin Pract Rheumatol 3: 570-579
Serhan CN et al. (2002) Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J Exp Med 196: 1025-1037
Serhan CN et al. (2004) Resolvins, docosatrienes, and neuroprotectins, novel omega-3-derived mediators, and their aspirin-triggered endogenous epimers: an overview of their protective roles in catabasis. Prostaglandins Other Lipid Mediat 73: 155-172
Serhan CN et al. (2008) Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol 8: 349-361
Hong S et al. (2003) Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. Autacoids in anti-inflammation. J Biol Chem 278: 14677-14687
Serhan CN et al. (2004) Resolvins, docosatrienes, and neuroprotectins, novel omega-3-derived mediators, and their endogenous aspirin-triggered epimers. Lipids 39: 1125-1132
Feige JN et al. (2006) From molecular action to physiological outputs: peroxisome proliferatoractivated receptors are nuclear receptors at the crossroads of key cellular functions. Prog Lipid Res 45: 120-159
Edwards IJ and O’Flaherty JT (2008) Omega-3 fatty acids and PPARã in cancer. PPAR Res 2008: 358052
Bordoni A et al. (2006) Polyunsaturated fatty acids: from diet to binding to PPARs and other nuclear receptors. Genes Nutr 1: 95-106
Calder PC (2008) Polyunsaturated fatty acids, inflammatory processes and inflammatory bowel diseases. Mol Nutr Food Res 52: 885-897
Schmitz G and Ecker J (2008) The opposing effects of n-3 and n-6 fatty acids. Prog Lipid Res 47: 147-155
Niino M et al. (2001) Amelioration of experimental autoimmune encephalomyelitis in C57BL/6 mice by an agonist of peroxisome proliferator-activated receptor-ã. J Neuroimmunol 116: 40-48
Diab A et al. (2002) Peroxisome proliferator-activated receptor-ã agonist 15-deoxy-ä12,14-prostaglandin J2 ameliorates experimental autoimmune encephalomyelitis. J Immunol 168: 2508-2515
Feinstein DL et al. (2002) Peroxisome proliferatoractivated receptor-ã agonists prevent experimental autoimmune encephalomyelitis. Ann Neurol 51: 694-702
Heneka MT et al. (2007) Drug insight: effects mediated by peroxisome proliferator-activated receptor-ã in CNS disorders. Nat Clin Pract Neurol 3: 496-504
Ghosh S et al. (1998) NF-êB and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16: 225-260
Hayden MS et al. (2006) NF-êB and the immune response. Oncogene 25: 6758-6780
Ghosh S and Hayden MS (2008) New regulators of NF-êB in inflammation. Nat Rev Immunol 8: 837-848
Novak TE et al. (2003) NF-êB inhibition by omega-3 fatty acids modulates LPS-stimulated macrophage TNF-á transcription. Am J Physiol Lung Cell Mol Physiol 284: L84-L89
Salvati S et al. (2008) Eicosapentaenoic acid stimulates the expression of myelin proteins in rat brain. J Neurosci Res 86: 776-784
Harris MA et al. (2001) Effects of conjugated linoleic acids and docosahexaenoic acid on rat liver and reproductive tissue fatty acids, prostaglandins and matrix metalloproteinase production. Prostaglandins Leukot Essent Fatty Acids 65: 23-29
Liuzzi GM et al. (2007) Inhibitory effect of polyunsaturated fatty acids on MMP-9 release from microglial cells—implications for complementary multiple sclerosis treatment. Neurochem Res 32: 2184-2193
Meade CJ et al. (1978) Reduction by linoleic acid of the severity of experimental allergic encephalomyelitis in the guinea pig. J Neurol Sci 35: 291-308
Hughes D et al. (1980) Linoleic acid therapy in severe experimental allergic encephalomyelitis in the guineapig: suppression by continuous treatment. Clin Exp Immunol 40: 523-531
Harbige LS et al. (1995) Prevention of experimental autoimmune encephalomyelitis in Lewis rats by a novel fungal source of ã-linolenic acid. Br J Nutr 74: 701-715
Harbige LS et al. (2000) The protective effects of omega-6 fatty acids in experimental autoimmune encephalomyelitis (EAE) in relation to transforming growth factor-beta 1 (TGF-â1) up-regulation and increased prostaglandin E2 (PGE2) production. Clin Exp Immunol 122: 445-452
Racke MK et al. (1991) Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-â1. J Immunol 146: 3012-3017
Swank RL (1955) Treatment of multiple sclerosis with low-fat diet; results of five and one-half years’ experience. AMA Arch Neurol Psychiatry 73: 631-644
Swank RL (1956) Treatment of multiple sclerosis with low-fat diet: result of seven years’ experience. Ann Intern Med 45: 812-824
Swank RL (1970) Multiple sclerosis: twenty years on low fat diet. Arch Neurol 23: 460-474
Swank RL and Dugan BB (1990) Effect of low saturated fat diet in early and late cases of multiple sclerosis. Lancet 336: 37-39
Swank RL and Goodwin J (2003) Review of MS patient survival on a Swank low saturated fat diet. Nutrition 19: 161-162
Cendrowski W (1986) Multiple sclerosis and MaxEPA. Br J Clin Pract 40: 365-367
Nordvik I et al. (2000) Effect of dietary advice and n-3 supplementation in newly diagnosed MS patients. Acta Neurol Scand 102: 143-149
Farinotti M et al. (2007) Dietary interventions for multiple sclerosis. Cochrane Database of Systematic Reviews 2007, Issue 1. Art. No.:CD004192. doi: 10.1002/14651858.CD004192.pub2
Millar JH et al. (1973) Double-blind trial of linoleate supplementation of the diet in multiple sclerosis. Br Med J 1: 765-768
Kurtzke JF (1961) On the evaluation of disability in multiple sclerosis. Neurology 11: 686-694
Millar JH et al. (1967) Long-term treatment of multiple sclerosis with corticotrophin. Lancet 2: 429-431
Bates D et al. (1977) Trial of polyunsaturated fatty acids in non-relapsing multiple sclerosis. Br Med J 2: 932-933
Bates D et al. (1978) Polyunsaturated fatty acids in treatment of acute remitting multiple sclerosis. Br Med J 2: 1390-1391
Kurtzke JF (1965) Further notes on disability evaluation in multiple sclerosis, with scale modifications. Neurology 15: 654-661
Paty DW et al. (1978) Linoleic acid in multiple sclerosis: failure to show any therapeutic benefit. Acta Neurol Scand 58: 53-58
Paty DW (1983) Double-blind trial of linoleic acid in multiple sclerosis. Arch Neurol 40: 693-694
Dworkin RH et al. (1984) Linoleic acid and multiple sclerosis: a reanalysis of three double-blind trials. Neurology 34: 1441-1445
Bates D et al. (1989) A double-blind controlled trial of long chain n-3 polyunsaturated fatty acids in the treatment of multiple sclerosis. J Neurol Neurosurg Psychiatry 52: 18-22
Weinstock-Guttman B et al. (2005) Low fat dietary intervention with omega-3 fatty acid supplementation in multiple sclerosis patients. Prostaglandins Leukot Essent Fatty Acids 73: 397-404
Harbige LS et al. (2008) Polyunsaturated fatty acids in the pathogenesis and treatment of multiple sclerosis. Proc Nutr Soc 67: E21
Goodin DS (2004) Disease-modifying therapy in MS: a critical review of the literature. Part I: analysis of clinical trial errors. J Neurol 251 (Suppl 5): v3-v11
Martinez-Yelamos S et al. (2006) Regression to the mean in multiple sclerosis. Mult Scler 12: 826-829 Acknowledgments The authors dedicate this article to the memory of Steven R Schwid, whose unparalleled commitment to the care of patients with multiple sclerosis and to discovering improved treatments for this disorder is an enduring inspiration. LR Mehta is supported by a Sylvia Lawry Physician Fellowship Award from the National Multiple Sclerosis Society. Competing interests The authors declared no competing interests.
Brunelleschi S et al. (2007) Minor polar compounds extra-virgin olive oil extract (MPC-OOE) inhibits NF-êB translocation in human monocyte/ macrophages. Pharmacol Res 56: 542-549
Falcetti E et al. (2007) IP receptor-dependent activation of PPARã by stable prostacyclin analogues. Biochem Biophys Res Commun 360: 821-827
Martinez-Gonzalez J et al. (2008) Oleanolic acid induces prostacyclin release in human vascular smooth muscle cells through a cyclooxygenase-2- dependent mechanism. J Nutr 138: 443-448
Bates EJ et al. (1993) Polyunsaturated fatty acids increase neutrophil adherence and integrin receptor expression. J Leukoc Biol 53: 420-426
Harbige LS (2003) Fatty acids, the immune response, and autoimmunity: a question of n-6 essentiality and the balance between n-6 and n-3. Lipids 38: 323-341
McFarland HF et al. (2002) The role of MRI as a surrogate outcome measure in multiple sclerosis. Mult Scler 8: 40-51
Simon JH (2003) Measures of gadolinium enhancement in multiple sclerosis. In Multiple Sclerosis Therapeutics, Edn. 2, 97-124 (Eds Cohen JA and Rudick RA) London: Martin Dunitz
Sormani MP et al. (2001) Clinical trials of multiple sclerosis monitored with enhanced MRI: new sample size calculations based on large data sets. J Neurol Neurosurg Psychiatry 70: 494-499
