Demyelination arrest and remyelination induced by glatiramer acetate treatment of experimental autoimmune encephalomyelitis

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1 Demyelination arrest and remyelination induced by glatiramer acetate treatment of experimental autoimmune encephalomyelitis Rina Aharoni*, Avia Herschkovitz*, Raya Eilam, Michal Blumberg-Hazan, Michael Sela*, Wolfgang Bruck, and Ruth Arnon* Departments of *Immunology and Veterinary Resources, The Weizmann Institute of Science, Rehovot 76100, Israel; QuantomiX Ltd., Ness Ziona 70400, Israel; and Institute for Neuropathology, Georg-August-University, Göttingen 37099, Germany Contributed by Michael Sela, May 19, 2008 (sent for review April 15, 2008) The interplay between demyelination and remyelination is critical in the progress of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). In the present study, we explored the capacity of glatiramer acetate (GA, Copaxone) to affect the demyelination process and/or lead to remyelination in mice inflicted by chronic EAE, using both scanning electron microscopy and immunohistological methods. Spinal cords of untreated EAE mice revealed substantial demyelination accompanied by tissue destruction and axonal loss. In contrast, in spinal cords of GA-treated mice, in which treatment started concomitantly with disease induction (prevention), no pathology was observed. Moreover, when treatment was initiated after the appearance of clinical symptoms (suppression) or even in the chronic disease phase (delayed suppression) when substantial demyelination was already manifested, it resulted in a significant decrease in the pathological damage. Detection of oligodendrocyte progenitor cells (OPCs) expressing the NG2 or O4 markers via colocalization with the proliferation marker BrdU indicated their elevated levels in spinal cords of GA-treated mice. The mode of action of GA in this system is attributed to increased proliferation, differentiation, and survival of OPCs along the oligodendroglial maturation cascade and their recruitment into injury sites, thus enhancing repair processes in situ. multiple sclerosis neuroprotection oligodendrocyte myelin repair Disseminated demyelination is the primary morphological hallmark characterizing multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), leading to axonal loss and neurological impairments (1, 2). It is therefore important to evaluate MS treatments for their neuroprotective capability to prevent demyelination and/or enhance remyelination. The interplay between pathological demyelination and the corresponding repair mechanism remyelination involves, on one hand, the inflammatory immune cells that mediate the damage and on the other hand, the myelinproducing cells, the oligodendrocytes. The latter are terminally differentiated cells with a limited capacity to respond to injury that are destroyed in the actively demyelinating lesions (3, 4). Accordingly, remyelination requires the recruitment of oligodendrocyte precursor cells (OPCs) by their proliferation and migration into the demyelinating area and their further differentiation into mature myelinating oligodendrocytes through distinct stages, characterized by morphological transformation and sequential expression of developmental markers. Thus, it has been demonstrated that in response to stimuli such as inflammation or demyelination, OPCs, characterized by bipolar morphology and by the presence of the early marker chondroitin sulfate proteoglycan, NG2, undergo proliferation and transform into multiprocessed preoligodendrocytes that acquire the later cell surface marker O4 (5, 6). Yet, despite this ability of the adult brain to retain the potential to generate oligodendrocytes with myelination capacity, remyelination in MS/EAE is incomplete and poorly sustained (3, 4). Glatiramer acetate (GA, Copaxone), an approved drug for the treatment of MS, has been shown to modulate the detrimental inflammation that mediates the demyelination process (7). The immunomodulatory effect of GA in EAE and in MS is attributed to its ability to induce specific Th2/3 cells (8, 9) that cross the blood brain barrier (BBB), accumulate in the CNS, and express in situ IL-10 and TGF-, as well as neurotrophic factors such as BDNF (10). Moreover, we recently demonstrated that GA treatment also affects the cells of the CNS and restores their impaired neurotrophic factor secretion, thus elevating the in situ levels of BDNF, neurotrophin 3 (NT3), and NT4 (11). The neuroprotective consequences of GA treatment were also manifested by a decrease in neuronal damage, and by an increase in neurogenesis of neuronal progenitors that migrate into injury sites and differentiate into mature neurons (12). In view of the immunomodulatory activity of GA, and its neuroprotective effects, it was of interest to investigate its ability to affect the primary target of the EAE/MS pathological process, the myelin. Previous studies indicated that coinjection of GA with encephalitogenic emulsion blocked EAE development and reduced its consequential demyelination (13). GA also prevented demyelination in the optic nerve when injected before or at disease induction (14). In the present study, we explored the potential of GA to interfere with demyelination. Furthermore, by applying GA treatment in the chronic EAE stage, we could examine whether GA induces remyelination even after extensive damage has occurred. For this purpose we used the recently developed wet scanning electron microscopy technique (wet SEM), which enables direct visualization of the myelin (15). We report here that GA treatment results in a decrease in myelin breakdown and tissue damage, and in stimulation of repair processes. The mode of action of GA in this system is attributed not only to the blockage of inflammation, but also to its effect on the expansion of cells from the oligodendrocyte lineage and their recruitment into injury sites, thus enhancing repair processes in situ. Results EAE manifestations and the effect of GA treatment were investigated in the myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE model, in which disease symptoms appear around day 15, with increased severity up to days 22 25, Author contributions: R. Aharoni, M.S., and R. Arnon designed research; R. Aharoni, A.H., R.E., and M.B.-H. performed research; R. Aharoni, A.H., R.E., W.B., and R. Arnon analyzed data; and R. Aharoni, M.S., and R. Arnon wrote the paper. Conflict of interest statement: Michael Sela and Ruth Arnon are among the inventors of Copaxone. To whom correspondence should be addressed. michael.sela@weizmann.ac.il by The National Academy of Sciences of the USA PNAS August 12, 2008 vol. 105 no cgi doi pnas

2 NEUROSCIENCE Fig. 1. The effect of GA on myelin visualized by wet SEM. Shown are series of wet SEM SC images (cervix) from representative mice, in magnifications of 800 (Center) and 1600 (Right), and their respective patterns of clinical daily scoring. (A) Prevention regimen: GA treatment started together with disease induction (eight daily injections). (B) Suppression regimen, started after the appearance of clinical symptoms (10 daily injections). (C) Delayed suppression regimen, started one month after disease induction (18 daily injections). In EAE mice, the myelin is reduced and accompanied by inflammation (white spots) and axonal loss (dark holes). In the SCs of EAE GA mice, the myelin is preserved and no pathology is observed. Arrows indicate the day of perfusion. GA treatment is indicated by a dark line. (Scale bars: 20 m, Center; 10 m, Right.) Aharoni et al. Fig. 2. Immunohistochemical characterization of SCs from EAE mice and the effect of GA treatment. SC sections from EAE induced YFP 2.2 mice, expressing YFP (green) on their neuronal population, were stained for myelin by using anti-mbp antibodies (yellow), overall cell nuclei by using Hoechst (blue), and T cells by using anti-cd3 antibodies (red). (A C) Corresponding coronal sections from: (A) An untreated EAE induced mouse, (B) An EAE induced mouse treated by GA (prevention regimen), and (C) an EAE induced mouse treated by GA (suppression regimen). Multiple demyelination sites are accompanied by fiber deterioration and overall cell/t cell infiltration in the EAE untreated mouse (indicated by arrows), in contrast to the minute damage detected in the GA treated mice. Scale bars; 250 m (Top), 100 m (Middle), 50 m (Bottom). (D) Treatment schedules and daily clinical score. (E) Quantitative analysis of myelin damages performed by measuring the area of MBP destruction in 16 sagittal sections along each SC (two mice per treatment group). Asterisk indicates significant reduction from untreated EAE mice. resulting in chronic disease. GA treatment was applied by daily injections at different disease stages: (i) starting immediately after disease induction (prevention), (ii) starting soon after the appearance of clinical manifestations (suppression), or (iii) in the chronic phase 10 days after disease was fully developed (delayed suppression). GA treatment by all three regimens ameliorated the clinical manifestations compared with unpnas 兩 August 12, 2008 兩 vol. 105 兩 no. 32 兩 11359

3 Table 1. Effect of GA treatment on SC histopathology visualized by wet SEM Treatment Mice tested, n Disease score at treatment initiation Disease score at perfusion Histopathological evaluation Naïve control EAE * EAE with GA prevention EAE with GA suppression EAE with GA delayed suppression A summary of a double blind evaluation of SC wet SEM images from GA- or vehicle-treated mice is shown. Histopathological evaluation criteria were as follows: 0, no pathology detectable; 1, mild: edema, mild subpial/meningeal inflammation; 2, moderate: parenchymal cell infiltration, mild tissue damage with demyelination; 3, severe: tissue destruction with parenchymal cell infiltration, demyelination, and axon damage. *Value is the average of 2, 2, 2, 2, 3, and 3. Value is the average of 0, 0, 0, 0, and 3. treated EAE mice (Figs. 1 and 2). Spinal cords (SC) of EAEuntreated vs. EAE-induced mice treated by GA (EAE GA) as well as naïve controls were subjected to both electron microscopy and immunohistochemistry for further evaluation. SEM Analysis of SC from EAE Mice and the Effect of GA Treatment. Electron microscopy was performed by application of the wet SEM technology, in which the analysis is done on wet tissue. SC were scanned at different white matter regions in both the cervix and the thorax. SEM images (30 36 images for each SC) were subjected to a histopathological evaluation in a blinded fashion, and the results (on a scale of 0 3) summarizing all of the animals tested (n 22) are presented in Table 1. A series of images (cervix) from representative mice of the three treatment regimens and their respective pattern of clinical daily scoring is demonstrated in Fig. 1. Whereas in naïve controls, the intact myelin structure emerged as sharp dark rings (Fig. 1A), in the SCs of EAE inflicted mice (clinical score 2 4, harvested 4 6 weeks after disease induction), the myelin was reduced in amount and degraded at multiple sites, indicative of the demyelination process (Fig. 1 A C). SCs of EAE untreated mice also revealed moderate to severe tissue destruction and axonal damage, accompanied by parenchymal cell infiltration that indicates the inflammatory disease course. The average histopathological score of the six EAE animals tested was 2.2. In contrast to the extensive pathology characteristic of the EAE untreated mice, in most (12 of 13) of the SCs of the GA-treated mice, no pathology was detected (Table 1) and their appearance was similar to that of naïve mice. This lack of pathology was observed in the mice in which treatment started concomitantly with EAE induction, thus preventing the disease and subsequent histological damage (Table 1 and Fig. 1A). Moreover, when treatment was initiated soon after the appearance of clinical symptoms during the suppression regimen (Fig. 1B) or even in the chronic disease phase (delayed suppression) (Fig. 1C) when injury was fully manifested, damage to myelin was hardly detectable (average histopathological scores 0 and 0.6 respectively) (Table 1). It should be noted that in the case of the delayed suppression regimen, treatment was applied for a longer period (18 daily injections, in comparison with 8 and 10 injections for prevention and suppression, respectively). The severity of the damage in these mice before treatment initiation can be appreciated by the tissue destruction observed in the SCs of untreated mice harvested at time points corresponding to the beginning of treatment (represented in Fig. 1C), which persisted throughout the later phase of the chronic disease (Fig. 1B). The only mouse in which pathological damage was found after GA treatment was one in which clinical symptoms persisted as well (grade 2). Parenchymal cell infiltration was observed in the SCs of GA-treated mice, but to a lesser extent than untreated mice. Taken together, the consequences of GA treatment in the various disease stages indicate that it reduces tissue damage, limits the breakdown of the myelin sheath, and improves tissue integrity even after injury has occurred. Immunohistochemical Characterization of SC from EAE Mice and the Effect of GA Treatment. To correlate these findings, myelin presence was visualized by using anti-myelin basic protein (MBP) antibodies in the SCs of YFP 2.2 transgenic mice, which selectively express YFP on their neuronal population (16) and are susceptible to MOG-induced EAE (12). As shown in Fig. 2A, multiple widespread areas of myelin damage were observed in various white matter regions of EAE inflicted mice (with clinical score 2 3, scarified one month after disease induction). The size of these demyelinating sites exceeded 6% of the overall area of MBP expression (quantified for 16 sagittal sections all along the SC, in two mice per treatment group) (Fig. 2E). In regions of myelin damage, YFP expression was obstructed, revealing sparse processes, axonal transection, and fiber deterioration, indicative of the axonal pathology that accompanies the demyelination process. Hoechst staining of cell nuclei as well as anti-cd3 staining of T cells demonstrated cellular infiltration into the lesion sites, consistent with the detrimental role of inflammation, in particular of T cells, in this disease. In the SCs of EAE induced mice treated with GA, either by the prevention (Fig. 2B) or by the suppression (Fig. 2C) regimens, considerably less damage was detected compared with the EAE untreated mice. This decreased damage was manifested in either a marked reduction or complete absence of the pathological manifestations, i.e., myelin damage, deteriorating fibers, and cellular/t cell infiltration sites. The effect of GA was especially prominent when GA was applied as a prevention treatment (94% reduction in demyelination), but a significant effect (74% reduction) was observed even when treatment started after pathological manifestations had been expressed (Fig. 2E). These results indicate a neuroprotective effect of GA in reducing the number and the size of SC lesions. Effect of GA Treatment on Oligodendrocyte Progenitor Cells in EAE Mice. To further understand the effect of GA on myelin after the pathological EAE process, we studied its effect on differentiation and proliferation of cells at two sequential stages in the oligodendroglial maturation cascade, identified by their phenotypic expression of the early marker NG2, which is characteristic of progenitor and preoligodendrocytes, and the later marker O4, which is expressed from the preoligodendrocyte stage all through maturation to myelin-producing oligodendrocytes. The proliferation of these lineage constituents was evaluated by their colocalization with BrdU (a thymidine analog incorporated into the DNA of dividing cells), which was injected into mice of all treatment groups concurrently with GA treatment. GA treatment (eight daily injections) was applied either as a prevention cgi doi pnas Aharoni et al.

4 Fig. 3. The effect of GA on NG2-expressing oligodendrocyte progenitor cells. (A) Quantitative analysis of double-labeled NG2 and BrdU cells in the SCs of naïve, EAE induced, and EAE induced mice treated with GA, in normalappearing white matter (nwm) or in damage regions (lesions), counted in a field of 0.03 mm 2 (25 50 regions along the cervix of each SC, 3 5 mice per treatment group). The respective experimental schedule illustrates GA and BrdU administration by eight injections either as prevention starting at disease induction on day 0 (experiment I) or as suppression starting one day after the appearance of clinical manifestations on day 14 (experiment II) and on day 19 (experiment III). The pound symbols indicate significant elevation from naïve mice, and the asterisks indicate significant elevation from untreated EAE mice. (B) The effect of GA on the morphology of OPCs stained for NG2 expression (orange) and BrdU incorporation (green). In untreated mice, NG2- expressing cells have bipolar progenitor morphology and multiprocessed morphology in GA treated mice. (C) SC sections from YFP 2.2 mice expressing YFP (green) on their neuronal population stained for NG2 (orange), demonstrating accumulation in sites of fiber aberration and multiprocessed preoligodendrocyte morphology after GA treatment. (Sagittal sections; scale bars: 25 m for B and C first to third rows and 10 m for C fourth row.) Fig. 4. The effect of GA on O4-expressing oligodendrocytes. (A) Quantitative analysis of double-labeled O4 and BrdU cells in the SCs of naïve, EAE induced, and EAE induced mice treated by GA in normal-appearing white matter (nwm) or in damage regions (lesions), counted in fields of 0.03 mm 2 (25 50 along the cervix of each SC, 3 5 mice per treatment group). The experimental schedule is illustrated in Fig. 3A. (B) SC sections from YFP 2.2 mice demonstrating elevation of O4-expressing cells in EAE induced mice treated with GA and their accumulation in a lesion. Scale bar, 25 m. regimen, starting at disease induction in experiment I, or as a suppression regimen, starting one day after the appearance of clinical manifestations, i.e., on day 14 in experiment II or on day 19 in experiment III (Fig. 3A). Quantitative analysis (25 50 regions of 0.03 mm 2 along the cervix, 3 5 mice per treatment group) (Fig. 3A) indicated that in normal-appearing white matter of both EAE and EAE GA mice, the number of NG2 /BrdU cells was similar to that of naïve mice. In contrast, in damaged white matter regions, a major elevation in the number of double-labeled NG2 /BrdU cells was observed, indicative of their proliferation and/or accumulation in response to the pathological damage. Yet these numbers gradually declined with time (2 weeks as compared with one day after completion of BrdU injections), demonstrating the consequence of disease progression. In mice treated with GA, the number of NG2 /BrdU -proliferating cells in lesion sites was even further elevated in comparison with untreated mice and was statistically significant for both the prevention and the suppression treatment groups. These differences were more pronounced 2 weeks after treatment termination (61% increase, experiment III) than closer to disease outbreak (28% increase, experiment II) when the number of NG2 /BrdU cells in EAE mice peaked. Notably, GA supported the survival of the NG2 / BrdU -proliferating cells because in the treated animals, most of them (73%) persisted for 2 weeks, whereas in untreated mice, only 53% of the NG2 /BrdU cells were present at the later time NEUROSCIENCE Aharoni et al. PNAS August 12, 2008 vol. 105 no

5 point. The effect of GA was also manifested in a significant elevation in the total number of NG2-expressing cells present in the lesion areas at the later time point ( cells per field in GA-treated mice, in comparison with cells per field in untreated mice, a 53% increase). Furthermore, whereas in untreated mice, the majority of the NG2 /BrdU cells had typical bipolar progenitor morphology, in EAE mice treated with GA, the NG2 /BrdU cells manifested mainly a multiprocessed morphology, indicative of the later preoligodendrocyte stage in the oligodendroglial maturation cascade (Fig. 3B). Thus, GA improved both the survival and the differentiation of oligodendrocyte progenitor cells in addition to its enhancing effect on their proliferation and recruitment into injury sites. Immunohistochemical analysis of NG2 expression in the SCs of YFP mice corroborated their accumulation in sites of fiber aberration, and their multiprocessed preoligodendrocyte morphology after GA treatment (Fig. 3C). GA injection into naïve mice (without EAE) did not result in significant elevation in the number of NG2 /BrdU cells when compared with naïve untreated mice (data not shown). Similar phenomena were observed for the later marker in the oligodendrocyte lineage, O4 (Fig. 4A). The effect of GA was exhibited as a higher elevation in the number of O4 /BrdU - proliferating cells in lesion sites (results from untreated mice for the suppression regimen both one day and 2 weeks after treatment were statistically significant). Moreover, in GA-treated mice, 77% of these cells survived 2 weeks after BrdU injection, compared with only 56% in the untreated mice. The increased proliferation and survival of the O4 /BrdU cells in GA-treated mice resulted in a 2-fold elevation in the treated mice compared with untreated mice at the later time point ( vs , respectively). Similar to the earlier marker NG2, this increase was manifested in a significant elevation of the total number of O4-expressing cells ( cells per field in the GA treated mice, compared with cells per field in EAE untreated mice, a 53% increase). As demonstrated in Fig. 4, in the YFP transgenic mice, the amount of O4-expressing cells in GA-treated mice was markedly increased in comparison to EAE untreated mice (Fig. 4B), and the cells were situated in areas of deteriorating YFP-expressing fibers, indicating their accumulation in lesion sites (Fig. 4C). The cumulative results obtained for both the NG2 and the O4 lineage markers indicate that GA treatment increases the proliferation, differentiation, and survival of OPCs, resulting in their higher accumulation in lesion sites. Discussion Recent years gave rise to considerable advances in the understanding of tissue injury in MS/EAE, but therapeutic strategies that enhance the intrinsic repair mechanism and induce effective neuroprotection and remyelination still lag behind. The major findings reported here indicate that peripheral immunomodulatory treatment can actually prevent the demyelination process and even induce tissue restoration in EAE. This effect was demonstrated by using a scanning electron microscopy technique for imaging wet biological specimens of the entire cross section of the mouse SC, which allowed rapid and highresolution imaging of the myelin that was further confirmed immunohistochemically. Both sets of results from EAE untreated mice revealed disseminated demyelination manifested by myelin reduction and degradation at multiple sites. Tissue injury accompanied demyelination, as demonstrated in electron microscopy by moderate-to-severe tissue destruction and axonal loss. In addition, the use of transgenic mice that selectively express YFP in the neuronal population of cells revealed fiber deterioration and axonal transection in demyelination sites. These findings indicate the pathological consequence of myelin loss that leads to the axonal impairment that is typical of the disease. The inflammatory disease course was confirmed by cell infiltration, of T cells in particular, into lesion sites. GA treatment of EAE induced mice abrogated the characteristic demyelination and the tissue destruction, resulting in the formation of normal-appearing SC tissue. Interestingly, the only mouse in which histological damage persisted after GA treatment was one in which the clinical response was less effective as well, indicating the relevance of the histological effect to the therapeutic consequence of GA. The various treatment regimens applied at different disease stages allowed further insight into the nature of this effect. Hence, the intact morphology obtained when treatment started concomitantly with EAE induction may reflect the prevention of disease development as well as its subsequent histological damage, similar to previous observations (13, 14). This effect could result from the immunomodulatory activity of GA that was previously shown to block pathogenic T cell activation and induce Th2/3 cells that down-regulate the detrimental inflammation (7 9). Yet, the effect of GA was not confined to anti-inflammation and damage prevention, because when treatment was initiated after the appearance of clinical symptoms or even in the chronic disease phase when substantial injury was definitely manifested (corroborated in untreated mice harvested at corresponding time points), it led to an almost complete elimination of the pathological damages. The consequences of GA treatment on chronic disease are of special significance, because this phase in EAE/MS is regarded as the stage in which exhausted self-compensating neuroprotective mechanisms fail and extensive degenerative processes become dominant (4, 17). These findings indicate that GA treatment induces not only prevention of demyelination and preservation of the neuronal tissue, but also genuine repair processes that may involve remyelination. Because mature oligodendrocytes are destroyed in the demyelinating regions by inflammatory and apoptotic processes, the major source of myelination and thus the extent of repair depends on the recruitment of OPCs into the lesions and on their differentiation to functional-myelin-producing cells (3 6). The detection of progenitor and preoligodendrocytes expressing the early marker NG2, and cells from the preoligodendrocyte stage all through their maturation to myelin-producing cells expressing O4, concomitantly with the proliferation marker BrdU, indicated that these oligodendrocyte lineage cells proliferated during the concurrent BrdU/GA injection period. It should be noted that EAE induction, as such, triggered the proliferation of oligodendrocyte lineage cells, in accord with previous studies demonstrating increased oligodendrocyte and neuronal progenitor proliferation after injury (4, 5, 12), indicative of a selfneuroprotective mechanism. Yet, the number of these oligodendrocytes declined with time so that only half of them survived 2 weeks after BrdU injection, demonstrating the persistence of impairment and the failure of self-neuroprotection to compensate for the damage. GA treatment augmented oligodendrocyte proliferation and prolonged their survival, because most of the NG2 /BrdU and O4 /BrdU cells persisted 2 weeks after BrdU injection, resulting in a 2-fold elevation in the level of O4 /BrdU cells, and in a significant expansion (53%) of the overall NG2 and O4 cells present in the lesion areas at the later time point. Of special interest is the morphological transformation from the earlier bipolar progenitor form to the more mature preoligodendrocyte multiprocessed form, suggesting an effect of GA on the differentiation along the oligodendroglial maturation cascade. These cumulative findings imply that the neuroprotective effect of GA on myelin is mediated through enhanced proliferation, differentiation, and survival of oligodendrocytes, resulting in their increased accumulation at the lesion sites cgi doi pnas Aharoni et al.

6 The effect of GA on oligodendrocytes can be attributed to the specific T cells generated by its injection that had been shown to cross the BBB, accumulate in the CNS, and express antiinflammatory cytokines such as IL-10 and TGF-, and the neurotrophic factor BDNF in situ (10) and in vitro (18). Furthermore, it was demonstrated that GA induces a bystander effect on the CNS resident cells to express beneficial cytokines/ neurotrophins and augments the in situ expression of BDNF, NT3, and NT4 (11). NT3, BDNF, and TGF- are key regulators of oligodendrocyte survival and development (3, 4). They are essential for the proliferation and recruitment of OPCs to the demyelinating lesions and for their subsequent differentiation into mature oligodendrocytes (19, 20). It is therefore significant that in the present study, cell infiltrations were observed in the SCs of GA-treated mice and their location was not associated with damage, suggesting the involvement of GA-induced cells in the neuroprotective effect of GA on myelin. It is of special significance that newly proliferated OPCs are attracted to damaged regions. Directed migration of progenitor cells of oligodendrocyte and neuronal origins toward injury and demyelinating sites has been demonstrated in MS and EAE (3, 4, 12). As presented here, in lesion sites of EAE untreated mice, the number of NG2/O4-expressing cells was higher than in normal-appearing regions, and they accumulated into areas of deteriorating YFP-expressing fibers. However, although the demyelination lesions in the GA-treated mice were less extensive, the number of progenitor cells recruited into them was significantly higher. These new oligodendrocytes constitute a pool for the replacement of dead or dysfunctional oligodendrocytes, enhancing in situ remyelination and repair. Materials and Methods Mice. C57BL/6 mice were purchased from Harlan. YFP 2.2 mice were kindly provided by Joshua R. Sanes (Washington University, St. Louis). Experiments were approved by the Institutional Animal Care and Use Committee. EAE. Disease was induced by MOG amino acids (Sigma) as previously described (11, 12). EAE was scored as follows: 0, no disease; 1, limp tail; 2, hind limb paralysis; 3, paralysis of four limbs; 4, moribund condition; and 5, death. GA Treatment. GA (Copaxone, Copolymer 1) from batch 242,990,599 with an average M r of 7.3 kda, was obtained from Teva Pharmaceutical Industries. GA treatment was applied by consecutive daily s.c. injections (2 mg per mouse) at different stages of disease: (i) at EAE induction, 8 injections (prevention); (ii) after appearance of clinical manifestations, 8 injections (suppression); (iii) during the chronic phase, 10 days after disease was fully developed, 18 injections (delayed suppression). BrdU Incorporation. BrdU (Sigma), a thymidine analog, was injected i.p. (50 mg/kg) concurrently with GA treatment. Perfusion and Sample Preparation. Mice were anesthetized and perfused transcardially with 2.5% paraformaldehyde. For electron microscopy, transversal SC sections of 300 nm from the thorax and the cervix were inserted into specimen capsules (15). For immunohistochemistry, coronal, and sagittal sections (16 m thick) were cut by sliding microtome (SM 2000r; Leica) through the entire SC. Scanning Electron Microscopy for Imaging Wet Biological Specimens (Wet SEM). SC cross-sections were captured at low resolution ( 40), identifying the dorsal, lateral, right, left, and ventral white-matter column, and then imaged at higher magnification ( 400 3,200) in different regions of the white matter. Wet SEM was performed by Quantomix (15). Histopathological Analysis. SC assessment was performed in a blinded manner on sets of photos from the thorax and the cervix of each animal. Evaluation criteria were: 0, no pathology detectable; 1, mild: edema, mild subpial/meningeal inflammation; 2, moderate: parenchymal cell infiltration, mild tissue damage with demyelination; or 3, severe: tissue destruction with parenchymal cell infiltration, demyelination, and axon damage. Immunohistochemistry. Staining was performed as described (11, 12) with the following antibodies: rabbit anti-chondroitin sulfate proteoglycan NG2 (Chemicon), mouse anti-o4 (Chemicon), rat anti-brdu (Serotec), mouse antimyelin basic protein (Abcam), and rat anti-cd3 (Serotec). Quantitative Analysis. Quantification of myelin damage was performed by measuring the sum of the MBP-distracted areas from the total area of positive MBP expression using Image-Pro 4.5 software in 16 coronal sections along each SC (two mice per treatment). Proliferating OPCs were quantified by counting immunopositive NG2- or O4-expressing cells and those with BrdU dual staining in a field of 0.03 mm 2 ( 40 magnification) (25 50 regions along the cervix, for at least six sagittal sections, from dorsal to ventral sides of each SC, 3 5 mice per treatment group). Statistical Analysis. The differences in areas of myelin damage between EAE untreated and EAE GA mice were analyzed by the two-tailed t test. The difference in the numbers of NG2 and O4 oligodendrocytes between EAE untreated and EAE GA mice in the lesion sites were analyzed by the twosample t test as the number of measurements 30. In normal-appearing white matter, the difference between naïve, EAE, and EAE GA were analyzed by one-way ANOVA followed by Fisher s least single distribution. The tests were performed by using Statistical Analysis System software (SAS). The level of significance for all of the tests was set at ACKNOWLEDGMENTS. This work was supported in part by a grant from Terry and Dr. Claude Oster, a special fund of the Eugene Applebaum Family Foundation, as well as by a grant from Teva Pharmaceutical Industries. NEUROSCIENCE 1. Lassmann H (1998) in: McAlpin s Multiple Sclerosis, eds Compston A, et al. (Churchill Livingstone, Oxford), pp Lassmann H (2007) Experimental models of multiple sclerosis. Rev Neurol 163: Baumann N, Phan-Dinh D (2001) Biology of oligodendrocyte and myelin in the mammalian central nervous system. Physiol Rev 81: Bruck W, Kuhlmann T, Stadelmann C (2003) Remyelination in multiple sclerosis. J Neurol Sci 206: Reynolds R, et al. 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