Demyelination Arrest and Remyelination Induced by Glatiramer Acetate Treatment of Experimental Autoimmune Encephalomyelitis

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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, Go¨ttingen 37099, Germany

Contributed by Michael Sela, May 19, 2008 (sent for review April 15, 2008) The interplay between demyelination and remyelination is critical myelination capacity, remyelination in MS/EAE is incomplete in the progress of multiple sclerosis (MS) and its animal model, and poorly sustained (3, 4). experimental autoimmune encephalomyelitis (EAE). In the present Glatiramer acetate (GA, Copaxone), an approved drug for the study, we explored the capacity of glatiramer acetate (GA, Cop- treatment of MS, has been shown to modulate the detrimental axone) to affect the demyelination process and/or lead to remy- inflammation that mediates the demyelination process (7). The elination in mice inflicted by chronic EAE, using both scanning immunomodulatory effect of GA in EAE and in MS is attributed electron microscopy and immunohistological methods. Spinal to its ability to induce specific Th2/3 cells (8, 9) that cross the cords of untreated EAE mice revealed substantial demyelination blood–brain barrier (BBB), accumulate in the CNS, and express accompanied by tissue destruction and axonal loss. In contrast, in in situ IL-10 and TGF-␤, as well as neurotrophic factors such as spinal cords of GA-treated mice, in which treatment started con- BDNF (10). Moreover, we recently demonstrated that GA comitantly with disease induction (prevention), no pathology was treatment also affects the cells of the CNS and restores their observed. Moreover, when treatment was initiated after the ap- impaired neurotrophic factor secretion, thus elevating the in situ pearance of clinical symptoms (suppression) or even in the chronic levels of BDNF, neurotrophin 3 (NT3), and NT4 (11). The disease phase (delayed suppression) when substantial demyelina- neuroprotective consequences of GA treatment were also man- tion was already manifested, it resulted in a significant decrease in ifested by a decrease in neuronal damage, and by an increase in the pathological damage. Detection of oligodendrocyte progenitor neurogenesis of neuronal progenitors that migrate into injury cells (OPCs) expressing the NG2 or O4 markers via colocalization sites and differentiate into mature neurons (12). with the proliferation marker BrdU indicated their elevated levels In view of the immunomodulatory activity of GA, and its in spinal cords of GA-treated mice. The mode of action of GA in this neuroprotective effects, it was of interest to investigate its ability system is attributed to increased proliferation, differentiation, and to affect the primary target of the EAE/MS pathological process, survival of OPCs along the oligodendroglial maturation cascade the myelin. Previous studies indicated that coinjection of GA and their recruitment into injury sites, thus enhancing repair with encephalitogenic emulsion blocked EAE development and processes in situ. reduced its consequential demyelination (13). GA also pre- vented demyelination in the optic nerve when injected before or multiple sclerosis ͉ neuroprotection ͉ oligodendrocyte ͉ myelin repair at disease induction (14). In the present study, we explored the potential of GA to interfere with demyelination. Furthermore, isseminated demyelination is the primary morphological by applying GA treatment in the chronic EAE stage, we could Dhallmark characterizing multiple sclerosis (MS) and its examine whether GA induces remyelination even after extensive animal model, experimental autoimmune encephalomyelitis damage has occurred. For this purpose we used the recently (EAE), leading to axonal loss and neurological impairments (1, developed wet scanning electron microscopy technique (wet 2). It is therefore important to evaluate MS treatments for their SEM), which enables direct visualization of the myelin (15). We neuroprotective capability to prevent demyelination and/or en- report here that GA treatment results in a decrease in myelin hance remyelination. The interplay between pathological demy- breakdown and tissue damage, and in stimulation of repair elination and the corresponding repair mechanism remyelina- processes. The mode of action of GA in this system is attributed tion involves, on one hand, the inflammatory immune cells that not only to the blockage of inflammation, but also to its effect mediate the damage and on the other hand, the myelin- on the expansion of cells from the oligodendrocyte lineage and producing cells, the oligodendrocytes. The latter are terminally their recruitment into injury sites, thus enhancing repair pro- differentiated cells with a limited capacity to respond to injury cesses in situ. that are destroyed in the actively demyelinating lesions (3, 4). Results Accordingly, remyelination requires the recruitment of oligodendrocyte precursor cells (OPCs) by their proliferation and EAE manifestations and the effect of GA treatment were migration into the demyelinating area and their further differ- investigated in the myelin oligodendrocyte glycoprotein (MOG) entiation into mature myelinating oligodendrocytes through 35–55 peptide-induced EAE model, in which disease symptoms distinct stages, characterized by morphological transformation appear around day 15, with increased severity up to days 22–25, and sequential expression of developmental markers. Thus, it has been demonstrated that in response to stimuli such as Author contributions: R. Aharoni, M.S., and R. Arnon designed research; R. Aharoni, A.H., inflammation or demyelination, OPCs, characterized by bipolar R.E., and M.B.-H. performed research; R. Aharoni, A.H., R.E., W.B., and R. Arnon analyzed morphology and by the presence of the early marker chondroitin data; and R. Aharoni, M.S., and R. Arnon wrote the paper. sulfate proteoglycan, NG2, undergo proliferation and transform Conflict of interest statement: Michael Sela and Ruth Arnon are among the inventors of into multiprocessed preoligodendrocytes that acquire the later Copaxone. cell surface marker O4 (5, 6). Yet, despite this ability of the adult §To whom correspondence should be addressed. E-mail: [email protected]. brain to retain the potential to generate oligodendrocytes with © 2008 by The National Academy of Sciences of the USA

11358–11363 ͉ PNAS ͉ August 12, 2008 ͉ vol. 105 ͉ no. 32 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804632105 Downloaded by guest on September 24, 2021 NEUROSCIENCE

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 Fig. 1. The effect of GA on myelin visualized by wet SEM. Shown are series sagittal sections along each SC (two mice per treatment group). Asterisk of wet SEM SC images (cervix) from representative mice, in magnifications of indicates significant reduction from untreated EAE mice. 800ϫ (Center) and 1600ϫ (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 resulting in chronic disease. GA treatment was applied by daily appearance of clinical symptoms (10 daily injections). (C) Delayed suppression injections at different disease stages: (i) starting immediately regimen, started one month after disease induction (18 daily injections). In after disease induction (prevention), (ii) starting soon after the EAE mice, the myelin is reduced and accompanied by inflammation (white spots) and axonal loss (dark holes). In the SCs of EAEϩGA mice, the myelin is appearance of clinical manifestations (suppression), or (iii)in preserved and no pathology is observed. Arrows indicate the day of perfusion. the chronic phase 10 days after disease was fully developed GA treatment is indicated by a dark line. (Scale bars: 20 ␮m, Center;10␮m, (delayed suppression). GA treatment by all three regimens Right.) ameliorated the clinical manifestations compared with un-

Aharoni et al. PNAS ͉ August 12, 2008 ͉ vol. 105 ͉ no. 32 ͉ 11359 Downloaded by guest on September 24, 2021 Table 1. Effect of GA treatment on SC histopathology visualized by wet SEM Mice Disease score at Disease score Histopathological Treatment tested, n treatment initiation at perfusion evaluation

Naïve control 3 0 0 0 EAE 6 2.3 3.6 2.2* EAE with GA prevention 2 0 0 0 EAE with GA suppression 6 2.3 0 0 EAE with GA delayed suppression 5 2.2 0.8 0.6†

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 EAE- various disease stages indicate that it reduces tissue damage, untreated vs. EAE-induced mice treated by GA (EAEϩGA) as limits the breakdown of the myelin sheath, and improves tissue well as naı¨ve controls were subjected to both electron microscopy integrity even after injury has occurred. and immunohistochemistry for further evaluation. Immunohistochemical Characterization of SC from EAE Mice and the SEM Analysis of SC from EAE Mice and the Effect of GA Treatment. Effect of GA Treatment. To correlate these findings, myelin pres- Electron microscopy was performed by application of the wet ence was visualized by using anti-myelin basic protein (MBP) SEM technology, in which the analysis is done on wet tissue. SC antibodies in the SCs of YFP 2.2 transgenic mice, which selec- were scanned at different white matter regions in both the cervix tively express YFP on their neuronal population (16) and are and the thorax. SEM images (30–36 images for each SC) were susceptible to MOG-induced EAE (12). As shown in Fig. 2A, subjected to a histopathological evaluation in a blinded fashion, multiple widespread areas of myelin damage were observed in and the results (on a scale of 0–3) summarizing all of the animals various white matter regions of EAE inflicted mice (with clinical tested (n ϭ 22) are presented in Table 1. A series of images score 2–3, scarified one month after disease induction). The size (cervix) from representative mice of the three treatment regi- of these demyelinating sites exceeded 6% of the overall area of mens and their respective pattern of clinical daily scoring is MBP expression (quantified for 16 sagittal sections all along the demonstrated in Fig. 1. Whereas in naı¨ve controls, the intact SC, in two mice per treatment group) (Fig. 2E). In regions of myelin structure emerged as sharp dark rings (Fig. 1A), in the myelin damage, YFP expression was obstructed, revealing sparse SCs of EAE inflicted mice (clinical score 2–4, harvested 4–6 processes, axonal transection, and fiber deterioration, indicative weeks after disease induction), the myelin was reduced in of the axonal pathology that accompanies the demyelination amount and degraded at multiple sites, indicative of the demy- process. Hoechst staining of cell nuclei as well as anti-CD3 elination process (Fig. 1 A–C). SCs of EAE untreated mice also staining of T cells demonstrated cellular infiltration into the revealed moderate to severe tissue destruction and axonal lesion sites, consistent with the detrimental role of inflamma- damage, accompanied by parenchymal cell infiltration that tion, in particular of T cells, in this disease. indicates the inflammatory disease course. The average his- In the SCs of EAE induced mice treated with GA, either by topathological score of the six EAE animals tested was 2.2. the prevention (Fig. 2B) or by the suppression (Fig. 2C) regi- In contrast to the extensive pathology characteristic of the mens, considerably less damage was detected compared with the EAE untreated mice, in most (12 of 13) of the SCs of the EAE untreated mice. This decreased damage was manifested in GA-treated mice, no pathology was detected (Table 1) and their either a marked reduction or complete absence of the patho- appearance was similar to that of naı¨ve mice. This lack of logical manifestations, i.e., myelin damage, deteriorating fibers, pathology was observed in the mice in which treatment started and cellular/T cell infiltration sites. The effect of GA was concomitantly with EAE induction, thus preventing the disease especially prominent when GA was applied as a prevention and subsequent histological damage (Table 1 and Fig. 1A). treatment (94% reduction in demyelination), but a significant Moreover, when treatment was initiated soon after the appear- effect (74% reduction) was observed even when treatment ance of clinical symptoms during the suppression regimen (Fig. started after pathological manifestations had been expressed 1B) or even in the chronic disease phase (delayed suppression) (Fig. 2E). These results indicate a neuroprotective effect of GA (Fig. 1C) when injury was fully manifested, damage to myelin was in reducing the number and the size of SC lesions. hardly detectable (average histopathological scores 0 and 0.6 respectively) (Table 1). It should be noted that in the case of the Effect of GA Treatment on Oligodendrocyte Progenitor Cells in EAE delayed suppression regimen, treatment was applied for a longer Mice. To further understand the effect of GA on myelin after the period (18 daily injections, in comparison with 8 and 10 injec- pathological EAE process, we studied its effect on differentia- tions for prevention and suppression, respectively). The severity tion and proliferation of cells at two sequential stages in the of the damage in these mice before treatment initiation can be oligodendroglial maturation cascade, identified by their pheno- appreciated by the tissue destruction observed in the SCs of typic expression of the early marker NG2, which is characteristic untreated mice harvested at time points corresponding to the of progenitor and preoligodendrocytes, and the later marker O4, beginning of treatment (represented in Fig. 1C), which persisted which is expressed from the preoligodendrocyte stage all throughout the later phase of the chronic disease (Fig. 1B). The through maturation to myelin-producing oligodendrocytes. The only mouse in which pathological damage was found after GA proliferation of these lineage constituents was evaluated by their treatment was one in which clinical symptoms persisted as well colocalization with BrdU (a thymidine analog incorporated into (grade 2). Parenchymal cell infiltration was observed in the SCs the DNA of dividing cells), which was injected into mice of all of GA-treated mice, but to a lesser extent than untreated mice. treatment groups concurrently with GA treatment. GA treat- Taken together, the consequences of GA treatment in the ment (eight daily injections) was applied either as a prevention

11360 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804632105 Aharoni et al. Downloaded by guest on September 24, 2021 Fig. 4. The effect of GA on O4-expressing oligodendrocytes. (A) Quantitative analysis of double-labeled O4 and BrdU cells in the SCs of naı¨ve,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 mm2 (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 NEUROSCIENCE 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 mm2 along the cervix, 3–5 mice per treatment group) (Fig. 3A) indicated that in normal-appearing white matter of both EAE and EAEϩGA mice, the number of NG2ϩ/BrdUϩ cells was similar to that of naı¨ve mice. In contrast, in damaged white matter regions, a ϩ ϩ Fig. 3. The effect of GA on NG2-expressing oligodendrocyte progenitor cells. major elevation in the number of double-labeled NG2 /BrdU (A) Quantitative analysis of double-labeled NG2 and BrdU cells in the SCs of cells was observed, indicative of their proliferation and/or ac- naı¨ve, EAE induced, and EAE induced mice treated with GA, in normal- cumulation in response to the pathological damage. Yet these appearing white matter (nwm) or in damage regions (lesions), counted in a numbers gradually declined with time (2 weeks as compared with field of 0.03 mm2 (25–50 regions along the cervix of each SC, 3–5 mice per one day after completion of BrdU injections), demonstrating the treatment group). The respective experimental schedule illustrates GA and consequence of disease progression. In mice treated with GA, BrdU administration by eight injections either as prevention starting at dis- ϩ ϩ ease induction on day 0 (experiment I) or as suppression starting one day after the number of NG2 /BrdU -proliferating cells in lesion sites the appearance of clinical manifestations on day 14 (experiment II) and on day was even further elevated in comparison with untreated mice and 19 (experiment III). The pound symbols indicate significant elevation from was statistically significant for both the prevention and the naı¨vemice, and the asterisks indicate significant elevation from untreated EAE suppression treatment groups. These differences were more mice. (B) The effect of GA on the morphology of OPCs stained for NG2 pronounced 2 weeks after treatment termination (61% increase, expression (orange) and BrdU incorporation (green). In untreated mice, NG2- experiment III) than closer to disease outbreak (28% increase, expressing cells have bipolar progenitor morphology and multiprocessed experiment II) when the number of NG2ϩ/BrdUϩ cells in EAE morphology in GA treated mice. (C) SC sections from YFP 2.2 mice expressing mice peaked. Notably, GA supported the survival of the NG2ϩ/ YFP (green) on their neuronal population stained for NG2 (orange), demon- ϩ strating accumulation in sites of fiber aberration and multiprocessed preoli- BrdU -proliferating cells because in the treated animals, most of godendrocyte morphology after GA treatment. (Sagittal sections; scale bars: them (73%) persisted for 2 weeks, whereas in untreated mice, ϩ ϩ 25 ␮m for B and C first to third rows and 10 ␮m for C fourth row.) only 53% of the NG2 /BrdU cells were present at the later time

Aharoni et al. PNAS ͉ August 12, 2008 ͉ vol. 105 ͉ no. 32 ͉ 11361 Downloaded by guest on September 24, 2021 point. The effect of GA was also manifested in a significant disease. The inflammatory disease course was confirmed by cell elevation in the total number of NG2-expressing cells present in infiltration, of T cells in particular, into lesion sites. GA treat- the lesion areas at the later time point (35.0 Ϯ 9.9 cells per field ment of EAE induced mice abrogated the characteristic demy- in GA-treated mice, in comparison with 22.8 Ϯ 8.3 cells per field elination and the tissue destruction, resulting in the formation of in untreated mice, a 53% increase). Furthermore, whereas in normal-appearing SC tissue. Interestingly, the only mouse in untreated mice, the majority of the NG2ϩ/BrdUϩ cells had which histological damage persisted after GA treatment was one typical bipolar progenitor morphology, in EAE mice treated in which the clinical response was less effective as well, indicating ϩ ϩ with GA, the NG2 /BrdU cells manifested mainly a multipro- the relevance of the histological effect to the therapeutic con- cessed morphology, indicative of the later preoligodendrocyte sequence of GA. stage in the oligodendroglial maturation cascade (Fig. 3B). Thus, The various treatment regimens applied at different disease GA improved both the survival and the differentiation of stages allowed further insight into the nature of this effect. oligodendrocyte progenitor cells in addition to its enhancing Hence, the intact morphology obtained when treatment started effect on their proliferation and recruitment into injury sites. concomitantly with EAE induction may reflect the prevention of Immunohistochemical analysis of NG2 expression in the SCs of disease development as well as its subsequent histological dam- YFP mice corroborated their accumulation in sites of fiber age, similar to previous observations (13, 14). This effect could aberration, and their multiprocessed preoligodendrocyte mor- result from the immunomodulatory activity of GA that was phology after GA treatment (Fig. 3C). GA injection into naı¨ve previously shown to block pathogenic T cell activation and mice (without EAE) did not result in significant elevation in the ϩ ϩ induce Th2/3 cells that down-regulate the detrimental inflam- number of NG2 /BrdU cells when compared with naı¨ve un- mation (7–9). Yet, the effect of GA was not confined to treated mice (data not shown). anti-inflammation and damage prevention, because when treat- Similar phenomena were observed for the later marker in the ment was initiated after the appearance of clinical symptoms or oligodendrocyte lineage, O4 (Fig. 4A). The effect of GA was ϩ ϩ even in the chronic disease phase when substantial injury was exhibited as a higher elevation in the number of O4 /BrdU - definitely manifested (corroborated in untreated mice harvested proliferating cells in lesion sites (results from untreated mice for at corresponding time points), it led to an almost complete the suppression regimen both one day and 2 weeks after treat- elimination of the pathological damages. The consequences of ment were statistically significant). Moreover, in GA-treated GA treatment on chronic disease are of special significance, mice, 77% of these cells survived 2 weeks after BrdU injection, because this phase in EAE/MS is regarded as the stage in which compared with only 56% in the untreated mice. The increased ϩ ϩ exhausted self-compensating neuroprotective mechanisms fail proliferation and survival of the O4 /BrdU cells in GA-treated and extensive degenerative processes become dominant (4, 17). mice resulted in a 2-fold elevation in the treated mice compared These findings indicate that GA treatment induces not only with untreated mice at the later time point (27.7 Ϯ 8.7 vs. 12.9 Ϯ prevention of demyelination and preservation of the neuronal 3.6, respectively). Similar to the earlier marker NG2, this in- tissue, but also genuine repair processes that may involve crease was manifested in a significant elevation of the total remyelination. number of O4-expressing cells (54.0 Ϯ 12.4 cells per field in the Because mature oligodendrocytes are destroyed in the demy- GA treated mice, compared with 35.3 Ϯ 10.4 cells per field in elinating regions by inflammatory and apoptotic processes, the EAE untreated mice, a 53% increase). As demonstrated in Fig. major source of myelination and thus the extent of repair 4, in the YFP transgenic mice, the amount of O4-expressing cells in GA-treated mice was markedly increased in comparison to depends on the recruitment of OPCs into the lesions and on their EAE untreated mice (Fig. 4B), and the cells were situated in differentiation to functional-myelin-producing cells (3–6). The areas of deteriorating YFP-expressing fibers, indicating their detection of progenitor and preoligodendrocytes expressing the accumulation in lesion sites (Fig. 4C). The cumulative results early marker NG2, and cells from the preoligodendrocyte stage obtained for both the NG2 and the O4 lineage markers indicate all through their maturation to myelin-producing cells expressing that GA treatment increases the proliferation, differentiation, O4, concomitantly with the proliferation marker BrdU, indi- and survival of OPCs, resulting in their higher accumulation in cated that these oligodendrocyte lineage cells proliferated dur- lesion sites. ing the concurrent BrdU/GA injection period. It should be noted that EAE induction, as such, triggered the proliferation of Discussion oligodendrocyte lineage cells, in accord with previous studies Recent years gave rise to considerable advances in the under- demonstrating increased oligodendrocyte and neuronal progen- standing of tissue injury in MS/EAE, but therapeutic strategies itor proliferation after injury (4, 5, 12), indicative of a self- that enhance the intrinsic repair mechanism and induce effective neuroprotective mechanism. Yet, the number of these oligoden- neuroprotection and remyelination still lag behind. The major drocytes declined with time so that only half of them survived 2 findings reported here indicate that peripheral immunomodu- weeks after BrdU injection, demonstrating the persistence of latory treatment can actually prevent the demyelination process impairment and the failure of self-neuroprotection to compen- and even induce tissue restoration in EAE. This effect was sate for the damage. GA treatment augmented oligodendrocyte demonstrated by using a scanning electron microscopy tech- proliferation and prolonged their survival, because most of the ϩ ϩ ϩ ϩ nique for imaging wet biological specimens of the entire cross NG2 /BrdU and O4 /BrdU cells persisted 2 weeks after section of the mouse SC, which allowed rapid and high- BrdU injection, resulting in a 2-fold elevation in the level of ϩ ϩ resolution imaging of the myelin that was further confirmed O4 /BrdU cells, and in a significant expansion (53%) of the ϩ ϩ immunohistochemically. Both sets of results from EAE un- overall NG2 and O4 cells present in the lesion areas at the treated mice revealed disseminated demyelination manifested by later time point. Of special interest is the morphological trans- myelin reduction and degradation at multiple sites. Tissue injury formation from the earlier bipolar progenitor form to the more accompanied demyelination, as demonstrated in electron mi- mature preoligodendrocyte multiprocessed form, suggesting an croscopy by moderate-to-severe tissue destruction and axonal effect of GA on the differentiation along the oligodendroglial loss. In addition, the use of transgenic mice that selectively maturation cascade. These cumulative findings imply that the express YFP in the neuronal population of cells revealed fiber neuroprotective effect of GA on myelin is mediated through deterioration and axonal transection in demyelination sites. enhanced proliferation, differentiation, and survival of oligo- These findings indicate the pathological consequence of myelin dendrocytes, resulting in their increased accumulation at the loss that leads to the axonal impairment that is typical of the lesion sites.

11362 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804632105 Aharoni et al. Downloaded by guest on September 24, 2021 The effect of GA on oligodendrocytes can be attributed to the BrdU Incorporation. BrdU (Sigma), a thymidine analog, was injected i.p. (50 specific T cells generated by its injection that had been shown to mg/kg) concurrently with GA treatment. cross the BBB, accumulate in the CNS, and express anti- inflammatory cytokines such as IL-10 and TGF-␤, and the Perfusion and Sample Preparation. Mice were anesthetized and perfused neurotrophic factor BDNF in situ (10) and in vitro (18). Fur- transcardially with 2.5% paraformaldehyde. For electron microscopy, trans- thermore, it was demonstrated that GA induces a bystander versal SC sections of 300 nm from the thorax and the cervix were inserted into specimen capsules (15). For immunohistochemistry, coronal, and sagittal sec- effect on the CNS resident cells to express beneficial cytokines/ tions (16 ␮m thick) were cut by sliding microtome (SM 2000r; Leica) through neurotrophins and augments the in situ expression of BDNF, the entire SC. NT3, and NT4 (11). NT3, BDNF, and TGF-␤ are key regulators of oligodendrocyte survival and development (3, 4). They are Scanning Electron Microscopy for Imaging Wet Biological Specimens (Wet SEM). essential for the proliferation and recruitment of OPCs to the SC cross-sections were captured at low resolution (ϫ40), identifying the demyelinating lesions and for their subsequent differentiation dorsal, lateral, right, left, and ventral white-matter column, and then imaged into mature oligodendrocytes (19, 20). It is therefore significant at higher magnification (ϫ400–3,200) in different regions of the white mat- that in the present study, cell infiltrations were observed in the ter. Wet SEM was performed by Quantomix (15). SCs of GA-treated mice and their location was not associated with damage, suggesting the involvement of GA-induced cells in Histopathological Analysis. SC assessment was performed in a blinded manner the neuroprotective effect of GA on myelin. on sets of 30–40 photos from the thorax and the cervix of each animal. Evaluation criteria were: 0, no pathology detectable; 1, mild: edema, mild It is of special significance that newly proliferated OPCs are subpial/meningeal inflammation; 2, moderate: parenchymal cell infiltration, attracted to damaged regions. Directed migration of progenitor mild tissue damage with demyelination; or 3, severe: tissue destruction with cells of oligodendrocyte and neuronal origins toward injury and parenchymal cell infiltration, demyelination, and axon damage. demyelinating sites has been demonstrated in MS and EAE (3, 4, 12). As presented here, in lesion sites of EAE untreated mice, Immunohistochemistry. Staining was performed as described (11, 12) with the the number of NG2/O4-expressing cells was higher than in following antibodies: rabbit anti-chondroitin sulfate proteoglycan NG2 normal-appearing regions, and they accumulated into areas of (Chemicon), mouse anti-O4 (Chemicon), rat anti-BrdU (Serotec), mouse anti- deteriorating YFP-expressing fibers. However, although the myelin basic protein (Abcam), and rat anti-CD3 (Serotec). demyelination lesions in the GA-treated mice were less extensive, the number of progenitor cells recruited into them was Quantitative Analysis. Quantification of myelin damage was performed by measuring the sum of the MBP-distracted areas from the total area of positive significantly higher. These new oligodendrocytes constitute a ϩ pool for the replacement of dead or dysfunctional oligodendro- MBP expression using Image-Pro 4.5 software in 16 coronal sections along each SC (two mice per treatment). Proliferating OPCs were quantified by cytes, enhancing in situ remyelination and repair. counting immunopositive NG2- or O4-expressing cells and those with BrdU dual staining in a field of 0.03 mm2 (ϫ40 magnification) (25–50 regions along Materials and Methods the cervix, for at least six sagittal sections, from dorsal to ventral sides of each Mice. C57BL/6 mice were purchased from Harlan. YFP 2.2 mice were kindly SC, 3–5 mice per treatment group). provided by Joshua R. Sanes (Washington University, St. Louis). Experiments were approved by the Institutional Animal Care and Use Committee. Statistical Analysis. The differences in areas of myelin damage between EAE untreated and EAEϩGA mice were analyzed by the two-tailed t test. The EAE. Disease was induced by MOG amino acids 35–55 (Sigma) as previously difference in the numbers of NG2ϩ and O4ϩ oligodendrocytes between EAE described (11, 12). EAE was scored as follows: 0, no disease; 1, limp tail; 2, hind untreated and EAEϩGA mice in the lesion sites were analyzed by the two- limb paralysis; 3, paralysis of four limbs; 4, moribund condition; and 5, death. sample t test as the number of measurements Ͼ30. In normal-appearing white matter, the difference between naı¨ve,EAE, and EAEϩGA were analyzed by GA Treatment. GA (Copaxone, Copolymer 1) from batch 242,990,599 with an one-way ANOVA followed by Fisher’s least single distribution. The tests were average Mr of 7.3 kDa, was obtained from Teva Pharmaceutical Industries. GA performed by using Statistical Analysis System software (SAS). The level of treatment was applied by consecutive daily s.c. injections (2 mg per mouse) at significance for all of the tests was set at Ͻ0.01. different stages of disease: (i) at EAE induction, 8 injections (prevention); (ii) NEUROSCIENCE after appearance of clinical manifestations, 8 injections (suppression); (iii) ACKNOWLEDGMENTS. This work was supported in part by a grant from Terry during the chronic phase, 10 days after disease was fully developed, 18 and Dr. Claude Oster, a special fund of the Eugene Applebaum Family Foun- injections (delayed suppression). dation, as well as by a grant from Teva Pharmaceutical Industries.

1. Lassmann H (1998) in: McAlpin’s Multiple Sclerosis, eds Compston A, et al. (Churchill 12. Aharoni R, Eilam R, Arnon R (2005) Neurogenesis and neuroprotection induced by Livingstone, Oxford), pp 323–358. peripheral immunomodulatory treatment of experimental autoimmune encephalo- 2. Lassmann H (2007) Experimental models of multiple sclerosis. Rev Neurol 163:651–655. myelitis. J Neurosci 25:8217–8228. 3. Baumann N, Phan-Dinh D (2001) Biology of oligodendrocyte and myelin in the mam- 13. Gilgum-Sherki Y, Panet H, Holdengreber V, Mosberg-Galili R, Offen D (2003) Axonal malian central nervous system. Physiol Rev 81:871–926. damage is reduced following glatiramer acetate treatment in C57/bl mice with chronic- 4. Bruck W, Kuhlmann T, Stadelmann C (2003) Remyelination in multiple sclerosis. induced experimental autoimmune encephalomyelitis. Neurosci Res 47:201–207. J Neurol Sci 206:181–185. 14. Maier K, et al. (2006) Effects of glatiramer acetate and interferon-␤ on neurodegen- 5. Reynolds R, et al. (2002) The response of NG2-expressing oligodendrocyte progenitors eration in a model of multiple sclerosis. Am J Pathol 169:1353–1364. to demyelination in MOG-EAE and MS. J Neurocytol 31:523–536. 15. Nyska A, et al. (2006) A new method of wet scanning electron microscopy for analysis 6. Polito A, Reynolds R (2005) NG2-expressing cells as oligodendrocyte progenitors in the of myelination in EAE mouse model of multiple sclerosis. Exp Toxicol Pathol 57:291– normal and demyelinated adult central nervous system. J Anat 207:707–716. 297. 7. Arnon R, Sela M (2003) Immunomodulation by the copolymer glatiramer acetate. JMol 16. Feng G, et al. (2000) Imaging neuronal subsets in transgenic mice expressing multiple Recognit 16:412–421. spectral variants of GFP. Neuron 28:41–51. 8. Aharoni R, Teitelbaum D, Sela M, Arnon R (1997) Copolymer 1 induces T cells of the T 17. Bjartmar C, Wujek JR, Trapp BD (2003) Axonal loss in the pathology of MS: Conse- helper type 2 that cross-react with myelin basic protein and suppress experimental quences for understanding the progressive phase of the disease. J Neurol Sci 15:165– autoimmune encephalomyelitis. Proc Natl Acad Sci USA 94:10821–10826. 9. Duda PW, et al. (2000) Glatiramer acetate (Copaxone) induces degenerate Th2-polarized 171. immune responses in patients with multiple sclerosis. J Clin Invest 105:967–976. 18. Kipnis J, et al. (2000) T cell immunity to copolymer 1 confers neuroprotection on the 10. Aharoni R, Kayhan B, Eilam R, Sela M, Arnon R (2003) Glatiramer acetate-speciﬁc T cells damaged optic nerve: Possible therapy for optic neuropathies. Proc Natl Acad Sci USA in the brain express T helper 2/3 cytokines and brain-derived neurotrophic factor in 97:7446–7451. situ. Proc Natl Acad Sci USA 100:14157–14162. 19. Franklin RJM, Hinks GL (1999) Understanding CNS remyelination: Clues from develop- 11. Aharoni R, et al. (2005) The immunomodulator glatiramer acetate augments the mental and regeneration biology. J Neurosci Res 58:207–213. expression of neurotrophic factors in brains of experimental autoimmune encepha- 20. Lalive PH, et al. (2005) TGF-beta-treated microglia induce oligodendrocyte precursor lomyelitis mice. Proc Natl Acad Sci USA 102:19045–19050. cell chemotaxis through the HGF-c-Met pathway. Eur J Immunol 35:727–737.

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