Mol. Cells 2015; 38(11): 1013-1021
Published online November 6, 2015
https://doi.org/10.14348/molcells.2015.0246
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: hcpark67@korea.ac.kr (HCP); sc2013.park@samsung.com (SCP)
Most of the axons in the vertebrate nervous system are surrounded by a lipid-rich membrane called myelin, which promotes rapid conduction of nerve impulses and protects the axon from being damaged. Multiple sclerosis (MS) is a chronic demyelinating disease of the CNS characterized by infiltration of immune cells and progressive damage to myelin and axons. One potential way to treat MS is to enhance the endogenous remyelination process, but at present there are no available treatments to promote remyelination in patients with demyelinating diseases.
Sulfasalazine is an anti-inflammatory and immune-modulating drug that is used in rheumatology and inflammatory bowel disease. Its anti-inflammatory and immunomodulatory properties prompted us to test the ability of sulfasalazine to promote remyelination. In this study, we found that sulfasalazine promotes remyelination in the CNS of a transgenic zebrafish model of NTR/MTZ-induced demyelination. We also found that sulfasalazine treatment reduced the number of macrophages/microglia in the CNS of demyelinated zebrafish larvae, suggesting that the acceleration of remyelination is mediated by the immunomodulatory function of sulfasalazine. Our data suggest that temporal modulation of the immune response by sulfasalazine can be used to overcome MS by enhancing myelin repair and remyelination in the CNS.
Keywords dorsal midline, enhancer, EphA7
Most axons in the vertebrate nervous system are surrounded by a lipid-rich membrane called myelin, which promotes rapid conduction of nerve impulses and protects against axonal damage. During development, myelin is produced by oligodendrocytes in the CNS and Schwann cells in the PNS (Emery, 2010). In addition to its role in forming myelin, a recent study has shown that oligodendrocytes provide metabolic support to neurons, which is crucial for axonal function and neuronal survival (Lee et al., 2012). Demyelination is the pathological process by which the myelin sheath is lost from around the axon. Normally, the CNS has the capacity to repair demyelinated axons through a process called remyelination, in which new myelin is synthesized to remyelinate demyelinated axons, enabling them to recover lost function. However, myelin is essential, and disruption of the myelin structure and failure to remyelinate in the CNS causes various neurodegenerative diseases including multiple sclerosis (MS).
MS is a chronic demyelinating disease of the CNS characterized by infiltration of immune cells and progressive damage to myelin and axons. MS is thought to be caused by the immune system inappropriately attacking the myelin in the brain and spinal cord, resulting in the demyelination of axons (Franklin, 2002; Franklin and Ffrench-Constant, 2008). It has been proposed that the enhancement of the endogenous remyelination process is one of the potential ways to efficiently treat MS, and several animal models of chemically induced demyelination such as cuprizone and lysolecitin have been used to screen drug candidates to promote remyelination (Imai et al., 2008; Lau et al., 2012; Moharregh-Khiabani et al., 2010; Ransohoff, 2012; Silvestroff et al., 2012). Although considerable progress has been achieved to promote remyelination in the demyelinated CNS, there are no available treatments to promote remyelination in patients with demyelinating diseases.
To investigate remyelination
The zebrafish embryos used in this study were
Metronidazole (Mtz, M1547, Sigma) was dissolved in EM containing 0.2% DMSO to 10 mM as previously described (Chung et al., 2013). For the ablation of myelinating glia,
Manually-dechorionated embryos were labeled with BrdU (Roche) by incubating them for 1 h 30min on ice in a solution of 10 mM BrdU and 15% DMSO in EM at 8 dpf. The embryos were then placed in EM, incubated for 1 h at 28.5°C, and fixed using 4% paraformaldehyde in PBS. Embryos were processed for immunohistochemistry, treated for 20 min with 2 M HCl, and were then processed for anti-BrdU immunohistochemistry. For immunohistochemistry, we used the following primary antibodies: mouse anti-BrdU (G3G4, 1:250, Developmental Studies Hybridoma Bank), rabbit anti-Sox10 (1:800, Lifespan bioscience), rat cd11b (1:100, BD bioscience), mouse anti-sodium channel (1:500, Sigma), and mouse anti-acetylated tubulin (1:1000, Sigma). Alexa 488-, 568-, 647-conjugated secondary antibodies were used for fluorescence detection (1:500, Molecular Probe). For histology section, embryos were embedded in 1.5% agar/5% sucrose. Sections (10 μm) were obtained by using a cryostat microtome (Zeiss).
Fluorescent
Tissues were prepared using standard procedures for TEM as described previously (Kim et al., 2002). Briefly, zebrafish larva were anesthetized with Tricaine (Sigma) and fixed with 10% paraformaldehyde/2.5% glutaraldehyde/0.1 M phosphate buffer, pH 7.4. Then larva were postfixed in 1% osmium tetroxide, dehydrated, embedded in Eponate-12 resin (Ted Pella). 1-μm-thick sections were obtained using Reichert-Jung Ultracut E ultramicrotome (Leica), stained with toluidine blue, and imaged using a Carl Zeiss Axio microscope. 60-nm-thick sections per block were collected on Formvar-coated slot grids and stained with uranyl acetate/lead citrate, and then recorded on an H-7500 TEM (Hitachi) (80 kV).
Adult brain injury was performed as described previously (Marz et al., 2011). Adult zebrafish were anesthetized with Tricaine (Sigma) and a BD U-100 insulin syringe (BD bioscience) was inserted vertically through the skull into the telencephalic region. Then brain damaged fish were transferred into a tank with fresh fish water and fixed in 4% paraformaldehyde at 4-day post injury.
To investigate remyelination
To examine the effectiveness of the NTR/MTZ system at inducing demyelination in the CNS and PNS via drug-dependent ablation of oligodendrocyte lineage cells, we first crossed
To investigate whether sulfasalazine promotes remyelination in the demyelinated zebrafish nervous system,
Next, we investigated whether sulfasalazine treatment promotes remyelination following the regeneration of myelinating glial cells in the CNS and PNS.
To investigate whether the regenerated myelinating glial cells in the sulfasalazine-treated larvae reconstructed a functional myelin sheath in the nervous system, we asked if the nodes of Ranvier structure reformed in the regenerated myelin sheath. To do so, we used an antibody against sodium channels (anti-panNavCh, NaCh), which is known to label node structures in axons (Voas et al., 2007). After immunohistochemistry with a NaCh antibody, we observed that NaCh+ node structures were regularly located in the PLL axons of the control larvae (Fig. 4A, arrows). However, NaCh+ node structures were rarely located in the PLL axons of the MTZ-treated
Our previous data showed that sulfasalazine treatment promoted regeneration of oligodendrocytes and Schwann cells and promoted subsequent remyelination in the NTR/MTZ-induced demyelination model. Therefore, we investigated the underlying mechanisms for the promotion of remyelination by sulfasalazine. To test whether sulfasalazine promoted proliferation of spinal cord precursors and thus the formation of oligodendrocyte precursor cells (OPCs) under normal conditions, we exposed wild-type
In the CNS of MS patients and in animal models of MS, substantial infiltration of macrophages and activation of microglia are observed and have been shown to be associated with disease severity (Lucchinetti et al., 2000; Prineas and Wright, 1978). In addition, inhibition of macrophage/microglia activation or a reduction in their density suppressed and attenuated progression of experimental autoimmune encephalomyelitis (EAE), a common immune-mediated animal model of MS (Bhasin et al., 2007; Heppner et al., 2005; Martiney et al., 1998). Altogether, these studies suggest that macrophages/microglia play a detrimental role in the remyelination process. Because sulfasalazine is thought to be an anti-inflammatory and immune-modulating drug (Hoult, 1986; Peppercorn, 1984), we hypothesized that sulfasalazine treatment promotes remyelination process by modulating the behavior macrophages/microglia in the demyelination animal model.
To test this idea, we first induced injury in the forebrain of adult zebrafish and treated the injured brain with either DMSO or sulfasalazine. Next, we compared the number of macrophages/microglia in the injured forebrain of DMSO- and sulfasalazine-treated adult zebrafish following labeling with an anti-CD11b antibody, which marks macrophages/microglia in zebra-fish (Fantin et al., 2010). In the injured forebrain of sulfasalazine-treated adult zebrafish, the number of anti-CD11b-positive macrophages/microglia clearly decreased around the lesion site (Figs. 6C?6E) compared with that in the DMSO-treated control zebrafish forebrain (Figs. 6A and 6B), indicating that sulfasalazine treatment reduced the number of macrophages/microglia in the injured forebrain.
To test whether sulfasalazine modulates macrophages/microglia in the demyelination animal model, we next labeled
Macrophages are myeloid cells derived from hematopoietic stem cells in the bone marrow and circulate the peripheral vasculature to survey tissue environments for damage or infection (Miron and Franklin, 2014). Microglia are the resident macrophages in the CNS and are generated from erythromyeloid precursors in the embryonic yolk sac (Rawji and Yong, 2013). In this study, we have shown that sulfasalazine treatment promotes oligodendrocyte and Schwann cell regeneration and subsequent remyelination in the nervous system of a transgenic zebrafish model of demyelination, and sulfasalazine function in remyelination is mediated by modulation of the immune response. Thus, sulfasalazine treatment reduces the number of macrophages/microglia in the CNS of the transgenic zebrafish model of demyelination, suggesting that modulation of the immune response, especially macrophages/microglia, is important for the remyelination process.
In the CNS of MS patients, infiltration of macrophages and activation of resident microglia are observed, and these immune cells have been shown to be associated with disease severity (Lucchinetti et al., 2000; Prineas and Connell, 1979). From studies using several models of CNS demyelination such as EAE, cupizone- and lysolecithin-induced demyelination, macrophages/microglia have been shown to play a negative role in remyelination (Franklin and Ffrench-Constant, 2008; Miron and Franklin, 2014; Rawji and Yong, 2013). Inhibition of macrophages/microglia activation or a reduction in their density suppressed and attenuated EAE progression (Bhasin et al., 2007; Heppner et al., 2005; Martiney et al., 1998). Administration of the microglia inhibitor minocycline to the cuprizone-induced demyelination model caused a decrease in demyelination and an improvement in motor coordination behavior (Pasquini et al., 2007; Skripuletz et al., 2010). In addition, treatment with corticosteroid enhanced remyelination associated with a reduction in the number of macrophages/microglia at the lesion site (Pavelko et al., 1998), and activation of macrophages/microglia using zymosan resulted in a loss of OPCs and oligodendrocytes in the lysolecithin-induced demyelination model (Schonberg et al., 2007). Altogether, these data indicate that macrophages/microglia play a negative role for remyelination in various models of demyelination in the CNS. Consistent with these studies, our data showed that reductions in macrophages/microglia are observed in the injured and demyelinated brain following sulfasalazine treatment, which resulted in the acceleration of the remyelination process (Fig. 6). This suggests that removal of macrophages/microglia from the demyelinated CNS promotes remyelination.
However, in contrast to its negative role in remyelination, several studies have shown that macrophages/microglia play a positive role in remyelination. First, in response to injury, OPCs near the lesion are activated from a normal quiescent state to a regenerative phenotype, which is the first step in remyelination, and macrophages/microglia are considered to be the major source of the factors that induce OPC activation (Franklin and Ffrench-Constant, 2008; Schonrock et al., 1998; Wilson et al., 2006). Second, after the activation and recruitment of OPCs to the demyelinated region, the next step in remyelination is the differentiation of OPCs into mature, myelinating oligodendrocytes. For the differentiation of OPCs, it is crucial to remove myelin debris that is generated during demyelination because CNS myelin contains proteins that inhibit OPC differentiation (Franklin and Ffrench-Constant, 2008; Kotter et al., 2006). In the cuprizone-induced demyelination model, removal of myelin debris by the phagocytic function of macrophages/microglia has been shown to be important for remyelination (Jurevics et al., 2002; Olah et al., 2012; Skripuletz et al., 2013). In the lysolecithin-induced demyelination model, depletion of macrophages reduced oligodendrocyte remyelination and delayed recruitment of OPCs to the lesion site by the lack of phagocytic function of macrophages, which is required for the removal of myelin debris (Kotter et al., 2001; 2005; 2006).
Because these reports and the discrepancies regarding whether the removal of macrophages/microglia promoted remyelination in our study, we reasoned that after demyelination, macrophages/microglia play a positive role in activating OPCs and removing myelin debris, but a prolonged presence of macrophages/microglia in a demyelinated region play a negative role in remyelination. In our experiment, we showed that the number of macrophages/microglia increased after demyelination by MTZ treatment. Thus, these cells have a chance to remove myelin debris and stimulate OPC activation for remyelination (Fig. 6F). Next, because we treated with sulfasalazine after demyelination occurred, sulfasalazine reduced the number of macrophages/microglia from the lesion after promoting recovery, and thus, the demyelinated CNS escaped from the negative effects caused by the sustained presence of macrophages/microglia. Our hypotheses are supported by the previous reports showing that the depletion of macrophages/microglia at the initial stage of demyelination significantly reduced remyelination but macrophage depletion at later stages in the remyelination phase does not change remyelination, suggesting that the beneficial role of macrophages/microglia was time-dependent (Kotter et al., 2001; 2005). Altogether, our data suggest that temporal regulation of the immune response of macrophages/microglia by sulfasalazine is important for the promotion of remyelination.
Mol. Cells 2015; 38(11): 1013-1021
Published online November 30, 2015 https://doi.org/10.14348/molcells.2015.0246
Copyright © The Korean Society for Molecular and Cellular Biology.
Suhyun Kim1,4, Yun-Il Lee2,4, Ki-Young Chang2, Dong-Won Lee1, Sung Chun Cho2, Young Wan Ha2, Ji Eun Na3, Im Joo Rhyu3, Sang Chul Park2,*, and Hae-Chul Park1,*
1Department of Biomedical Sciences, Korea University, Ansan 425-707, Korea, 2Well Aging Research Center, Samsung Advanced Institute of Technology (SAIT), Suwon 443-803, Korea, 3Department of Anatomy, College of Medicine, Korea University, Seoul 136-705, Korea, 4These authors contributed equally to this work.
Correspondence to:*Correspondence: hcpark67@korea.ac.kr (HCP); sc2013.park@samsung.com (SCP)
Most of the axons in the vertebrate nervous system are surrounded by a lipid-rich membrane called myelin, which promotes rapid conduction of nerve impulses and protects the axon from being damaged. Multiple sclerosis (MS) is a chronic demyelinating disease of the CNS characterized by infiltration of immune cells and progressive damage to myelin and axons. One potential way to treat MS is to enhance the endogenous remyelination process, but at present there are no available treatments to promote remyelination in patients with demyelinating diseases.
Sulfasalazine is an anti-inflammatory and immune-modulating drug that is used in rheumatology and inflammatory bowel disease. Its anti-inflammatory and immunomodulatory properties prompted us to test the ability of sulfasalazine to promote remyelination. In this study, we found that sulfasalazine promotes remyelination in the CNS of a transgenic zebrafish model of NTR/MTZ-induced demyelination. We also found that sulfasalazine treatment reduced the number of macrophages/microglia in the CNS of demyelinated zebrafish larvae, suggesting that the acceleration of remyelination is mediated by the immunomodulatory function of sulfasalazine. Our data suggest that temporal modulation of the immune response by sulfasalazine can be used to overcome MS by enhancing myelin repair and remyelination in the CNS.
Keywords: dorsal midline, enhancer, EphA7
Most axons in the vertebrate nervous system are surrounded by a lipid-rich membrane called myelin, which promotes rapid conduction of nerve impulses and protects against axonal damage. During development, myelin is produced by oligodendrocytes in the CNS and Schwann cells in the PNS (Emery, 2010). In addition to its role in forming myelin, a recent study has shown that oligodendrocytes provide metabolic support to neurons, which is crucial for axonal function and neuronal survival (Lee et al., 2012). Demyelination is the pathological process by which the myelin sheath is lost from around the axon. Normally, the CNS has the capacity to repair demyelinated axons through a process called remyelination, in which new myelin is synthesized to remyelinate demyelinated axons, enabling them to recover lost function. However, myelin is essential, and disruption of the myelin structure and failure to remyelinate in the CNS causes various neurodegenerative diseases including multiple sclerosis (MS).
MS is a chronic demyelinating disease of the CNS characterized by infiltration of immune cells and progressive damage to myelin and axons. MS is thought to be caused by the immune system inappropriately attacking the myelin in the brain and spinal cord, resulting in the demyelination of axons (Franklin, 2002; Franklin and Ffrench-Constant, 2008). It has been proposed that the enhancement of the endogenous remyelination process is one of the potential ways to efficiently treat MS, and several animal models of chemically induced demyelination such as cuprizone and lysolecitin have been used to screen drug candidates to promote remyelination (Imai et al., 2008; Lau et al., 2012; Moharregh-Khiabani et al., 2010; Ransohoff, 2012; Silvestroff et al., 2012). Although considerable progress has been achieved to promote remyelination in the demyelinated CNS, there are no available treatments to promote remyelination in patients with demyelinating diseases.
To investigate remyelination
The zebrafish embryos used in this study were
Metronidazole (Mtz, M1547, Sigma) was dissolved in EM containing 0.2% DMSO to 10 mM as previously described (Chung et al., 2013). For the ablation of myelinating glia,
Manually-dechorionated embryos were labeled with BrdU (Roche) by incubating them for 1 h 30min on ice in a solution of 10 mM BrdU and 15% DMSO in EM at 8 dpf. The embryos were then placed in EM, incubated for 1 h at 28.5°C, and fixed using 4% paraformaldehyde in PBS. Embryos were processed for immunohistochemistry, treated for 20 min with 2 M HCl, and were then processed for anti-BrdU immunohistochemistry. For immunohistochemistry, we used the following primary antibodies: mouse anti-BrdU (G3G4, 1:250, Developmental Studies Hybridoma Bank), rabbit anti-Sox10 (1:800, Lifespan bioscience), rat cd11b (1:100, BD bioscience), mouse anti-sodium channel (1:500, Sigma), and mouse anti-acetylated tubulin (1:1000, Sigma). Alexa 488-, 568-, 647-conjugated secondary antibodies were used for fluorescence detection (1:500, Molecular Probe). For histology section, embryos were embedded in 1.5% agar/5% sucrose. Sections (10 μm) were obtained by using a cryostat microtome (Zeiss).
Fluorescent
Tissues were prepared using standard procedures for TEM as described previously (Kim et al., 2002). Briefly, zebrafish larva were anesthetized with Tricaine (Sigma) and fixed with 10% paraformaldehyde/2.5% glutaraldehyde/0.1 M phosphate buffer, pH 7.4. Then larva were postfixed in 1% osmium tetroxide, dehydrated, embedded in Eponate-12 resin (Ted Pella). 1-μm-thick sections were obtained using Reichert-Jung Ultracut E ultramicrotome (Leica), stained with toluidine blue, and imaged using a Carl Zeiss Axio microscope. 60-nm-thick sections per block were collected on Formvar-coated slot grids and stained with uranyl acetate/lead citrate, and then recorded on an H-7500 TEM (Hitachi) (80 kV).
Adult brain injury was performed as described previously (Marz et al., 2011). Adult zebrafish were anesthetized with Tricaine (Sigma) and a BD U-100 insulin syringe (BD bioscience) was inserted vertically through the skull into the telencephalic region. Then brain damaged fish were transferred into a tank with fresh fish water and fixed in 4% paraformaldehyde at 4-day post injury.
To investigate remyelination
To examine the effectiveness of the NTR/MTZ system at inducing demyelination in the CNS and PNS via drug-dependent ablation of oligodendrocyte lineage cells, we first crossed
To investigate whether sulfasalazine promotes remyelination in the demyelinated zebrafish nervous system,
Next, we investigated whether sulfasalazine treatment promotes remyelination following the regeneration of myelinating glial cells in the CNS and PNS.
To investigate whether the regenerated myelinating glial cells in the sulfasalazine-treated larvae reconstructed a functional myelin sheath in the nervous system, we asked if the nodes of Ranvier structure reformed in the regenerated myelin sheath. To do so, we used an antibody against sodium channels (anti-panNavCh, NaCh), which is known to label node structures in axons (Voas et al., 2007). After immunohistochemistry with a NaCh antibody, we observed that NaCh+ node structures were regularly located in the PLL axons of the control larvae (Fig. 4A, arrows). However, NaCh+ node structures were rarely located in the PLL axons of the MTZ-treated
Our previous data showed that sulfasalazine treatment promoted regeneration of oligodendrocytes and Schwann cells and promoted subsequent remyelination in the NTR/MTZ-induced demyelination model. Therefore, we investigated the underlying mechanisms for the promotion of remyelination by sulfasalazine. To test whether sulfasalazine promoted proliferation of spinal cord precursors and thus the formation of oligodendrocyte precursor cells (OPCs) under normal conditions, we exposed wild-type
In the CNS of MS patients and in animal models of MS, substantial infiltration of macrophages and activation of microglia are observed and have been shown to be associated with disease severity (Lucchinetti et al., 2000; Prineas and Wright, 1978). In addition, inhibition of macrophage/microglia activation or a reduction in their density suppressed and attenuated progression of experimental autoimmune encephalomyelitis (EAE), a common immune-mediated animal model of MS (Bhasin et al., 2007; Heppner et al., 2005; Martiney et al., 1998). Altogether, these studies suggest that macrophages/microglia play a detrimental role in the remyelination process. Because sulfasalazine is thought to be an anti-inflammatory and immune-modulating drug (Hoult, 1986; Peppercorn, 1984), we hypothesized that sulfasalazine treatment promotes remyelination process by modulating the behavior macrophages/microglia in the demyelination animal model.
To test this idea, we first induced injury in the forebrain of adult zebrafish and treated the injured brain with either DMSO or sulfasalazine. Next, we compared the number of macrophages/microglia in the injured forebrain of DMSO- and sulfasalazine-treated adult zebrafish following labeling with an anti-CD11b antibody, which marks macrophages/microglia in zebra-fish (Fantin et al., 2010). In the injured forebrain of sulfasalazine-treated adult zebrafish, the number of anti-CD11b-positive macrophages/microglia clearly decreased around the lesion site (Figs. 6C?6E) compared with that in the DMSO-treated control zebrafish forebrain (Figs. 6A and 6B), indicating that sulfasalazine treatment reduced the number of macrophages/microglia in the injured forebrain.
To test whether sulfasalazine modulates macrophages/microglia in the demyelination animal model, we next labeled
Macrophages are myeloid cells derived from hematopoietic stem cells in the bone marrow and circulate the peripheral vasculature to survey tissue environments for damage or infection (Miron and Franklin, 2014). Microglia are the resident macrophages in the CNS and are generated from erythromyeloid precursors in the embryonic yolk sac (Rawji and Yong, 2013). In this study, we have shown that sulfasalazine treatment promotes oligodendrocyte and Schwann cell regeneration and subsequent remyelination in the nervous system of a transgenic zebrafish model of demyelination, and sulfasalazine function in remyelination is mediated by modulation of the immune response. Thus, sulfasalazine treatment reduces the number of macrophages/microglia in the CNS of the transgenic zebrafish model of demyelination, suggesting that modulation of the immune response, especially macrophages/microglia, is important for the remyelination process.
In the CNS of MS patients, infiltration of macrophages and activation of resident microglia are observed, and these immune cells have been shown to be associated with disease severity (Lucchinetti et al., 2000; Prineas and Connell, 1979). From studies using several models of CNS demyelination such as EAE, cupizone- and lysolecithin-induced demyelination, macrophages/microglia have been shown to play a negative role in remyelination (Franklin and Ffrench-Constant, 2008; Miron and Franklin, 2014; Rawji and Yong, 2013). Inhibition of macrophages/microglia activation or a reduction in their density suppressed and attenuated EAE progression (Bhasin et al., 2007; Heppner et al., 2005; Martiney et al., 1998). Administration of the microglia inhibitor minocycline to the cuprizone-induced demyelination model caused a decrease in demyelination and an improvement in motor coordination behavior (Pasquini et al., 2007; Skripuletz et al., 2010). In addition, treatment with corticosteroid enhanced remyelination associated with a reduction in the number of macrophages/microglia at the lesion site (Pavelko et al., 1998), and activation of macrophages/microglia using zymosan resulted in a loss of OPCs and oligodendrocytes in the lysolecithin-induced demyelination model (Schonberg et al., 2007). Altogether, these data indicate that macrophages/microglia play a negative role for remyelination in various models of demyelination in the CNS. Consistent with these studies, our data showed that reductions in macrophages/microglia are observed in the injured and demyelinated brain following sulfasalazine treatment, which resulted in the acceleration of the remyelination process (Fig. 6). This suggests that removal of macrophages/microglia from the demyelinated CNS promotes remyelination.
However, in contrast to its negative role in remyelination, several studies have shown that macrophages/microglia play a positive role in remyelination. First, in response to injury, OPCs near the lesion are activated from a normal quiescent state to a regenerative phenotype, which is the first step in remyelination, and macrophages/microglia are considered to be the major source of the factors that induce OPC activation (Franklin and Ffrench-Constant, 2008; Schonrock et al., 1998; Wilson et al., 2006). Second, after the activation and recruitment of OPCs to the demyelinated region, the next step in remyelination is the differentiation of OPCs into mature, myelinating oligodendrocytes. For the differentiation of OPCs, it is crucial to remove myelin debris that is generated during demyelination because CNS myelin contains proteins that inhibit OPC differentiation (Franklin and Ffrench-Constant, 2008; Kotter et al., 2006). In the cuprizone-induced demyelination model, removal of myelin debris by the phagocytic function of macrophages/microglia has been shown to be important for remyelination (Jurevics et al., 2002; Olah et al., 2012; Skripuletz et al., 2013). In the lysolecithin-induced demyelination model, depletion of macrophages reduced oligodendrocyte remyelination and delayed recruitment of OPCs to the lesion site by the lack of phagocytic function of macrophages, which is required for the removal of myelin debris (Kotter et al., 2001; 2005; 2006).
Because these reports and the discrepancies regarding whether the removal of macrophages/microglia promoted remyelination in our study, we reasoned that after demyelination, macrophages/microglia play a positive role in activating OPCs and removing myelin debris, but a prolonged presence of macrophages/microglia in a demyelinated region play a negative role in remyelination. In our experiment, we showed that the number of macrophages/microglia increased after demyelination by MTZ treatment. Thus, these cells have a chance to remove myelin debris and stimulate OPC activation for remyelination (Fig. 6F). Next, because we treated with sulfasalazine after demyelination occurred, sulfasalazine reduced the number of macrophages/microglia from the lesion after promoting recovery, and thus, the demyelinated CNS escaped from the negative effects caused by the sustained presence of macrophages/microglia. Our hypotheses are supported by the previous reports showing that the depletion of macrophages/microglia at the initial stage of demyelination significantly reduced remyelination but macrophage depletion at later stages in the remyelination phase does not change remyelination, suggesting that the beneficial role of macrophages/microglia was time-dependent (Kotter et al., 2001; 2005). Altogether, our data suggest that temporal regulation of the immune response of macrophages/microglia by sulfasalazine is important for the promotion of remyelination.
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