Demyelination and Schwann cell responses adjacent to injury epicenter cavities following chronic human spinal cord injury

The natural history of post-traumatic demyelination and myelin repair in the human spinal cord is largely unknown and has remained a matter of speculation. A wealth of experimental studies indicate that mild to moderate contusive injuries to the mammalian spinal cord evolve into a cavity with a preserved rim of white matter in which a population of segmentally demyelinated axons persists. It is believed that such injured axons have abnormal conduction properties. Theoretically, such axons might show improved function if myelin repair occurred. Schwann cells can remyelinate axons affected by multiple sclerosis, but little evidence exists that such repair can occur spontaneously following traumatic human SCI. Therefore, it is important to determine if chronic demyelination is present following human spinal cord injury. There are no previous reports that have conclusively demonstrated demyelination in the human spinal cord following traumatic spinal cord injury using immunohistochemical techniques. Immunohistochemical methods were used to study the distribution of peripheral and central myelin proteins as well as axonal neurofilament at the injury epicenter in 13 postmortem chronically injured human spinal cords 1-22 years following injury. Of these seven could be assessed by our methods. We found that some axonal demyelination can be detected even a decade following human SCI and indirect evidence that invading Schwann cells contributed to restoration of myelin sheaths around some spinal axons.     

Number of oligodendrocyte progenitors recruited to the lesioned spinal cord is modulated by the levels of the cell cycle regulatory protein p27Kip-1

Remyelination is a critical step for recovery of function after demyelination and defines the ability to generate new myelin. This repair process is dependent on the presence of resident oligodendrocyte progenitors (OLPs) that have been shown to remyelinate axons after demyelination. We have previously shown that the levels of the cell cycle inhibitor p27Kip-1 modulate the number of neonatal cortical OLPs. We now asked whether this cell cycle molecule plays also a role in regulating the number of adult OLP in the spinal cord after demyelination induced by lysolecithin injection. The proliferative response of OLP in the spinal cord of injected wild-type (wt) and p27Kip-1 null mice was evaluated 3 days after lesion. In vivo labeling with bromodeoxyuridine (BrdU) was used to identify cells in S phase. Double immunofluorescence for the OLP marker NG2, and for BrdU was used to count the number of proliferating progenitors. Consistent with a role of p27Kip-1 in regulating the number of adult OLP in the injured spinal cord, a larger number of proliferating OLPs was observed in p27Kip-1null mice compared with wild-type controls. These cells were able to differentiate as assessed by the presence of MBP+ cells in the spinal cord 14 days after injury. We conclude that the cellular levels of the cell cycle inhibitor p27Kip-1 modulate the repair response of OLPs to injury in the adult spinal cord.     

Ultrastructural pathology of nerve fibers in calcium-induced myelopathy

Calcium has been proposed as a mediator of nerve fiber degeneration following traumatic injury of the spinal cord. It induces a spongy, necrotizing myelopathy similar in its evolution to that observed in experimental spinal cord trauma. The current study was undertaken to determine the ultrastructural changes in the central nervous system (CNS) nerve fibers associated with calcium-induced myelopathy. A 10% calcium chloride (CaCl2) solution (pH 7.4) was slowly dripped on the dorsal surface of the surgically exposed lower thoracolumbar spinal cord of adult male Sprague-Dawley rats. The posterior and lateral columns of the spinal cords were fixed and processed for electron microscopy. Controls consisted of tissue from normal and sham-operated animals, as well as those receiving equal volumes and osmolarities of sodium chloride (NaCl), magnesium chloride (MgCl2), and potassium chloride (KCl) at the same pH. In the CaCl2 treated animals, spongiosis of increasing severity developed in white matter, as the result of periaxonal, adaxonal and intramyelinic swelling. Vesicular demyelination was consistently observed, beginning within one hour (h) and progressing with increasing severity up to 24-72 h. Axonal changes included pleomorphic spheroids, granular degeneration and intra-axonal calcification. The ultrastructural changes in the nerve fibers provoked by calcium were indistinguishable from those previously reported in experimental spinal cord trauma. These observations strengthen the hypothesis that calcium initiates the nerve fiber degeneration following spinal cord injury.     

Vitamin B12 extensive thoracic myelopathy: clinical, radiological and prognostic aspects. Two cases report and literature review

The myelopathy caused by vitamin B12 deficiency is known as subacute combined degeneration. It is rare, but a well known cause of demyelination of the dorsal columns of the spinal cord. The magnetic resonance imaging is characterized by an increased signal on T2-weighted images involving the posterior columns of cervical and thoracic cord. There have been few cases in literature with extensive lesions (more than seven levels) of the thoracic spinal cord. The clinical and radiological improvements are possible if the replacement of vitamin B12 is initiated precocious. We present two rare cases of extensive thoracic myelopathy due to vitamin B12 deficiency. The first is a young woman with complete clinical recovery and important radiologic improvement after early treatment. In addition, the second case is an older man with partial response to the treatment. Those cases illustrate the importance of considering vitamin B12 deficiency in any patient, who presents with myelopathy.     

Protease activated receptor 2 controls myelin development,resiliency and repair

Oligodendrocytes are essential regulators of axonal energy homeostasis and electrical conduction and emerging target cells for restoration of neurological function. Here we investigate the role of protease activated receptor 2 (PAR2), a unique protease activated G protein‐coupled receptor, in myelin development and repair using the spinal cord as a model. Results demonstrate that genetic deletion of PAR2 accelerates myelin production, including higher proteolipid protein (PLP) levels in the spinal cord at birth and higher levels of myelin basic protein and thickened myelin sheaths in adulthood. Enhancements in spinal cord myelin with PAR2 loss‐of‐function were accompanied by increased numbers of Olig2‐ and CC1‐positive oligodendrocytes, as well as in levels of cyclic adenosine monophosphate (cAMP), and extracellular signal related kinase 1/2 (ERK1/2) signaling. Parallel promyelinating effects were observed after blocking PAR2 expression in purified oligodendrocyte cultures, whereas inhibiting adenylate cyclase reversed these effects. Conversely, PAR2 activation reduced PLP expression and this effect was prevented by brain derived neurotrophic factor (BDNF), a promyelinating growth factor that signals through cAMP. PAR2 knockout mice also showed improved myelin resiliency after traumatic spinal cord injury and an accelerated pattern of myelin regeneration after focal demyelination. These findings suggest that PAR2 is an important controller of myelin production and regeneration, both in the developing and adult spinal cord.     

SB209670, a potent endothelin receptor antagonist, prevents or delays axonal degeneration after spinal cord injury

We developed a rat spinal cord transection injury model and investigated whether endogenous endothelin takes part in axonal degeneration after injury, by using a potent nonselective endothelin receptor antagonist, SB209670. Light microscopic analysis showed that axonal degeneration of the spinal cord was clearly observed one week after injury, supported by immunohistochemical study with anti-neurofilament antibody. Electron microscopic observation showed enlargement and shrinking of spinal axons in the injured sites one week after injury. Application of SB209670 to the lesion sites markedly inhibited axonal damage after injury. These results suggest that endogenous endothelin plays a role in axonal degeneration after spinal cord injury and that SB209670 prevents or delays the axonal degeneration after CNS damage.     

Chronic encephalomyelitis presenting as chronic progressive myelopathy.

Chronic progressive myelopathy is a clinical entity with few neuropathological studies. The most frequent diagnosis in necropsied cases is multiple sclerosis. A case of chronic progressive myelopathy is described with a 16-year course in a woman who was 28 years old at the onset of the disease. At necropsy, there was chronic inflammation in the central nervous system with predominant involvement of the spinal cord with diffuse myelin loss and axonal degeneration.     

Central nervous system and non-central nervous system antigen vaccines exacerbate neuropathology caused by nerve injury

Previously, we showed that autoimmune (central nervous system myelin-reactive) T cells exacerbate tissue damage and impair neurological recovery after spinal cord injury. Conversely, independent studies have shown T cell-mediated neuroprotection after spinal cord injury or facial nerve axotomy (FNAx). The antigen specificity of the neuroprotective T cells has not been investigated after FNAx. Here, we compared the neuroprotective capacity of autoimmune and non-autoimmune lymphocytes after FNAx. Prior to axotomy, C57BL/6 mice were immunized with myelin basic protein, myelin oligodendrocyte glycoprotein (MOG) or ovalbumin (a non-self antigen) emulsified in complete Freund's adjuvant (CFA). FNAx mice receiving injections of phosphate-buffered saline (PBS) only (unimmunized) or PBS/CFA emulsions served as controls. At 4 weeks after axotomy, bilateral facial motor neuron counts were obtained throughout the facial motor nucleus using unbiased stereology (optical fractionator). The data show that neuroantigen immunizations and 'generic' lymphocyte activation (e.g. PBS/CFA or ovalbumin/CFA immunizations) exacerbated neuron loss above that caused by FNAx alone. We also found that nerve injury potentiated the effector potential of autoimmune lymphocytes. Indeed, prominent forelimb and hindlimb motor deficits were accompanied by disseminated neuroinflammation and demyelination in FNAx mice receiving subencephalitogenic immunization with MOG. FNAx or neuroantigen (MOG or myelin basic protein) immunization alone did not cause these pathological changes. Thus, irrespective of the antigens used to trigger an immune response, neuropathology was enhanced when the immune system was primed in parallel with nerve injury. These data have important implications for therapeutic vaccination in clinical neurotrauma and neurodegeneration.     

E6020, a synthetic TLR4 agonist,accelerates myelin debris clearance,Schwann cell infiltration,and remyelination in the rat spinal cord

Oligodendrocyte progenitor cells (OPCs) are present throughout the adult brain and spinal cord and can replace oligodendrocytes lost to injury, aging, or disease. Their differentiation, however, is inhibited by myelin debris, making clearance of this debris an important step for cellular repair following demyelination. In models of peripheral nerve injury, TLR4 activation by lipopolysaccharide (LPS) promotes macrophage phagocytosis of debris. Here we tested whether the novel synthetic TLR4 agonist E6020, a Lipid A mimetic, promotes myelin debris clearance and remyelination in spinal cord white matter following lysolecithin‐induced demyelination. In vitro, E6020 induced TLR4‐dependent cytokine expression (TNFα, IL1β, IL‐6) and NF‐κB signaling, albeit at ～10‐fold reduced potency compared to LPS. Microinjection of E6020 into the intact rat spinal cord gray/white matter border induced macrophage activation, OPC proliferation, and robust oligodendrogenesis, similar to what we described previously using an intraspinal LPS microinjection model. Finally, a single co‐injection of E6020 with lysolecithin into spinal cord white matter increased axon sparing, accelerated myelin debris clearance, enhanced Schwann cell infiltration into demyelinated lesions, and increased the number of remyelinated axons. In vitro assays confirmed that direct stimulation of macrophages by E6020 stimulates myelin phagocytosis. These data implicate TLR4 signaling in promoting repair after CNS demyelination, likely by stimulating phagocytic activity of macrophages, sparing axons, recruiting myelinating cells, and promoting remyelination. This work furthers our understanding of immune–myelin interactions and identifies a novel synthetic TLR4 agonist as a potential therapeutic avenue for white matter demyelinating conditions such as spinal cord injury and multiple sclerosis.     

神经干细胞移植促进鼠脊髓损伤后髓鞘结构的修复

目的 观察神经干细胞移植治疗对鼠脊髓损伤后髓鞘结构修复的作用并探讨其作用机制。方法 制备鼠T10脊髓损伤模型,体外培养、诱导鼠神经干细胞,定量评价神经干细胞移植对脊髓损伤后髓鞘结构修复的影响。结果 与对照组相比,神经干细胞移植组明显地增强了蛋白前脂蛋白信使核糖核酸(PLP mRNA)的表达,促进了髓鞘碱性蛋白(MBP)性的髓鞘再生和髓鞘结构的修复。结论 神经干细胞移植通过增强髓鞘的再生而促进了脊髓损伤后髓鞘结构的修复,是急性脊髓损伤一种有效的治疗方案。     

Chemokine antagonist infusion promotes axonal sparing after spinal cord contusion injury in rat

Spinal cord injury produced by mechanical contusion causes the onset of acute and chronic degradative events. These include blood brain barrier disruption, edema, demyelination, axonal damage and neuronal cell death. Posttraumatic inflammation after spinal cord injury has been implicated in the secondary injury that ultimately leads to neurologic dysfunction. Studies after spinal cord contusion have shown expression of several chemokines early after injury and suggested a role for them in the ordered recruitment of inflammatory cells at the lesion site (McTigue et al. [1998] J. Neurosci. Res. 53:368-376; Lee et al., [2000] Neurochem Int). We have demonstrated previously that infusion of the broad-spectrum chemokine receptor antagonist (vMIPII) in the contused spinal cord initially attenuates leukocyte infiltration, suppresses' gliotic reaction and reduces neuronal damage after injury. These changes are accompanied by increased expression of bcl-2, the endogenous apoptosis inhibitor, and reduced neuronal apoptosis (Ghirnikar et al. [2000] J. Neurosci. Res. 59:63-73). We demonstrate that 2 and 4 weeks of vMIPII infusion in the contusion-injured spinal cord also results in decreased hematogenous infiltration and is accompanied by reduced axonal degeneration in the gray matter. Luxol fast blue and MBP immunoreactivity indicated reduced myelin breakdown in the dorsal and ventral funiculi. Increased neuronal survival in the ventral horns of vMIPII infused cords was seen along with increased bcl-2 staining in them. Immunohistochemical identification of fiber phenotypes showed increased presence of calcitonin gene related peptide, choline acetyl transferase and tyrosine hydroxylase positive fibers as well as increased GAP43 staining in treated cords. These results suggest that sustained reduction in posttraumatic cellular infiltration is beneficial for tissue survival. A preliminary report of this study has been published (Eng et al. [2000] J. Neurochem. 74(Suppl):S67B). In contrast to vMIPII, infusion of MCP-1 (9-76), a N-terminal analog of the MCP-1 chemokine showed only a modest reduction in cellular infiltration at 14 and 21 dpi without significant tissue survival after spinal cord contusion injury. Comparing data on tissue survival obtained with vMIPII and MCP-1 (9-76) further validate the importance of the use of broad-spectrum antagonists in the treatment of spinal cord injury. Controlling the inflammatory reaction and providing a growth permissive environment would enhance regeneration and ultimately lead to neurological recovery after spinal cord injury. J. Neurosci. Res. 64:582-589, 2001. Published 2001 Wiley-Liss, Inc.     

Immunological myelin disruption does not alter expression of regeneration-associated genes in intact or axotomized rubrospinal neurons

The inability of axotomized neurons to regenerate within the CNS has been partially attributed to a number of inhibitory factors associated with CNS myelin that are extrinsic to the severed neurons. However, some neurons are capable of limited regeneration after injury and this ability has been shown to correlate with the expression of certain regeneration-associated genes (RAGs) intrinsic to injured neurons. It has therefore been postulated that neutralization of inhibitory factors, as well as the induction of an appropriate neuronal cell body response, would facilitate improved regrowth of injured CNS axons. In previous studies we have shown that immunological removal of myelin from the spinal cord facilitates axonal regeneration by rubrospinal neurons, as indicated by retrograde transport of a fluorescent dye placed distal to the site of injury. Here, we investigated whether the immunological focal removal of spinal cord myelin, following a thoracic spinal cord injury, concomitantly stimulated an increase in the expression of RAGs in rubrospinal neurons. In situ hybridization for Talpha-1 tubulin and GAP-43 at days 7, 14, and 21 revealed no significant increase in gene expression in rubrospinal neurons following immunological demyelination. The ability of various neuronal populations to sprout or slowly regrow without expressing the previously characterized cell body response is reviewed. We conclude that the recently demonstrated regeneration of rubrospinal tract, after immunologically directed spinal cord demyelination, is the result of either axonal sprouting or slow axonal regrowth without the increased expression of RAGs characteristic for fast axon regeneration.     

Ataxia in Jack Russell terriers

Summary This paper describes two cases of a progressive incoordination in Jack Russell terriers. Histologically the entity was characterised by widespread Wallerian-type degeneration of the brain and cord, together with focal symmetrical demyelination of the dorsolateral and ventromedial columns of the cord. One dog also showed severe ballooning of myelin sheaths in the dorsal and ventral spinal nerves and oedema and fibrosis of the sciatic nerve. Both dogs also showed degenerative changes of the central auditory pathways.     

The neuropathology of chronic relapsing experimental allergic encephalomyelitis induced in the Lewis rat by inoculation with whole spinal cord and treatment with cyclosporin A

Summary Chronic relapsing experimental allergic encephalomyelitis was induced in Lewis rats by inoculation with guinea-pig spinal cord and complete Freund's adjuvant followed by treatment with low-dose cyclosporin A. In most animals, tail and limb weakness developed in a relapsing remitting pattern but in some these signs were persistent or progressive from onset. Histological studies during the early stages of clinically active disease (< 25 days after inoculation) revealed inflammation and primary demyelination in the central nervous system (CNS), particularly the spinal cord, and in the peripheral nervous system (PNS), specifically the ventral and dorsal roots and dorsal root ganglia. Animals studied in the later stages of clinically active disease (> 28 days after inoculation) had extensive spinal cord demyelination but minimal PNS demyelination. In these animals, large plaques of demyelination with gliosis and prominent plasma cells occurred particularly in the thoracic spinal cord, and lesions of different ages were present within the spinal cord. CNS and PNS remyelination by oligodendrocytes and Schwann cells, respectively, was present in all animals studied later than 18 days after inoculation (the time of the first remission, if it occurred). In both early and late clinically active disease electron microscopy revealed macrophages invading and destroying CNS myelin sheaths. Active demyelination was sometimes found in regions of CNS remyelination, suggesting that remyelinated fibres were being attacked. Axonal degeneration occurred in the spinal cord. During clinical remission there was CNS and PNS remyelination and much less inflammation; however, active demyelination still occurred to a limited degree.Supported by the National Health and Medical Research Council of Australia, University of Queensland Foundation and Clive and Vera Ramaciotti Foundations is gratefully acknowledged. During this work Glenn Stanley was a recipient of a Research Training Fellowship of the National Multiple Sclerosis Society of Australia     

Central nervous system demyelination and remyelination in multiple sclerosis and viral models of disease

The mechanisms of myelin injury and repair were studied in acute multiple sclerosis lesions and in a murine model of demyelination induced by a virus. Injury to oligodendrocytes resulting in degeneration of inner glial loops and inner myelin lamellae (dying-back oligodendrogliopathy) was observed by electron microscopy in brain biopsies of acute demyelinating lesions. Attempts at central nervous system remyelination as manifested by thinly myelinated axons and proliferation of oligodendrocytes were observed at the edge of many acute plaques. To develop therapeutic strategies to inhibit demyelination or promote remyelination, mice infected intracranially with Theiler's virus (a picornavirus) were studied. Experimental manipulation of Theiler's virus-infected mice by treatment during chronic demyelinating disease with immunoglobulins directed at normal spinal cord antigens or with monoclonal antibodies which deplete CD4 or CD8-positive T cells reslted in augmentation of new myelin synthesis. These observations suggest that disturbances in the myelinating function of oligodendrocytes, events not accompanied by death of these cells, may be among the earliest pathological events in multiple sclerosis. Experiments using the Theiler's virus model of demyelination indicate that manipulation of the immune response has the potential to promote central nervous system remyelination and functional recovery in multiple sclerosis.     

The mechanism of Naringin-enhanced remyelination after spinal cord injury

Our previous study revealed that intragastric administration of naringin improved remyelination in rats with spinal cord injury and promoted the recovery of neurological function of the injured spinal cord.This study sought to reveal the mechanisms by which naringin improves oligodendrocyte precursor cell differentiation and maturation,and promotes remyelination.Spinal cord injury was induced in rats by the weight-drop method.Naringin was intragastrically administered daily(20,40 mg/kg) for 4 weeks after spinal cord injury induction.Behavioral assessment,histopathological staining,immunofluorescence spectroscopy,ultrastructural analysis and biochemical assays were employed.Naringin treatment remarkably mitigated demyelination in the white matter,increased the quality of myelinated nerve fibers and myelin sheath thickness,promoted oligodendrocyte precursor cell differentiation by upregulating the expression of NKx2.2 and 2′3′-cyclic nucleotide 3′-phosphodiesterase,and inhibited β-catenin expression and glycogen synthase kinase-3β(GSK-3β) phosphorylation.These findings indicate that naringin treatment regulates oligodendrocyte precursor cell differentiation and promotes remyelination after spinal cord injury through the β-catenin/GSK-3β signaling pathway.     

Demyelination in the spinal cord of murine globoid cell leukodystrophy (the twitcher mouse)

Summary Chronologic events of demyelination were investigated in the spinal cord of the twicher mouse, an authentic murine model of human globoid cell leukodystrophy (GLD) from 5 to 45 days postnatal. There was very little evidence of myelin degeneration before day 25 although clustered or scattered globoid cells were already noted in the dorsal columns and intramedullary portion of the ventral roots.Globoid cells contained typical cytoplasmic inclusions and in those which were found adjacent to degenerating myelin and naked axons, myelin debris were conspicuous in their cytoplasm.Vesiculation of myelin and a feature of globoid cells stripping myelin lamellae were noted in the area of demyelination. Myelin and oligodendroglial degeneration became pronounced throughout the spinal white matter after day 40 but globoid cells tended to be more concentrated in the dorsal columns.Our observations suggest that the emergence of globoid cells in GLD is in response to the changes in biochemical environment (i.e., excessive presence of galactosylceramide in the tissue?), and these cells appear to have a role as phagocytic cells in removing myelin lamellae.Supported in part by research grants NS-03356, NS-10803, and HD-01799 and a training grant for experimental neuropathology, NS-07098, from NINCDS     

Galectin-3/MAC-2 in experimental allergic encephalomyelitis

The removal of degenerating myelin by phagocytosis is central to pathogenesis and repair in traumatized and diseased nervous system. Galectin-3/MAC-2 is a differentiation and activation marker of murine and human monocytes/macrophages/microglia. Galectin-3/MAC-2, along with MAC-1 that mediates myelin phagocytosis, marks an in vivo activation state in macrophages, which are involved in myelin degeneration and phagocytosis in injured mouse peripheral nerves. In contrast, high levels of MAC-1 but extremely low levels of Galectin-3/MAC-2 are expressed in vivo in injured CNS where myelin degeneration and phagocytosis progress extremely slowly. The present study was aimed at testing whether an activation state marked by Galectin-3/MAC-2 is present in vivo in the CNS of EAE mice concomitant with autoimmune induced myelin degeneration and phagocytosis. EAE was inflicted by mouse spinal cord homogenate. Demyelination was assessed by light microscopy and Galectin-3/MAC-2, MAC-1, and F4/80 expression by immunocytochemistry. We presently document that Galectin-3/MAC-2 expression is up regulated, along with MAC-1 and F4/80, in spinal cords and optic nerves of EAE mice in areas of demyelination and myelin degeneration, in myelin phagocytosing microglia and macrophages. Copolymer 1 (Glatiramer acetate) suppresses EAE, demyelination, and Galectin-3/MAC-2 expression. EAE pathogenesis thus involves a state of activation in microglia and macrophages characterized by the expression Galectin-3/MAC-2 along with MAC-1. Furthermore, the in vivo responses to injury and autoimmune challenge in the CNS differ in the activation pattern of microglia and macrophages with regard to Galectin-3/MAC-2 expression and the corresponding occurrence of myelin degeneration and phagocytosis.     

Morphological characteristics of central demyelination and remyelination: A single-fiber study

The characteristics of demyelination and remyelination in the central nervous system of the cat were examined using quantitative single-fiber analysis. Internodal length, fiber diameter, and nodal gap length were measured in single fibers teased from the spinal cord of normal animals and of animals with transient experimental cord compression. Demyelination was primarily paranodal, but longer extents of myelin loss occurred. New myelin sheaths were formed by oligodendrocytes and organized into segments bounded by nodes. The internodal length remained inappropriately short for fiber diameter 6 months after compression. The findings demonstrate that the spinal cord is capable of remyelinating after injury, but whether this contributes to functional recovery remains unknown.