Induction of metallothionein in astrocytes and microglia in the spinal cord from the myelin-deficient jimpy mouse

Jimpy is a shortened life-span murine mutant whose genetic disorder results in severe pathological alterations in the CNS, including hypomyelination, oligodendrocyte death and strong astroglial and microglial reaction. The knowledge of metallothionein (MT) regulation in the CNS and especially of MT presence in specific glial cell types under pathological conditions is scarce. In the present study, immunocytochemical detection of MT-I+II has been performed in spinal cord sections from 10–12- and 20–22-day-old jimpy and normal animals. The identification of MT-positive glial cells was achieved through double labeling combining MT immunocytochemistry and selective markers for oligodendrocytes, astrocytes and microglia. MT was found in glial cells and was present in the spinal cord of jimpy and normal mice at both ages, but there were remarkable differences in MT expression and in the nature of MT-positive glial cells depending on the type of mouse. The number of MT-positive cells was higher in jimpy than in normal spinal cords. This was apparent in all spinal cord areas, although it was more pronounced in white than in the gray matter and at 20–22 days than at 10–12 days. The mean number of MT-positive glia in the jimpy white matter was 1.9-fold (10–12 days) and 2.4-fold (20–22 days) higher than in the normal one. Astrocytes were the only parenchymal glial cells that were positively identified as MT-producing cells in normal animals. Interestingly, MT in the jimpy spinal cord was localized not only in astrocytes but also in microglial cells. The occurrence of MT induction in relation to reactive astrocytes and microglia, and its role in neuropathological conditions is discussed.     

The microglial reaction in spinal cords of jimpy mice is related to apoptotic oligodendrocytes

Jimpy is a shortened life-span murine mutant whose genetic disorder results in a severe hypomyelination in the central nervous system associated with a variety of glial abnormalities, including oligodendrocyte death. In this study, we report that oligodendrocyte death in jimpy occurs through an apoptotic mechanism, as demonstrated by in situ labeling of nuclear DNA fragmentation. Compared to those of normal littermates, the spinal cords of jimpy mice showed a significantly higher number of apoptotic cells. Our observations also corroborate that specific glial cell death in jimpy is restricted to oligodendrocytes, as evidenced by double labeling for DNA fragmentation and MBP immunocytochemistry. Cells labeled for DNA fragmentation were always negative for astroglial or microglial markers. Apoptotic oligodendrocytes were not aggregated into clusters and were ubiquitously distributed throughout the jimpy spinal cord, although were more numerous in white matter than in gray matter. We found no physical association between astrocytes and dying cells in jimpy. Microglial cells, however, were found closely attached to and even surrounding apoptotic cells. The possible role of microglial cells in relation to apoptotsis is discussed.     

A Quantitative Spatial Analysis of the Blood–Spinal Cord Barrier: I. Permeability Changes after Experimental Spinal Contusion Injury

Blood–spinal cord barrier (BSB) permeability was measured using quantitative autoradiography following contusion injury to the rat spinal cord. Permeability was assessed by calculating blood-to-tissue transfer constants (K_ivalues) for the vascular tracer [¹⁴C]-α-aminoisobutyric acid (AIB) in injured (3, 7, 14, and 28 days postinjury), laminectomy control, and uninjured control animals. Permeability was quantitated using four separate imaging techniques in gray and white matter throughout the rostro-caudal extents of the forming lesion. Away from the epicenter, gray matter permeability was further differentiated within discrete spinal lamina using computerized templates. Regardless of the type of analysis used, increased AIB permeability (K_ivalues) was noted at all survival times in all tissue regions with respect to both uninjured and laminectomy control groups. The data indicate a large increase in individualK_ivalues throughout the dorsoventral axis of the spinal cord at 3 days postinjury (≈6–9 ml/kg/min). By 7 days,K_ivalues were quantitatively smaller (≈4–5 ml/kg/min) in all regions compared with 3-day tissues. Despite further attenuation of AIB uptake in the gray matter at 14 and 28 days postinjury, circumferential white matter tracts showed a secondary increase in permeability compared to 7-day tissue. Permeability in the white matter at 14–28 days postinjury (≈5–6 ml/kg/min) was comparable to that at 3 days postinjury (6–7 ml/kg/min). Measurements of the axial distribution of AIB permeability indicate increased BSB permeability over several segments rostral and caudal to the lesion epicenter (≈3 cm in both directions). Secondary elevations of AIB transfer in the spinal white matter between 14 and 28 days were colocalized with zones of immunohistochemically defined microglial clusters. The known plasticity of this cell type in response to changes in the extracellular microenvironment suggests that the spinal white matter at later survival times (14–28 days postinjury) is an area of dynamic vascular and/or axonal reconstruction. The implications of increased permeability to both tissue injury and neural regeneration are discussed.     

Synaptogenesis in the intermediate gray region of the lumbar spinal cord in the postnatal rat

Mid-thoracic spinal cord transection produces dramatically different behavioral results depending upon a rat's age at the time of surgery. The present study was initiated to determine whether the synaptic development in the gray matter of the normal, developing spinal cord differs before and after the period when maximal behavioral recovery occurs. The L6 segments from 10 groups of animals, 0–30 days of age, taken at 3 day intervals (4 animals/group) were studied by light microscopy. Areal measurements of the gray matter were made using an integrating x-y tablet interfaced to a computer. Cell size, cell density and area of neuropil were evaluated in the lateral portions of the intermediate gray matter, laminae VI and VII. Electron microscopic analyses of synaptogenesis were performed on material from the same region in animals 3, 12, 15, 21 and 30 days old using similar morphometric methods while taking note of vesicle, junctional, and mitochondrial morphology. A 60% increase in area of neuropil paralleled a linear increase, of comparable magnitude, in area of the gray matter until 15 days of age when both curves reached a plateau. Neuronal perikaryal size remained constant ( 200 sq. μm in plane of nucleolus) throughout development and so could not have contributed to the increase in area of gray matter. Areal measurements of the size and counts of the number of vesicle containing profiles demonstrated a 50% increase in density of axon terminals between 3 and 12 days of age and a steady decline thereafter. The size of vesicle-containing profiles in laminae VI and VII remained constant at a small value ( 0.35 sq. μm) until 12 days of age, showed rapid growth to 0.54 sq. μm between 12 and 15 days of age, followed by a more moderate increase in sectional area after 15 days. These results suggest that during the period when recovery of function follows spinal injury, synaptogenesis in the intermediate gray region of the lumbar spinal cord proceeds rapidly, while at stages when little recovery of function follows spinal transection, synaptogenesis is essentially complete.     

Oligodendroglial structures and distribution shown by carbonic anhydrase immunostaining in the spinal cords of developing normal and shiverer mice

The spinal cords of young and adult normal and dysmyelinating mutant (shiverer) mice were immunostained with anticarbonic anhydrase to investigate the distribution of oligodendroglial populations into the gray- and white-matter regions in the developing normal and mutant animals; the morphology of oligodendrocytes and their processes at the light microscopic level in gray matter and white matter; and the apparent gliosis in the gray matter, as well as the white matter, of the mutants. Immunocytochemistry and enzyme assays revealed consistent increases in carbonic anhydrase antigenicity and specific activity in controls and mutants between the ages of approximately 15 days and approximately 60 days. As shown previously in adult animals, oligodendroglia in larger than normal proportions were situated at the periphery of the "white-matter" columns, as compared to gray matter, in the shiverers, with, however, significant numbers of oligodendroglia were heterogeneous with respect to shapes, configuration of processes, and intensity of carbonic anhydrase immunostaining. In the shiverer "white matter" the oligodendrocytes were smaller than normal, and their shapes and arrangement were relatively irregular. In the normal gray matter short oligodendroglial processes appeared to be associated with neuronal perikarya, and those processes were more pronounced at approximately 90 days than at approximately 20 days of age. Background staining in normal gray matter suggested that oligodendroglial processes were, in addition, tightly wound around many axons. In shiverer gray matter the oligodendrocytes were smaller, and their processes appeared to be wrapped more loosely around smaller numbers of conspicuous axons and to be associated less frequently with neuronal perikarya. This finding suggests that the deficiency in the myelin basic protein in the mutant may affect interactions between oligodendrocytes and neurons in the gray matter as well as in the white matter. The astrocytic "marker," glial fibrillary acidic protein, was detected in gray and white matter of shiverers as young as 16 days, and the differences from carbonic anhydrase localization supported the conclusion that the processes enwrapping axons in the shiverer mouse CNS are derived from oligodendrocytes, not astrocytes.     

Lysophosphatidylcholine induces rapid recruitment and activation of macrophages in the adult mouse spinal cord

Lysophosphatidylcholine (LPC) can induce rapid breakdown and removal of myelin from the adult mammalian CNS. In this paper we report the detailed characterization of the immune cell response as well as changes in the expression of cell adhesion molecules and the permeability of the blood-brain barrier after microinjection of LPC into the adult mouse spinal cord. T cells and neutrophils were seen in the spinal cord 6-12 h after LPC injection, but not in PBS-injected mice. Mac-1+ monocytes were also seen at 6 h and 12 h in the white and gray matter of mice injected with LPC and PBS but were significantly greater in the white matter after LPC injections. At later time points LPC induced an increase in the number of activated Mac-1+ macrophages that displayed a variety of morphologies in the white and gray matter. These cells were not present in PBS-injected control mice. LPC also induced widespread microglial activation in the white and gray matter. The number of these Mac-1+ microglia reduced drastically at 96 h after LPC injection suggesting that they may have transformed into Mac-1+ phagocytic cells with a different morphology. These LPC-induced changes in immune cells were accompanied by significant increases in VCAM-1+ and ICAM-1+ blood vessels in the spinal cord. In addition, LPC induced a rapid and widespread disruption of the blood-brain barrier, as compared to PBS injected mice. Therefore, LPC can induce an early and transient T cell and neutrophil response in the CNS. These cells likely promote the rapid influx of monocytes followed by widespread and effective activation of macrophages that mediate rapid phagocytosis of myelin debris.     

Microglial development is altered in immature spinal cord by exposure to radiation

Previous studies in this laboratory have documented that the microglial environment of the immature spinal cord is altered by exposure to ionizing radiation. As a result, the lumbosacral spinal cord is markedly depleted of both oligodendrocytes and astrocytes, while leaving axons and the overall cytoarchitecture intact. The status of the microglia in the irradiated region is unknown and is of interest given the interactions between microglia and astrocytes recently elucidated by others. This study uses both in vivo and in vitro approaches to examine the microglial population in normal and irradiated immature spinal cord. The lectin, Griffonia (Bandeiraea) simplicifolia, was selected since it marks microglia both in paraffin embedded sections and in cell cultures. Light microscopic examination of spinal cord sections revealed a reduced microglial population in the irradiated region when compared to littermate controls, and a change in morphology of the remaining microglia to that described by others as ‘‘activated’’. Cultures prepared from lumbosacral spinal cords harvested from 3-day-old rats within 2–4 hr following irradiation were compared with cultures derived from their non-irradiated littermates after 8 days in vitro. Cultures from the irradiated spinal cords revealed trends similar to those observed in vivo, i.e. a reduced microglial population and altered morphology. Although all glial cell types were reduced in cultures from irradiated spinal cords, the few microglia present were usually positioned atop astrocytes. The consistency of reduction in all glial populations in this model shows the microglia to be a novel microenvironment for further studies of roles of microglia within the spinal cord.     

Localization of glial cell antigens in the brains of young normal mice and the dysmyelinating mutant mice, jimpy and shiverer

Tissue sections from the brains of normal, jimpy, and shiverer mice were immunostained by the peroxidase antiperoxidase method for carbonic anhydrase (CA) and the putative astrocytic "markers" glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP). The cells in normal gray matter that immunostained with anti-CA and anti-GS were similar to one another in size and process elaboration. In the normal gray matter there were relatively few GFAP-positive astrocytes. When present, these cells resembled the CA- and GS-positive cells; however, the GFAP appeared to be concentrated in the astroglial processes, as distinguished from the cell bodies. Glial cell processes, immunostained for CA or GS, surrounded blood vessels and unstained neurons in the normal gray matter. The glial cells in shiverer gray matter were similar to those in the normal gray matter. When stained for GS or GFAP, the glial cells in the jimpy gray matter appeared to be somewhat hypertrophied, and when the glial cells in this mutant were stained for CA, the nuclei appeared to be swollen. It was concluded that some of the CA-positive cells in the gray matter of the normal and of each mutant mouse brain could be astrocytes. The patterns of immunostaining in the white matter emphasized the different complements of glial cells in the mutants. In the normal and shiverer mouse corpus callosum, CA, in particular, was detected only in the oligodendrocytes, their processes, and myelin. However, the data concerning the jimpy mouse suggested that the few CA-positive cells in the corpus callosum of that mutant could be astrocytes.     

An Upregulation of SIAH1 After Spinal Cord Injury in Adult Rats

E3 ubiquitin ligase SIAH1 is a protein associated with the onset of nontumorigenicy in revertant tumorigenic cell lines and with several apoptotic processes. However, its role in the injury of the central nervous system remains unknown. In this study, we performed acute spinal cord injury (SCI) in adult rats and investigated the protein expression and cellular localization of SIAH1 in the spinal cord. Western blot analysis revealed that SIAH1 was low expressed in normal spinal cord. It increased at 8 h after SCI, peaked at 1 day, remained for another 3 days, then declined to basal levels at 5 days after injury. Immunohistochemistry further confirmed that SIAH1 immunoactivity was expressed at low levels in gray and white matters in normal condition and increased after SCI. Double immunofluorescence staining showed that SIAH1 was coexpressed with NeuN (neuronal marker), CNPase (oligodendroglial marker), GFAP (astroglial marker), and CD11b (microglial marker) at 1 day post-injury and was also coexpressed with active caspase-3 in neurons and glial cells after injury. In addition, double immunofluorescence staining indicated that p-c-Jun NH2-kinase (JNK) coexpressed with SIAH1 in neurons and glial cells. Coimmunoprecipitation further showed that p-JNK and SIAH1 precipitated with each other in the damaged spinal cord. Taken together, these data suggest SIAH1 involvement in the injury response of the adult spinal cord of the rats.     

Expression of the jimpy gene in the spinal cords of heterozygous female mice. I. An early myelin deficit followed by compensation

The jimpy mutation results in severe hypomyelination throughout the CNS of hemizygous male mice. In the female carrier of the jimpy gene, partial hypomyelination is predicted as a consequence of genetic mosaicism resulting from random X-chromosome inactivation. The spinal cord of the female carrier was studied morphologically to determine if hypomyelination is present, the manner in which a possible myelin deficit is expressed, and the extent, if any, of compensation. The spinal cords of 14- to 15-d-old heterozygotes were found to be hypomyelinated. A deficit of 31% in the amount of myelin as compared to controls was detected in these young carriers by point-counting stereology. By the end of the first month the deficit was 12%, and after the fifth month complete recovery had occurred. These results demonstrate that the neuroglial cells are capable of compensating totally for the jimpy defect over a several month period. The reduction in the amount of myelin at 2 weeks postnatally is due to the ensheathment of fewer axons than normal and the formation of myelin sheaths that are thinner than normal. It is not due to a significant reduction in amount of axoplasm and a corresponding decrease in amount of myelin. This finding indicates that overall brain development is not retarded but that expression of the jimpy gene selectively affects the glial cells. Our morphologic studies also suggest that the neuron is not the target of the jimpy mutation. One line of evidence for this statement is that virtually all the axons are partially ensheathed, a condition that should not occur if 50% of the neurons are defective in the mosaic. The coexistence of both normal and defective cells within the same cell population and the apparent sparing of the neuron makes the female carrier of the jimpy gene an excellent model for studying mechanisms of compensation and plasticity of neuroglial cells.     

Oligodendroglial cell death in jimpy mice: an explanation for the myelin deficit

Neuroglial cell death was investigated in 3 white matter tracts of jimpy and normal mice. In normal animals, glial death during development ranged from 0.5 to 2.7% of the total glial population. The number of dying glial cells was significantly higher in jimpy animals at times corresponding with oligodendroglial proliferation and the onset of myelination in each tract. At certain ages, over 10% of the glial population were pyknotic at the light-microscopic level. Dying glial cells that were identified ultrastructurally presented the characteristics of oligodendrocytes. Premature death of oligodendrocytes presents a simple explanation for the gross deficits of myelin in jimpy animals. The shortened life span of the jimpy oligodendrocyte may preclude the elaboration of a normal myelin sheath. The jimpy model may prove to be a valuable tool in delineating a role for normal neuroglial cell death during the development of the nervous system.     

Oligodendrogenesis is differentially regulated in gray and white matter of jimpy mice

The factors that regulate oligodendrogenesis have been studied extensively in optic nerve, where oligodendrocyte production and myelination quickly follow colonization of the nerve by progenitor cells. In contrast, oligodendrocyte production in the cerebral cortex begins approximately 1 week after progenitor cell colonization and continues for 3-4 weeks. This and other observations raise the possibility that oligodendrogenesis is regulated by different mechanisms in white and gray matter. The present study examined oligodendrocyte production in the developing cerebral cortex of jimpy (jp) and jimpy(msd) (msd) mice, which exhibit hypomyelination and oligodendrocyte death due to mutations in and toxic accumulations of proteolipid protein, the major structural protein of CNS myelin. Proliferation of oligodendrocyte progenitors and production of myelinating oligodendrocytes was reduced in jp cerebral cortex when compared to wild-type (wt) and msd mice. The incidence of oligodendrocyte cell death was similar in jp and msd cortex, but total dying oligodendrocytes were greater in msd. We confirm previous reports of increased oligodendrocyte production in white matter of both jp and msd mice. The jp mutation, therefore, reduces oligodendrocyte production in cerebral cortex but not in white matter. These data provide additional evidence that oligodendrogenesis is differentially regulated in white matter and gray matter and implicate PLP/DM20 as a modulator of these differences.     

A defect in the cell cycle of neuroglia in the myelin deficient jimpy mouse

One h after receiving a single pulse of [3H]thymidine, spinal cords from jimpy and control animals were examined for labeled cells and for cells in mitosis. Although the number of labeled cells was significantly higher in jimpy spinal cords at 14 and 20 days postnatal, the number of mitotic cells was not. In normal animals, the ratio of the number of labeled cells to the number of mitotic cells (L:M) was close to 4:1 regardless of the age of the animal or the labeling index. In jimpy animals, the L:M ratio was always close to 8:1. Previous ultrastructural studies have shown that the great majority of [3H]thymidine-labeled cells in jimpy central nervous system (CNS) at these ages are oligodendrocytes. Therefore, the unusually high L:M ratio indicates that there is an abnormality in some aspect of the jimpy oligodendroglial cell cycle.     

Enrichment of rat oligodendrocyte progenitor cells by magnetic cell sorting

The embryonic, neonatal, as well as adult rat spinal cords harbor a pool of neural stem cells (NSCs), which may be easily isolated and used to replace neuronal cell loss or remyelinate damaged axons following various neurodegenerative disorders. In the present study we have used magnetic cell sorting (MACs) technology to generate enriched oligodendroglial cell populations from the embryonic (E16) rat spinal cord. Target cells were separated by positive selection, using specific A2B5 antibody-labeled MicroBeads achieving optimal recovery and high purity of pro-oligodendroglial cells. Based on immunocytochemical analyses for oligodendroglial developmental markers (A2B5, NG2, RIP and MBP) we were able to characterize and quantify oligodendroglial progenitors (OPCs) and mature oligodendroglial cells in: (i) unseparated heterogeneous population of NSCs, or in (ii) antigen–antibody separated NSCs. Our results showed that MACs technology enable us to gain enriched OPCs from heterogeneous population of spinal NSCs, resulting in a 58–61% of mature oligodendrocytes content (MBP+, RIP+) in comparison to 6–12% of oligodendroglial cells acquired from unseparated population. In addition, the enriched OPCs could be cultured in vitro for several >8 passages, giving rise to a high number of newly formed spheres, as well as high expansion potential. These experiments indicate that MACs technology provide a feasible approach for experimental cell enrichment of desired oligodendroglial progeny, which may be used in future trials for cell-based therapies to treat spinal cord injury.     

Abnormal expression of the proliferating cell nuclear antigen (PCNA) in the spinal cord of the hypomyelinated Jimpy mutant mice

In the present study, assessment of the expression of the proliferating cell nuclear antigen (PCNA), a nuclear acidic protein necessary for DNA replication that is expressed through the cell cycle, was used to investigate the proliferative capability of glial cells in the hypomyelinated Jimpy mutant mice. Spinal cords from 10–12 and 20–22 day Jimpy and normal animals were used for quantitative microscopic image analysis. Simultaneous demonstration of cycling cells and oligodendroglia, astroglia or microglia was achieved through the sequential combination of PCNA immunostaining and selective markers for these glial cells. Our results revealed that the density of PCNA-positive cells was higher in Jimpy than in normal spinal cords, this difference being more pronounced at 20–22 days than at 10–12 days and more so in white than in gray matter. In addition, Jimpy glial cells exhibited an abnormal PCNA expression, as demonstrated by quantification of the intensity of nuclear immunostaining. In comparison to normal animals, the percentage of PCNA-positive cells showing intensely stained nuclei was higher in Jimpy. About 50% of PCNA-positive cells in the Jimpy white matter were identified as cells from the oligodendrocyte line, 30% were microglial cells and 20% were astrocytes. The expression of PCNA in relation to the proliferative capability and possible cell cycle abnormalities of the different glial cell types in Jimpy is discussed.     

Differential expression of neuropsin and protease M/neurosin in oligodendrocytes after injury to the spinal cord

Neuropsin and protease M/neurosin are serine proteases expressed by neurons and glial cells, and serve a variety of functions in the central nervous system (CNS). The current study demonstrates changes in the expression of these proteases following hemisection of the mouse spinal cord. Within unlesioned spinal cord, neuropsin mRNA expression was occasionally observed in the gray but not white matter, while the level of protease M/neurosin mRNA was higher in the white matter. After injury to the spinal cord, neuropsin mRNA expression was induced in the white matter in the area immediately adjacent to the lesion, peaking at 4 days post-injury and disappearing by 14 days. Enhanced expression of protease M/neurosin mRNA was observed throughout the white and gray matter surrounding the lesion, peaking at 4 days and persisting for 14 days. Neuropsin mRNA was expressed predominantly by CNPase-positive oligodendrocytes. Furthermore, most of these cells were also associated with immunoreactivity for protease M/neurosin protein. Within unlesioned spinal cord, most protease M/neurosin mRNA-expressing cells were CNPase-positive oligodendrocytes, and a substantial fraction of these cells also showed immunoreactivity for NG2, a marker for oligodendrocyte progenitors. After injury, protease M/neurosin mRNA expression within NG2-positive cells was significantly decreased, while the constitutive expression in CNPase-positive oligodendrocytes appeared to be preserved. These findings suggest that each subpopulation of oligodendrocytes based on the expression of neuropsin and protease M/neurosin has different roles in the response of the spinal cord to injury as well as in normal homeostasis.     

Apoptotic and anti-apoptotic mechanisms following spinal cord injury

A number of studies have provided evidence that cell death from moderate traumatic spinal cord injury (SCI) is regulated, in part, by apoptosis that involves the caspase family of cysteine proteases. However, little or no information is available about anti-apoptotic mechanisms mediated by the inhibitors of apoptosis (IAP) family of proteins that inhibit cell death pathways. In the present study, we examined caspase and IAP expression in spinal cords of rats subjected to moderate traumatic injury. Within 6 h after injury, caspase-8 and-9 (2 initiators of apoptosis) were predominantly present in gray matter neurons within the lesion epicenter. By 3 days following spinal cord injury (SCI), caspase-8 and-9 immunoreactivity was localized to gray and white matter cells, and by 7 days following SCI, both upstream caspases were expressed in cells within white matter or within foamy macrophages in gray matter. Caspase-3, an effector caspase, was evident in a few fragmented cells in gray matter at 24 h following injury and then localized to white matter in later stages. Thus, distinct patterns of caspase expression can be found in the spinal cord following injury. XIAP, cIAP-1, and cIAP-2, members of the IAP family, were constitutively expressed in the cord. Immunoblots of spinal cord extracts revealed that the processed forms of caspases-8 and-9 and cleavage of PARP are present as early as 6 h following trauma. The expression of caspases corresponded with the detection of cleavage of XIAP into 2 fragments following injury. cIAP-1 and cIAP-2 expression remained constant during early periods following SCI but demonstrated alterations by 7 days following SCI. Our data are consistent with the idea that XIAP may have a protective role within the spinal cord, and that alteration in cleavage of XIAP may regulate cell death following SCI.     

Rumpshaker: an X-linked mutation causing hypomyelination: developmental differences in myelination and glial cells between the optic nerve and spinal cord.

The X-linked mutation rumpshaker (rsh), which is probably an allele of jimpy (jp), causes hypomyelination in the CNS of mice. This study examines the developmental expression of the morphology, glial cells, and immunostaining of myelin proteins in the optic nerve and spinal cord. The optic nerve contains varying numbers of amyelinated and myelinated fibres. The majority of such sheaths are of normal thickness whereas in the spinal cord most axons are associated with a disproportionately thin sheath which changes little in thickness during development. In the optic nerve glial cell numbers are elevated in mutants during early and peak myelination but then fall slightly below normal in adults. In contrast, the number of glial cells is consistently elevated after 16 days of age in the spinal cord. The majority of the alterations to total glial cells are due to corresponding changes in the oligodendrocyte population. Immunostaining intensity is somewhat reduced for myelin basic protein (MBP) and the C-terminal common to proteolipid protein (PLP) and DM-20 and profoundly decreased for the PLP-specific peptide. Glial fibrillary acidic protein (GFAP) is increased in rsh. It is probable that some of the variation in myelination between optic nerve and cord in rsh is related to the difference in axon diameter in the two locations, as there are adequate numbers of oligodendrocytes at the time of myelination. However, the effect of the mutation on cell development in the brain and the spinal cord may be different. The immunostaining indicates a marked deficiency in PLP in myelin but suggests that DM-20 levels may be relatively normal. rsh shows several major differences from jp and other X-linked myelin mutants, particularly in relation to oligodendrocyte numbers, and will be useful to elucidate the role of the PLP gene in influencing oligodendrocyte differentiation and survival.     

Mild cerebral hypoxia–ischemia produces a sub-acute transient inflammatory response that is less selective and prolonged after a substantial insult

Cerebral ischemia initiates various injurious processes including neuroinflammatory responses such as activation of microglia and increases in cytokine and nitric oxide release. Evidence primarily from in vitro studies, indicates that neuroinflammatory effects can be either beneficial or harmful, possibly related to stimulus strength. We investigated using in vivo models, the effect of a mild or substantial cerebral hypoxia–ischemia on: cerebral microglial/macrophage activation (ED1), pro-inflammatory cytokines (tumor necrosis factor-alpha), nitrosative stress (nitrotyrosine) and permanent brain damage. A mild insult produced a transient (1–2 days post) increase in activated microglia/macrophages within subcortical white and not gray matter but transiently increased cytokine or nitrotyrosine expression in cortex and not white matter. There was also prolonged scattered cell death in cortex and white matter over weeks along with loss of myelin/axons and cortical atrophy at 4 weeks post-insult. In contrast, a substantial insult produced white and gray matter necrosis, cyst formation and atrophy, along with increases in tumor necrosis factor and nitrotyrosine staining within both white and gray matter starting at 1–2 days post-insult. Microglial/macrophage staining was increased starting at 1-week post a substantial insult and remained elevated for weeks thereafter.Thus, a transient neuroinflammatory response occurs following a mild insult whereas prolonged scattered cell death occurs for weeks, particularly in white matter. Insult severity also affects the progression of the neuroinflammatory response, which is prolonged after a substantial insult. Effective therapy will need to be customized for insult severity and timing; and, monitoring the injury processes with imaging or biomarkers may help guide treatment.     

Physiological, biochemical and histological changes in skeletal muscle, neuromuscular junction and spinal cord of rats rendered paraplegic by subarachnoidal administration of 6-aminonicotinamide

Exposure of rats to the nicotinamide analog, 6-aminonicotinamide, produces an irreversible rigid paralysis of the hind limbs. The present study describes electrophysiological alterations that occur in extensor digitorum longus (extensor) and soleus muscles of rats treated with a single subarachnoid injection of 6-aminonicotinamide and compares these changes with cellular and biochemical changes that occur in the spinal cord. Injection of 50 μ of 6-aminonicotinamide into the subarachnoid space at L2–L4 of adult female rats produced rigid paralysis of both hind limbs in 24–36 h. In extensor muscles, the membrane potential decreased significantly about 48 h after injection, but this partial depolarization took somewhat longer in the soleus muscles. Spontaneous transmitter release, as studied by frequency and amplitude of miniature endplate potentials, was significantly affected from day 7 after injection. At the onset of paralysis, 6-phosphogluconate levels were increased more than 1000-fold in the lumbar region of the spinal cord and returned to control levels by 14 days after injection. This metabolite thus serves as a biochemical marker for the presence of the 6-aminonicotinamide analog of NADP in tissues. Beginning at 3 days an increase in small phagocytic cells was noted in both the cervical and lumbar regions of the cord. These proliferating cells were highly localized to the anterior horn region where maximal numbers were detected at 8 days. Few viable motor neurons were present in the lumbar region of the cord at this time. There was a close correlation between the accumulation of small cells and the increased β-glucuronidase activity. Despite the marked cellular alterations in the spinal cord at 8 days there were no changes in the total extractable lipid in gray and white matter. Cellular changes were maintained in the lumbar region for as long as 540 days. At this time most of the electrophysiological properties of the extensor muscle were restored to normal, suggesting that collateral sprouts from the residual motor neurons may have reinnervated the denervated muscle fibers.