Differential effects of distinct central nervous system regions on cell migration and axonal extension of neural precursor transplants

Transplantation of neural precursor cells (NPCs) is a promising therapeutic strategy in CNS injury. However, the adult CNS lacks instructive signals present during development and, depending on the region and type of transplant, may be inhibitory for neuron generation and axonal growth. We examined the effects of the white matter in different regions of the adult CNS on the properties of NPC transplants with respect to cell survival, differentiation, migration, and axonal growth. NPCs were prepared from day 13.5 embryonic spinal cord of transgenic rats that express the human placental alkaline phosphatase (AP) reporter. These NPCs were injected unilaterally into the cervical spinal cord white matter and into the corpus callosum of adult rats and were analyzed immunohistochemically 2 weeks later. NPCs survived in both regions and differentiated into astrocytes, oligodendrocytes, and neurons, with no apparent differences in survival or phenotypic composition. However, in the spinal cord white matter, graft‐derived cells, identified as precursors and glial cells, migrated from the injection site rostrally and caudally, whereas, in the corpus callosum, graft‐derived cells did not migrate and remained at the injection site. Importantly, graft‐derived neurons extended axons from the grafting site along the corpus callosum past the midline, entering into the contralateral side of the corpus callosum. These results demonstrate dramatic differences between white matter regions in the spinal cord and brain with respect to cell migration and axonal growth and underscore the importance of considering the effects of the local CNS environment in the design of effective transplantation strategies. © 2012 Wiley Periodicals, Inc.     

Metastasis-associated mts1 (S100A4) protein in the developing and adult central nervous system

We have found recently that white matter astrocytes in the spinal cord constitutively express immunoreactivity for Mts1 (S100A4) protein and that this expression is up-regulated ipsilaterally after sciatic nerve or dorsal root injury. Here, we have studied the expression pattern of Mts1 throughout the rat central nervous system (CNS). We found Mts1 immunoreactivity in myelinated tracts such as the olfactory tract, optic nerve, corpus callosum, internal capsule, fimbria, and spinal cord funiculi but not in cerebellar white matter. Mts1-immunoreactive (IR) cells were consistently astrocytic (glial fibrillary acidic protein positive). In addition to myelinated tracts, Mts1 immunoreactivity was also present in a few nonmyelinated or poorly myelinated areas, such as pituitary gland, olfactory bulb, and around the lateral ventricle. Based on location, three Mts1-IR astrocyte groups were distinguished: 1) astrocytes at the surfaces of the CNS, i.e., adjacent to the cerebrospinal fluid, organized perpendicularly to the bundles of axonal tracts; 2) astrocytes located in parallel to, and inserted between, axonal bundles; and 3) clusters of astrocytes around the lateral ventricle and in the olfactory bulb. We further analyzed the relationship between Mts1 immunoreactivity and the development of CNS fiber tracts by combining staining for Mts1 and myelin basic protein (MBP). Mts1 immunoreactivity appeared postnatally in recently myelinated areas. During the development of corpus callosum and the optic tract, Mts1 immunoreactivity was concentrated at the frontier of myelination. The developmental expression pattern suggests a role of Mts1-IR astrocytes in the maturation of myelinated fiber tracts. The preferential localization of Mts1 to the subpial region in the mature CNS suggests that Mts1 participates in astrocyte-mediated CNS-cerebrospinal fluid exchange.     

Angiotensin receptor-like immunoreactivity in adult brain white matter astrocytes and oligodendrocytes.

Most of the physiological effects of brain angiotensins are currently believed to be mediated by angiotensin receptors located principally on neurons. However, numerous studies in vitro have demonstrated the presence of functional angiotensin receptors on brain astrocytes, raising the possibility that glial cells may also participate in mediating the effects of the central renin-angiotensin system. Nevertheless, it is uncertain whether these cells in situ express angiotensin receptors, raising questions about the physiological significance of results observed in cell cultures. We have examined the distribution of angiotensin receptor-like immunoreactivity in glial cells in white matter tracts in the adult CNS, using a panel of antisera to the AT1 and AT2 angiotensin receptors. Antiserum preadsorption and/or Western blot demonstrated the specificity of the antisera in brain tissue. In immunohistochemical experiments, the AT1 antisera selectively labeled AT1-expressing neurons in the piriform cortex, whereas the AT2 antiserum stained cells in the trigeminal motor nucleus, these being nuclei known to express AT1 and AT2 receptors, respectively. Using double-label immunohistochemistry, we observed AT1- and AT2-immunoreactive astrocytes and oligodendrocytes in white matter tracts, which include the rat cerebellar white matter, periventricular white matter, and optic nerve, in addition to the bovine corpus callosum and human subcortical white matter. In contrast, astrocytes in the gray matter region of the cerebral cortex were not found to be angiotensin receptor-like immunoreactive. These results demonstrate the presence of AT1 and/or AT2 angiotensin receptor-like immunoreactivity in brain white matter macroglial cells in situ and support the idea that glial cells may play a more important role in the central renin-angiotensin system than previously thought.     

Molecular heterogeneity of oligodendrocytes in chicken white matter.

The classical studies by Del Rio Hortega (Mem. Real. Soc. Espan. Hist. Nat. 14:40-122, 1928) suggest that the oligodendrocyte population includes four morphological subtypes. Recent data from the cat and the rat show that the anatomy of oligodendrocytes related to early myelinating prospective large fibers differs from that of oligodendrocytes related to late myelinating prospective small fibers. After application of a polyclonal antiserum to cryostat sections from the chicken CNS, we noted that glial cells in the spinal cord white matter had become labeled. Analysis of the occurrence and cellular localization of this immunoreactivity--the T4-O immunoreactivity--in the CNS of the adult chicken showed that T4-O immunoreactive cells are enriched in the ventral funiculus and superficially in the lateral funiculus of the spinal cord, where they are co-localized with large fibers. Double staining with T4-O antiserum and anti-GFAP or the lectin BSI-B4 revealed that T4-O immunoreactive cells are not astrocytes or microglia. Staining with anti-HSP108, a general marker for avian oligodendrocytes, showed that T4-O immunoreactivity defines an oligodendroglial subpopulation. A search for T4-O immunoreactivity in spinal cord white matter of some other vertebrates revealed that T4-O immunoreactive cells are not present in sections from fish, frog, turtle, rat, and rabbit spinal cord white matter. These results suggest the presence of a fiber size-related molecular heterogeneity among chicken white matter oligodendrocytes.     

Immunocytochemical localization of 5'-nucleotidase in oligodendroglia and myelinated fibers in the central nervous system of adult and young rats

Rat central nervous system (CNS) tissue sections were immunostained by the peroxidase-anti-peroxidase (PAP) method using a rabbit serum directed against rat liver 5'-nucleotidase. In paraffin sections from the brains of 60-day-old rats 5'-nucleotidase immunoreactivity occurred in the same white-matter regions as myelin-basic protein immunoreactivity and histological staining of myelin. The immunostaining of cerebral white matter for 5'-nucleotidase was more intense and wide-spread at the age of 120 days than at 60 days, and the choroid plexus and blood vessels were stained consistently. In the paraffin sections from the brains of younger (20-day-old) rats the staining of 5'-nucleotidase in the white matter was faint and patchy. In paraffin sections from spinal cord, 5'-nucleotidase immunoreactivity was observed throughout the lateral white-matter columns and, frequently, in the cell bodies of interfascicular oligodendroglia. Interfascicular oligodendroglia also showed 5'-nucleotidase immunoreactivity in vibratome sections from the CNS tissue of young and adult rats. The findings were consistent with histochemical and biochemical evidence for 5'-nucleotidase in rat brain myelin and oligodendroglia, with substantial increases in activity in the myelin as rats develop from the ages of 20 to 120 days. 5'-Nucleotidase immunoreactivity was not observed in any astrocytes or in oligodendrocytes in the gray matter; however, the enzyme may occur in those glial cells at levels lower than were detectable using the present method.     

Localization of pore-forming subunit of the ATP-sensitive K(+)-channel,Kir6.2, in rat brain neurons and glial cells

Kir6.2, a subunit of the ATP-sensitive K(+) channel (K(ATP)), was localized in adult rat brain by immunohistochemistry and in situ hybridization. The Kir6.2 mRNA was widely expressed in most rat brain neuronal populations and nuclei examined, intensely in the mitral cell layer and tufted cells of the olfactory bulb, pontine nucleus, pontine reticular nucleus, motor and spinal trigeminal nuclei and cuneate nuclei of the brain stem, moderately in the neocortex and cerebellar Purkinje cells, and weakly in the granular cell layer of the olfactory bulb and the granular layer of the cerebellum. In addition, glial cells also expressed the Kir6.2 gene weakly in the corpus callosum and cerebellar white matter. This wide localization of the gene was quite similar to that of Kir6.2 protein. Double stainings with anti-GFAP and anti-Kir6.2 antibodies were performed in this study. Glial cells showing immunoreactivity to both anti-Kir6.2 and anti-GFAP were confirmed to be astrocytes, and those showing only immunoreactivity to anti-Kir6.2 but not to anti-GFAP were presumed to be oligodendrocytes and confirmed by immunoelectron microscopy. Thus, it may be concluded that both oligodendrocytes and astrocytes contain Kir6.2. Under the electron microscope, we showed in vivo for the first time that the immunoreactive products were localized in the endoplasmic reticulum and Golgi apparatus as well as the plasma membranes of neurons and glial cells.     

Grid-mapped freeze-fracture analysis of gap junctions in gray and white matter of adult rat central nervous system,with evidence for a “panglial syncytium” that is not coupled to neurons

In white matter regions of the brain and spinal cord of adult mammals, gap junctions previously were observed linking astrocytes to astrocytes, as well as to oligodendrocytes and ependymacytes. The resulting “functional syncytium” was proposed to modulate the ion fluxes that occur during electrical activity of the associated axons. Gap junctions also have been reported linking neurons with glia, and functional neuronal-glial coupling has been postulated. To investigate the glial syncytium and the neuron-to-glia coupling hypotheses, we used “grid-mapped freeze fracture,” conventional thin-section electron microscopy, and light microscope immunocytochemistry to examine and characterize neurons and glia in gray and white matter of adult rat brain and spinal cord. We have obtained quantitative evidence for the abundance and widespread distribution of gap junctions interlinking the three primary types of macroglia throughout both gray and white matter of the mammalian central nervous system (CNS), thereby extending the concept to that of a functional panglial syncytium. In contrast to previous reports, we show that of more than 400 gap junctions in which both participating cells were identified, none were between neurons and glia. Thus, neuronal coupling and glial coupling involved separate and distinct pathways. Finally, putative water channels (i.e., “square arrays”) were confirmed to be abundant and in close association with gap junctions in astrocytes and ependymacytes. Because the astrocyte “intermediaries” extend cytoplasmic conduits throughout gray and white matter of brain and spinal cord, from the ependymal layer to the pia-glial limitans, and from oligodendrocytes surrounding axons to astrocyte endfeet surrounding capillaries, the proposed panglial syncytium, with its abundance of water channels and intercellular ion channels, is optimally positioned and equipped to modulate water and ion fluxes across broad regions of the CNS. J. Comp. Neurol. 388:265–292, 1997. © 1997 Wiley-Liss, Inc.     

Cell proliferation and replacement following contusive spinal cord injury

After spinal cord injury (SCI), about 50% of the oligodendrocytes and astrocytes in the residual white matter at the injury site are lost by 24 h. However, chronically after SCI, the density of oligodendrocytes is normal. Previous studies have shown that the adult rat spinal cord contains a pool of proliferating glial progenitors whose progeny could help restore cell density after injury. To study proliferation in response to injury, we performed SCI on adult female rats at the T8 level, using a standardized contusion model. Animals received bromodeoxyuridine (BrdU) injections during the first week after SCI, and were perfused within 2 h for acute studies, and at 6 weeks for chronic studies. The tissue was analyzed using immunohistochemical detection of BrdU and cell marker antigens. We demonstrate that cell proliferation in the residual white matter is increased at 1-7 days after SCI, peaking on day 3. Dividing cells include oligodendrocytes, astrocytes, microglia/macrophages, and a high proportion of NG2(+) glial precursors. By 6 weeks, some cells that had been labeled 2-4 days after SCI were still present. Double immunohistochemistry showed that while very few of these cells expressed NG2 or the microglia/macrophage marker OX42, about 50% expressed CC1 or glial fibrillary acidic protein (GFAP), markers of mature oligodendrocytes and astrocytes, respectively. The post-injury environment represented by residual white matter is thus permissive to the differentiation of glial precursors. Cells that are stimulated to divide during the first week after SCI develop chronically into mature phenotypes that replace macroglia lost after injury.     

Alterations in the oligodendrocyte lineage, myelin, and white matter in adult mice lacking the chemokine receptor CXCR2

Oligodendrocyte precursor cell (OPC) proliferation and migration are critical for the development of myelin in the central nervous system (CNS). Previous studies showed that localized expression of the chemokine CXCL1 signals through the receptor CXCR2 to inhibit the migration and enhance the proliferation of spinal cord OPCs during development. Here, we report structural and functional alterations in the adult CNS of Cxcr2-/- mice. In Cxcr2-/- adult mice, we observed regional alterations in the density of oligodendrocyte lineage cells in Cxcr2-/- adult mice, with decreases in the cortex and anterior commissure but increases in the corpus callosum and spinal cord. An increase in the density and arborization of spinal cord NG2 positive cells was also observed in Cxcr2-/- adult mice. Compared with wild-type (WT) littermates, Cxcr2-/- mice exhibited a significant decrease in spinal cord white matter area, reduced thickness of myelin sheaths, and a slowing in the rate of central conduction of spinally elicited evoked potentials without significant changes in axonal caliber or number. Biochemical analyses showed decreased levels of myelin basic protein (MBP), proteolipid protein (PLP), and glial fibrillary acidic protein (GFAP). In vitro studies showed reduced numbers of differentiated oligodendrocytes in Cxcr2-/- spinal cord cultures. Together, these findings indicate that the chemokine receptor CXCR2 is important for the development and maintenance of the oligodendrocyte lineage, myelination, and white matter in the vertebrate CNS.     

Expression of 48-kilodalton intermediate filament-associated protein in differentiating and in mature astrocytes in various regions of the central nervous system

In this paper we present evidence that the 48-kD intermediate filament-associated protein (IFAP) is expressed relatively late in maturation of astrocytes, after they have acquired the glial fibrillary acidic protein (GFAP). In the astrocytes of white matter in the cerebellum the GFAP is detected at P3, whereas the 48-kD IFAP is detected only at P11. In the periventricular region and the hippocampus the 48-kD IFAP was detected at P6, long after the appearance of GFAP. In adult mice the 48-kD IFAP was observed in GFAP-positive astrocytes in the white matter of cerebellum, spinal cord, brainstem, and corpus callosum as well as in GFAP-positive cells in the grey matter of cerebral cortex and spinal cord. The 48-kD IFAP was not, however, detected in radial glia and their derivatives, in Bergmann glia or in Müller glia. Thus, not all the GFAP-positive astroglia express the 48-kD IFAP. Similarly, 48-kD IFAP was not detected in cells which were GFAP-negative.     

Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage

Proliferating populations of undifferentiated neural stem cells were isolated from the embryonic day 14 rat cerebral cortex or the adult rat subventricular zone. These cells were pluripotent through multiple passages, retaining the ability to differentiate in vitro into neurons, astrocytes, and oligodendrocytes. Two weeks to 2 months after engraftment of undifferentiated, BrdU-labeled stem cells into the normal adult spinal cord, large numbers of surviving cells were seen. The majority of the cells differentiated with astrocytic phenotype, although some oligodendrocytes and undifferentiated, nestin-positive cells were detected; NeuN-positive neurons were not seen. Labeled cells were also engrafted into the contused adult rat spinal cord (moderate NYU Impactor injury), either into the lesion cavity or into the white or gray matter both rostral and caudal to the injury epicenter. Up to 2 months postgrafting, the majority of cells either differentiated into GFAP-positive astrocytes or remained nestin positive. No BrdU-positive neurons or oligodendrocytes were observed. These results show robust survival of engrafted stem cells, but a differentiated phenotype restricted to glial lineages. We suggest that in vitro induction prior to transplantation will be necessary for these cells to differentiate into neurons or large numbers of oligodendrocytes.     

Immunohistochemical localization of aspartoacylase in the rat central nervous system

Aspartoacylase (ASPA; EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartate (NAA) to generate free acetate in the central nervous system (CNS). Mutations in the gene coding ASPA cause Canavan disease (CD), an autosomal recessive neurodegenerative disease that results in death before 10 years of age. The pathogenesis of CD remains unclear. Our working hypothesis is that deficiency in the supply of the NAA-derived acetate leads to inadequate lipid/myelin synthesis during development, resulting in CD. To explore the localization of ASPA in the CNS, we used double-label immunohistochemistry for ASPA and several cell-specific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (approximately 37 kD) on Western blots of rat brain homogenate. ASPA colocalized throughout the brain with CC1, a marker for oligodendrocytes, with 92-98% of CC1-positive cells also reactive with the ASPA antibody. Many cells were labeled with ASPA antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively fewer cells were labeled in gray matter, including cerebral cortex. No astrocytes were labeled for ASPA. Neurons were unstained in the forebrain, although small numbers of large reticular and motor neurons were faintly to moderately stained in the brainstem and spinal cord. Many ascending and descending neuronal fibers were moderately stained for ASPA in the medulla and spinal cord. Microglial-like cells showed faint to moderate staining with the ASPA antibodies throughout the brain by the avidin/biotin-peroxidase detection method, and colocalization studies with labeled lectins confirmed their identity as microglia. The predominant immunoreactivity in oligodendrocytes is consistent with the proposed role of ASPA in myelination, supporting the case for acetate supplementation as an immediate and inexpensive therapy for infants diagnosed with CD.     

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.     

Serotonin 2A receptor-like immunoreactivity is detected in astrocytes but not in oligodendrocytes of rat spinal cord

The distribution of the serotonin 2A (5-HT2A) receptor in glial cells in the white matter of rat spinal cord was immunohistochemically examined with specific antibodies against the 5-HT2A receptor. 5-HT2A receptor-like immunoreactivity was detected in astrocytes that were identified by an antibody against the glial fibrillary acidic protein. In contrast, 5-HT2A receptor-like immunoreactivity was not observed in oligodendrocytes.     

Positioned to inhibit: netrin-1 and netrin receptor expression after spinal cord injury

Netrin-1 regulates axon extension during embryonic development and is expressed by neurons and myelinating oligodendrocytes in the adult CNS. To investigate the potential role of netrin-1 after spinal cord injury, we examined the expression of netrin-1 and netrin receptors after sagittal myelotomy in adult rats. This lesion targets spinal commissural projections, which respond to netrin-1 during development. Netrin-1 mRNA and protein levels were dramatically reduced at the site of injury and reduced expression persisted for at least 7 months. Neither netrin-1 protein nor mRNA was associated with the glial scar, but netrin-1 was expressed by neurons and oligodendrocytes immediately adjacent to the lesion. The post-injury distribution detected is similar to that reported for myelin-associated inhibitors of axon regeneration, such as Nogo, and is distinct from the distribution of inhibitors associated with a glial scar. DCC and UNC-5 homologue (UNC5H) expression also was reduced after injury. Although UNC5H levels recovered, DCC expression at the site of injury remained approximately 50% of pre-injury values at 7 months. Increased UNC5H immunoreactivity was associated with fibers in the superficial layers of the dorsal horn and in fibers located in white matter adjacent to the lesion. The dominant expression of UNC5H on axons and neurons in the spinal cord after injury and the persistent expression of netrin-1 by oligodendrocytes surrounding the lesion are consistent with the hypothesis that netrin-1 is a myelin-associated inhibitor of axonal regeneration after spinal cord injury.     

Astrocytes, oligodendrocytes, and Schwann cells share a common antigenic determinant that cross-reacts with myelin basic protein: identification with monoclonal antibody

We have produced a monoclonal antibody against myelin basic protein that reacts with astrocytes, oligodendrocytes, and Schwann cells. This antibody was generated by fusion of mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from adult rat corpus callosum. The antibody was characterized via solid-phase radioimmunoassay, immunoblot of SDS-PAGE, and by indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes, astrocytes, and Schwann cells. Myelin basic protein (MBP) was shown previously to be present only in myelin producing cells in CNS and PNS (oligodendroglia and Schwann cells) and not in astrocytes. The binding of this monoclonal antibody to all 3 cell types suggests that these cells share a common epitope. This epitope may be related to a common progenitor cell.     

The trophic factors S-100beta and basic fibroblast growth factor are increased in the forebrain reactive astrocytes of adult callosotomized rat.

S-100 is a calcium-binding protein that is predominantly found in astrocytes of the central nervous system. In the present study, we investigated the temporal and spatial changes of S-100beta immunoreactivity after a stereotaxic mechanical lesion of the adult rat corpus callosum performed with an adjustable wire knife. Rats were killed 7, 14 and 28 days after surgery. S-100beta immunoreactivity was found within the cytoplasm and processes of quiescent putative astrocytes that were observed throughout the gray and white matters of the forebrain of sham-operated rats. Following callosotomy, the S-100beta immunoreactive profiles showed increased size and thick processes, as well as increased amount of S-100beta immunoreactivity. Unbiased stereologic analysis revealed a sustained and widespread increase of the Areal Fraction of S-100beta immunoreactive profiles in the medial and lateral regions of the white matter of callosotomized rats at the studied time-intervals. In the cerebral cortex of callosotomized rats, the estimated total number of S-100beta immunoreactive profiles was also increased 7 and 14 days after the lesion. Since the cellular and temporal changes in S-100beta immunoreactivity were closely similar to those described for basic fibroblast growth factor (bFGF) following brain lesions, we co-localized the S-100beta and bFGF immunoreactivities after callosotomy. bFGF immunoreactivity was found in the nuclei of S-100beta immunoreactive glial profiles throughout the forebrain regions of the sham-operated rats. bFGF immunoreactivity was increased in the nuclei of reactive S-100beta immunoreactive putative astrocytes in the forebrain white matter and in the cerebral cortex of callosotomized rats. These results indicate that after transection of the corpus callosum of adult rats, the reactive astrocytes may exert paracrine trophic actions through S-100beta and bFGF. Interactions between S-100beta and bFGF may be relevant to the events related to neuronal maintenance and repair following brain injury.     

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.