Gliosis in the spinal cords of rats with experimental allergic encephalomyelitis: immunostaining of carbonic anhydrase and vimentin in reactive astrocytes

Spinal cord sections from rats sensitized to develop experimental allergic encephalomyelitis (EAE) were immunostained with antibodies against glial fibrillary acidic protein (GFAP), carbonic anhydrase, and vimentin, to see whether the latter two antigens could be detected in GFAP-positive reactive astrocytes. Sixteen days after sensitization (16 dpi) there was intense carbonic anhydrase immunostaining in GFAP-positive cells in the spinal cords of EAE rats, particularly in the white matter. At 13 and 20 dpi carbonic anhydrase immunostaining in astrocytes was less intense, and in the spinal cord white matter of control animals carbonic anhydrase was not detected in the few GFAP-positive cells. In the spinal cords of EAE rats vimentin immunostaining was observed in inflammatory cells and astrocytes. In the latter, GFAP and carbonic anhydrase were colocalized with vimentin. The data suggest that carbonic anhydrase expression in astrocytes is an acute response to injury and that vimentin can be detected in astrocytes, as well as inflammatory cells, as early as 16 dpi.     

Glial fibrillary acidic protein and S-100 protein in human hepatic encephalopathy: Immunocytochemical demonstration of dissociation of two glia-associated proteins

Summary Immunocytochemical staining patterns for glial fibrillary acidic protein (GFAP) and S-100 protein (S100P) were compared in cerebral cortex, basal ganglia and white matter of eight cases with hepatic encephalopathy (HE), including four cases of Wilson's disease and four of liver cirrhosis, and of eight age-matched controls, using the peroxidase-antiperoxidase method on adjacent paraffin sections. The majority of Alzheimer type II glia (Alzg II) showed prominent immunoreactivity for S100P but not for GFAP, resembling normal astrocytes of protoplasmic type; Alzg II might be interpreted as being peculiar types of reactive astrocytes retaining characteristics of protoplasmic astrocytes. A small number of Alzg II cells showed slight perinuclear immunoreactivity for GFAP; some lacked both markers. This suggests a spectrum of metabolic changes in these two proteins in Alzg II. GFAP-positive Alzg II cells were restricted to basal ganglia and white matter adjacent to grey matter, indicating that expression of GFAP in Alzg II might be modulated by local factors. Alzheimer type I cells and Opalski cells in Wilson's disease were immunoreactive for both proteins, confirming their astroglial origin and different character from that of Alzg II. In morphometric comparison, the proportion of GFAP-positive glial cells decreased in the cortex (P<0.001) but not significantly in the white matter (0.05<P<0.1), confirming earlier data that the prominent reduction of GFAP in HE brains is restricted to the grey matter. In the putamen, the proportion of GFAP-positive glia decreased in acquired HE (0.01<P<0.05) but not in Wilson's disease, probably resulting from prominent fibrous glial poliferation related to severe parenchymal damage in Wilson's disease. In contrast, glial cells immunoreactive for S100P did not significantly change (P>0.1) in any of the areas studied, indicating an intact glial metabolism of S100P in HE. This clear dissociation between GFAP and S100P defines Alzg II as a peculiar glial reaction with a rather selective deficit of GFAP metabolism (gliofibrillary dystrophy).     

The distribution of glial fibrillary acidic protein and vimentin in postnatal marmoset (Callithrix jacchus) brain

Antisera to glial fibrillary acidic protein (GFAP) and vimentin were used to elucidate the distribution of these intermediate filament proteins in postnatal marmoset brains of various ages. The ependyma of the lateral ventricles was unique in being equally immunoreactive for both GFAP and vimentin at all ages. Vimentin alone was consistently demonstrated in endothelial and leptomeningeal cells at all ages. In neonates, vimentin immunoreactivity greatly exceeded that of GFAP and was located primarily in radial glia in the subependymal plate of the anterior cerebrum. Their vimentin-positive processes formed thick fascicles in the corpus callosum but separated into fine fibres on entering the cortex. GFAP immunoreactivity in these cells and processes was very limited. With age, GFAP-positive cells increased in number and displayed the typical stellate appearance of astroglia. The vimentin-positive radial glial population decreased considerably during this period and by 6 months had virtually disappeared. The GFAP reaction in adult brain was even more widespread, largely due to the increased number of positive astrocytes in the white matter. Vimentin immunoreactivity in the adult was greatly diminished and positive radial glia were not detectable. A major change in intermediate filament protein expression, therefore, occurs in the early postnatal period and probably reflects phases in the differentiation of radial glial precursors into astrocytes.     

Differentiation of radial glia from radial precursor cells and transformation into astrocytes in the developing rat spinal cord

Radial glial cell origins and functions have been studied extensively in the brain; however, questions remain relating to their origin and fate in the spinal cord. In the present study, radial glia are investigated in vivo using the neuroepithelial markers nestin and vimentin and the gliogenic markers GLAST, BLBP, 3CB2, and glial fibrillary acidic protein (GFAP). This has revealed heterogeneity among nestin/vimentin-positive precursor cells and suggests a lineage progression from neuroepithelial cell through to astrocyte in the developing spinal cord. A population of self-renewing radial cells, distinct from an earlier pseudo-stratified neuroepithelium, that resemble radial glial cells in morphology but do not express GLAST, BLBP, or 3CB2, is revealed. These radial cells arise directly from the spinal cord neuroepithelium and are probably the progenitors of neurons and the earliest appearing radial glial cells. GLAST/BLBP-positive radial glia first appear in the ventral cord at E14, and these cells gradually transform through one or more intermediate stages into differentiated astrocytes. Few if any neurons appear to be derived from radial glial cells, which are instead the major sources of astrocytes in the spinal cord. Evidence for the nonradial glial cell origins of some white matter astrocytes is also presented.     

Spatiotemporal distribution of neuronal calcium sensor-1 in the developing rat spinal cord

The present study revealed the localization of neuronal calcium sensor (NCS)-1 immunoreactivity (IR) in the developing rat spinal cord. The NCS-1 IR first appeared at embryonic day 12 in the peripheral nerves and their somata. Intense NCS-1 IR was expressed in ascending and descending tracts in the white matter during the late prenatal period, which gradually decreased to the faint level during postnatal development. Intense NCS-1 IR was colocalized with growth associated protein (GAP)-43 IR in the marginal zone and with the glutamate-aspartate transporter (GLAST) IR in the radial processes traversing the marginal zone. In the adult rat white matter, radially oriented astrocytes and astrocytes in the glia limitans were double-labeled for NCS-1 and glial fibrillary acidic protein (GFAP), whereas small dots on finger-like dendritic projections were double-labeled for NCS-1 and synaptophysin. In the developing gray matter, the NCS-1 IR appeared at embryonic day 12 and gradually increased in the neuronal somata and neuropil, reaching a plateau after the end of the 4th postnatal week. The small dots in neuropil were colabeled for NCS-1 and GFAP or NCS-1 and synaptophysin in the adult rat gray matter. These results strongly suggest that NCS-1 is involved in axogenesis and synaptogenesis in the developing rat spinal cord. NCS-1 can serve as a Ca(2+)-sensor not only in neurons but also in radial glial cells or even in radially oriented astrocytes in the adult rat spinal cord.     

Manipulation of the amygdala noradrenergic system impairs extinction of passive avoidance

The presence and distribution of gliofibrillary acidic protein (GFAP) was studied in the postnatal spinal cord of the rat. On birth GFAP can be seen in astrocytic bodies and their processes of ventral columns; perivascular glial membrane was initially seen within white and gray matter; most of the fibers were transversally oriented. There are many mitotic cells, some of them with GFAP in their cytoplasm, while others lack it, thus providing strong evidence to identify two cellular populations as astrocytes and, possibly, oligodendrocytes and to state that neuroglial cells differentiate to astrocytes before myelination gliosis. The glia limitans membrane is continuous from the 5th day onwards and in 17-day-old animals the astrocytic framework of the spinal cord has reached its adult appearance.     

Temporal progressive antigen expression in radial glia after contusive spinal cord injury in adult rats

In the development of the CNS, radial glial cells are among the first cells derived from neuroepithelial cells. Recent studies have reported that radial glia possess properties of neural stem cells. We analyzed the antigen expression and distribution of radial glia after spinal cord injury (SCI). Sprague-Dawley rats had a laminectomy at Th11-12, and spinal cord contusion was created by compression with 30 g of force for 10 min. In the injury group, rats were examined at 24 h and 1, 4, and 12 weeks after injury. Frozen sections of 20-microm thickness were prepared from regions 5 and 10 mm rostral and caudal to the injury epicenter. Immunohistochemical staining was performed using antibodies to 3CB2 (a specific marker for radial glia), nestin, and glial fibrillary acidic protein (GFAP). At 1 week after injury, radial glia that bound anti-3CB2 MAb had spread throughout the white matter from below the pial surface. From 4 weeks after injury, 3CB2 expression was also observed in the gray matter around the central canal, and was especially strong around the ependymal cells and around blood vessels. In double-immunohistochemical assays for 3CB2 and GFAP or 3CB2 and nestin, coexpression was observed in subpial structures that extended into the white matter as arborizing processes and around blood vessels in the gray matter. The present study demonstrated the emergence of radial glia after SCI in adult mammals. Radial glia derived from subpial astrocytes most likely play an important role in neural repair and regeneration after SCI.     

Developmental expression of glial fibrillary acidic protein mRNA in the rat brain analyzed by in situ hybridization

Glial fibrillary acidic protein (GFAP) accumulates in astrocytes during development. We have characterized the increase in GFAP mRNA during development of the rat brain by using Northern blotting and in situ hybridization histochemistry and have found a caudal to rostral gradient of expression, consistent with overall brain maturation. GFAP mRNA was first observed at embryonic day 16 (E16) in the glial limitans of the ventral hindbrain. During brain development message levels increased rostrally and by postnatal day 5 (P5) the entire glial limitans showed a positive signal which persisted into adulthood. GFAP mRNA was also found to accumulate in a caudal to rostral direction within the Purkinje cell layer of cerebellum beginning shortly after birth. By P5 the entire layer was positive and signal in this region could be localized to Bergmann glia by P15. A transient elevation in GFAP mRNA was apparent during the second postnatal week in cerebellum and cerebrum. Using in situ hybridization, a peak in message levels was observed at P15 and could be localized primarily to the deep white matter of cerebellum, to the corpus callosum, and to certain hippocampal fiber tracts. The pattern of GFAP expression in these regions is consistent with the differentiation of interfascicular glia and the appearance of type-2 astrocytes during the initial events of myelination. GFAP mRNA levels in white matter were greatly reduced in the adult. The pronounced regional differences in GFAP mRNA expression during development may reflect the differentiation of subpopulations of astrocytes.     

Gliogenesis in rat spinal cord: evidence for origin of astrocytes and oligodendrocytes from radial precursors

We have examined glial cell lineages during rat spinal cord development by using a variety of antibodies that react with immature and mature glia. Radial glia in embryonic cord bound 1) A2B5, an antibody that reacts with a glial precursor cell population in optic nerve; 2) AbR24, which is directed against GD3 ganglioside and binds to immature neuroectodermal cells and to developing oligodendrocytes in forebrain and cerebellum; and 3) an antibody to the intermediate filament, vimentin. With time, two different populations emerged, both of which seemed to be derivatives of radial cells. One cell type expressed the astrocyte intermediate filament, GFAP, in addition to vimentin. GFAP-containing cells eventually took on the forms of astrocytes in gray and white matter. The other type expressed carbonic anhydrase, an enzyme characteristic of oligodendrocytes and enriched in myelin. Carbonic anhydrase-positive cells eventually developed into small cells with oligodendrocyte morphology. Our observations suggest a common lineage for astrocytes and oligodendrocytes from radial cells during spinal cord gliogenesis.     

Expression of neurotrophins in the adult spinal cord in vivo

Potential roles of trophins in the normal and injured spinal cord are largely undefined. However, a number of recent studies suggest that adult spinal cord expresses neurotrophin receptors and responds to the neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3), particularly after injury. The data indicate that trophins may enhance regrowth after damage and may represent a new therapeutic approach to injury. Neurotrophins are reportedly present in the spinal cord, but the cellular localization is unknown. This information is critical to begin delineating mechanisms of actions. To approach this problem, we examined whether spinal cord glia express BDNF and NT3 in vivo and have begun to define cellular distribution. Specific antibodies directed against the neurotrophins were utilized to visualize neurotrophin protein. Initial studies indicated that small cells in the white matter of adult rat spinal cord express BDNF and NT3. Large neurotrophin-positive neurons were also identified in the ventral cord. To identify the neurotrophin-positive cells, co-localization studies were performed utilizing neurotrophin polyclonal antisera together with monoclonal antibodies directed against the astrocyte marker, glial fibrillary acidic protein (GFAP). In the white matter of adult spinal cord, GFAP-positive and GFAP-negative cells expressed BDNF and NT3. Our study suggests that astrocyte and non-astrocyte cells provide trophic support to the adult spinal cord.     

GFAP-positive and myelin marker-positive glia in normal and pathologic environments

The data herein demonstrate that in addition to the well-characterized myelin marker-positive, glial fibrillary acidic protein (GFAP)-negative, membrane sheet-bearing oligodendrocytes, another type of myelin marker-positive, process-bearing glia exists in normal and pathologic conditions. This second type of myelin marker-positive glia expresses GFAP, and therefore these cells have been referred to as mixed phenotype glia. Although mixed phenotype glia have been documented previously, their identity and function have remained a mystery. The goal of this immunocytochemical study was to further characterize these cells. Using the MBPlacZ transgenic mouse in which beta-galactosidase is under the control of the myelin basic protein (MBP) gene promoter, GFAP-positive/beta-galactosidase-positive and myelin/oligodendrocyte-specific protein (MOSP)-positive/beta-galactosidase-positive cells were detected in subcortical white matter and in perivascular locations within cerebral white and gray matter. In cultures prepared from highly enriched myelin marker-positive immature glia, mixed phenotype glia were detected that were GFAP-positive and either MOSP-, MBP-, O1-, and O4-positive. The expression of multiple myelin markers by mixed phenotype glia may suggest that these cells are of oligodendrocyte origin. Increased numbers of MOSP-positive/GFAP-positive mixed phenotype glia were detected in sections from adult hypomyelinated brain from shiverer, quaking, and PKU mice compared to myelinated control adult mouse brain. Similarly, cultures from control brain exposed to elevated pH for 2-3 weeks showed dramatically increased numbers of mixed phenotype glia (80%) compared to control (<10%). Increased numbers of mixed phenotype glia also were detected in shiverer cultures (40%). Since increases in the number of mixed phenotype glia occur in shiverer, quaking, and PKU mouse brain, these data suggest that mixed phenotype glia contribute to gliosis in pathologic white matter.     

Upregulation of glial clusterin in brains of patients with AIDs

Since clusterin (CLU) production in reactive astrocytes may be neuroprotective, we examined its distribution in AIDS brains where brain injury and reactive astrocytosis are common. The relative area and number of CLU-positive astrocytes, as well as their percent total of all white matter glia, significantly increased in AIDS brains with and without HIV encephalitis (P<0.05). Proliferation markers were absent. In contrast, the relative area and number of GFAP-positive astrocytes and their percent of all white matter glia, increased in some cases but the mean increases were not significant. Clusterin is sensitive marker of glial reactivity in AIDS brains and its enhanced expression was not dependent on increases in GFAP.     

Developmental changes of glial fibrillary acidic protein and myelin basic protein in perinatal leukomalacia: relationship to a predisposing factor

Focal white matter necrosis is frequently seen in the brains of infants with perinatal cerebral hypoperfusion. Periventricular leukomalacia (PL) occurs in the deep white matter of premature and term neonates and subcortical leukomalacia (SL) in the subcortical white matter of young infants. Using immunoperoxidase methods in normal infants, glia positive for glial fibrillary acidic protein (GFAP) were found first in the deep zones of white matter and with increasing age they became more prominent in the subcortical zone. They increased diffusely in the deep or subcortical zones of the cases of PL or SL, respectively. The number of myelin basic protein-positive glia is much larger than that of GFAP-positive glia in the cases of old PL. These findings suggest that an increased number of positive glia may be a reaction to hypoxic, ischemic, or toxic insults, or this shifting, transient increase of positive glia in cerebral white matter may be one of several predisposing factors leading to perinatal leukomalacia. Furthermore, positive staining of GFAP and MBP for reactive astrocytes in old PL suggests that at a certain stage of gliogenesis both GFAP and myelin basic protein may be present within the same cell.     

Prenatal exposure to ethanol alters the postnatal development and transformation of radial glia to astrocytes in the cortex

Postmitotic neurons migrate from a zone(s) near the ventricles to the neocortex. During this migration, neurons associate with radial glia. After serving their role as guides for neuronal migration, the radial glia transform into astrocytes. Prenatal exposure to ethanol causes abnormal neuronal migration. We examined the effects of gestational exposure to ethanol on radial glia and astrocytes. Radial glia were stained immunohistochemically with the antibody RAT-401, and astrocytes were labeled with an antibody directed against glial-fibrillary acidic protein (GFAP). The subjects were the offspring of rats fed an ethanol-containing liquid. diet (Et), pair-fed a liquid control diet (Ct), or fed chow and water (Ch). During the first postnatal week, radial glial fibers (in Et-treated rats and controls) stretched from the ventricular surface through the developing. cerebral wall to the pial surface. In the Et-treated rats, the radial processes were less dense and more poorly fasciculated than they were in the Ch-and Ct-treated rats. Moreover, by postnatal day (P) 5, there was a significant reduction in RAT-401 immunostaining in the Et-treated rats, particularly in the superficial cortex. A similar reduction in control rats did not begin until P10. In all three treatment groups, GFAP-immunoreactive astrocytes were in the cortex throughout the period from P1 to P45. In neonates, GFAP-positive cells were distributed in the marginal zone (layer I) and the intermediate zone (the white matter). The number of GFAP-positive cells in the cortical plate increased steadily with time so that, by P26, GFAP-immunoreactive astrocytes were distributed evenly through all cortical laminae. Interestingly, between P5 and P12, the number of astrocytes was significantly greater in Et-treated rats than in controls. Thus prenatal exposure to ethanol induces the premature loss of RAT-401-positive processes and the precocious increase in GFAP immunostaining. These ethanol-induced changes in glial development indicate that ethanol accelerates the transformation of radial glia into astrocytes. Moreover, the ethanol-induced premature degradation of the network of radial glial fibers may underlie the migration of late-generated neurons to ectopic sites. © 1993 Wiley-Liss, Inc.     

Glial expression of fibroblast growth factor-9 in rat central nervous system.

We examined the expression of fibroblast growth factor (FGF)-9 in the rat central nervous system (CNS) by immunohistochemistry and in situ hybridization studies. FGF-9 immunoreactivity was conspicuous in motor neurons of the spinal cord, Purkinje cells, and neurons in the hippocampus and cerebral cortex. In addition to the neuronal localization of FGF-9 immunoreactivity that we reported previously, the present double-label immunohistochemistry clearly demonstrated that the immunoreactivity was present in glial fibrillary acidic protein (GFAP)-positive astrocytes preferentially present in the white matter of spinal cord and brainstem of adult rats and in CNPase-positive oligodendrocytes that were arranged between the fasciculi of nerve fibers in cerebellar white matter and corpus callosum of both adult and young rats. There was a tendency for FGF-9 immunoreactivity in oligodendrocytes to be more pronounced in young rats than in adult rats. The variation of oligodendrocyte FGF-9 immunoreactivity in adult rats was also more pronounced than that in young rats. With in situ hybridization, FGF-9 mRNA was observed in astrocytes in the white matter of rat spinal cord and oligodendrocytes in the white matter of cerebellum and corpus callosum of adult and young rats. The expression of FGF-9 mRNA in glial cells was lower than in neurons, and not all glial cells expressed FGF-9. In the present study, we demonstrated that FGF-9 was expressed not only in neurons but also in glial cells in the CNS. FGF-9 was considered to have important functions in adult and developing CNS.     

Proliferation and differentiation of reactive nestin＋/GFAP＋ cells in an adult rat model of compression-induced spinal cord injury

BACKGROUND： Studies have demonstrated that astrocytes may possess similar properties to neural stem cells/neural precursor cells and have the potential to differentiate into neurons. OBJECTIVE： To observe neuroepithelial stem cell protein （nestin） and glial fibrillary acidic protein （GFAP） expression following spinal cord injury, and to explore whether nestin＋/GFAP＋ cells, which are detected at peak levels in gray and white matter around the ependymal region of the central canal in injured spinal cord, possess similar properties of neural stem cells. DESIGN, TIME AND SETTING： A randomized, controlled experiment. The study was performed at the Key Laboratory of Environment and Genes Related to Diseases （Xi＇an Jiaotong University）, Ministry of Education between January 2004 and December 2006. MATERIALS： Rabbit anti-rat nestin, β-tubulinⅢ, mouse anti-rat GFAP, galactocerebroside （GaLC） antibodies were utilized, as well as flow cytometry. METHODS： A total of 60 male, Sprague Dawley rats, aged 8 weeks, were randomly assigned to control （n = 12） and model （n = 48） groups. The spinal cord injury model was established in the model group by aneurysm clip compression, while the control animals were not treated. The gray and white matter around the ependymal region of the central canal exhibited peak expression of nestin＋/GFAP＋ cells. These cells were harvested and prepared into single cell suspension, followed by primary and passage cultures. The cells were incubated with serum-containing neural stem cell complete medium. MAINOUTCOME MEASURES： Nestin and GFAP expression in injured spinal cord was determined using immunohistochemistry and double-labeled immunofluorescence at 1, 3, 5, 7, 14, 28, and 56 days post-injury. In addition, cell proliferation and differentiation were detected using immunofluorescence cytochemistry and flow cytometry. RESULTS： Compared with the control group, the model group exhibited significantly increased nestin and GFAP expression （P 〈 0.05）, which reached peak levels between 3 and 7 days. The majority of cells in the ependymal region around the central canal were nestin＋/GFAP- cells, while the gray and white matter around the ependymal region were full of nestin＋/GFAP＋ cells, with an astrocytic-like appearance. A large number of nestin＋/GFAP＋cells were observed in the model group cell culture, and the cells formed clonal spheres and displayed strong nestin-positive immunofluorescence staining. Following induced differentiation, a large number of GaLC-nestin, β-tubulin Ⅲ-nestin, and GFAP-nestin positive cells were observed. However, no obvious changes were seen in the control group. Cells in S stage, as well as the percentage of proliferating cells, in the model group were significantly greater than in the control group （P 〈 0.01）, CONCLUSION： Spinal cord injury in the adult rat induced high expression of nestin＋/GFAP＋ in the gray and white matter around the ependymal region of the central canal. These nestin＋/GFAP＋ cells displayed the potential to self-renew and differentiate into various cells. The cells could be neural stem cells of the central nervous system.     

Axonal and Nonneuronal Cell Responses to Spinal Cord Injury in Mice Lacking Glial Fibrillary Acidic Protein

We have examined the regeneration of corticospinal tract fibers and expression of various extracellular matrix (ECM) molecules and intermediate filaments [vimentin and glial fibrillary acidic protein (GFAP)] after dorsal hemisection of the spinal cord of adult GFAP-null and wild-type littermate control mice. The expression of these molecules was also examined in the uninjured spinal cord. There was no increase in axon sprouting or long distance regeneration in GFAP−/− mice compared to the wild type. In the uninjured spinal cord (i) GFAP was expressed in the wild type but not the mutant mice, while vimentin was expressed in astrocytes in the white matter of both types of mice; (ii) laminin and fibronectin immunoreactivity was localized to blood vessels and meninges; (iii) tenascin and chondroitin sulfate proteoglycan (CSPG) labeling was detected in astrocytes and the nodes of Ranvier in the white matter; and (iv) in addition, CSPG labeling which was generally less intense in the gray matter of mutant mice. Ten days after hemisection there was a large increase in vimentin⁺cells at the lesion site in both groups of mice. These include astrocytes as well as meningeal cells that migrate into the wound. The center of these lesions was filled by laminin⁺/fibronectin⁺cells. Discrete strands of tenascin-like immunoreactivity were seen in the core of the lesion and lining its walls. Marked increases in CSPG labeling was observed in the CNS parenchyma on either side of the lesion. These results indicate that the absence of GFAP in reactive astrocytes does not alter axonal sprouting or regeneration. In addition, except for CSPG, the expression of various ECM molecules appears unaltered in GFAP−/− mice.     

Astrocytic reactivity and intermediate filament metabolism in experimental autoimmune encephalomyelitis: The effect of suppression with prazosin

In either actively or passively transferred experimental autoimmune encephalomyelitis (EAE), increased immunocytochemical staining of glial fibrillary acidic protein (GFAP) in astrocytes was detected early in the disease process in both the gray and white matter of the spinal cord. Staining was not restricted to areas of perivascular mononuclear infiltration, and was observed at all levels of the cord. This enhanced staining pattern was delayed in rats in which clinical signs of EAE had been suppressed by treatment with the alpha 1-adrenoceptor antagonist prazosin. This glial reaction in EAE was not accompanied by increased GFAP synthesis, as measured by in vitro labeling of spinal cord slices, nor an increase in GFAP content, as measured by densitometry of intermediate filament fractions separated by polyacrylamide gel electrophoresis. Total protein synthesis was increased, with vimentin being labeled especially heavily; in prazosin-treated EAE animals, the increase in total protein synthesis was reduced and delayed.     

Connexin43 and astrocytic gap junctions in the rat spinal cord after acute compression injury

To examine the possible role of interastrocytic gap junctions in the maintenance of tissue homeostasis after spinal cord damage, we initiated studies of the astrocytic gap junctional protein connexin43 (Cx43) in relation to temporal and spatial parameters of neuronal loss, reactive gliosis, and white matter survival in a rat model of traumatic spinal cord injury (SCI). Cx43 immunolocalization in normal and compression-injured spinal cord was compared by using two different sequence-specific anti-Cx43 antibodies that have previously exhibited different immunorecognition properties at lesion sites in brain. At 1- and 3-day survival times, gray matter areas with mild to moderate neuronal depletion exhibited a loss of immunolabeling with one of the two antibodies. At the lesion epicenter, these areas consisted of a zone that separated normal staining distal to the lesion from intensified labeling seen with both antibodies immediately adjacent to the lesion. Loss of immunoreactivity with only one of the two antibodies suggested masking of the corresponding Cx43 epitope. By 7 days post-SCI, Cx43 labeling was absent with both antibodies in all regions extending up to 1 mm from the lesion site. Reactive astrocytes displaying glial fibrillary acidic protein (GFAP) appeared by 1 day and were prominent by 3 days post-SCI. Their distribution in white and gray matter corresponded closely to that of Cx43 staining at 1 day, but less so at 3 days when GFAP-positive profiles were present at sites where Cx43 labeling was absent. By 7 days post-SCI, Cx43 again co-localized with GFAP-positive cells in the surviving subpial rim, and with astrocytic processes on radially oriented vascular profiles investing the central borders of the lesion. The results indicate that alterations in Cx43 cellular localization and Cx43 molecular modifications reflected by epitope masking, which were previously correlated with gap junction remodeling following excitotoxin-induced lesions in brain, are not responses limited to exogenously applied excitotoxins; they also occur in damaged spinal cord and are evoked by endogenous mechanisms after traumatic SCI. The GFAP/Cx43 co-localization results suggest that during their transformation to a reactive state, spinal cord astrocytes undergo a transitional phase marked by altered Cx43 localization or expression. J. Comp. Neurol. 382:199-214, 1997. © 1997 Wiley-Liss, Inc.