Characterization of cellular and neurological damage following unilateral hypoxia/ischemia.

Rodent models of stroke are often used to investigate the mechanisms that lead to ischemic neuronal damage. In this study, we used a model of cerebral hypoxia with ischemia to produce unilateral damage in C57Bl/6 mice. Lesion volume, ascertained by TTC staining, increased with longer durations of hypoxia. Additionally, cresyl violet, TUNEL, and FluoroJade staining showed a statistically significant increase in cellular damage in the ipsilateral cortex, CA1 pyramidal layer, and dentate gyrus of the hippocampus of ipsilateral hypoxic/ischemic tissue versus sham tissue. Astrocyte reactivity, determined by GFAP staining, was significantly higher in the ipsilateral H/I cortex and contralateral hippocampus compared to sham cortex and hippocampus, respectively. Increased microglia activation was evident in the H/I-treated cortex and hippocampus versus sham cortex and hippocampus, particularly within areas undergoing degeneration. To examine whether this model produces motor deficits, a battery of tests were administered before and after hypoxia. Following 45 min H/I, locomotor activity, rotarod performance and performance on an inverted wire hang test were all significantly decreased. These data indicate that the histological evidence of neuronal damage is consistent with functional deficits and suggest that this model may be useful for investigating strategies designed to protect neurons from hypoxia/ischemia-induced damage.

The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar.

Chondroitin sulfate proteoglycans (CS-PGs) expressed by reactive astrocytes may contribute to the axon growth-inhibitory environment of the injured CNS. The specific potentially inhibitory CS-PGs present in areas of reactive gliosis, however, have yet to be thoroughly examined. In this study, we used immunohistochemistry, combined immunohistochemistry-in situ hybridization, immunoblot analysis, and reverse transcription-PCR to examine the expression of specific CS-PGs by reactive astrocytes in an in vivo model of reactive gliosis: that is, the glial scar, after cortical injury. Neurocan and phosphacan can be localized to reactive astrocytes 30 d after CNS injury, whereas brevican and versican are not expressed in the chronic glial scar. Neurocan is also expressed by astrocytes in primary cell culture. Relative to the amount present in cultured astrocytes or uninjured cortex, neurocan expression increases significantly in the glial scar resulting from cortical injury, including the re-expression of the neonatal isoform of neurocan. In contrast, phosphacan protein levels are decreased in the glial scar compared with the uninjured brain. Because these CS-PGs are capable of inhibiting neurite outgrowth in vitro, our data suggest that phosphacan and neurocan in areas of reactive gliosis may contribute to axonal regenerative failure after CNS injury.

Expression of full-length trkB receptors by reactive astrocytes after chronic CNS injury.

Growth factors, including members of the neurotrophin family, are expressed by neuronal and glial elements following injury to the CNS. In order to assess the capacity for glial cells to respond to neurotrophins at sites of chronic injury, full-length trkB receptors were localized following implantation of a nitrocellulose filter into the cerebral cortex for 30 days. Northern analysis demonstrated that filter implants contained cells expressing transcripts for full-length and truncated trkB receptors, in contrast to the predominant expression of truncated trkB receptors by cultured astrocytes. In situ hybridization and immunohistochemistry using probes to the trkB kinase domain colocalized full-length receptors with GFAP-immunopositive reactive astrocytes adjacent to and within the filter implant. In contrast, OX-42-immunopositive microglia/macrophages were not stained for full-length trkB. These data indicate that reactive astrocytes can express functional trkB receptors following a chronic insult to the cerebral cortex and support the hypothesis that neurotrophins may regulate astrocytic responses to injury.

Injury-induced proteoglycans inhibit the potential for laminin-mediated axon growth on astrocytic scars.

Following injury to the adult CNS, the expression of a number of extracellular matrix molecules increases in regions of reactive gliosis. This glial matrix includes certain chondroitin sulfate proteoglycans (CS-PGs) which have been correlated with an inhibition of axon outgrowth. In order to test the influence of glial associated CS-PGs on neurite elongation directly, we sought to determine whether enzymatic modification of injury-induced CS-PGs could enhance neurite outgrowth across the surface of intact glial scars formed in vivo after implanting nitrocellulose filters into the cortex of adult rats. This gliotic tissue was subsequently explanted in vitro and used as a substrate for growing embryonic retinal neurons. Treatment of adult explants with chondroitinase ABC led to a significant increase in mean neurite length over the scar surface. Heparitinase treatment caused a much smaller, although significant, increase in neurite outgrowth. This suggested that more than one type of PG was present or that a single PG with both CS and HS side chains was upregulated. Western analysis revealed that a PG(s) with a core protein between 180 and 400 kDa was found to be relatively more abundant in areas of reactive gliosis induced to form in adult rather than neonatal animals. Simultaneous treatment of adult glial scars with chondroitinase and antibodies to the beta 1, beta 2 chain of laminin partially reversed the growth-enhancing effect of enzymatic digestion alone. These data demonstrate that the increase in neurite outgrowth along the surface of reactive astrocytes following enzymatic modification of injury-induced PGs was due, in part, to the presence of laminin. Thus, in this model of gliosis, particular PGs may act as inhibitors of neurite outgrowth by attenuating the potential for axon elongation that could occur due to the concomitant expression of growth-promoting molecules in regions of reactive gliosis.

beta-Amyloid of Alzheimer's disease induces reactive gliosis that inhibits axonal outgrowth.

Pathological lesions in the brains of patients with Alzheimer's disease (AD) are characterized by dense deposits of the protein beta-amyloid. The link between the deposition of beta-amyloid in senile plaques and AD-associated pathology is, at present, controversial since there have been conflicting reports on whether the 39-43 amino acid beta-amyloid sequence is toxic or trophic to neurons. In this report, we show that beta-amyloid peptide when presented as an insoluble substrate which mimics its conformation in vivo can induce cortical glial cells in vitro and in vivo to locally deposit chondroitin sulfate containing proteoglycan. In vitro the proteoglycan-containing matrix deposited by glia on beta-amyloid blocks the usual ability of the peptide to allow cortical neurons to adhere and grow. Chondroitin sulfate-containing proteoglycan was also found in senile plaques of human AD tissue. We suggest that an additional effect of beta-amyloid in the brain, which compounds the direct effects of beta-amyloid on neurons, is mediated by the stimulation of astroglia to become reactive. Once in the reactive state, glial cells deposit large amounts of growth-inhibitory molecules within the neuropil which could impair neuronal process survival and regeneration leading to neurite retraction and/or dystrophy around senile plaques in AD.

Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes.

The extracellular matrix (ECM) molecules chondroitin-6-sulfate proteoglycan (CS-PG) and cytotactin/tenascin (CT), present on subpopulations of astroglia or their precursors during development, can inhibit neurite outgrowth in vitro. However, it is not known whether these molecules are expressed within the mature CNS following injury, where they could contribute to regenerative failure. Thus, the expression of various ECM molecules that affect axon growth was examined in areas of reactive gliosis caused by implanting a piece of nitrocellulose into the cortex of neonatal and adult animals. The expression of these molecules was compared to the amount of neurite outgrowth that occurred in vitro when the damaged CNS tissue from animals of various ages was removed intact and used as a substrate in explant culture. The results demonstrate that the growth-promoting molecules laminin, collagen type IV, and fibronectin were present around the implant in all experimental groups. In comparison, CS-PG and CT were present within and around the area of the lesion only in adult animals. In vivo, these molecules were colocalized with intensely glial fibrillary acidic protein (GFAP)-positive astrocytes in and immediately adjacent to the scar, but not with other equally intensely GFAP-positive astrocytes in the cortex away from the site of injury. CT and CS-PG were present in gray matter areas of the cortex that had been directly damaged during the implant procedure and in the corpus callosum when lesioned during implantation. In vitro, the glial tissue removed from the lesion site of neonatal animals supported neurite outgrowth, while scars removed from adult animals did not. The inability of the adult glial scar tissue to support neurite outgrowth was best correlated with the expression of CS-PG and CT, suggesting that these molecules may be involved in limiting the growth of regenerating axons in the CNS after injury.

Cross-species septohippocampal transplants: ultrastructure of Thy-1.2-labeled donor fibers into the dentate gyrus.

A naturally occurring species-specific membrane marker was used to identify unambiguously transplanted septal cells and their fibers which have grown into host tissue. Cell suspensions of the septum/basal forebrain region of C57Bl/6 mouse embryos were transplanted into the dentate gyrus of Sprague-Dawley rats that had received a fornix lesion. The membranes of the mouse contained Thy-1.2, while the membranes of the rat contained Thy-1.1. An antibody to Thy-1.2 clearly identified the donor tissue and did not react with the Thy-1.1 of the host's membranes. The ultrastructure of the immunoreactively labeled tissue confirmed previous biochemical findings on the distribution of Thy-1 and showed Thy-1.2 immunoreactivity on axons and dendrites, microtubules, some mitochondrial membranes, and the surface membranes of cell bodies. Within the transplant, a few glial profiles showed immunoreactive fibrils, but most glial profiles within the transplant and all glial profiles outside the transplant were not immunoreactive. Astrocyte fibers enclosed the outgrowing labeled fibers to form fascicles, but did not penetrate the fascicle. There was no other distinctive association of astrocytic profiles with immunoreactive fibers. Dendrites grew for long distances into the host's molecular layer. Many immunoreactive dendritic profiles formed synapses with unlabeled terminal profiles from the host. The host synapses on the long dendrites of the transplanted neurons may form an important source of input for the initiation of physiological activity in the new circuits established by the transplant. A few labeled (donor) synaptic terminals were observed in the molecular layer, but Thy-1.2-labeled dendritic profiles were much more prominent than labeled axonal profiles.

Increase in acetylcholinesterase in the molecular layer of the dentate gyrus in the absence of septal inputs following selective granule cell lesions.

Granule cell lesions cause an increase in acetylcholinesterase (AChE) staining in the molecular layer of the dentate gyrus. The source of this response was examined by combining granule cell lesions with lesions of the fornix-fimbria, thereby removing the cholinergic input from the septum to the hippocampus. The increased AChE staining was present in animals with granule cell lesions regardless of whether the fornix was lesioned or intact. The increase in AChE staining occurred without a corresponding increase in choline acetyltransferase staining. These findings suggest that an AChE-positive, but non-cholinergic, sprouting response occurred within the dentate gyrus following selective lesions of the granule cells. The source of this sprouting may be from AChE-positive hilar interneurons.

Interactions between donor and host tissue following cross-species septohippocampal transplants.

Interactions between donor and host tissues following xenogeneic transplantation were studied using the neural cell surface antigen, Thy 1.2, as a marker for the donor tissue. Dissociated septal cells from Thy 1.2-positive fetal mice were transplanted to the dentate gyrus of Thy 1.2-negative adult rats. At post-transplantation survival times between 1 and 5 months, an antibody to Thy 1.2 was used to identify donor tissue. The results demonstrate that the donor tissue was capable of migrating and developing within the host following transplantation. Thy 1.2-positive cells and processes were consistently found within the supragranular, infragranular, and molecular layers of the dentate gyrus, and occasionally within the hilus, suggesting that mechanisms existed within the host which influenced the development of the transplanted tissue. Additionally, the survival and growth of the Thy 1.2-positive neurons differed from previous reports describing the growth of acetylcholinesterase (AChE)-positive fibers from xenogeneic transplants. This finding suggested that in addition to growing within the host, xenogeneic transplants may also stimulate a compensatory sprouting response from the host.