Distant microglial and astroglial activation secondary to experimental spinal cord lesion

This paper analysed whether glial responses following a spinal cord lesion is restricted to a scar formation close to the wound or they might be also related to widespread paracrine trophic events in the entire cord. Spinal cord hemitransection was performed in adult rats at the thoracic level. Seven days and three months later the spinal cords were removed and submitted to immunohistochemistry of glial fibrillary acidic protein (GFAP) and OX42, markers for astrocytes and microglia, as well as of basic fibroblast growth factor (bFGF), an astroglial neurotrophic factor. Computer assisted image analysis was employed in the quantification of the immunoreactivity changes. At the lesion site an increased number of GFAP positive astrocytes and OX42 positive phagocytic cells characterized a dense scar formation by seven days, which was further augmented after three months. Morphometric analysis of the area and microdensitometric analysis of the intensity of the GFAP and OX42 immunoreactivities showed reactive astrocytes and microglia in the entire spinal cord white and gray matters 7 days and 3 months after surgery. Double immunofluorescence demonstrated increased bFGF immunostaining in reactive astrocytes. The results indicated that glial reaction close to an injury site of the spinal cord is related to wounding and repair events. Although gliosis constitutes a barrier to axonal regeneration, glial activation far from the lesion may contribute to neuronal trophism and plasticity in the lesioned spinal cord favoring neuronal maintenance and fiber outgrowth.     

Growth-associated protein-43 and ephrin B3 induction in the brain of adult SIV-infected rhesus macaques

Understanding the mechanisms of neuronal regeneration and repair in the adult central nervous system is a vital area of research. Using a rhesus lentiviral encephalitis model, we sought to determine whether recovery of neuronal metabolism after injury coincides with the induction of two important markers of synaptodendritic repair: growth-associated protein-43 (GAP-43) and ephrin B3. We examined whether the improvement of neuronal metabolism with combined anti-retroviral therapy (cART) after simian immunodeficiency virus (SIV) infection in rhesus macaques involved induction of GAP-43, also known as neuromodulin, and ephrin B3, both implicated in axonal pathfinding during neurodevelopment and regulation of synapse formation, neuronal plasticity, and repair in adult brain. We utilized magnetic resonance spectroscopy to demonstrate improved neuronal metabolism in vivo in adult SIV-infected cART animals compared to untreated and uninfected controls. We then assessed levels of GAP-43, ephrin B3, and synaptophysin, a pre-synaptic marker, in three brain regions important for cognitive function, cortex, hippocampus, and putamen, by quantitative real-time RT-PCR and immunohistochemistry. Here we demonstrate that (1) GAP-43 mRNA and protein are induced with SIV infection, (2) GAP-43 protein is higher in the hippocampus outer molecular layer in SIV-infected animals that received cART compared to those that did not, and (3) activated microglia and infiltrating SIV-infected macrophages express abundant ephrin B3, an important axonal guidance molecule. We propose a model whereby SIV infection triggers events that lead to induction of GAP-43 and ephrin B3, and that short-term cART results in increased magnitude of repair mechanisms especially in the hippocampus, a region known for high levels of adult plasticity.     

Relationship of calpain-mediated proteolysis to the expression of axonal and synaptic plasticity markers following traumatic brain injury in mice

The role of neuronal plasticity and repair on the final functional outcome following traumatic brain injury (TBI) remains poorly understood. Moreover, the relationship of the magnitude of post-traumatic secondary injury and neurodegeneration to the potential for neuronal repair has not been explored. To address these questions, we employed Western immunoblotting techniques to examine how injury severity affects the spatial and temporal expression of markers of axonal growth (growth-associated protein GAP-43) and synaptogenesis (pre-synaptic vesicular protein synaptophysin) following either moderate (0.5 mm, 3.5 M/s) or severe (1.0 mm, 3.5 M/s) lateral controlled cortical impact traumatic brain injury (CCI-TBI) in young adult male CF-1 mice. Moderate CCI increased GAP-43 levels at 24 and 48 h post-insult in the ipsilateral hippocampus relative to sham, non-injured animals. This increase in axonal plasticity occurred prior to maximal hippocampal neurodegeneration, as revealed by de Olmos silver staining, at 72 h. However, moderate CCI-TBI did not elevate GAP-43 expression in the ipsilateral cortex where neurodegeneration was extensive by 6 h post-TBI. In contrast to moderate injury, severe CCI-TBI failed to increase hippocampal GAP-43 levels and instead resulted in depressed GAP-43 expression in the ipsilateral hippocampus and cortex at 48 h post-insult. In regards to injury-induced changes in synaptogenesis, we found that moderate CCI-TBI elevated synaptophysin levels in the ipsilateral hippocampus at 24, 48, 72 h and 21 days, but this effect was not present after severe injury. Together, these data highlights the adult brain's ability for axonal and synaptic plasticity following a focal cortical injury, but that severe injuries may diminish these endogenous repair mechanisms. The differential effects of moderate versus severe TBI on the post-traumatic plasticity response may be related to the calpain-mediated proteolytic activity occurring after a severe injury preventing increased expression of proteins required for plasticity. Supporting this hypothesis is the fact that GAP-43 is a substrate for calpain along with our data demonstrating that calpain-mediated degradation of the cytoskeletal protein, alpha-spectrin, is approximately 10 times greater in ipsilateral hippocampal tissue following severe compared to moderate CCI-TBI. Thus, TBI severity has a differential effect on the injury-induced neurorestorative response with calpain activation being one putative factor contributing to neuroregenerative failure following severe CCI-TBI. If true, then calpain inhibition may lead to both neuroprotective effects and an enhancement of neuronal plasticity/repair mechanisms post-TBI.     

Failure to express GAP-43 leads to disruption of a multipotent precursor and inhibits astrocyte differentiation

The nervous system-specific protein GAP-43 is significantly upregulated in neurons and glia that are differentiating. In P19 EC cells that do not express GAP-43, neurogenesis is inhibited; many immature neurons apoptose and the survivors do not mature morphologically. Here we show that the initial defect is in an early precursor with characteristics of a neural stem cell, which failed to respond normally to retinoic acid (RA). As a consequence, its progeny had altered cell fates: In addition to the neuronal defects previously reported, RC1-labeled radial glia failed to exit the cell cycle, accumulated, and failed to acquire GFAP immunoreactivity. However, leukemia inhibitory factor (LIF) could stimulate GFAP expression suggesting that astrocytes not derived from radial glia are less affected by absence of GAP-43. Differentiation of radial glia-derived astrocytes was also inhibited in glial cultures from GAP-43 (-/-) cerebellum, and in GAP-43 (-/-) telencephalon in vivo, differentiation of astrocytes derived from both radial and nonradial glia lineages were both affected: In the glial wedge, GFAP-labeled radial glia-derived astrocytes were reduced consistent with the interpretation that they may be unable to deflect GAP-43 (-/-) commissural axons toward the midline. At the midline, both radial and nonradial glia-derived astrocytes were also decreased although it fused normally. The results demonstrate that GAP-43 expressed in multipotent precursors is required for appropriate cell fate commitment, and that its absence affects astrocyte as well as neuronal differentiation.     

GAP-43 gene expression during development: persistence in a distinctive set of neurons in the mature central nervous system

GAP-43 is a rapidly transported axonal protein most prominently expressed in regenerating and developing nerves. However, the low level persistence of GAP-43 in the adult CNS where growth and regenerative capacity are minimal may additionally indicate a role for this molecule in neuronal remodeling. Previous studies have revealed GAP-43 immunoreactivity in neurites throughout many regions of the CNS. To identify the CNS neurons that express GAP-43 at different stages of development, we utilized in situ hybridization and immunocytochemistry; the latter was performed with an antibody that recognizes GAP-43 immunoreactivity in both perikarya and neurites. In the perinatal period GAP-43 is expressed in all neurons. Subsequently its expression becomes progressively restricted such that by maturity most neurons no longer express detectable levels, although GAP-43 expression is still moderately high in the adult entorhinal cortex, and strikingly high in the adult hippocampus and olfactory bulb. In light of current notions about the function of GAP-43, it is tempting to speculate that this anatomy denotes neurons engaged in structural remodeling and functional plasticity.     

Reactive astrocytes in neonate brain upregulate intermediate filament gene expression in response to axonal injury

We have examined the injury response of astrocytes in the immature hamster brain in this study, focusing on alterations in the expression of glial fibrillary acidic protein (GFAP) and vimentin. In the adult CNS these two type III intermediate filament (IF) proteins have been shown to undergo robust increases in expression in response to axonal injury. Since injury to the immature CNS reportedly elicits less glial scar formation than adult brain injury, we examined the possibility that immature astrocytes respond differently than adult astrocytes to CNS injury with respect to IF gene expression. In situ hybridization using a ³⁵S-labeled cDNA GFAP probe was done on brainstem sections obtained 2,7 and 14 days after unilateral transection of the corticospinal tract in P8 hamster pups. The results indicated that substantial increases in GFAP mRNA were associated with the degenerating portion of the corticospinal tract by 2 days after axotomy and that the levels remained elevated for at least 14 days. Double-label immunofluorescence studies of this material suggested that GFAP as well as vimentin protein levels were also increased in many astrocytes in and around the degenerating corticospinal tract 2–14 days after axotomy. Most of the reactive astocytes in the degenerating regions exhibited increases in GFAP and vimentin immunostaining but some vimentin-negative GFAP-positive reactive astrocytes were also observed, particularly in regions surrounding the actual degenerative zones. The results from these experiments revealed that immature astrocytes have the potential for altering their normal developmental program of GFAP and vimentin expression after injury and mount a response that is qualitatively similar to that of astrocytes after CNS injury in the adult animal.     

Growth-associated protein (GAP-43)-like immunoreactivity in primary and permanent tooth pulp nerve fibers of the cat.

GAP-43-like immunoreactivity in developing and mature incisor and canine tooth pulp nerve fibers in the cat was examined with fluorescence immunohistochemistry and pre-embedding immunogold electron microscopy. As expected, pulpal and periodontal nerve fibers in primary teeth aged 2-3 weeks showed strong immunoreactivity. Double-labeling experiments demonstrated that 50-70% of primary pulpal GAP-43-positive nerve fibers showed CGRP-like immunoreactivity. However, in adult permanent teeth the vast majority of pulpal nerve fibers also displayed intense GAP-43-like immunoreactivity both when surrounding pulpal blood vessels and in the subodontoblast/odontoblast region. There was a high degree (90-95%) of simultaneous expression of GAP-43-like immunoreactivity and CGRP-like immunoreactivity in adult permanent pulps. Immunogold GAP-43 labeling was mainly associated with the cytoplasmic side of axonal membranes. However, occasional examples of immunolabeled Schwann cells were also found. High levels of GAP-43 in normal mature permanent pulpal nerves may facilitate neural plasticity after dental wear or injury.     

Immunohistochemical localization of GAP-43 in rat and human sympathetic nervous system — effects of aging and diabetes

The neuronal 43 kDa growth associated peptide (GAP-43) is expressed in conditions of embryonic growth, axonal regeneration, and, to a limited degree, within the central nervous system as an indicator of synaptic plasticity. Although much is known about the expression of GAP-43 in cultured sympathetic neurons, information concerning the existence, immunolocalization and response of GAP-43 to experimental injury is not available for intact sympathetic ganglia in vivo. In this study we have characterized the in situ distribution and identity of GAP-43 in adult rat and human prevertebral and paravertebral sympathetic ganglia using immunohistochemical and biochemical methods. Antisera to GAP-43 intensely labeled intraganglionic presynaptic axons and synapses terminating on neurons of normal adult rat and human sympathetic ganglia in situ. There was minimal GAP-43 immunoreactivity of principal sympathetic neuron perikarya, proximal dendrites and initial axonal segments. The immunohistologic appearance of GAP-43 was unchanged in the ganglia of aged and diabetic rats and elderly humans, conditions in which presynaptic terminal axons and synapses show evidence of chronic degeneration, regeneration and neuroaxonal dystrophy, an unusual ultrastructural alteration which may represent disordered synaptic plasticity. Radioimmunoassay of ganglionic GAP-43 is comparable in young adult, aged and diabetic rat prevertebral or paravertebral sympathetic ganglia. Double immunolocalization of NPY (which labeled markedly swollen dystrophic axons) and GAP-43 in human sympathetic ganglia using a sequential immunogold-silver/fluorescence technique demonstrated that typical dystrophic axons contain little GAP-43.     

Immunohistochemical localization of GAP-43 in rat and human sympathetic nervous system--effects of aging and diabetes.

The neuronal 43 kDa growth associated peptide (GAP-43) is expressed in conditions of embryonic growth, axonal regeneration, and, to a limited degree, within the central nervous system as an indicator of synaptic plasticity. Although much is known about the expression of GAP-43 in cultured sympathetic neurons, information concerning the existence, immunolocalization and response of GAP-43 to experimental injury is not available for intact sympathetic ganglia in vivo. In this study we have characterized the in situ distribution and identity of GAP-43 in adult rat and human prevertebral and paravertebral sympathetic ganglia using immunohistochemical and biochemical methods. Antisera to GAP-43 intensely labeled intraganglionic presynaptic axons and synapses terminating on neurons of normal adult rat and human sympathetic ganglia in situ. There was minimal GAP-43 immunoreactivity of principal sympathetic neuron perikarya, proximal dendrites and initial axonal segments. The immunohistologic appearance of GAP-43 was unchanged in the ganglia of aged and diabetic rats and elderly humans, conditions in which presynaptic terminal axons and synapses show evidence of chronic degeneration, regeneration and neuroaxonal dystrophy, an unusual ultrastructural alteration which may represent disordered synaptic plasticity. Radioimmunoassay of ganglionic GAP-43 is comparable in young adult, aged and diabetic rat prevertebral or paravertebral sympathetic ganglia. Double immunolocalization of NPY (which labeled markedly swollen dystrophic axons) and GAP-43 in human sympathetic ganglia using a sequential immunogold-silver/fluorescence technique demonstrated that typical dystrophic axons contain little GAP-43.