Differential expression of fibronectin, tenascin-C and NCAMs in cultured hippocampal astrocytes activated by kainate, bacterial lipopolysaccharide or basic fibroblast growth factor

Different reports demonstrated that reactive glial cells express increased amounts of adhesion and matrix molecules. Despite a wealth of information on the expression of these molecules during development and after lesion, very little is known of how this expression is regulated. In the present report we used Western blots and immunocytochemistry to investigate the expression of neural cell adhesion molecule (NCAM), fibronectin and tenascin-C in cultured astrocytes from rat hippocampus. The effects of three different extracellular signals were analyzed: the glutamatergic receptor agonist kainic acid, the basic fibroblast growth factor (bFGF) and the bacterial lipopolysaccharide. Each treatment had a specific pattern of glial activation and differentially modified the expression of these proteins. Treatment of astrocytes with kainic acid resulted in an increase of tenascin-C, a decrease of fibronectin and a shift of NCAMs isoforms: NCAM 140 and PSA-NCAM (polysialic acid-rich NCAMs) were increased while NCAM 120 was decreased. bFGF increased fibronectin, tenascin-C and NCAM 120, while decreasing PSA-NCAM. Finally, the treatment of astrocytes with lipopolysaccharide induced a significant increase of fibronectin, tenascin-C and NCAM 120 but did not modify the expression of NCAM 140 and PSA-NCAM. These data suggest different mechanisms for modulation of cell surface interactions. They suggest that glial activation by bFGF and lipopolysaccharide are associated with an increase of the adhesive properties, while kainate action is rather associated with a decrease of the adhesiveness of astrocytes.     

Differential regulation of ciliary neurotrophic factor and its receptor in the rat hippocampus following transient global ischemia

To investigate a potential role of ciliary neurotrophic factor (CNTF) in transient global ischemia, we have studied the postischemic regulatory changes in the expression of CNTF and its receptor, the ligand-binding alpha-subunit (CNTFRalpha). Immunoblot analysis demonstrated CNTF levels were slightly upregulated already during the first day after ischemia and then increased markedly by more than 10-fold until 2 weeks postischemia. Immunoreactivity for CNTF became detectable 1 day after ischemia and was localized in reactive astrocytes. The intensity of the immunolabeling was maximal in CA1 during the phase of neuronal cell death (days 3-7 postischemia) and in the deafferented inner molecular layer of the dentate gyrus. Upregulation of CNTF expression was less pronounced in CA3 and absent in the stratum lacunosum moleculare and the outer molecular layer of the dentate gyrus and thus did not simply correlate with astroliosis as represented by upregulation of glial fibrillary acidic protein (GFAP). As shown by in situ hybridization, expression of CNTFRalpha mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Following ischemia, reactive astrocytes, identified by double labeling with antibodies to GFAP, transiently expressed CNTFRalpha mRNA with a maximum around postischemic day 3. This astrocytic response was most pronounced in CA1 and in the hilar part of CA3. These results show that CNTF and its receptor are differentially regulated in activated astrocytes of the postischemic hippocampus, indicating that they are involved in the regulation of astrocytic responses and the neuronal reorganizations occurring after an ischemic insult.     

Splicing factor NSSR1 reduces neuronal injury after mouse transient global cerebral ischemia

This study focuses on the function of NSSR1, a splicing factor, in neuronal injury in the ischemic mouse brain using the transient global cerebral ischemic mouse model and the cultured cells treated with oxygen‐glucose deprivation (OGD). The results showed that the cerebral ischemia triggers the expression of NSSR1 in hippocampal astrocytes, predominantly the dephosphorylated NSSR1 proteins, and the Exon3 inclusive NCAM‐L1 variant and the Exon4 inclusive CREB variant. While in the hippocampus of astrocyte‐specific NSSR1 conditional knockdown (cKD) mice, where cerebral ischemia no longer triggers NSSR1 expression in astrocytes, the expression of Exon3 inclusive NCAM‐L1 variant and Exon4 inclusive CREB variant were no longer triggered as well. In addition, the injury of hippocampal neurons was more severe in astrocyte‐specific NSSR1 cKD mice compared with in wild‐type mice after brain ischemia. Of note, the culture media harvested from the astrocytes with overexpression of NSSR1 or the Exon3 inclusive NCAM‐L1 variant, or Exon4 inclusive CREB variant were all able to reduce the neuronal injury induced by OGD. The results provide the evidence demonstrating that: (1) Splicing factor NSSR1 is a new factor involved in reducing ischemic injury. (2) Ischemia induces NSSR1 expression in astrocytes, not in neurons. (3) NSSR1‐mediated pathway in astrocytes is required for reducing ischemic neuronal injury. (4) NCAM‐L1 and CREB are probably mediators in NSSR1‐mediated pathway. In conclusion, our results suggest for the first time that NSSR1 may provide a novel mechanism for reducing neuronal injury after ischemia, probably through regulation on alternative splicing of NCAM‐L1 and CREB in astrocytes. GLIA 2015;63:826–845     

Transient upregulation of the AT2 receptor mRNA level after global ischemia in the rat brain

The present study examined changes in angiotensin receptors (AT1 and AT2) and angiotensinogen mRNA level after global ischemia in the rat brain. The AT2 mRNA level increased by three-fold in both the cortex and hippocampus, which are known to be sensitive to ischemic injury, 3 h after ischemia. The increase thus appeared only during the early reperfusion period. In the striatum, amygdala and cerebellum, the level increased moderately 3 h and/or 24 h after ischemia; there was no change in the hypothalamus. On the other hand, the AT1A and AT1B receptor mRNA levels were not altered in the cortex or hippocampus during the early reperfusion period, even 3 h and 24 h after ischemia. There was no significant alteration in angiotensinogen mRNA level 3 h or 24 h after ischemia. These results suggest that the transient upregulation of AT2 receptor mRNA occurs in the cortex and hippocampus after injury and these changes may be in some way related to the molecular events which lead to delayed neuronal cell death.     

Expression of glial cell line-derived neurotrophic factor induced by transient forebrain ischemia in rats.

This study examined the expression of glial cell line-derived neurotrophic factor (GDNF) mRNA and the cellular localization of GDNF production in rats subjected to transient forebrain ischemia induced by four-vessel occlusion. Transient forebrain ischemia induced GDNF mRNA expression in the hippocampus from 3 h to 3 days after the ischemic episode, with peak expression at 6 h. The GDNF mRNA increase in the cerebral cortex was similar to that in the hippocampus, whereas no increase in GDNF mRNA was observed in the striatum and brainstem. Western blot analysis showed that GDNF in the hippocampal CA1 region was increased slightly from 3 to 24 h after the ischemia, and then subsequently declined to below the baseline level. In the hippocampus, GDNF was evenly produced in pyramidal neurons of both sham-operated rats and normal rats, as determined by immunohistochemistry. Interestingly, we found that ischemia-induced reactive astrocytes, as well as surviving neurons, produced GDNF in 3-7 days after the ischemia. On the other hand, in other regions, such as the cerebral cortex, striatum, and brainstem, there was no change in GDNF-positive cells secondary to ischemia. These findings suggest that expression of GDNF mRNA is regulated in part via ischemia-induced neuronal degeneration. They also suggest that ischemia-induced reactive astrocytes may produce GDNF to protect against neuronal death. Therefore, GDNF may play an important role in ischemia-induced neuronal death in the brain.     

Basic FGF in adult rat brain: cellular distribution and response to entorhinal lesion and fimbria-fornix transection.

Basic fibroblast growth factor (bFGF) is a potent trophic factor for neurons and astrocytes and recently has been implicated in the pathology of Alzheimer's disease. In order to better understand the role of bFGF in normal brain function and during pathology, we have analyzed its anatomical distribution and its response to injury in the CNS. Double-staining immunohistochemistry showed that bFGF immunoreactivity was localized in astrocytes, in select neuronal populations, and occasionally in microglial cells throughout the normal rat brain. Neuronal populations that showed bFGF immunoreactivity included septohippocampal nucleus, cingulate cortex, subfield CA2 of the hippocampus, cerebellar Purkinje cells, cerebellar deep nuclei, facial nerve nucleus, and the motor and spinal subdivisions of the trigeminal nucleus and facial nerve nucleus. The pattern of bFGF immunoreactivity in the hippocampus was examined following entorhinal cortex lesion, or fimbria-fornix transection. After entorhinal cortex lesion, bFGF immunoreactivity increased in the outer molecular layer of the dentate gyrus ipsilateral to the lesion. The lesion effect on bFGF immunoreactivity was expressed as an increase in the number of bFGF astrocytes, as an increase in the intensity of bFGF immunoreactivity within astrocytes, and as an increase of bFGF immunoreactivity in the surrounding extracellular matrix, relative to the contralateral side. The time course and pattern of reorganization paralleled the sprouting of septal cholinergic terminals in response to the same type of lesion, suggesting that bFGF may play an important role in lesion-induced plasticity. After transection of the fimbria-fornix, chronic infusion of bFGF appeared to preserve NGF receptors on neurons within the medial septal complex and, as previously reported, prevent the death of medial septal neurons. Therefore, it appears that bFGF infusion, which has been shown to increase the synthesis of NGF by astrocytes (Yoshida and Gage, 1991), also helps enable neurons to respond to NGF. This suggests that after injury bFGF may participate in a cascade of neurotrophic events, directly and indirectly facilitating neuronal repair and/or promoting neuronal survival.     

Presence of apolipoprotein E immunoreactivity in degenerating neurones of mice is dependent on the severity of kainic acid-induced lesion

Apolipoprotein E (apoE) is a major apolipoprotein in the central nervous system (CNS) that may play a role in various CNS disorders. ApoE is primarily localised in astrocytes, but neuronal apoE mRNA expression has been demonstrated in normal and diseased human brain, as well as in ischaemic rat brain. To obtain further insight into the role of apoE in neuronal degeneration in the CNS and conditions of neuronal apoE localisation, we have investigated in mice the distribution of apoE following neuronal injury induced by kainic acid (n=35, 25 or 35 mg kainic acid/kg BW). Consecutive series of brain sections were immunostained for apoE and markers for astroglia (GFAP) and microglia/macrophage cells (CR3). Degenerating neurones were identified with a silver-degeneration staining technique. The intensity and cellular distribution of apoE-immunoreactivity (apoE-ir) was dependent on the severity of neuronal injury. Mice that developed mild neuronal degeneration, restricted to a subset of neurones in the hippocampus, showed increased apoE-ir in astrocytes concomitant with increased GFAP-ir and mild microgliosis. In these mice, no neuronal apoE-ir was detected. In contrast, mice developing severe neuronal injury in the hippocampus - frequently also showing degeneration in other brain regions including cortex, thalamus, striatum and amygdala - showed intense apoE-ir in degenerating neurones. Surrounding the lesion, apoE-ir was increased in neuropil recurrently whereas GFAP-ir astrocytes disappeared. Thus, in mice apoE accumulates in degenerating neurones in conditions of severe neuronal injury putatively in association with disruption of the glial network.     

Effects of neonatal stress on markers of synaptic plasticity in the hippocampus: Implications for spatial memory

Early stressful adverse situations may increase the vulnerability to cognitive deficits and psychiatric disorders, such as depression. Maternal separation (MS) has been used as an animal model to study changes in neurochemistry and behavior associated with exposure to early‐life stress. This study investigated the effects of neonatal stress (MS) on the expression of synaptic plasticity markers in the hippocampus and a purported relationship to cognitive processes. Spatial learning (Morris water maze) significantly increased the expression of total levels of the neural cell adhesion molecule (NCAM), as well as its three major isoforms (NCAM‐120, ‐140, and ‐180) both in the control and MS groups. Interestingly, these increases in NCAM expression after learning were lower in MS animals when compared with control rats. MS induced a significant decrease in total levels of NCAM, and specifically, in the NCAM‐140 isoform expression. In the hippocampus of MS rats there was a significant decrease in brain‐derived neurotrophic factor and synaptophysin mRNA densities. Cell proliferation, measured as BrdU‐positive cells, was also decreased in the dentate gyrus of MS rats. Altogether these results suggest that MS can alter normal brain development, providing a potential mechanism by which early environmental stressors may influence vulnerability to show cognitive impairments later in life. © 2009 Wiley‐Liss, Inc.     

Changes in spinal GDNF, BDNF, and NT-3 expression after transient spinal cord ischemia in the rat

Previous studies have demonstrated that the expression of several growth factors including glial cell-derived neurotrophic factor (GDNF), brain-derived growth factor (BDNF), and neurotrophin-3 (NT-3) play an important role in defining neuronal survival after brain ischemia. In the present study, using a well-defined model of transient spinal ischemia in rat, we characterized the changes in spinal GDNF, BDNF, and NT-3 expression as defined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence coupled with deconvolution microscopy. In control animals, baseline levels of GDNF, BDNF, and NT-3 (74 +/- 22, 3,600 +/- 270, 593 +/- 176 pg/g tissue, respectively) were measured. In the ischemic group, 6 min of spinal ischemia resulted in a biphasic response with increases in tissue GDNF and BDNF concentrations at the 2-hr and 72-hr points after ischemia. No significant differences in NT-3 concentration were detected. Deconvolution analysis revealed that the initial increase in tissue GDNF concentration corresponded to a neuronal upregulation whereas the late peak seen at 72 hr corresponded with increased astrocyte-derived GDNF synthesis. Increased expression of BDNF was seen in neurons, astrocytes, and oligodendrocytes. These data suggest that the early increase in neuronal GDNF/BDNF expression likely modulates neuronal resistance/recovery during the initial period of postischemic reflow. Increased astrocyte-derived BDNF/GDNF expression corresponds with transient activation of astrocytes and may play an active role in neuronal plasticity after non-injurious intervals of spinal ischemia.     

Differential expression of Musashi1 and nestin in the adult rat hippocampus after ischemia

Both nestin and the neural RNA-binding protein Musashi1 (Msi1) are expressed in neural stem cells in the subventricular zone. Neurogenesis in the hippocampus has received much attention, so we evaluated the expression of Msi1 and nestin in the adult rat hippocampus after transient forebrain ischemia. Both Msi1 and nestin were induced in the reactive astrocytes after ischemia, especially in the CA1 region, until 35 days after ischemia. Induction of both molecules suggested that reactive astrocytes might have immature characteristics. In the subgranular zone (SGZ) of the hippocampal dentate gyrus, Msi1-positive cells formed clusters after ischemia. These cells were labeled by bromodeoxyuridine (BrdU) but did not express glial fibrillary acidic protein. In contrast, very few nestin-positive cells were labeled by BrdU. Our results suggest that neuronal progenitor cells in the SGZ expressed Msi1 but not nestin.     

Dexamethasone enhances upregulation of nerve growth factor mRNA expression in ischemic rat brain.

Nerve growth factor (NGF) is well-established as a trophic factor that plays a crucial role in neuroregeneration and plasticity after brain insults. Dexamethasone (DEX), a powerful glucocorticoid steroid, has long been used in the clinical management of neurological disorders. We examined the relationship between NGF and DEX after an ischemic insult to the brain. In situ hybridization was used to measure NGF mRNA expression in the rat hippocampus after 20 min of transient forebrain ischemia. Immunostaining for NGF protein was performed using the avidin-biotin peroxidase method. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was also used to study the astrocyte reaction in the hippocampal CA1 area. Ischemic brain from rats not treated with DEX had a 2 and 3 fold increase in NGF mRNA compared to sham-operated rats at 4 and 6 h after ischemia, respectively. The NGF mRNA expression returned to basal levels 12 h to 7 days post-ischemia. Treatment with DEX potentiated the ischemia-induced increase of NGF mRNA to 4 times that of sham-operated rats at 6 h following reperfusion and NGF protein expression was similarly elevated. Additionally, the number of GFAP positive astrocytes in the CA1 region in the ischemic rats was markedly increased. These data suggest that DEX may play a role in modulating NGF mRNA expression in the hippocampal neuronal response to brain ischemia.     

Up-regulation of the peripheral-type benzodiazepine receptor expression and [(3)H]PK11195 binding in gerbil hippocampus after transient forebrain ischemia

In mammalian CNS, the peripheral-type benzodiazepine receptor (PTBR) is localized on the outer mitochondrial membrane within the astrocytes and microglia. The main function of PTBR is to transport cholesterol across the mitochondrial membrane to the site of neurosteroid biosynthesis. The present study evaluated the changes in the PTBR density, gene expression and immunoreactivity in gerbil hippocampus as a function of reperfusion time after transient forebrain ischemia. Between 3 to 7 days of reperfusion, there was a significant increase in the maximal binding site density (B(max)) of the PTBR antagonist [(3)H]PK11195 (by 94-156%; P < 0.01) and PTBR mRNA levels (by 1.8- to 2.9-fold; P < 0.01). At 7 days of reperfusion, in the hippocampal CA1 (the brain region manifesting selective neuronal death), PTBR immunoreactivity increased significantly. Increased PTBR expression after transient forebrain ischemia may lead to increased neurosteroid biosynthesis, and thus may play a role in the ischemic pathophysiology.     

VEGF mRNA Induction Correlates With Changes in the Vascular Architecture Upon Spinal Cord Damage in the Rat

The multiple cellular and molecular processes induced by injury to the central nervous system (CNS) are still poorly understood. In the present study, we investigated the response of the vasculature and the expression of mRNA for the angiogenic vascular endothelial growth factor (VEGF) following X-irradiation of the spinal cord in the newborn and following traumatic spinal cord injury in the adult rat. Both lesion models induced changes in the density and the distribution pattern of blood vessels: while X-irradiation led to a permanent local increase in vascular density in the fibre tracts of the exposed segments, a transient local sprouting of vessels was induced upon traumatic spinal cord injury. In situ hybridization showed that an increase of VEGF mRNA anticipated and overlapped with the vascular responses in both lesion models. In addition to the temporal correlation of VEGF expression and vascular sprouting, there was a clear correlation in the spatial distribution patterns. Following X-irradiation, the expression of VEGF mRNA was restricted to the fibre tracts, precisely the areas where the changes in the vasculature were observed later on. Upon transection in the adult animal, VEGF was mainly detectable at the border of the lesion area, where the transient increase in vascular density could be observed. Interestingly, according to the type of lesion applied, astrocytes (X-irradiation) or inflammatory cells (presumably microglial cells or macrophages; traumatic lesion) are the cellular sources of VEGF mRNA. Our results strongly indicate that VEGF is crucially involved in mediating vascular changes following different types of injury in the CNS.     

The modulations of NCAM polysialylation state that follow transient global ischemia are brief on neurons but enduring on glia

To investigate the role of polysialylated neural cell adhesion molecule (NCAM PSA)-mediated plasticity after injury, we examined the temporal and spatial expression of NCAM PSA immunoreactivity in the medial temporal lobe following global ischemia. Male Mongolian gerbils were subjected to bilateral common carotid artery occlusion for 5 min and killed at increasing times post-occlusion. The well-characterized delayed CAl pyramidal cell death was observed 5-7 days post-occlusion. At post-occlusion days 1-2 there was a small but significant increase of NCAM PSA-positive hippocampal granule cells followed by an equally significant decrease at post-occlusion day 5. In contrast, a substantial increase in glial PSA expression was observed in all hippocampal regions at 1-7 days post-occlusion that was associated generally with stellate astroglia and specifically with the radial processes of glia traversing the granule cell layer of the dentate gyrus. Administration of the glutamate antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-ben-zo(F)quinoxaline significantly blocked the ischemia-induced modulation of neuronal and glial NCAM PSA expression. Astroglial NCAM polysialylation became attenuated by 35 days post-occlusion except in the CAI area of cell death. The temporal and regional pattern of polysialylated NCAM expression in the ischemic gerbil hippocampus implicates this neuroplastic marker in mechanisms of neurotrophic-dependent repair/remodeling that ensue following transient interruption of blood flow.     

Cell cycle protein expression in proliferating microglia and astrocytes following transient global cerebral ischemia in the rat

Cerebral ischemia induces microglial and astroglial activation, which may play a crucial role in the development of ischemic neuronal damage. In this study, we examined the role of cell cycle proteins in glial proliferation in the hippocampus following 10min of global cerebral ischemia in the rat. Proliferating cells were identified with immunostaining for proliferating cell nuclear antigen (PCNA), and glial cells were visualized with immunostaining for microglial response factor-1 (microglia/macrophages) and glial fibrillary acidic protein (astrocytes). Expression of cyclin D1 and cyclin-dependent kinase-4 was also examined with double label immunohistochemistry. Proliferating cells in the CA1 region after ischemia consisted of microglia and much fewer astrocytes. Microglial activation and proliferation (7.6-fold increase in number after 7 days) were preceded by an increase in PCNA-positive microglia; 83% of microglia were PCNA-positive after 2 days. Astrocytes increased by 1.8-fold after 7 days, and only 6% of astrocytes became PCNA-positive by day 7. Cyclin D1 and cyclin-dependent kinase-4 immunoreactivity appeared in these glial cells in parallel with the expression of PCNA. The findings suggest that the accumulation of brain macrophages elicited by transient cerebral ischemia is caused predominantly by activation and proliferation of resident microglia through the upregulation of cell cycle proteins.