GFAP Protein Expression Research Articles

Experimental study of repairing acute spinal cord injury with transplantation of mesenchymal stem cells genetically modified by NGF in rats

Objective To investigate mesenchymal stem cells genetically modified by nerve growth factor to repair acute spinal cord injury in rats. Methods Fifty six Wistar rats of inbred strain were randomly divided into sham operation group cell transplantation group and simple injury group. The spinal cord injury model was prepared according to the modified Allen's method. NGFβ (hNGFβ) and GFP genes were transfected into MSCs by replication-deficient recombinant adenovirus vector (Ad-hNGFβ) and replication-deficient recombinant retrovirus vector (Rt-GFP) respectively. GFP positive MSCs were transplanted into intradural space of injured spinal cord at 7 days after spina coral injury. Spinal cord was dissected at 24 h, 1 and 2 weeks after transplantation. To observe the expression of GFAP and nestin and the distribution of MSCs after transplantation following the spina corol injury. Results MSCs migrated to the injured parenchyma In transplantation group, the expression of GFAP and NGF protein was higher than in the control group (P<0.05), the BBB score in transplantation group was higher than that in the control group (P<0.05). Conclusion The MSCs transplantation repaired the injured spinal cord to some extent.

Obesity induces functional astrocytic leptin receptors in hypothalamus

The possible role of astrocytes in the regulation of feeding has been overlooked. It is well-established that the endothelial cells constituting the blood-brain barrier transport leptin from blood to brain and that hypothalamic neurons respond to leptin to induce anorexic signaling. However, few studies have addressed the role of astrocytes in either leptin transport or cellular activation. We recently showed that the obese agouti viable yellow mouse has prominent astrocytic expression of the leptin receptor. In this study, we test the hypothesis that diet-induced obesity increases astrocytic leptin receptor expression and function in the hypothalamus. Double-labelling immunohistochemistry and confocal microscopic analysis showed that all astrocytes in the hypothalamus express leptin receptors. In adult obese mice, 2 months after being placed on a high-fat diet, there was a striking increase of leptin receptor (+) astrocytes, most prominent in the dorsomedial hypothalamus and arcuate nucleus. Agouti viable yellow mice with their adult-onset obesity showed similar changes, but the increase of leptin receptor (+) astrocytes was barely seen in ob/ob or db/db mice with their early-onset obesity and defective leptin systems. The marked leptin receptor protein expression in the astrocytes, shown with several antibodies against different receptor epitopes, was supported by RT-PCR detection of leptin receptor-a and -b mRNAs in primary hypothalamic astrocytes. Unexpectedly, the protein expression of GFAP, a marker of astrocytes, was also increased in adult-onset obesity. Real-time confocal imaging showed that leptin caused a robust increase of calcium signalling in primary astrocytes from the hypothalamus, confirming their functionality. The results indicate that metabolic changes in obese mice can rapidly alter leptin receptor expression and astrocytic activity, and that leptin receptor is responsible for leptin-induced calcium signalling in astrocytes. This novel and clinically relevant finding opens new avenues in astrocyte biology.

Inflammatory responses of the substantia nigra after acute hypoxia in neonatal rats

The neocortex and the striatum are the brain regions most known to be particularly vulnerable to acute insults like hypoxia or ischemia. In this work, we assess the possibility of cellular damage to the substantia nigra (SN) after hypoxia-reoxygenation in the new born rat. The aim of the present paper was to evaluate the expression of growth factor IGF-I, and growth factor binding proteins IGFBP-3 and IGFBP-5 genes and induction of NOS family members (nNOS, eNOS and iNOS) and TNF-α genes together with glia activation, in the SN at 5 and 48 h after severe hypoxia in the 7 day-old rat, a model for the term human fetus. At early time, while IGFs remain unchanged, we found a transient increase in eNOS and nNOS. Two days after the injury, nNOS expression remained high, iNOS and TNF-α increased and also GFAP protein expression was observed together with a profusion of reactive astrocytes distributed throughout the SN. This study on the acute effects of hypoxia on the developing brain provides additional insights into the vulnerability of the SN, a brain region involved in neurodegenerative pathologies.

Characterization of spinal HSP72 induction and development of ischemic tolerance after spinal ischemia in rats

Induction of heat shock protein (HSP72) has been implicated in the development of ischemic tolerance in several tissue organs including brain and spinal cord. In the present study, using an aortic balloon occlusion model in rats, we characterized the effect of transient noninjurious (3 or 6 min) or injurious intervals (10 min) of spinal ischemia followed by 4–72 h of reflow on spinal expression of HSP72 and GFAP protein. In a separate group of animals, the effect of ischemic preconditioning (3 or 6 min) on the recovery of function after injurious interval of spinal ischemia (10 min) was studied. After 3 min of ischemia, there was a modest increase in HSP72 protein immunoreactivity in the dorsal horn neurons at 12 h after reperfusion. After 6 min of ischemia, a more robust and wide spread HSP72 protein expression in both dorsal and ventral horn neurons was detected. The peak of the expression was seen at 24 h after ischemia. At the same time point, a significant increase in spinal tissue GFAP expression was measured with Western blots and corresponded morphologically with the presence of activated astrocytes in spinal segments that had been treated similarly. After 10 min of ischemia and 24 h of reflow, a significant increase in spinal neuronal HSP72 expression in perinecrotic regions was seen. Behaviorally, 3 min preconditioning ischemia led to the development of a biphasic ischemic tolerance (the first at 30 min and the second at 24 h after preconditioning) and was expressed as a significantly better recovery of motor function after exposure to a second 10-min interval of spinal ischemia. After 6 min ischemic preconditioning, a more robust ischemic tolerance at 24 h after preconditioning then seen after 3-min preconditioning was detected. These data indicate that 3 min of spinal ischemia represents a threshold for spinal neuronal HSP72 induction, however, a longer sublethal interval (6 min) of preconditioning ischemia is required for a potent neuronal HSP72 induction. More robust neurological protection, seen after 6 min of preconditioning ischemia, also indicates that HSP72 expression in spinal interneurons seen at 24 h after preconditioning may represent an important variable in modulating ischemic tolerance observed during this time frame.

Neuropathologic study of long term cyanide administration to goats

Cyanogenic glycosides, which release cyanide, are present in several plant species of high importance for animal production, such as cassava and sorghum. Several human neurological diseases have been associated with chronic cyanide exposure. On the other hand, these effects in ruminants are almost unknown. Thus, the objective of the present study was to determine the long-term lesions of the central nervous system (CNS) caused by daily administration of potassium cyanide (KCN) to goats. Thirty-four male goats were divided into five groups, respectively treated orally with 0 (control), 0.3, 0.6, 1.2 or 3.0 mg KCN/kg/day for 5 months. At the end of the experiment, the whole CNS of each animal was collected for histopathology and immunohistochemistry for apoptotic markers (BAX, BCl2 and CPP32) and for glial fibrillary acid protein (GFAP; vimentin). The results showed the presence of spheroids in the pons, medulla oblongata, and ventral horn of the spinal cord, gliosis and spongiosis in medulla oblongata, gliosis in the pons, and damaged Purkinje cells in the cerebellum from goats that received the higher cyanide dose. In goats from the 1.2 mg KCN/kg group we observed congestion and hemorrhage in the cerebellum, and spheroids in the spinal cord. Gliosis was confirmed by GFAP protein expression. Immunohistochemistry for apoptotic markers and typical apoptotic morphology suggested apoptosis did not participate in the pathogenesis of the observed lesions. Thus, chronic cyanide exposure can promote neurophatological lesions also in goats, and this species can be a useful ruminant model to study the neurotoxic effects of long-term cyanide exposure.

Neurogenic differentiation of murine and human adipose-derived stromal cells

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone. As early as 1–3 h after neuronal induction, the phenotype of ADAS cells changed towards neuronal morphology. Following neuronal induction, muADAS cells displayed immunocytochemical staining for GFAP, nestin and NeuN and huADAS cells displayed staining for intermediate filament M, nestin, and NeuN. Following neuronal induction of murine and human ADAS cells, Western blot analysis confirmed GFAP, nestin, and NeuN protein expression. Pretreatment with EGF and basic FGF augmented the neuronal differentiation of huADAS cells. The neuronal differentiation of stromal cells from adipose tissue has broad biological and clinical implications.

Expression of connexin genes in hippocampus of kainate-treated and kindled rats under conditions of experimental epilepsy

We have analyzed whether the expression of connexin genes is altered in the hippocampus of kindled and kainate-treated rats, i.e., animal models of human temporal lobe epilepsy. We have tested this hypothesis by analyzing mRNA, protein abundance and cellular location of connexins (Cx) 43, 36, 32 and 30. The expression of glial fibrillary acid protein and mRNA was also monitored both in kainate-treated and kindled rats, in order to take into account reactive gliosis under these conditions. We found significantly increased expression of GFAP mRNA (100%) and protein (178%) in kainate-treated rats 4 weeks after kainate application, whereas in kindled rats only moderate increases of GFAP mRNA and protein were detected 2–3 weeks (group 2) or 4–6 weeks (group 1) after the last stage 5 induced seizure. Under gliotic conditions, connexins 43 and 30 mRNA or protein expression in astrocytes of kainate-treated rats were nearly unaffected. Cx36 mRNA expression (presumably in neurons) was significantly reduced (44%), whereas abundance of Cx36 protein was only slightly reduced. In both groups of kindled rats, Cx30 and Cx43 mRNA or protein expression were either slightly decreased or unchanged. Again, Cx36 mRNA and protein expression were reduced by about half in group 2. Immunofluorescence analysis of Cx43, Cx36 and Cx30 expression revealed that 4 weeks after the last kainate administration or kindling, cellular localization of these connexins was indistinguishable from control animals.

Astrocytic demise precedes delayed neuronal death in focal ischemic rat brain

Active neuronal–glial interaction is important in the maintenance of brain homeostasis and is vital for neuronal survival following brain injury. The time course of post-ischemic astroglial dysfunction and neuronal death was studied in the spontaneously hypertensive rat (SHR) brain following permanent middle cerebral artery occlusion (MCAO). In situ hybridization with 35 S -labeled riboprobes for GFAP and GLUT3 was used to monitor mRNA expression in glia and neurons. Astrocytic proteins GFAP, vimentin, S100, Glutathione- S-Transferase Y b (GST Y b) and neuronal protein TG2 were detected by immunofluorescence. Cells were co-stained with in situ end labeling (ISEL) to detect DNA fragmentation, a hallmark of cell death. GFAP mRNA expression declined rapidly in the ischemic region of the cortex and was almost absent by 12 h. Immunohistochemical studies revealed a parallel decline in the corresponding protein: a reduction in GFAP staining was apparent in the infarct after 3 h and by 24 h, there was essentially no remaining GFAP. Three other glial proteins (vimentin, S100 and GST Y b) disappeared from infarct over a similar time course. A few ISEL positive cells were observed in the infarct at 6 h, but maximal detection was not seen until 24–48 h. Most of the ISEL-positive cells were neurons, identified by co-staining with the neuronal marker TG2. Few cells expressing GFAP or other glial markers were positive at any time point. Neuronal GLUT3 mRNA declined more slowly than GFAP mRNA in the ischemic core and disappeared during the period of neuronal death. Concurrent with the loss of GFAP mRNA and protein expression in the infarct, there was a rapid rise in GFAP mRNA in the peri-infarct region of ipsilateral hemisphere and proximal region of the contralateral hemisphere. This was followed by the enhanced GFAP protein expression characteristic of reactive astrocytes, but over a significantly slower time course. These studies show that MCAO leads to a rapid decline of GFAP mRNA and glial proteins, which appears to precede the decline in neuronal mRNA and neuronal death within the infarct. Early astroglial dysfunction may play a critical role in determining the outcome of acute hypoxic–ischemic injury by compromising neuronal–glial interactions.

Glutamate metabolism is down-regulated in astrocytes during experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) was induced in SJL/J mice by adoptive transfer of MBP-reactive T cells in order to investigate the role of astrocytes in pathology. GFAP protein and mRNA expression (analyzed using semiquantitative Western blot and RT-PCR techniques) were upregulated in the spinal cord of mice, which had developed a complete paralysis of hind- and fore-limbs and tail (grade 4 EAE), thus establishing that reactive gliosis occurred under these experimental conditions. Within the same samples and using similar techniques, we found that glutamine synthetase (GS) and glutamate dehydrogenase (GDH) expression were dramatically reduced. These two astrocytic enzymes are responsible for degradation of glutamate, the most abundant excitatory neurotransmitter in the brain. Since elevated levels of glutamate may be neurotoxic, we propose that the decreased capacity of astrocytes to metabolize glutamate may contribute to EAE pathology.

METHOTREXATE INCREASES GFAP mRNA AND PROTEIN EXPRESSION IN CULTURED ASTROCYTES

METHOTREXATE INCREASES GFAP mRNA AND PROTEIN EXPRESSION IN CULTURED ASTROCYTES

Transferrin receptor molecules in human cell lines derived from the CNS malignant gliomas: Immunohistochemical and flow-cytometric study.

The ten human cell lines derived from glioma tissues were characterized for the aspects of the phenotypic expression of GFAP and S-100 protein, DNA-ploidy, doublhg time, and response to DBc-AMP and LAK cells. The expression of the TfR was evaluated on these established cell lines with correlation to their phenotypic markers and characteristics. As a result of immunohistochemical studies on frozen materials using a monoclonal anti-Tfit antibody, the high expression of the TfR was related to the doubling time (36-48 hr), but not to the phenotypic expression of glial markers (GFAP and S-100 protein) and other characteristics. And thus, rapidly proliferating astrocytic cell lines including C6 and U251 showed the high level of the TfR in the cells.For the flow cytometry (FCM), the cell harvest with trypsin abolished the extracellular part of TfR molecules from the cell membrane, and thus, single cell preparation with trypsin was not appropriate for the study. On the other hand, EDTA (0.02%) with mechanical shaking provided a preservation of the TfR, molecules for FCM study. The series of FCM analysis demonstrated that newly synthesized (or recycled) TfR molecules appear in the extracellular matrix of the cell membrane by 12 hr and then increase in quantity up to 28 hr after trypsin digestion.The results suggest that the TfR molecules are functioning in rapidly proliferating cells derived from the central nervous system as described previously in other types of tissue.