Hippocampal Kindling in the Rat Is Associated with Time-Dependent Increases in the Concentration of Glial Fibrillary Acidic Protein

The effect of hippocampal kindling on the regional brain concentration of total glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes, was studied in partially kindled rats, and in fully kindled rats after a post-kindling period of 24 h, 1 week, and 2 months. GFAP concentration was measured in arbitrary units by dot-blots. In the hippocampus, dentate gyrus, basolateral amygdala, pyriform cortex, and entorhinal cortex, limbic structures which are known to be involved in the kindling process, there was an increase in GFAP concentration which was maximal in the fully kindled animals studied after 24 h. In most brain areas, GFAP concentration was still elevated 1 week post-kindling, but had declined to control level 2 months post-kindling. A significant increase in GFAP was also found in septum, ventral pallidum/accumbens nucleus, and primary motor cortex of kindled rats with a post-kindling period of 24 h, whereas in several other brain regions GFAP was unchanged. These results suggest that astrocyte activation, indicative of degenerative changes in nearby neurons, is a transient and regional phenomenon in kindling occurring only during the development of the kindled state.     

NDRG2与GFAP在不同脑区星形胶质细胞的表达与分布

目的:观察NDRG2(N-myc下游调节基因2)与GFAP(胶质纤维酸性蛋白)在不同脑区星形胶质细胞的表达与分布。方法:利用免疫荧光NDRG2与GFAP双标技术以及Western Blot技术观察皮层、海马及纹状体等不同脑区星形胶质细胞NDRG2和GFAP的表达与分布。结果:免疫荧光结果显示NDRG2阳性细胞广泛而均匀地分布于不同脑区,并与GFAP存在较好的共定位;NDRG2与GFAP标记的星形胶质细胞形态不尽相同。Western Blot结果显示NDRG2在皮层中表达比海马和纹状体多,而GFAP在海马中表达比皮层和纹状体多。结论:NDRG2广泛表达于不同脑区星形胶质细胞,并于GFAP存在较好的共定位。     

Chronic Administration of Lithium Chloride Increases Immunodetectable Glial Fibrillary Acidic Protein in the Rat Hippocampus

Abstract: We studied the effect of treating rats with lithium salts on the content and in vitro phosphorylation rate of the astrocyte cell marker, glial fibrillary acidic protein (GFAP), in brain slices. Rats were fed a diet incorporating lithium chloride until the concentration of Li⁺ in serum reached 0.6–1.2 m M , a range similar to that achieved in clinical practice. Hippocampal tissue was analyzed for immunoreactive GFAP by a dot assay, and slices of hippocampus and caudate nucleus were labeled with [³²P]-phosphate to determine the in vitro rate of phosphorylation of GFAP. Compared with controls, the level of immunoreactive GFAP in the hippocampus from lithium-treated rats was increased 34%, and GFAP in hippocampal slices incorporated 39% more ³²P. This effect of lithium was apparently not confined to the hippocampus because the in vitro rate of phosphorylation of GFAP in caudate slices was also increased in the treated rats.     

Decrease of Glial Fibrillary Acidic Protein in Rat Frontal Cortex Following Aluminum Treatment

Aluminum lactate was injected either intraperitoneally or stereotactically into the lateral cerebral ventricles of rats. Rats were killed at various times after treatment, and frontal cortex, hippocampus, and striatum were dissected out. Microtiter plate-based sandwich ELISA and immunohistochemistry were used to measure the glial fibrillary acidic protein (GFAP) concentration. GFAP levels were significantly decreased in frontal cortex 7 days after a single lateral ventricular injection of aluminum lactate and 14 days following systemic treatment. In contrast, neither hippocampus nor striatum exhibited any significant changes in the content of this astrocytic intermediate filament protein after aluminum treatment. Levels of a predominantly astroglial enzyme, glutamine synthetase, were also selectively reduced in the frontal cortex following intraventricular injection of aluminum. This depression exhibited a regional and temporal specificity similar to that found for GFAP. These results suggest a selective and progressive diminution of astrocytic responsivity in frontal cortex following either systemic or intraventricular aluminum dosing. The depression of GFAP levels reported here, which was found in the rat cerebral cortex 7-14 days after aluminum treatment in a species that does not form neurofilamentous aggregates, may reflect extended impairment of astrocytic function and suggests that these cells may be the primary targets of aluminum neurotoxicity.     

Changes in the Solubility of Glial Fibrillary Acidic Protein After Ischemic Brain Damage in the Mouse

Abstract: In the present study, changes in the content of glial fibrillary acidic protein (GFAP) in mouse cortex were investigated at different time intervals after unilateral middle cerebral artery occlusion. The GFAP content was assessed semiquantitatively by ELISA and immunoblotting. GFAP immunoreactivity was determined for each animal separately in protein fractions obtained from the ipsilateral, lesioned cortex and the contralateral, unlesioned cortex. Changes in the GFAP content of the lesioned cortex with respect to that of the unlesioned cortex were calculated for each fraction individually. GFAP was detectable in all protein fractions with a significant amount recovered from the aqueous extracts. A pronounced increase in the GFAP content of the lesioned cortex was observed. As measured by ELISA, this increase was maximal 5 days after injury and significantly more pronounced for the soluble and the Triton X-100-soluble protein fractions (mean increase 7 days after lesion, 281.4 and 240.2%, respectively) than for the crude cytoskeletal fraction (mean increase, 153.3%). A small and transient increase in GFAP immunoreactivity was also found in all protein fractions prepared from the contralateral, unlesioned cortex. These results were confirmed by immunoblotting.     

碘过量对后代鼠脑NSE和GFAP表达的影响

目的观察碘过量对后代鼠神经元特异性烯醇化酶和胶质纤维酸性蛋白表达的影响。方法将断乳后一个月Wistar大鼠随机分为五组（NI、5HI、10HI、50HI和100HI）,饮用不同浓度的碘水,饲养3个月后雌雄合笼,取第二代60日龄鼠,测量血清甲状腺激素值,以NSE和GFAP为观察指标,研究后代鼠大脑的发育情况。结果各碘过量组甲状腺激素水平呈下降趋势,100HI组呈明显甲低状态;NSE的免疫组化和形态计量学显示：海马CA3区NSE阳性细胞的NA、VV,和灰度值随碘过量的严重程度而呈下降趋势,在100HI组呈明显的统计学差异。各组GFAP阳性星形胶质细胞阳性显色强弱未见明显不同。结论大鼠对碘摄入量的增高,只有在100HI组可以观察到以NSE表达降低为主要特点的脑发育障碍,其发病机理可能与碘过量所致的甲状腺功能低下有关。     

Properties of astrocytes cultured from GFAP over-expressing and GFAP mutant mice

Alexander disease is a fatal leukoencephalopathy caused by dominantly-acting coding mutations in GFAP. Previous work has also implicated elevations in absolute levels of GFAP as central to the pathogenesis of the disease. However, identification of the critical astrocyte functions that are compromised by mis-expression of GFAP has not yet been possible. To provide new tools for investigating the nature of astrocyte dysfunction in Alexander disease, we have established primary astrocyte cultures from two mouse models of Alexander disease, a transgenic that over-expresses wild type human GFAP, and a knock-in at the endogenous mouse locus that mimics a common Alexander disease mutation. We find that mutant GFAP, as well as excess wild type GFAP, promotes formation of cytoplasmic inclusions, disrupts the cytoskeleton, decreases cell proliferation, increases cell death, reduces proteasomal function, and compromises astrocyte resistance to stress.     

Analysis of Cyclic AMP-Dependent Changes in Intermediate Filament Protein Phosphorylation and Cell Morphology in Cultured Astroglia

Receptor agonists that increase cyclic AMP levels in cultured astroglia have been shown to increase 32P-labeling of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin in these cells. Experiments were designed to determine if the increase in 32P-labeling resulted from either an increase in the turnover or net number of phosphates associated with the intermediate filament proteins and if the phosphorylation of these proteins causally affected astroglial morphology. Time course experiments indicated that 6-8 h were required to reach steady-state 32P-labeling of both GFAP and vimentin. Treatment with forskolin (10 microM) after steady-state 32P-labeling increased GFAP and vimentin phosphorylation fourfold and twofold, respectively, and also induced a morphological change from polygonal to process-bearing cells within 20-30 min of drug addition. Cells incubated in media containing brain extract (30%) for 24 h at 37 degrees C and then 3 h at 23 degrees C underwent changes from polygonal to process-bearing cells with no apparent increase in the phosphorylation of either GFAP or vimentin. Treatment of process-bearing cells (induced by brain extract) or polygonal cells with 10 microM forskolin at 23 degrees C resulted in a three- to fourfold increase in GFAP phosphorylation without significant morphological changes. These results suggest that forskolin stimulation of GFAP and vimentin increases net number of phosphates associated with these intermediate filament proteins and that the resulting increase in phosphorylation can be dissociated from morphological changes.     

Biochemical Correlates of Epilepsy in the El Mouse: Analysis of Glial Fibrillary Acidic Protein and Gangliosides

The E1 (epileptic) mouse is considered a model for complex partial seizures in humans. Seizures in E1 mice begin around 7-8 weeks of age and persist throughout life. To determine if astrocytic gliosis was present in adult seizing E1 mice, the distribution of glial fibrillary acidic protein (GFAP) was studied in the hippocampus using an antibody to GFAP. The mean number of GFAP-positive cells per square millimeter of hippocampus was approximately 15- to 40-fold higher in adult E1 mice than in nonseizing control C57BL/6J (B6) mice or in young nonseizing E1 mice. Relative GFAP concentration (expressed per milligram of total tissue protein) in hippocampus and cerebellum was estimated by densitometric scanning of peroxidase-stained western blots. GFAP concentration was 2.7-fold greater in hippocampus of adult seizing E1 mice than in the control B6 mice. No differences in GFAP content were detected between the strains in the cerebellum. Because gangliosides can serve as cell surface markers for changes in neuronal cytoarchitecture, they were analyzed to determine if the gliotic response in E1 mice was associated with changes in neural composition. Although the total ganglioside concentration of hippocampus, cerebral cortex, and cerebellum was similar in adult E1 and control B6 mice, a synaptic membrane enriched ganglioside, GD1a, was elevated in the adult E1 cerebral cortex and hippocampus. The findings indicate that E1 mice express a type of gliosis that is not accompanied by obvious neuronal loss.     

Adrenalectomy Blocks the Compensatory Increases in UT-A1 and AQP2 in Diabetic Rat Kidney

In normal rats we showed that glucocorticoids participate in the downregulation of UT-A1 protein abundance in the inner medullary tip and in lowering of basal and vasopressin-stimulated facilitated urea permeability in terminal IMCDs. To examine the relevance of this response to a rat model of human disease, we studied rats with uncontrolled diabetes mellitus (DM) induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We found that at 3 days of DM, UT-A1 protein abundance is downregulated in the inner medullary tip compared to pair-fed control rats, while DM for more than 7 days caused an increase in UT-A1. To test whether adrenal steroids could be a mechanism contributing to the latter increase, we studied adrenalectomized rats (ADX), ADX rats given STZ to induce diabetes (ADX + STZ), and ADX + STZ rats receiving exogenous aldosterone or dexamethasone. In contrast to control rats, UT-A1 protein abundance was not increased by prolonged DM in the ADX rats. Aquaporin 2 (AQP2) was not increased in the inner medullas of 10-day DM rats either. However, UT-A1 protein abundance was significantly reduced in the inner medullary tips from both diabetic aldosterone-treated (40 ± 2%) and dexamethasone-treated (43 ± 2%) ADX rats compared to diabetic ADX rats without steroid replacement. AQP2 was unaffected by steroid hormone treatments. Thus, both mineralocorticoids and glucocorticoids downregulate UT-A1 protein abundance in rats with uncontrolled diabetes mellitus for 10 days. These results suggest that: 1) the increase in UT-A1 observed in DM is dependent upon having adrenal steroids present; and 2) adrenal steroids are not sufficient to enable the compensatory rise in UT-A1 to a steroid-deficient diabetic animal.     

Lentivirus-mediated microRNA-26a-modified neural stem cells improve brain injury in rats with cerebral palsy

This study is launched to investigate the effect of lentivirus-mediated microRNA-26a (miR-26a)-modified neural stem cells (NSCs) in brain injury in rats with cerebral palsy (CP). The successfully constructed miR-26a lentivirus expression vector and empty vector virus were used to modify NSCs. The model of CP with ischemia and anoxia was established in rats. NSCs and miR-26a-NSCs were stereoscopically injected into the cerebral cortex of the modeled rats, respectively. The survival and migration of NSCs infected with recombinant lentivirus expressing green fluorescence in vivo was observed under a light microscope. The neurobehavioral functions, morphology, and ultrastructure of cerebral cortex and hippocampus, apoptosis of brain cells, expression of apoptosis-related protein caspase-3 and Bax, together with the expression of the glial fibrillary acidic protein (GFAP) in cerebral cortex and hippocampus were determined. Expression of miR-26a in NSCs infected with plVTHM-miR-26a increased significantly. After NSCs transplantation, the neurobehavioral status of CP rats was improved, the degree of brain pathological injury was alleviated, the apoptotic index of cells in cerebral cortex and hippocampus and the expression of the apoptotic protein (caspase-3 and Bax) were decreased, the expression of GFAP were significantly decreased. After miR-26a-NSCs transplantation, these aforementioned results further improved or decreased. Our study suggests that miR-26a-modified NSCs mediated by lentivirus can improve brain injury, inhibit apoptosis of brain cells and activation of astrocytes in CP rats.     

Effects of chronic ethanol treatment on glial fibrillary acidic protein expression in adult rat optic nerve: an immunocytochemical study

Glial fibrillary acidic protein (GFAP) is used as a marker of astrocyte response to various central nervous system injuries. In the present study, the effects of chronic ethanol administration on GFAP immunoreactivity were evaluated in astrocytes of the adult optic nerve head. The results demonstrated that ethanol exposure significantly and dramatically increases GFAP immunoreactivity and the number of immunoreactive astrocytes (p<0.001). In addition, GFAP immunoreactive cells in the optic nerve showed extensive hypertrophy (p<0.001).     

Adrenalectomy Attenuates Kainic Acid-Induced Spectrin Proteolysis and Heat Shock Protein 70 Induction in Hippocampus and Cortex

Abstract: Glucocorticoids have been shown to exacerbate the damaging effects of a variety of neurotoxic insults in the hippocampus and other brain areas. Evidence suggests that the endangering effects of glucocorticoids may be due to augmenting the cascade of events, such as elevations in intracellular calcium levels, because of excitatory amino acid (EAA) receptor stimulation. A potential mechanism responsible for EAA-induced neuronal damage is activation of calcium-sensitive proteases, such as calpain, which then proteolytically degrade cytoskeleton structural proteins, such as spectrin. The present study was designed to determine if glucocorticoids can regulate the spectrin proteolysis produced by the EAA agonist, kainic acid. Rats were adrenalectomized (ADX) or sham operated and 7 days later injected with kainic acid (10 mg/kg). Twenty-four hours later rats were killed and tissues obtained for western blot analyses of the intact spectrin molecule and the proteolytically derived breakdown products. Kainic acid produced an approximate sevenfold increase in the 145–155-kDa spectrin breakdown products in the hippocampus relative to ADX or sham rats injected with vehicle. ADX attenuated the kainic acid-induced increase in breakdown products by 43%. In a similar way, kainic acid produced a large 10-fold increase in spectrin breakdown products in the frontal cortex, which was also significantly attenuated (?80%) by ADX. Induction of heat shock protein 70 (hsp70) by neurotoxic insults has been suggested to be a sensitive indicator of cellular stress in neurons. Kainic acid induced large amounts of hsp70 in both hippocampus and frontal cortex of sham-operated rats that was markedly attenuated (85–95%) by ADX. There was a strong positive correlation between the amount of spectrin proteolysis and the degree of hsp70 induction in both the hippocampus and frontal cortex. In contrast, kainic acid did not significantly produce spectrin proteolysis and induced only a very modest and inconsistent increase of hsp70 in the hypothalamus. This is consistent with the observation that the hypothalamus is relatively insensitive to the neurotoxic effects of systemically administered kainic acid. The dose of kainic acid (10 mg/kg) used in this experiment produces a 10-fold elevation in circulating corticosterone levels at both 1 and 3 h after administration. These results suggest that part of the endangering effects of glucocorticoids on hippocampal and cortical neurons may be due to augmentation of calpain-induced spectrin proteolysis. The attenuation of kainic acid-induced synthesis of hsp70 by ADX indicates that the cellular stress produced by EAAs is regulated in part by glucocorticoids. In addition, the elevation in endogenous corticosterone levels produced by kainic acid appears to be a significant factor contributing to the neuronal damage produced by this agent.     

Ganglioside Modulation of Cyclic AMP-Dependent Protein Kinase and Cyclic Nucleotide Phosphodiesterase In Vitro

The expression of glial fibrillary acidic protein (GFAP)-mRNA during mouse brain development and in astroglial primary cultures has been investigated by using two approaches: Northern-blot evaluation using a specific cDNA probe, and cell-free translation associated with immunoprecipitation. During brain maturation (4-56 days postnatal), the GFAP-mRNA underwent a biphasic evolution. An increase was observed between birth and day 15 (i.e., during the period of astroglial proliferation), which was followed by a decrease until day 56 (i.e., during astroglial cell differentiation). At older stages (300 days), an increase was observed, which might reflect gliosis. During astroglial in vitro development (7-32 days in culture), the GFAP-mRNA showed similar variations. An increase, observed during the period of astroglial proliferation (7-18 days), was followed by a decrease which occurred in parallel to marked changes in cell shape, cell process outgrowth, and the organization and accumulation of gliofilaments. During the same culture period (7-32 days), alpha-tubulin mRNA, which was used as an internal standard, did not vary significantly. These results show that the increase of the GFAP protein and of gliofilaments observed both in vivo and in vitro during astroglial differentiation cannot be ascribed to an accumulation of the GFAP-mRNA. It might be that more than one mechanism regulates the levels of free and polymerized GFAP and of its encoding mRNA.     

Astrogliosis in the hippocampus and cortex and cognitive deficits in rats with streptozotocin-induced diabetes: Effects of melatonin

We examined, using a Western blot technique, the contents and compositions of a specific neuronal protein, NCAM, and of an astrocyte marker, GFAP, in the hippocampus and cortex of rats with streptozotocin (STZ)-induced diabetes and compared these indices with those in control (intact) animals and STZ-diabetic rats treated with melatonin. Behavioral cognitive indices manifested in the passive avoidance test (PAT) and Morris water maze (MWM) learning performance were also estimated in the above groups of animals. As was found, STZ-diabetic rats demonstrated clear cognitive deficits according to the values of the retention latency in the PAT and time of reaching the escape platform in the MWM performance. In these animals, the GFAP content was elevated, and the amount of degraded products of this protein increased, as compared with the control. Simultaneously, considerable down-regulation of the NCAM expression and modifications of NCAM isoform composition were found in diabetic animals. In addition, significantly increased levels of lipid peroxidation (according to the amounts of malondialdehyde + 4-hydroxyalkenals) were measured in the cortex and hippocampus of rats with stable diabetic hyperglycemia. All the above-mentioned shifts were significantly smoothed or even nearly completely compensated in the case of treatment of STZ-diabetic rats with melatonin (10 mg/kg per day). The role of diabetes-related changes in the amount and composition of specific neural and glial proteins in the development of cognitive deficits, the involvement of oxidative stress in the mechanisms of the respective shifts, and possible mechanisms of the neuroprotective effect of melatonin with respect to diabetes-related pathological biochemical and behavioral shifts are discussed. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 105–111, March–April, 2008.     

Glial Fibrillary Acidic Protein Increases in the Spinal Cord of Lewis Rats with Acute Experimental Autoimmune Encephalomyelitis

Glial fibrillary acidic protein (GFAP) in the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) was quantitated by densitometry of both stained gels and immunoblots of electrophoretically separated cytoskeletal proteins. The experimental period ranged from 7 to 65 days postinoculation (dpi). Greater than 92% of the total spinal cord GFAP was recovered in the Triton-insoluble cytoskeletal pellet; less than 2% was truly soluble. GFAP increased gradually and significantly with time, reaching a level one-and-a-half to two times greater than that of controls by 35 dpi and remaining elevated at 65 dpi. In EAE animals, GFAP was 33% of the total Triton-insoluble protein (excluding histones and other small basic proteins) at 7 dpi, rising to 48% at 65 dpi. Increases in vimentin were also noted, following a time course similar to that of GFAP. An increase in immunocytochemical staining of GFAP was noticeable at 10 dpi and became marked at 14 dpi, a time before GFAP levels had increased significantly. Thus, enhanced staining at the peak of the disease cannot be explained simply by an increase in antigen protein. Other possible explanations, such as an increase in soluble GFAP content, proteolytic degradation, or modifications in the immunochemical properties of GFAP in EAE animals, were ruled out. Both the biochemical and immunocytochemical increases in GFAP persisted through 65 dpi, even though the animals recovered from clinical signs at approximately 18 dpi.     

Effects of vitamin E against aluminum neurotoxicity in rats

The present study examined the protective effects of vitamin E against aluminum-induced neurotoxicity in rats. Wistar rats were given daily aluminum via their drinking water containing 1600 mg/liter aluminum chloride for six weeks. Aluminum induced a significant increase in lipid peroxidation (LPO) in hippocampus and frontal cortex. Furthermore, aluminum caused marked elevation in the levels of the glial markers (glial fibrillary acidic protein (GFAP) and S100B) and proinflammatory cytokines (TNF-alpha and IL-1beta) in both brain areas. Vitamin E treatment reduced the contents of glial markers and cytokines and the levels of LPO. In conclusion, this study demonstrates that vitamin E ameliorates glial activation and reduces release of proinflammatory cytokines induced by aluminum.     

Rapid Increases in Glial Fibrillary Acidic Protein mRNA and Protein Levels in the Copper-Deficient, Brindled Mouse

The brindled mouse (MObr/y) carries an X-linked mutation that produces severe copper deficiency. Affected males suffer profound deficits in oxidative metabolism. We have examined astrocyte pathology in MObr/y during development and have found marked changes in the metabolism of glial fibrillary acidic protein (GFAP). Immunocytochemistry with anti-GFAP antisera revealed a marked increase in staining at postnatal day 12 (P12), compared to heterozygous female and unaffected male littermates, particularly in neocortex and thalamus. Septum, hypothalamus, and striatum showed little change. Western blot analysis revealed increased levels of GFAP in MObr/y forebrain and cerebellum. Levels of GFAP mRNA were determined by Northern blotting with a mouse GFAP cDNA probe. At P10, mRNA levels were normal, but increased to 8-10 times normal by P12. Levels at P15 remained similarly elevated. Thus, immunostaining and protein determinations correlate with mRNA elevations. Astrocytes can alter GFAP mRNA and protein levels over a relatively short time. Counts of neocortical cells did not reveal differences in cell numbers between MObr/y and controls, indicating that the observed changes reflect increased cellular levels and not a large increase in the numbers of astrocytes.