Melatonin reduces glial reactivity in the hippocampus, cortex, and cerebellum of streptozotocin-induced diabetic rats

Hyperglycemia plays a critical role in the development and progression of diabetic neuropathy. One of the mechanisms by which hyperglycemia causes neural degeneration is via the increased oxidative stress that accompanies diabetes. Metabolic and oxidative insults often cause rapid changes in glial cells. Key indicators of this response are increased synthesis of glial fibrillary acidic protein (GFAP) and S100B, both astrocytic markers. In the present study, we examined glial reactivity in hippocampus, cortex, and cerebellum of streptozotocin (STZ)-induced diabetic rats by determining the expression of GFAP and S-100B and we evaluated the effect of melatonin on the glial response. Western blot measurement of contents in brain regions after 6 weeks of STZ-induced diabetes indicated significant increases in these constituents compared with those in nondiabetic controls. Administration of melatonin prevented the upregulation of GFAP in all brain regions of diabetic rats. Using GFAP immunohistochemistry, we observed an increase in GFAP immunostaining in the hippocampus of STZ-diabetic rats relative to levels in the control brains. Treatment with melatonin resulted in an obvious reduction of GFAP-immunoreactive astrocytes in hippocampus. Like GFAP, S100B levels also were increased in all three brain areas of diabetic rats, an effect also reduced by melatonin treatment. Finally, the levels of lipid peroxidation products were elevated as a consequence of diabetes, with this change also being prevented by melatonin. These results suggest that diabetes causes increased glial reactivity possibly due to elevated oxidative stress, and administration of melatonin represents an achievable adjunct therapy for preventing gliosis.     

Quantification of glial fibrillary acidic protein in developing sheep brain cortex, cerebellum and stem

1. The glial fibrillary acidic protein (GFAP) content of foetal, young (lamb) and adult sheep brain white (stem and cerebellum) and grey (cortex) matter-enriched regions has been determined by means of an improved ELISA using one layer of anti-human GFAP monoclonal antibody. 2. The order of GFAP concentration in brain regions was as follows: brain stem greater than cerebellum greater than cortex. 3. Postnatal brain development accounts for an increase of GFAP in all the regions. The most important increase in GFAP was observed in the adult brain and was proportionally more significant in the grey matter-enriched cortex.     

Qualitative and quantitative comparison of brain proteins in Alzheimer's disease

In human brain extracts, most proteins of pathological interest in Alzheimer's disease are insoluble and their analysis is often performed on denatured and reduced samples by immunoblotting after electrophoresis on polyacrylamide gel in presence of sodium dodecyl sulfate. Because we needed to accurately compare the concentration of several proteins in brain extracts to investigate the etiology of the disease, the quantitative aspect of immunoblotting was assessed and the results compared for a soluble component with those obtained by electroimmunoassay. Glial fibrillary acidic protein (GFAP) and Tau proteins were analysed by immunoblotting in brain homogenates treated with the Laemmli sample buffer from 10 control and 25 Alzheimer's disease brains. The linearity of densitometric measures of dilutions for one given sample was demonstrated. A 8 to 16-fold GFAP increase in Alzheimer brain was established. With regard to Tau proteins it was possible to show the presence of two pathological Tau variants (Tau 64 and 69) in all the Alzheimer brain homogenates, furthermore, the amount of Tau 64 and 69 was proportional to the presence of neurofibrillary degeneration. As far as alpha 1-antichymotrypsin is concerned, we showed, in a second set of brain samples (14 control and 12 Alzheimer brains), discrepancies between the results obtained by immunoblotting and by electroimmunoassay while for a given sample linearity of immunoblotting measures of dilutions of this sample was demonstrated. Quantitation by immunoblotting of such components which can be quantified using other procedures is uncertain whereas the interest of immunoblotting is undoubted for the insoluble proteins in the brain extracts.     

Neuroprotection by α-Lipoic Acid in Streptozotocin-Induced Diabetes

Glial cells provide structural and metabolic support for neurons, and these cells become reactive to any insult to the central nervous system. The streptozotocin (STZ) rat model was used to study glial reactivity and the prevention of gliosis by alpha-lipoic acid (alpha-LA) administration. The expression of glial fibrillary acidic protein (GFAP), S100B protein, and neuron specific enolase (NSE) was determined as well as lipid peroxidation (LPO) and glutathione (GSH) levels in some brain tissues. Western blot analyses showed GFAP, S100B, and NSE levels significantly increased under STZ-induced diabetes in brain, and LPO level increased as well. Administration of alpha-LA reduced the expression both of glial and neuronal markers. In addition, alpha-LA significantly prevented the increase in LPO levels found in diabetic rats. GSH levels were increased by the administration of alpha-LA. This study suggests that alpha-LA prevents neural injury by inhibiting oxidative stress and suppressing reactive gliosis.     

Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala after systemic kainic acid administration

Cell specific markers were quantified in the hippocampus, the amygdala/pyriform cortex, the frontal cerebral cortex and the striatum of the rat brain after systemic administration of kainic acid. Neuron specific enolase (NSE) reflects loss of neurons, glial fibrillary acidic protein (GFAP) reflects reactive gliosis, and brain levels of serum proteins measures blood-brain-barrier permeability. While the concentration of NSE remained unaffected in the frontal cerebral cortex and the striatum, their GFAP content increased during the first three days. In the hippocampus and amygdala, NSE levels decreased significantly. GFAP levels in the hippocampus were unaffected after one day and decreased in the amygdala/pyriform cortex. After that, GFAP increased strikingly until day 9 or, in the case of amygdala/pyriform cortex, even longer. This biphasic time course for GFAP was accompanied by a decrease of S-100 during days 1-9 followed by a significant increase at day 27 above the initial level. The regional differences in GFAP and S-100 could result from the degree of neuronal degeneration, the astrocytic receptor set-up and/or effects on the blood-brain barrier.     

重组反义GFAP逆转录病毒对星形胶质细胞在胶质瘢痕增生中作用的研究

本文研究了脑穿刺损伤后伤灶组织中大胶质细胞的变化、性激素对脑损伤后星形胶质细胞反应的影响,以及反义胶质原纤维酸性蛋白（ＧＦＡＰ）逆转录病毒表达载体对Ａｓｔ形态结构,反应性胶质化及胶质瘢痕形成的作用。结果表明,胶质瘢痕中增生的大胶质细胞主要是Ａｓｔ,ＧＦＡＰ对维持Ａｓｔ的形态结构及功能具有重要作用;少突胶质细胞在胶质瘢痕形成过程中不是反应活跃的细胞成分;性激素对Ａｓｔ的反应性胶质化有一定程度的抑制作     

Basic fibroblast growth factor in Alzheimer's disease

We have examined the presence of basic fibroblast growth factor (FGF) in normal and in Alzheimer brains, studied the distribution of the mitogen by immunohistochemical techniques, measured the quantities of growth factor in selected areas of the brain (Brodmann areas 10/11 and 20/21), characterized the molecular forms by Western blotting and determined its sites of synthesis by in situ hybridization. Although the same molecular forms of basic FGF are found in control and Alzheimer brains, basic FGF is increased in the brains of Alzheimer's patients. Furthermore, basic FGF is not distributed in an identical fashion to normal and Alzheimer brains, but is found in association with the lesions that characterize this disease. In normal controls (n = 5), basic FGF was found to be widely distributed throughout the three brain regions examined (prefrontal cortex, hippocampus, and hypothalamus). Immunoreactivity was observed within astrocytes in both the grey and white matter, as well as within neuronal perikarya. Brain tissues that were obtained from Alzheimer patients (N = 4) showed a substantial increase in the overall specific staining of astrocytes and neurons, particularly in areas of reactive gliosis. Focal concentration of immunoreactive basic FGF was evident within the neuritic plaques, and could be clearly seen in association with the neurofibrillary tangles present within neuronal perikarya. The possibility that basic FGF expression in the CNS is linked to the pathogenesis of the disease is discussed.     

Analysis of the Hippocampal Proteome in ME7 Prion Disease Reveals a Predominant Astrocytic Signature and Highlights the Brain-restricted Production of Clusterin in Chronic Neurodegeneration

Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ^TM/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.     

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.     

FABP7 expression in normal and stab-injured brain cortex and its role in astrocyte proliferation

Reactive gliosis, in which astrocytes as well as other types of glial cells undergo massive proliferation, is a common hallmark of all brain pathologies. Brain-type fatty acid-binding protein (FABP7) is abundantly expressed in neural stem cells and astrocytes of developing brain, suggesting its role in differentiation and/or proliferation of glial cells through regulation of lipid metabolism and/or signaling. However, the role of FABP7 in proliferation of glial cells during reactive gliosis is unknown. In this study, we examined the expression of FABP7 in mouse cortical stab injury model and also the phenotype of FABP7-KO mice in glial cell proliferation. Western blotting showed that FABP7 expression was increased significantly in the injured cortex compared with the contralateral side. By immunohistochemistry, FABP7 was localized to GFAP(+) astrocytes (21% of FABP7(+) cells) and NG2(+) oligodendrocyte progenitor cells (62%) in the normal cortex. In the injured cortex there was no change in the population of FABP7(+)/NG2(+) cells, while there was a significant increase in FABP7(+)/GFAP(+) cells. In the stab-injured cortex of FABP7-KO mice there was decrease in the total number of reactive astrocytes and in the number of BrdU(+) astrocytes compared with wild-type mice. Primary cultured astrocytes from FABP7-KO mice also showed a significant decrease in proliferation and omega-3 fatty acid incorporation compared with wild-type astrocytes. Overall, these data suggest that FABP7 is involved in the proliferation of astrocytes by controlling cellular fatty acid homeostasis.     

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。     

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.     

Changes in iNOS,GFAP and NR1 Expression in Various Brain Regions and Elevation of Sphingosine-1-phosphate in Serum after Immobilized Stress

Several studies have been suggested that long-term exposure to stress has detrimental effects on various brain functions and leads to neurodegenerative changes. However, the precise mechanism by which stress induces brain damage or neurodegenerative change is still a matter of debate. This study investigated the damage of neuronal cells involving in the expression of iNOS, NR1, and GFAP in various brain regions and characterized the change of sphingolipid metabolites as a biomarker of physiological change in serum after 3 weeks of repeated immobilization. In this report, the expression of iNOS, GFAP and NR1 in the brain of rats exposed to chronic immobilization stress was investigated. The expression of iNOS, GFAP and NR1 was elevated in the cortex and hippocampal area after 3 weeks of repeated immobilization. Immunoreactivity for GFAP and vimentin, as a marker of reactive gliosis, was also elevated in the cortex and hippocampus. The level of sphingolipids was measured in order to assess the changes in sphingolipid metabolites in the serum of rats exposed to stress. Interestingly, the level of So-1-P was increased in the plasma of rats subjected to 6-h immobilization stress than repeated immobilization. To further investigate the modulating effect of increased So-1-P in various brain regions, So-1-P was infused into the lateral cerebroventricle at a rate of 100 pmol/10 μl/h for 7 days. The expression of iNOS and NR1 was elevated in the cortex, hippocampus, striatum, and cerebellum after So-1-P infusion into the cerebroventricle, while the level of GFAP was elevated in the hippocampus and striatum. Interestingly, the expression levels of iNOS, GFAP, and NR1 were increased by the direct application of So-1-P to cultured cortical cells. These results suggest that NO production via iNOS expression, the NR1 expression, the activation of astrocytes, and the elevation of So-1-P may cause neurodegenerative changes in rats subjected to chronic immobilization and that the elevation of So-1-P by stress exposure would be one of the stress signal molecules.     

14-3-3 expression in denervated hippocampus after entorhinal cortex lesion assessed by culture-derived isotope tags in quantitative proteomics

Activation of astrocytes accompanies many brain pathologies. Reactive astrocytes have a beneficial role in acute neurotrauma but later on might inhibit regeneration. 2D-gel electrophoresis and mass spectrometry were applied to study the proteome difference in denervated hippocampus in wildtype mice and mice lacking intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin (GFAP-/-Vim-/-) that show attenuated reactive gliosis and enhanced posttraumatic regeneration. Proteomic data and immunohistochemical analyses showed upregulation of the adapter protein 14-3-3 four days postlesion and suggested that 14-3-3 upregulation after injury is triggered by reactive gliosis. Culture-derived isotope tags (CDIT) and mass spectrometry demonstrated that 14-3-3 epsilon was the major isoform upregulated in denervated hippocampus and that its upregulation was attenuated in GFAP-/-Vim-/- mice and thus most likely connected to reactive gliosis.     

Expression of neuronal plasticity markers in hypoglycemia induced brain injury

The expression of neuroplasticity markers was analyzed in four brain regions, namely cerebral hemispheres (CH), cerebellum (CB), brain stem (BS) and diencephalon (DC) from insulin-induced hypoglycemic young adult rats. Significant decrease in neural cell adhesion molecule (NCAM) isoforms and growth-associated protein-43 (GAP-43) was observed following hypoglycemic injury from majority of brain regions studied. The glial fibrillary acidic protein (GFAP) level increased significantly in cerebral hemispheres and diencephalon regions, whereas, synaptophysin level increased in cerebellum, brain stem and diencephalon regions. The selective downregulation of the neuronal plasticity marker proteins (GAP-43 and NCAM), and enhanced expression of GFAP and synaptophysin suggests that in acute hypoglycemia, mechanisms other than energy failure may also contribute to neuronal cell damage in the brain.     

Immunochemical and biochemical characterization of tau proteins in normal and Alzheimer's disease brains with Alz 50 and Tau-1

Microtubule-associated protein tau was characterized in 5 Alzheimer and 5 control brains using two monoclonal antibodies, Alz 50 and Tau-1. Quantitative analysis of immunoblots with the antibodies showed that both homogenate and supernatant fractions (12,000 x g) from Alzheimer brains contained 38-65% less tau immunoreactivity compared to normal brains. The reduction was found in all brain regions studied (frontal and temporal lobes and thalamus) and in both gray and white matter. In partially purified tau preparations, the yield of protein was lower in Alzheimer (by 35%) than in control brain. Incubation of brain proteins, transferred onto nitrocellulose paper, with alkaline phosphatase had either no effect or slightly increased the antibody binding to tau proteins from both brain tissues. Immunoblots of tau-enriched preparations subjected to two-dimensional gel electrophoresis showed no major changes in the staining pattern of tau isoforms in Alzheimer samples except for a weaker reactivity of the basic isovariants as compared to non-Alzheimer samples. The elution volume of tau from Alzheimer brain supernatant on a Sepharose CL-6B column was similar to that from non-Alzheimer brain and equal to that of aldolase (Mr = 158,000). Our data suggest that most of tau proteins from both types of brain have similar biochemical properties. The reduction in tau reactivity in Alzheimer tissue may be due to a reduction in neuronal cell population or incorporation of soluble tau into stable structures such as neurofibrillary tangles, since the tangles have been shown to react with anti-tau antibodies.     

Glial Fibrillary Acidic Protein and the Mesolimbic Dopamine System: Regulation by Chronic Morphine and Lewis-Fischer Strain Differences in the Rat Ventral Tegmental Area

Abstract— In this study we demonstrate that a 51-kDa phosphoprotein, previously identified as morphine regulated and showing different basal levels among rat strains, is glial fibrillary acidic protein (GFAP). Chronic morphine increased levels of GFAP immunoreactivity by >70% in the ventral tegmental area (VTA) of outbred Sprague-Dawley rats. This increase in GFAP content was not observed in rats that were treated concomitantly with morphine and naltrexone, an opiate receptor antagonist, and did not occur in response to a single acute injection with morphine. No alterations in GFAP levels were observed in response to chronic morphine in several other regions of the CNS studied, including the substantia nigra, locus coeruleus, cerebral cortex, and spinal cord. There were also inherent differences in levels of GFAP immunoreactivity in the VTA of drug-naive Fischer 344 and Lewis rats, two inbred rat strains that differ in their relative preference for morphine and other drugs of abuse. The VTA of drug-naive Lewis rats contained more than twofold higher levels of GFAP compared with drug-naive Fischer rats. This strain difference was also apparent in the locus coeruleus but not in several other brain regions or in spinal cord. Because the mesolimbic dopamine system is thought to play a critical role in mediating the reinforcing properties of opiates and other drugs of abuse, it is possible that the opiate induction of GFAP and inherent Lewis versus Fischer strain differences in GFAP levels in the VTA may be related to the reinforcing and/or addictive properties of opiates mediated by this brain region, as well as to genetic differences in drug preference.     

Glial Fibrillary Acidic Protein in the Cytoskeletal and Soluble Protein Fractions of the Developing Rat Brain

The distribution of glial fibrillary acidic protein (GFAP) into cytoskeletal and soluble protein fractions during development of the rat brain has been studied by quantitative immunoblotting and enzyme-linked immunosorbent assay (ELISA). These assays indicate that cytoskeletal GFAP accounts for nearly all the total GFAP in the adult rat brain, and that the developmental increase in the GFAP content of the rat brain is due to accumulation of GFAP into the cytoskeleton. A small and constant amount of the total GFAP was detected in the soluble protein fraction. This GFAP had an apparent molecular mass (Mr) similar to that of the highest Mr form of GFAP detected in the cytoskeletal fraction. In contrast to the assays for cytoskeletal GFAP, no significant increase in the GFAP concentration of the soluble protein fraction could be measured during development. Sensitive, calibrated immunoblotting of cytoskeletal and soluble protein with [125I]protein A confirmed these findings, and showed that both cytoskeletal and soluble GFAP are first detected during the same period of foetal rat brain development. A finite and reproducible amount of lower Mr forms of GFAP were observed in the cytoskeletal fraction even when prepared in the presence of stringent proteolytic inhibitors. These presumed proteolytic degradation products of GFAP increased in abundance during development, parallel to the increase in cytoskeletal GFAP content of the rat brain. However, the abundant proteolytic degradation products of GFAP found in the cytoskeletal fraction were not detected in the soluble protein fraction at any age studied.(ABSTRACT TRUNCATED AT 250 WORDS)