Acquisition of neuronal and glial markers by neural crest-derived cells in the mouse intestine

Enteric neurons and glia arise from the neural crest. The phenotype of crest-derived cells was examined as they differentiated into neurons or glia in the mouse small and large intestine. Previous studies have shown that undifferentiated enteric crest-derived cells are Phox2b(+)/Ret(+)/p75(+)/Sox10(+), and at embryonic day (E) 10.5, about 10-15% of the crest-derived cells in the small intestine have started to differentiate into neurons. In the current study, by E12.5 and E14.5, about 25% and 47%, respectively, of Phox2b(+) cells in the small intestine were immunoreactive to the pan-neuronal protein, ubitquitin hydrolase (PGP9.5), and the percentage did not change dramatically from E14.5 onward. The differentiation of crest-derived cells into neurons in the colon lagged behind that in the small intestine by several days. Differentiating enteric neurons showed high Ret, low p75, and undetectable Sox10 immunostaining. Glial precursors were identified by the presence of brain-specific fatty acid binding protein (B-FABP) and detected first in the fore- and rostral midgut at E11.5. Glial precursors appeared to be B-FABP(+)/Sox10(+)/p75(+) but showed low Ret immunostaining. S100b was not detected until E14.5. Adult glial cells were B-FABP(+)/Sox10(+)/p75(+)/S100b(+). A nucleic acid stain (to identify all ganglion cells) was combined with immunostaining for PGP9.5 and S100b to detect neurons and glial cells, respectively, in the postnatal intestine. At postnatal day 0, fewer than 5% and 10% of cells in myenteric ganglia of the small and large intestine, respectively, were neither PGP9.5(+) nor S100b(+). Because some classes of neurons are not present in significant numbers until after birth, the expression of PGP9.5 by developing enteric neurons appeared to precede the expression of neuron type-specific markers.     

Inhibition of vascular endothelial growth factor receptor 2 activity in experimental brain contusions aggravates injury outcome and leads to early increased neuronal and glial degeneration

Angiogenesis following traumatic brain injuries (TBIs) may be of importance for post-traumatic reparative processes and the development of secondary injuries. We have previously shown expression of vascular endothelial growth factor (VEGF), a major regulator of endothelial cell proliferation, angiogenesis and vascular permeability, and VEGF receptors (VEGFR1 and 2) after TBI in rat. In the present work we tried to further elucidate the role of VEGF after TBI by performing specific VEGFR2 activity inhibition. In rats subjected to VEGFR2 blockage we report an increased haemorrhagic area (P < 0.05), early increase in serum levels of neural injury marker neuron-specific enolase (P < 0.05) and glial injury marker S100beta (P < 0.05), and increased numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling- (TUNEL-) and FluoroJade B- (P < 0.05) positive cells, all increases preceding the known VEGF/VEGFR vascular response in brain trauma. An increase in lesion area, as measured by decreased microtubuli-associated protein 2 expression (P < 0.05) and increased glial fibrillary acidic protein reactivity (P < 0.05), could also be demonstrated. In addition, vascular density, as measured by von Willebrandt factor-positive cells, was decreased (P < 0.05). No differences in post-traumatic inflammatory response, as measured by stainings for macrophages, granulocytes and intracellular adhesion molecules, were shown between the groups. Taken together, our findings point towards VEGF/VEGFR2 up-regulation after TBI as being an important endogenous cytoprotective mechanism in TBI. The possible importance of VEGF on the vascular, neuronal and glial compartments of the neurovascular unit after TBI is discussed.     

Patients with suspected benign tumors and glial fibrillary acidic protein autoantibody: an analysis of five cases

Abstract

Aim: To investigate the clinical features of five glial fibrillary acidic protein (GFAP) antibody positive patients with suspected benign tumors and explore its underlying pathogenesis.

Materials and methods: Overall, 1018 serum and cerebrospinal fluid (CSF) samples were tested by indirect immunofluorescence assay and data from five patients with suspected tumors and positive for GFAP autoantibody in the CSF were analyzed retrospectively.

Results: The positive rate of GFAP antibody in the serum and CSF was 3.93% by indirect immunofluorescence assay. Tumors were diagnosed before or after neurologic onset in 5 of 40 patients (12.5%) and no deterioration of the tumors was found during the long-term follow-up. Of the five patients, one patient suffered a thyroid nodule, one patient had a small nodule in the left lung, two patients suffered meningiomas, and one patient had a suspicious eosinophilic granuloma.

Conclusion: GFAP autoimmunity may be a paraneoplastic immune response with a low frequency of tumor in Chinese patients with GFAP astrocytopathy.     

The relationship between glial distortion and neuronal changes following intestinal ischemia and reperfusion

Background Damage to mucosal epithelial cells, muscle cells and enteric neurons has been extensively studied following intestinal ischemia and reperfusion (I/R). Interestingly, the effects of intestinal I/R on enteric glia remains unexplored, despite knowledge that glia contribute to neuronal maintenance. Here, we describe structural damage to enteric glia and associated changes in distribution and immunoreactivity of the neuronal protein Hu. Methods The mouse small intestine was made ischemic for 3 h and reperfused from 1 to 12 h. Immunohistochemical localisation of glial fibrillary acidic protein (GFAP), Hu and TUNEL were used to evaluate changes. Key Results At all time points glial cells became distorted, which was evident by their altered GFAP immunoreactivity, including an unusual appearance of bright perinuclear GFAP staining and the presence of GFAP globules. The numbers of neurons per ganglion area were significantly fewer in ganglia that contained distorted glia when compared with ganglia that contained glia of normal appearance. The distribution of Hu immunoreactivity was altered at all reperfusion time points. The presence of vacuoles and Hu granules in neurons was evident and an increase in nuclear Hu, relative to cytoplasmic Hu, was observed in ganglia that contained both normal and distorted glial cells. A number of neurons appeared to lose their Hu immunoreactivity, most noticeably in ganglia that contained distorted glial cells. TUNEL reaction occurred in a minority of glial cells and neurons. Conclusions & Inferences Structural damage to gliofilaments occurs following I/R and may be associated with damage to neighboring neurons.     

Inhibitory effect of synthetic small interfering RNAs on glial fibrillary acidic expression in astrocytes*☆

BACKGROUND： Glial fibritlary acidic protein （GFAP） expression highly correlates with spinal glial scar formation, and is regarded as an important target for scar therapy. Efficient inhibition of expression could benefit recovery from spinal cord injury. OBJECTIVE： To investigate the inhibitory effects of synthetic small interfering RNAs （siRNAs） on astrocytic GFAP expression in rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment at the cellular and molecular level was performed at the First Hospital of Dalian Medical University between June 2005 and February 2006. MATERIALS： A total of 100 seven-day-old, Sprague Dawley rats were selected. GAPDH siRNA was purchased from Ambion, USA, And TransMessengerTM Transfection Reagent from DAKO, Carpinteria, CA. METHODS： Rat astrocytes were isolated and cultured. Three pairs of 21-nucleotide （nt） siRNAs specific to rats GFAP mRNA, 401,404 and 854, were synthesized and transfected in primary astrocytes at 1, 2, 3, and 4 g/L using TransMessengerTM Transfection Reagent. Non-transfected astrocytes served as the blank group. Cells transfected with siRNA were regarded as the negative control group, with GAPDH siRNA as the positive control group, and 401 siRNA, 404 siRNA, and 854 siRNA as experimental groups. MAIN OUTCOME MEASURES： GFAP mRNA and protein expression were assessed by RT-PCR and Western blot, respectively, at 24, 48, and 72 hours of culture. RESULTS： GFAP mRNA expression in the positive control group was significantly less than the negative control group （P 〈 0.01）. GFAP mRNA expression in astrocytes from three pairs of siRNA was significantly less than the blank group after 48 hours （P 〈 0.01 ）, while no differences were detected between the negative control and blank groups （P 〉 0.05）. GFAP protein expression was remarkably less in siRNA-transfected astrocytes compared to the blank control （P 〈 0.01 ）. CONCLUSION： Transfected siRNAs could significantly inhibit GFAP gene expression in astrocyt     

Colocalization of taurine and glial fibrillary acidic protein immunoreactivity in mouse hippocampus induced by short-term ethanol exposure

Morphological changes of the hippocampus were investigated in mice exhibiting signs of intoxication following short-term exposure to 6% ethanol. These alterations were examined by a double immunofluorescent study using antibodies to taurine and anti-glial fibrillary acidic protein (GFAP) antibody. Antibody-labeled taurine was localized mainly in the astrocytes and endothelial cells of control mice. Ethanol administration resulted in a significant increase in the accumulation of taurine and GFAP immunoreactivity (IR) in the stratum lacunosum-moleculare (sl-m) of the hippocampus. Specifically, the cell bodies of taurine-positive astrocytes were hypertrophied, their processes were elongated in the pericapillary region, and some colocalized with GFAP-IR cells. Furthermore, quantitative analysis revealed that the merged area in ethanol-treated mice was twice that (71.6% vs. 35.8%) of control mice. Since taurine is involved in various neuroprotective functions, the present observations suggest that the expression of taurine IR in reactive astrocytes after ethanol exposure might play an important role in neuroprotective processes.     

Inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cAMP-induced differentiation of rat C6 glial cell line

Dioxin is suspected to cause adverse effects on the development of the central nervous system (CNS). To investigate the neurotoxic effects of dioxin on the differentiation of astrocytes, rat C6 glial cell line was used as a model, because these cells are induced to express astrocyte markers and to change the cell morphology toward an astrocytic phenotype by increasing intracellular cAMP levels. When C6 cells were simultaneously exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and N(6),O(2')-dibutylyl cAMP (dbcAMP), the expression of cytochrome P-450 1A1 (CYP1A1) was dramatically increased, and the expression of aryl hydrocarbon receptor (AhR) was moderately decreased in a dose-dependent manner. In addition, extension of astrocytic processes was inhibited by 1 nM TCDD that did not reduce cell viability. TCDD also inhibited the induction of glial fibrillary acidic protein (GFAP) expression in a dose-dependent manner, until the end of a 72-hr exposure period. This inhibition was restored by the addition of an antagonist of AhR, alpha-naphthoflavone. These results indicate that TCDD inhibits astrocytic differentiation of C6 cells, which may be mediated by an AhR-dependent pathway.     

Protective effects of melatonin against ethanol-induced reactive gliosis in hippocampus and cortex of young and aged rats

Evidence has been accumulated indicating that chronic ethanol consumption leads to direct or indirect changes in the viability of central nervous system cells. The effects of aging and chronic ethanol consumption on glial markers [glial fibrillary acidic protein (GFAP) and S100B] and oxidant and antioxidant status of rats were studied. Furthermore, protective effects of melatonin against aging and alcohol consumption were also assayed. Chronic ethanol administration to young and aged rats produced an increase in lipid peroxidation, and a decline in glutathione (GSH) levels, which was significantly reversed by the co-administration of melatonin. Lipid peroxidation status was markedly affected in aged rats treated with alcohol compared to the young rats. An age-related increase in GFAP and S100B levels were found in the cortex and hippocampus. Long-term alcohol exposure resulted in distinct elevation in GFAP content in young rats (P < 0.01) while there was less increase in the cortex of aged rats (P < 0.05). In old rats, hippocampal GFAP levels were not significantly changed by alcohol treatment (P > 0.05). Co-administration of melatonin with alcohol significantly reduced GFAP contents both in the hippocampus (P < 0.01) and cortex (P < 0.001) of aged rats. No significant effects of alcohol treatment were found on the levels of neuron-specific enolase (NSE) in aged rats. This finding suggests that melatonin exerts its protective effect on injured nervous tissues by scavenging free radicals and stabilizing glial activity against the damaging effects of ethanol and aging. Furthermore, this work suggests that the signal to initiate gliosis is mediated, at least indirectly, by free radical formation.     

Colonization of the bowel by the precursors of enteric glia: studies of normal and congenitally aganglionic mutant mice

The terminal portion of the ls/ls mouse is congenitally aganglionic because the precursors of enteric neurons fail to enter this region. This animal was studied in order to gain insight into the origin of enteric glia and into the process by which the precursors of these cells colonize the gut. In control (CD-1) mice, immunoreactivity of the glial marker, glial fibrillary acidic protein, appeared for the first time in the fetal bowel at day E16 and, in adults, was much more intense within intraenteric neural elements than in nerves outside the bowel. Glial fibrillary acidic protein developed in tissue cultures of fetal intestine explanted before the protein appeared in situ, and before the bowel became innervated by extrinsic nerves; thus, the precursors of cells able to elaborate glial fibrillary acidic protein must have been present, but unrecognizable, in the original explants. This explant assay demonstrated that these glial precursors were present in all regions of the bowel of control mice, but not in the presumptive aganglionic bowel of ls/ls mice. The nerves (of extrinsic origin) in the aganglionic tissue of ls/ls mice showed a high level of immunoreactive glial fibrillary acidic protein; nevertheless, their ultrastructure was typical of peripheral nerve, not enteric plexus, and they contained Schwann cells, not enteric glia. These observations support the view that enteric glia are derived from the single wave of neural crest colonists that populates the enteric nervous system before the gut receives its extrinsic innervation. These glial precursors, like neuronal precursors, tend to be excluded from the presumptive aganglionic ls/ls bowel. In contrast, Schwann cells grow into the abnormal ls/ls gut with the extrinsic innervation. The enteric microenvironment appears to promote the expression of glial fibrillary acidic protein in both enteric glia and Schwann cells; however, even within the bowel, Schwann cells retain their characteristic morphology. It is thus probable that the normal enteric nervous system contains supporting cells of separate lineages, enteric glia and Schwann cells.     

Heparan sulfate modifies the effects of basic fibroblast growth factor on glial reactivity

Our previous studies have shown that injection of basic fibroblast growth factor (bFGF) into a brain wound enhances astrocyte hypertrophy and macrophage-microglia proliferation in areas adjacent to the lesion. In the present study, designed to test the effects of co-administration of bFGF and heparan sulfate (HS), rats received injections of 200 ng bFGF, 200 ng bFGF with 50 μg HS, or 50 μg HS into a brain wound. Glial proliferation and astrocyte hypertrophy were evaluated in seven non-overlapping subfields in the mid-cortex including the wound edge. Our results show that bFGF-HS, compared to bFGF or HS alone, enhanced the total area of GFAP staining in all subfields except the one nearest to the wound edge. The combination of bFGF and HS did not increase total glial or astrocyte proliferation. We propose that the observed effects resulted from a greater diffusion of bFGF-HS complex into the brain parenchyma, where it bypassed low-affinity binding sites that would otherwise sequester free bFGF. Our results suggest that bFGF-HS complex, compared to bFGF alone, may gain entry into the brain more readily, reach higher concentrations and be more effective as a neurotrophic agent.     

An immunohistochemical study of glial fibrillary acidic (GFA) protein and S-100 protein in the colon affected by Hirschsprung's disease

Summary The supportive cells of the enteric nervous system were examined in gut tissues from 15 patients with Hirschsprung's disease by means of immunohistochemistry, utilizing antisera to glial fibrillary acidic (GFA) protein and S-100 protein. In the normoganglionic segment, GFA protein immunoreactivity was predominantly found in association with the myenteric plexus and to a lesser extent in the submucous plexus. On the other hand, the extrinsic, hypertrophic nerve fasciculi were selectively immunostained with GFA protein antiserum throughout the entire length of the aganglionic intestinal walls from all children studied. The large fasciculi were numerous in the distal aganglionic segment and commonly appeared in the intermuscular zone and submucosal connective tissue. Both small-and mediumsized nerve fasciculi with GFA protein immunoreactivity were also encountered within the circular muscle layer of the proximal aganglionic segment. A subpopulation of supportive cells within the hypertrophic nerve fasciculi showed immunoreactivity for GFA protein, while all supportive elements of these fasciculi were stained for S-100 protein. The intrinsic nerve fibers within the circular muscle layer of normoganglionic segments were stained for S-100 protein, but not for GFA protein. The present study supports our previous findings that two types of supportive cells can be differentiated by immunohistochemistry in the enteric nervous system, utilizing antisera to GFA protein and S-100 protein. It is also concluded that the demonstration of GFA protein by immunohistochemical methods favors the diagnosis of aganglionic colons with Hirschsprung's disease, since GFA protein immunoreactivity is confined to the extrinsic, hypertrophic nerve fasciculi characteristic of aganglionic bowels.     

A glial fibrillary acidic protein (GFA)-containing cell clone derived from mouse cerebella transformed ‘in vitro’ by SV-40

A clonal cell line containing the glial fibrillary acidic protein (GFA) was derived from cultures of 14 day post-natal C57Bl mouse cerebella, transformed by SV-40. All the cells are GFA-positive — even in sparse exponential cultures — have an astroglial morphology and contain the intranuclear SV-40 T-antigen.     

Expression of vimentin and glial fibrillary acidic protein in ethylnitrosourea-induced rat gliomas and glioma cell lines

Summary The expression of glial fibrillary acidic protein (GFAP) and vimentin was investigated immuno-histochemically in 104 experimental gliomas induced by transplancental application of ethylnitrosourea (ENU) in CDF rats. Immunoreactivity for vimentin was prominent in many astrocytic tumor cells and especially in small glioma cells forming anaplastic medulloblastoma-like foci in many tumors. The majority of tumor cells in oligodendroglial tumors were vimentin negative, except for some of the large polymorphous oligodendrogliomas which contained intermingled vimentin positive glioma cells. GFAP immunoreactivity was detectable only in a low fraction of tumor astrocytes and in a few exceptional cases some oligodendroglial tumor cells stained positive. Immunohistochemistry with antibodies against neurofilaments and cytokeratins revealed no staining in tumor cells of ENU-induced gliomas, while all oligoden-drogliomatous tumors stained positive for HNK-1. Immunocytological and immunoblot investigations of the two rat glioma cell clones RG2 and F98, which are both derived from ENU-induced gliomas, showed a prominent expression of vimentin in monolayer cultures and in syngeneic intracerebral transplantation tumors. F98 additionally demonstrated a fraction of GFAP positive cells especially in confluent cultures and in intracerebral tumors. RG2, on the other hand, exhibited virtually no GFAP immunoreactivity in culture but showed individual GFAP positive tumor cells in intracerebral tumors. Our results revealed a more precise picture of the cellular differentiation in ENU-induced rat gliomas and in two widely used glioma cell lines. They underline the heterogeneity of experimental rat gliomas which may comprise cells at different stages of differentiation towards the oligodendroglial or astroglial phenotype.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

Endothelin-3 and glial fibrillary acidic protein expression in the frontal and parietal cortex of type-2 diabetic mice following ischemia-reperfusion injury☆

BACKGROUND： Patients with type-2 diabetes mellitus exhibit higher levels of plasma endothelin-1 （ET-1）. However, very few reports exist regarding altered endothelin-3 （ET-3） and ET-1 concentrations in brain tissue. OBJECTIVE： To observe expression changes of ET-3 and glial fibrillary acidic protein （GFAP） in the frontal and parietal cortex of type-2 diabetic mice following ischemia-reperfusion injury, with various reperfusion durations. DESIGN, TIME AND SETTING： Randomized controlled animal study. The experiment was conducted in the Xiangya Medical College of Central South University and the Third Xiangya Hospital between February 2002 and January 2003. MATERIALS： Sixty-six, adult, male, Kunming mice, weighing （30 ± 5） g, as well as rabbit anti-ET-3 polyclonal and rabbit anti-GFAP polyclonal antibodies, were provided by the Neurobiology Institute of Second Military Medical University in Japan. METHODS： Sixty-six mice were randomly divided into five groups： diabetes mellitus （DM, n = 6）, diabetes mellitus with ischemia-reperfusion （DM/IR, n = 24）, ischemia-reperfusion （IR, n = 24）, sham operation （SO, n = 6）, and control （n = 6）. MAIN OUTCOME MEASURES： Following ischemia-reperfusion for 1, 3, 5, and 10 days, respectively, expression of ET- 3 and GFAP was immunohistochemically measured in the frontal and parietal cortex. RESULTS： All 66 mice were included in the final result analysis. In the IR and DM/IR groups, ET-3- and GFAP-positive neurons increased in the frontal and parietal cortex in response to one day reperfusion, peaked at five days, and decreased at 10 days. ET-3 and GFAP expression was significantly greater in the DM/IR group after reperfusion for 1 day compared to the IR group. However, at other time points, there were no significant differences between the two groups. CONCLUSION： Brain ischemia-reperfusion injury results in overexpression of ET-3 and activation of astrocytes. Diabetes increases the number of ET-3- and GFAP-positive astrocytes in b     

Endothelin-3 and glial fibrillary acidic protein expression in the frontal and parietal cortex of type-2 diabetic mice following ischemia-reperfusion injury

BACKGROUND: Patients with type-2 diabetes mellitus exhibit higher levels of plasma endothelin-! (ET-I). However, very few reports exist regarding altered endothelin-3 (ET-3) and ET-1 concentrations in brain tissue. OBJECTIVE: To observe expression changes of ET-3 and glial fibrillary acidic protein (GFAP) in the frontal and parietal cortex of type-2 diabetic mice following ischemia-reperfusion injury, with various reperfusion durations. DESIGN, TIME AND SETTING: Randomized controlled animal study. The experiment was conducted in the Xiangya Medical College of Central South University and the Third Xiangya Hospital between February 2002 and January 2003. MATERIALS: Sixty-six, adult, male, Kunming mice, weighing (30±5) g, as well as rabbit anti-ET-3 polyclonal and rabbit anti-GFAP polyclonal antibodies, were provided by the Neurobiology Institute of Second Military Medical University in Japan. METHODS: Sixty-six mice were randomly divided into five groups: diabetes mellitus (DM, n=6), diabetes mellitus with ischemia-reperfusion (DM/IR, n = 24), ischemia-reperfusion (IR, n = 24), sham operation (SO, n = 6), and control (n = 6). MAIN OUTCOME MEASURES: Following ischemia-reperfusion for 1, 3, 5, and 10 days, respectively, expression of ET- 3 and GFAP was immunohistochemically measured in the frontal and parietal cortex. RESULTS: All 66 mice were included in the final result analysis. In the IR and DM/IR groups, ET-3- and GFAP-positive neurons increased in the frontal and parietal cortex in response to one day reperfusion, peaked at five days, and decreased at 10 days. ET-3 and GFAP expression was significantly greater in the DM/IR group after reperfusion for 1 day compared to the IR group. However, at other time points, there were no significant differences between the two groups. CONCLUSION: Brain ischemia-reperfusion injury results in overexpression of ET-3 and activation of astrocytes. Diabetes increases the number of ET-3- and GFAP-positive astrocytes in brain tissue of ischemia-reperfusion mice with the same reperfusion duration.     

Inhibitory effect of synthetic small interfering RNAs on glial fibrillary acidic expression in astrocytes

BACKGROUND: Glial fibrillary acidic protein (GFAP) expression highly correlates with spinal glial scar formation, and is regarded as an important target for scar therapy. Efficient inhibition of expression could benefit recovery from spinal cord injury. OBJECTIVE: To investigate the inhibitory effects of synthetic small interfering RNAs (siRNAs) on astrocytie GFAP expression in rats. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment at the cellular and molecular level was performed at the First Hospital of Dalian Medical University between June 2005 and February 2006. MATERIALS: A total of 100 seven-day-old, Sprague Dawley rats were selected. GAPDH siRNA was purchased from Ambion, USA, And TransMessengerTM Transfection Reagent from DAKO, Carpinteria, CA. METHODS: Rat astrocytes were isolated and cultured. Three pairs of 21-nucleotide (nt) siRNAs specific to rats GFAP mRNA, 401,404 and 854, were synthesized and transfected in primary astrocytes at 1, 2, 3, and 4 g/L using TransMessengerTM Transfection Reagent. Non-transfected astrocytes served as the blank group. Cells transfected with siRNA were regarded as the negative control group, with GAPDH siRNA as the positive control group, and 401 siRNA, 404 siRNA, and 854 siRNA as experimental groups. MAIN OUTCOME MEASURES: GFAP mRNA and protein expression were assessed by RT-PCR and Western blot, respectively, at 24, 48, and 72 hours of culture. RESULTS: GFAP mRNA expression in the positive control group was significantly less than the negative control group (P<0.01). GFAP mRNA expression in astrocytes from three pairs of siRNA was significantly less than the blank group after 48 hours (P<0.01 ), while no differences were detected between the negative control and blank groups (P>0.05). GFAP protein expression was remarkably less in siRNA-transfected astroeytes compared to the blank control (P < 0.01). CONCLUSION: Transfected siRNAs could significantly inhibit GFAP gene expression in astrocytes after 72 hours in culture.     

Neuronal modulation of Schwann cell glial fibrillary acidic protein (GFAP)

Adult rat sciatic nerves contain cytoskeletal peptides that resemble CNS glial fibrillary acidic protein (GFAP) in immunoreactivity and molecular weight. Immunohistological examination of teased nerve fascicles indicated that these peptides are expressed selectively by Schwann cells related to small axons. Radiolabelled mouse and rat CNS GFAP cDNA probes hybridized with a single, 2.7 kb RNA band in Northern blots prepared from total RNA from both rat sciatic nerve and rat brain. Sciatic nerve GFAP mRNA was detectable by this means in adult, 2 month, or 21 day postnatal rats, but not in 3,6, or 10 day postnatal rats. Sciatic nerve transection caused a marked reduction in the level of GFAP mRNA in the axotomized distal stump. We conclude that Schwann cell synthesis of GFAP is developmentally regulated and that Schwann cells, unlike astroglia, require continued trophic input from small axons in order to express GFAP.     

Prognostic value of biochemical markers of brain damage and oxidative stress in post-surgical aneurysmal subarachnoid hemorrhage patients

The aim of this study is to determine effective biochemical markers and optimal sampling timing for prediction of neurological prognosis in post-surgical aneurysmal subarachnoid hemorrhage (SAH) patients. Subjects were a sequential group of SAH patients admitted to our centre who underwent aneurysm clipping before Day 3 and who received a cerebrospinal fluid (CSF) drain. CSF samples from 32 patients were collected on Days 3, 7, and 14. Neurological outcome was assessed by neurosurgeons using the Glasgow outcome scale (GOS) at 6 months after onset. CSF levels of neuron-specific enolase (NSE), S100B, and glial fibrillary acidic protein (GFAP) were determined using enzyme-linked immunosorbent assay, and the CSF concentrations of malondialdehyde (MDA) were determined using spectrophotometric assay. In univariate analysis, S100B on Days 3 and 14, GFAP on Days 3 and 7, and MDA on Day 14 were significantly higher in the poor outcome group (GOS 1-4) than in the good outcome group (GOS 5). In multivariate analysis, only MDA on Day 14 was identified as a significant predictor of poor neurological outcome at 6 months after onset. The area under the receiver-operating characteristic (ROC) curve for MDA on Day 14 was 0.841. For a threshold of 0.3 μM, sensitivity and specificity were 0.875 and 0.750, respectively. Our findings suggest that these biochemical markers, especially MDA, show significant promise as predictors of neurological outcome in clinical practice.     

Localization of factor VIII/von willebrand factor and glial fibrillary acidic protein in the hemangioblastoma: Implications for stromal cell histogenesis

Summary The histogenesis of hemangioblastoma stromal cells is unresolved. Ultrastructural observations suggest that the stromal cells, endothelial cells, and pericytes that compose this neoplasm are all derived from angiogenic mesenchyme. The expression of factor VIII/von Willebrand factor (FVIII/vWF), a specific marker for endothelial cells, and of glial fibrillary acidic protein (GFAP), a specific marker for glial cells, was examined in 16 hemangioblastomas using the peroxidase-antiperoxidase immunohistochemical method. Endothelial cell staining for FVIII/vWF was intense in 14 tumors, weak in one, and absent in another. There was no stromal cell staining in any of the neoplasms. Process-bearing, GFAP-positive cells were observed near the tumor margin in 13 cases, and deeper in the neoplasm in 8. In two of these tumors there were also occasional GFAP-positive cells that lacked processes and had a vacuolated cytoplasm. Virtually all of the GFAP-positive cells were interpreted as trapped astrocytes rather than stromal cells. The lack of expression of FVIII/vWF by the stromal cells indicates that they are antigenically distinct from endothelial cells. Several alternatives for stromal cell histogenesis remain open. The stromal cells may be derived from endothelial cells that have undergone antigenic loss, or from angiogenic mesenchymal cells that do not express FVIII/vWF. Alternatively, the stromal cells may originate from nonangiogenic mesenchymal cells derived from the mesoderm or neuroectoderm.Supported by Grants CA-11898 and CA-22790 from the National Cancer Institute, HL-24066 from the National Heart, Lung, and Blood Institute, and IM-150 from the American Cancer Society. R.D.M. was supported by Training Grant 5T32 GM-0740304 from The National Institute of General Medical Services     

Loss of serotonin uptake sites and immunoreactivity in rat cortex after dexfenfluramine occur without parallel glial cell reactions

The frontal cortices of rats which received eitherd,l- ord-fenfluramine (DFEN) for 4 days were examined 18 h to 2 weeks following treatment for changes in synaptosomal uptake of serotonin (5HT), paroxetine binding, 5HT-immunoreactivity (5HT-IR), and both astrocytic (GFAP) and microglial markers. Additional rats received intracerebroventricular injections of the neurotoxin 5,7-dihydroxytryptamine (DHT). Consistent with previous reports,d,l- and DFEN produced dose-dependent losses of both 5HT uptake and paroxetine binding, and loss of 5HT-IR which coincided with an abnormal or ‘swollen’ appearance of immunoreactive axon processes. Recovery of these serotonergic indices was greatest following the lowest doses of DFEN, but was absent after 5,7-DHT treatment. No evidence for an increase in GFAP synthesis or microglial activation was observed in frontal cortices of rats treated with either DFEN or 5,7-DHT. We conclude that the presence of swollen 5HT-IR axons in the cortices of both the 5,7-DHT and DFEN groups is insufficient to trigger the glial responses often associated with neuronal degeneration. Thus, it remains to be determined if swollen 5HT-IR axons are a prelude to neurodegeneration, or whether they represent reversible changes in axonal immunochemistry associated with decreases in 5HT levels. The implications of the data for the clinical safety of DFEN are briefly discussed.