Osteopontin is indispensable for activation of astrocytes in injured mouse brain and primary culture

ABSTRACT

Objectives: Osteopontin (OPN) is an inflammatory cytokine inducer involved in cell proliferation and migration in inflammatory diseases or tumors. To investigate the function of OPN in astrocyte activation during brain injury, we compared OPN-deficient (OPN/KO) with wild-type (WT) mouse brains after stab wound injury and primary culture of astrocytes.

Methods: Primary cultures of astrocytes were prepared from either WT or OPN/KO postnatal mouse brains. Activation efficiency of astrocytes in primary culture was accessed using Western blotting by examining the protein levels of glial fibrillary acidic protein (GFAP) and tenascin-C (TN-C), which are markers for reactive astrocytes, following lipopolysaccharide (LPS) stimulation. Furthermore, the stab wound injury on the cerebral cortex as a brain traumatic injury model was used, and activation of astrocytes and microglial cells was investigated using immunofluorescent analysis on fixed brain sections.

Results: Primary cultures of astrocytes prepared from WT or OPN/KO postnatal mouse brains showed that only 25% of normal shaped astrocytes in a flask were produced in OPN/KO mice. The expression levels of both GFAP and TN-C were downregulated in the primary culture of astrocytes from OPN/KO mice compared with that from WT mice. By the immunofluorescent analysis on the injured brain sections, glial activation was attenuated in OPN/KO mice compared with WT mice.

Discussion: Our data suggest that OPN is essential for proper astrocytic generation in vitro culture prepared from mouse cerebral cortex. OPN is indispensable for astrocyte activation in the mouse brain injury model and in LPS stimulated primary culture.

Abbreviations: AQP4: aquaporin 4; BBB: blood brain barrier; BrdU: bromo-deoxy uridine; CNS: central nervous system; GFAP: glial fibllirary acidic protein; IgG: immunoglobulin G; LPS: lipopolysaccharide; OPN: osteopontin; OPN/KO: osteopontin-deficient; TN-C: tenascin-C     

The two patterns of reactive astrocytosis in postischemic rat brain

The distribution and time course of postischemic astrocyte hypertrophy and hyperplasia and the relationship to neuronal viability or necrosis was studied in rats subjected to 30 min of carotid and vertebral artery occlusion followed by reperfusion from 3 h to 5 weeks. Intermediate filaments (IFs) were evaluated by electron microscopy, IF proteins by immunohistochemistry, and astrocyte division by [3H]thymidine uptake. Glial fibrillary acidic protein (GFAP) increased in damaged and nondamaged brain regions by 2 days and was associated with cell enlargement, increases in IF, and transformation of GFAP-negative into GFAP-positive glia. Cell hypertrophy and increased GFAP persisted only in regions of neuronal necrosis whereas the number and size of GFAP-positive astrocytes returned to control levels in nondamaged regions by 2 weeks. Astrocyte hyperplasia was not seen until 3 days and was confined to damaged brain regions. Vimentin-positive astrocytes were numerous by 2 days in damaged brain and remained only in those regions at 5 weeks. The data demonstrate that reactive astrocytosis develops in undamaged brain, but is reversible with prolonged survival, whereas reactive astrocytosis that accompanies structural brain damage persists for prolonged periods and is associated with hyperplasia, as well as hypertrophy. In addition, the results show that astrocyte expression of vimentin is more specific than GFAP in identifying regions of permanent ischemic injury during the early postischemic period.     

An inhibitory effect of interferon gamma on the injury-induced astrocyte proliferation in the postmitotic rat brain

Influence of interferon gamma (IFNγ) on astrocytes proliferating in response to unilateral injury of the cerebral hemisphere was investigated in 30-day-old rats. The brain injury was followed by an immediate injection of IFNγ into the lesion cavity. On 1st or 2nd day following injury the animals were injected with [³H]thymidine and killed 4 h after the injection. The proliferating astrocytes were visualised by combination of immunocytochemical staining for glial fibrillary acidic protein (GFAP) and autoradiography. Thereafter, numbers of GFAP-immunopositive and autoradiographically-labeled astrocytes located within the region of injury were counted. On the day 1 after injury no effect of IFNγ administration was seen. However, on day 2 the average 43% reduction of the number of proliferating astrocytes was recorded. The reduction showed no dose-dependent changes. This in vivo evidence of IFNγ-induced suppression of astrocyte proliferation was considered in relation to other determinants of astrocyte reactivity existing in the injured brain.     

Degeneration and proliferation of astrocytes in the mouse dentate gyrus after pilocarpine-induced status epilepticus

Astrocytes are relatively resistant to injury compared to neurons and oligodendrocytes. Here, we report transient region-specific loss of astrocytes in mice early after pilocarpine-induced status epilepticus (SE). In the dentate hilus, immunoreactivity for glial acidic fibrillary protein (GFAP) was decreased, and the number of healthy appearing GFAP- or S100beta-positive cells was significantly reduced (> or =65%) 1 and 3 days after pilocarpine-induced SE. Many remaining GFAP-positive cells were shrunken, and 1 day after SE electron microscopy revealed numerous electron-dense degenerating astrocyte processes and degenerating glial somata in the hilus. Degeneration of GFAP-expressing cells may be linked to hilar neuronal death, because we did not observe loss of astrocytes after kainate-induced SE, after which hilar neurons remained intact. Ten days after SE, hilar GFAP immunoreactivity had returned, partially from GFAP-positive cells in the hilus. Unlike control mice, many GFAP-positive hilar processes originated from cell bodies located in the subgranular zone (SGZ). To investigate whether proliferation contributes to hilar repopulation, we injected 5-bromo-2'-deoxyuridine (BrdU) 3 days after SE. Five hours later and up to 31 days after SE, many BrdU/GFAP colabeled cells were found in the hilus and the SGZ, some with hilar processes, indicating that proliferation in both areas contributes to generation of hilar astrocytes and astrocyte processes. In contrast to pilocarpine-induced SE in mice, astrocyte degeneration was not found after pilocarpine-induced SE in rats. These findings demonstrate astrocyte degeneration in the mouse dentate hilus specifically in the mouse pilocarpine epilepsy model, followed by astrogenesis leading to hilar repopulation.     

Memory impairment is associated with the loss of regular oestrous cycle and plasma oestradiol levels in an activity-based anorexia animal model

Objectives: Patients with anorexia nervosa (AN) suffer from neuropsychological deficits including memory impairments. Memory partially depends on 17β-oestradiol (E2), which is reduced in patients with AN. We assessed whether memory functions correlate with E2 plasma levels in the activity-based anorexia (ABA) rat model. Methods: Nine 4-week-old female Wistar rats were sacrificed directly after weight loss of 20–25% (acute starvation), whereas 17 animals had additional 2-week weight-holding (chronic starvation). E2 serum levels and novel object recognition tasks were tested before and after starvation and compared with 21 normally fed controls. Results: Starvation disrupted menstrual cycle and impaired memory function, which became statistically significant in the chronic state (oestrous cycle (P?<?0.001), E2 levels (P?=?0.011) and object recognition memory (P =?0.042) compared to controls). E2 reduction also correlated with the loss of memory in the chronic condition (r?=?0.633, P =?0.020). Conclusions: Our results demonstrate that starvation reduces the E2 levels which are associated with memory deficits in ABA rats. These effects might explain reduced memory capacity in patients with AN as a consequence of E2 deficiency and the potentially limited effectiveness of psychotherapeutic interventions in the starved state. Future studies should examine whether E2 substitution could prevent cognitive deficits and aid in earlier readiness for therapy.     

Cognitive impairment among different clinical courses of multiple sclerosis

Abstract

Objective: Motoneurons are the focus of most investigations of amyotrophic lateral sclerosis (ALS), while the astrocyte reaction is regarded as a phenomenon secondary to neuron degeneration. Since astroglial reactivity differed in different studies of human and animal ALS models and often varied from case to case, we examined and compared astrocyte reactivity within the anterior horns of the spinal cord in a transgenic rat model of familial ALS and in human sporadic ALS (sALS) cases.

Methods: Routine histological staining and immunohistochemical reactions to cytoskeletal proteins [neurofilaments, glial fibrillary acidic protein (GFAP), vimentin and tau] and proliferative markers (proliferating cell nuclear antigen and Ki-67).

Results: In human sALS cases and in rats at the early pre-symptomatic and symptomatic stages of the disease, the astroglial reaction was very weak, although there was visible evidence of the morphological manifestations of motoneuron degeneration. Poor immunoreactivity to the GFAP and vimentin antigens and increased expression of tau protein were observed in astrocytes, particularly in the rat model. The astrocyte reaction was evident during a short 'transient' phase of the disease in animals, between the asymptomatic and paretic stages. Proliferating cell nuclear antigen immunoreactivity in glial and neuronal nuclei was observed only in animal material.

Conclusions: Abnormalities in astrocyte cytoskeletal proteins are characteristic features for ALS, both in acquired and congenital forms of the disease. The cytoskeletal aberrations may lead to astroglial dysfunction and disturbances in glutamate uptake that may in turn increase the degeneration of motoneurons.     

Human intravenous immunoglobulins suppress seizure activities and inhibit the activation of GFAP-positive astrocytes in the hippocampus of picrotoxin-kindled rats

We previously showed that human intravenous immunoglobulin (IVIG) can lower seizure severity and prolong seizure latency in picrotoxin-kindled rats. The aim of this study was to further characterize the effects of IVIG on seizure activity and investigate its influence on astrocytes in the hippocampus of picrotoxin-kindled rats. A rat kindling model was established by peritoneal injections of picrotoxin for 21 days in Wistar rats. Seventy-five rats were equally divided into five groups: picrotoxin, IVIG pretreatment, IVIG post-treatment, normal saline control, and IVIG control. Seizure severity was evaluated according to a six-stage classification. The number and morphology of glial fibrillary acidic protein (GFAP)-positive astrocytes were studied by immunohistochemistry using the anti-GFAP antibody. The cross-sectional area and grayscale of GFAP-positive astrocytes were also determined. In picrotoxin-kindled rats, pretreatment with IVIG appeared to inhibit full kindling rates, and it significantly reduced the number of GFAP-positive cells in the hippocampus (p < .001). IVIG also significantly (p < .001) attenuated the increase in the cross-sectional area and grayscale of GFAP-positive astrocytes in the hippocampus. Our results suggest that by suppressing the expression of GFAP, IVIGs may reduce seizure activity and inhibit the activation of GFAP-positive astrocytes in picrotoxin-kindled rats.     

Amygdala astrocyte reduction in subjects with major depressive disorder but not bipolar disorder

Altshuler LL, Abulseoud OA, Foland‐Ross L, Bartzokis G, Chang S, Mintz J, Hellemann G, Vinters HV. Amygdala astrocyte reduction in subjects with major depressive disorder but not bipolar disorder.
Bipolar Disord 2010: 12: 541–549. © 2010 The Authors. Journal compilation © 2010 John Wiley & Sons A/S. Objectives: Several magnetic resonance imaging studies have found changes in amygdala volumes in adults with mood disorders. The cellular basis for these changes has not been explored in detail. Specifically, it is not known whether differences in the density and/or volume of neurons or glial cells contribute to tissue volume changes seen on magnetic resonance images. Methods: Postmortem amygdala samples were obtained from the Stanley Foundation Neuropathology Consortium from subjects diagnosed with bipolar disorder (n = 10), major depressive disorder (n = 11), and schizophrenia (n = 9), and from normal controls (n = 14). Samples were first stained with glial fibrillary acidic protein (GFAP) and counter‐stained with hematoxylin to ascertain neuron and glia (astrocyte) densities. Results: No significant differences in neuronal densities were found between groups. However, a reduction in the density of GFAP immunoreactive astrocytes was observed in the amygdala of subjects with major depressive disorder compared to the bipolar disorder, schizophrenia, and normal control postmortem samples. Conclusions: A decrease in density of GFAP immunoreactive astrocytes in the amygdala of depressed subjects is consistent with prior histologic reports and might contribute to amygdala volume reductions reported in several in vivo neuroimaging studies.     

Thalidomide alleviates bone cancer pain by down-regulating expressions of NF-κB and GFAP in spinal astrocytes in a mouse model

Abstract

Aim: Thalidomide is one of the first line therapies in cancer pain management. Previous study has shown that thalidomide decreases the expression of tumor necrosis factor alpha in the mouse spinal cord. However, the exact cellular and molecular mechanism underlying the effect of thalidomide remains unclear. Here, we investigated the effect of thalidomide on the expression level of NF-κB as well as glial fibrillary acidic protein (GFAP) in the spinal cord astrocyte in a mice model.

Materials and methods: MC57G fibrosarcoma cells were intramedullary injected into the right femurs of C57/BL mice to induce behaviors related to bone cancer pain. Postoperative thalidomide was administered intraperitoneally to the mice at dose of 100?mg/kg/day for 7?days. The effect of thalidomide on pain hypersensitivity was checked by behavioral testing. The expression levels of NF-κB and GFAP in spinal cord were evaluated by using Western blotting and Immunohistochemistry.

Results: Compared with the controls, the tumor-bearing mice showed substantial pain-related behaviors. Furthermore, the expression levels of both NF-κB and GFAP increased significantly in the spinal cord astrocytes of the tumor-bearing mice. Treating the tumor-bearing mice with thalidomide results in a dramatic reduction in pain behaviors and a significant decrease of NF-κB and GFAP expressions.

Conclusions: Thalidomide alleviates the pain behaviors probably by down-regulating the expression of NF-κB and GFAP.     

Serotonin uptake by astrocytes in situ.

Co-localization of glial fibrillary acidic protein (GFAP) and radioactivity was examined after intraventricular injection of [3H]5-HT in adult rat brains. Radioactivity localized over GFAP-positive astrocytes was seen, especially when image-enhancing techniques were applied to the data. Also slices prepared from astrogliotic hippocampi of rats pretreated with kainic acid showed a twofold increased uptake of [3H]5-HT compared to control slices. This indicates that the uptake of [3H]5-HT seen in primary astrocyte cultures also occurs for astrocytes in situ. Also, as with astrocyte cultures, only some of the GFAP(+) astrocytes in situ showed localization of radioactivity, supporting the concept of intraregional heterogeneity of astrocyte functions.     

Immunohistochemical analysis of brain lesions using S100B and glial fibrillary acidic protein antibodies in arundic acid- (ONO-2506) treated stroke-prone spontaneously hypertensive rats

Stroke-prone spontaneously hypertensive rats (SHRSP) used as a model of essential hypertension cause a high incidence of brain stroke on the course of hypertension. Incidences and sizes of brain lesions are known to relate to the astrocyte activities. Therefore, relation between brain damage and the expression profile of the astrocytes was investigated with morphometric and immunohistochemical analyses using astrocyte marker antibodies of S100B and glial fibrillary acidic protein (GFAP) with or without arundic acid administration, a suppressor on the activation of astrocytes. Arundic acid extended the average life span of SHRSP. An increase in brain tissue weight was inhibited concomitant with a lower rate of gliosis/hemosiderin deposit/scarring in brain lesions. S100B- or GFAP-positive dot and filamentous structures were decreased in arundic acid-treated SHRSP, and this effect was most pronounced in the cerebral cortex, white matter, and pons, and less so in the hippocampus, diencephalon, midbrain, and cerebellum. Blood pressure decreased after administration of arundic acid in the high-dose group (100 mg/kg/day arundic acid), but not in the low-dose group (30 mg/kg/day). These data indicate that arundic acid can prevent hypertension-induced stroke, and may inhibit the enlargement of the stroke lesion by preventing the inflammatory changes caused by overproduction of the S100B protein in the astrocytes.     

Chronic stress-induced disruption of the astrocyte network is driven by structural atrophy and not loss of astrocytes

Chronic stress is well recognized to decrease the number of GFAP⁺ astrocytes within the prefrontal cortex (PFC). Recent research, however, has suggested that our understanding of how stress alters astrocytes may be incomplete. Specifically, chronic stress has been shown to induce a unique form of microglial remodelling, but it is not yet clear whether astrocytes also undergo similar structural modifications. Such alterations may be significant given the role of astrocytes in modulating synaptic function. Accordingly, in the current study we have examined changes in astrocyte morphology following exposure to chronic stress in adult rats, using three-dimensional digital reconstructions of astrocytes. Our analysis indicated that chronic stress produced profound atrophy of astrocyte process length, branching and volume. We additionally examined changes in astrocyte-specific S100β, which are both a putative astrocyte marker and a protein whose expression is associated with astrocyte distress. While we found that S100β levels were increased by stress, this increase was not correlated with atrophy. We further established that while chronic stress was associated with a decrease in astrocyte numbers when GFAP labelling was used as a marker, we could find no evidence of a decrease in the total number of cells, based on Nissl staining, or in the number of S100β⁺ cells. This finding suggests that chronic stress may not actually reduce astrocyte numbers and may instead selectively decrease GFAP expression. The results of the current study are significant as they indicate stress-induced astrocyte-mediated disturbances may not be due to a loss of cells but rather due to significant remodeling of the astrocyte network.     

Wistar大鼠实验性自身免疫性脑脊髓炎胶质纤维酸性蛋白变化的研究

目的通过建立实验性自身免疫性脑脊髓炎(EAE)模型研究Wistar大鼠脑内神经脱髓鞘后星形胶质细胞损伤随时间变化的规律。方法对Wistar大鼠经足垫注射豚鼠脊髓匀浆制作EAE模型,并于不同时间点将其处死,取脑组织进行免疫组化染色及图像分析检测脑组织中胶质纤维酸性蛋白(GFAP)水平并与健康对照组进行比较。结果GFAP阳性细胞随损伤时间呈先上升后下降的变化趋势,于发病后第7天表达最高,第21天恢复正常。其中发病后第7、14天GFAP阳性细胞突起增长、增粗,染色加深。结论EAE模型中星形胶质细胞可能参与了脑组织损伤后的修复过程,其标志性蛋白GFAP水平随损伤时间呈规律性改变。     

Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast‐derived iPS cells using zinc‐finger nuclease technology

Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast‐derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human‐derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc‐finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte‐specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno‐associated virus integration site 1 (AAVS1) locus in disease and control‐derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP‐iPS astrocytes exhibited a well‐described functional astrocytic activity in vitro characterized by neuron‐dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP‐iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP‐iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte‐driven disease pathogenesis and therapy. GLIA 2016;64:63–75     

MicroRNA‐365 modulates astrocyte conversion into neuron in adult rat brain after stroke by targeting Pax6

Reactive astrocytes induced by ischemia can transdifferentiate into mature neurons. This neurogenic potential of astrocytes may have therapeutic value for brain injury. Epigenetic modifications are widely known to involve in developmental and adult neurogenesis. PAX6, a neurogenic fate determinant, contributes to the astrocyte‐to‐neuron conversion. However, it is unclear whether microRNAs (miRs) modulate PAX6‐mediated astrocyte‐to‐neuron conversion. In the present study we used bioinformatic approaches to predict miRs potentially targeting Pax6, and transient middle cerebral artery occlusion (MCAO) to model cerebral ischemic injury in adult rats. These rats were given striatal injection of glial fibrillary acidic protein targeted enhanced green fluorescence protein lentiviral vectors (Lv‐GFAP‐EGFP) to permit cell fate mapping for tracing astrocytes‐derived neurons. We verified that miR‐365 directly targets to the 3′‐UTR of Pax6 by luciferase assay. We found that miR‐365 expression was significantly increased in the ischemic brain. Intraventricular injection of miR‐365 antagomir effectively increased astrocytic PAX6 expression and the number of new mature neurons derived from astrocytes in the ischemic striatum, and reduced neurological deficits as well as cerebral infarct volume. Conversely, miR‐365 agomir reduced PAX6 expression and neurogenesis, and worsened brain injury. Moreover, exogenous overexpression of PAX6 enhanced the astrocyte‐to‐neuron conversion and abolished the effects of miR‐365. Our results demonstrate that increase of miR‐365 in the ischemic brain inhibits astrocyte‐to‐neuron conversion by targeting Pax6, whereas knockdown of miR‐365 enhances PAX6‐mediated neurogenesis from astrocytes and attenuates neuronal injury in the brain after ischemic stroke. Our findings provide a foundation for developing novel therapeutic strategies for brain injury.     

Ammonia-induced brain swelling and neurotoxicity in an organotypic slice model

Abstract

Objectives: Acute liver failure (ALF) produces cerebral dysfunction and edema, mediated in part by elevated ammonia concentrations, often leading to coma and death. The pathophysiology of cerebral edema in ALF is incompletely understood. In vitro models of the cerebral effects of ALF have predominately consisted of dissociated astrocyte cultures or acute brain slices. We describe a stable long-term culture model incorporating both neural and glial elements in a three-dimensional tissue structure offering significant advantages to the study of astrocytic-neuronal interactions in the pathophysiology of cerebral edema and dysfunction in ALF.

Methods: We utilized chronic organotypic slice cultures from mouse forebrain, applying ammonium acetate in iso-osmolar fashion for 72 hours. Imaging of slice thickness to assess for tissue swelling was accomplished in living slices with optical coherence tomography, and confocal microscopy of fluorescence immunochemical and histochemical staining served to assess astrocyte and neuronal numbers, morphology, and volume in the fixed brain slices.

Results: Ammonia exposure at 1-10 mM produced swelling of immunochemically identified astrocytes, and at 10 mM resulted in macroscopic tissue swelling, with slice thickness increasing by about 30%. Astrocytes were unchanged in number. In contrast, 10 mM ammonia treatment severely disrupted neuronal morphology and reduced neuronal survival at 72 hours by one-half.

Discussion: Elevated ammonia produces astrocytic swelling, tissue swelling, and neuronal toxicity in cerebral tissues. Ammonia-treated organotypic brain slice cultures provide an In vitro model of cerebral effects of conditions relevant to ALF, applicable to pathophysiological investigations.     

Brain volume reduction predicts weight development in adolescent patients with anorexia nervosa

BackgroundAcute anorexia nervosa (AN) is associated with marked brain volume loss potentially leading to neuropsychological deficits. However, the mechanisms leading to this brain volume loss and its influencing factors are poorly understood and the clinical relevance of these brain alterations for the outcome of these AN-patients is yet unknown.MethodsBrain volumes of 56 female adolescent AN inpatients and 50 healthy controls (HCs) were measured using MRI scans. Multiple linear regression analyses were used to determine the impact of body weight at admission, prior weight loss, age of onset and illness duration on volume loss at admission and to analyse the association of brain volume reduction with body weight at a 1-year follow-up (N = 25).ResultsCortical and subcortical grey matter (GM) and cortical white matter (WM) but not cerebellar GM or WM were associated with low weight at admission. Amount of weight loss, age of onset and illness duration did not independently correlate with any volume changes. Prediction of age-adjusted standardized body mass index (BMI-SDS) at 1-year follow-up could be significantly improved from 34% of variance explained by age and BMI-SDS at admission to 47.5–53% after adding cortical WM, cerebellar GM or WM at time of admission.ConclusionWhereas cortical GM changes appear to be an unspecific reflection of current body weight (“state marker”), cortical WM and cerebellar volume losses seem to indicate a longer-term risk (trait or “scar” of the illness), which appear to be important for the prediction of weight rehabilitation and long-term outcome.     

Troxerutin cerebroprotein hydrolysate injection ameliorates neurovascular injury induced by traumatic brain injury – via endothelial nitric oxide synthase pathway regulation

Abstract

Background: Neurovascular dysfunction caused by traumatic brain injury (TBI) is characterized by cerebralvascular damage, blood–brain barrier (BBB) breakdown, brain edema, etc. This study was designed to assess the protective role of 5 days troxerutin cerebroprotein hydrolysate (TCH) injection treatment against TBI, as well as the potential mechanism.

Methods: The weight-drop model of TBI in male Sprague-Dawley rats was chosen to induce TBI model, rats either with TCH or a vehicle via intraperitoneal injection were examined 3 days after TBI.

Results: TCH resulted in alleviation of neurological deficits, reduction of infarct volume, improvement of regional cerebral blood flow (rCBF), amelioration of neuronal death, astrocyte proliferation, endothelial cell loss, and BBB dysintegrity. These effects of TCH treatment against TBI were through endothelial nitric oxide synthase (eNOS) coupling/decoupling status adjustment, which not only increased nitric oxide (NO) level, but also decreased peroxynitrate level expression.

Conclusions: All the results indicated that TCH injection has multifaceted protective effects of neurovascular unit (NVU) against TBI via eNOS pathway regulation.     

P-glycoprotein expression in brain capillary endothelial cells after focal ischaemia in the rat

Abstract

P-glycoprotein (P-gp) is expressed not only in tumour cells but also in some normal tissues including brain capillaries. We investigated whether or not P-gp was expressed in the capillary, endothelial cells of a rat focal ischaemic brain. The brains were immunohistochemically studied for Factor VIII, glial fibrillary acidic protein (GFAP), and P-gp. Endothelial γ-glutamyl transpeptidase (γ-GTP) activity; which is thought to be induced by glial cells, was also studied histochemically. The P-gp positive endothelial cells disappeared in the ischaemic lesion by post-ischaemic day 3. Factor Vlll-positive regenerating capillaries were first observed on day 3 without P-gp expression.. The P-gp positive endothelial cells began to reappear on day 5, and were detected in all the endothelial cells by day 8. The P-gp expression in endothelial cells showed a similar pattern as that of y-GTP, and seemed to correlate with GFAP-positive reactive astrocytes. The newly-formed brain capillaries thus appeared to have a potential to express P-gp in abnormal pathogenic conditions as cerebral infarction, and our present study also suggested that P-gp in the brain capillaries might therefore be expressed in conjunction with glial cells. [Neurol Res 1994; 16: 217–223]     

Lactation Reduces Glial Activation Induced by Excitotoxicity in the Rat Hippocampus

Motherhood induces a series of adaptations in the physiology of the female, including an increase of maternal brain plasticity and a reduction of cell damage in the hippocampus caused by kainic acid (KA) excitotoxicity. We analysed the role of lactation in glial activation in the hippocampal fields of virgin and lactating rats after i.c.v. application of 100 ng of KA. Immunohistochemical analysis for glial fibrillary acidic protein (GFAP) and ionised calcium binding adaptor molecule 1 (Iba-1), which are markers for astrocytes and microglial cell-surface proteins, respectively, revealed differential cellular responses to KA in lactating and virgin rats. A significant astrocyte and microglial response in hippocampal areas of virgin rats was observed 24 h and 72 h after KA. By contrast, no increase in either GFAP- or Iba-1-positive cells was observed in response to KA in the hippocampus of lactating rats. Western blot analysis of GFAP showed an initial decrease at 24 h after KA treatment, with an increase at 72 h in the whole hippocampus of virgin but not of lactating rats. The number of GFAP-positive cells was increased by lactation in the dentate gyrus of the hippocampus but not in CA1 and CA3 areas. The present results indicate that lactating rats exhibit diminished responses of astrocyte and microglial cells in the hippocampus to damage induced by KA, supporting the notion that the maternal hippocampus is resistant to excitotoxic insults.