大鼠轻型颅脑损伤后星形胶质细胞与神经元的病理改变

目的 探讨轻型颅脑损伤（TBI）后神经元及星形胶质细胞改变的病理生理过程。方法 将24只成年SD大鼠随机分为轻型TBI组（n=18）和假手术组（n=6）,轻型TBI组又分为伤后3 h（n=6）、伤后24 h（n=6）、伤后72 h（n=6）三亚组。采用液压冲击法制作轻型TBI模型。采用胶质纤维酸性蛋白（GFAP）染色检测星形胶质细胞,采用Fluoro-Jade B（FJ-B）荧光染色检测变性神经元。结果 与假手术组相比,轻型TBI后3 h、24 h、72 h邻近顶叶皮质、海马CA2/3区GFAP阳性细胞数量均明显减少（P<0.05）;缺失区周围星形胶质细胞肿胀增生明显。FJ-B阳性神经元在损伤后3 h无明显增加（P>0.05）,伤后24 h皮层区FJ-B阳性神经元显著增加（P<0.05）,伤后72 h海马区FJ-B阳性神经元显著增加（P<0.05）。伤后72 h伤侧皮层区与海马区GFAP阳性细胞数和FJ-B阳性细胞数呈显著负相关（r=-0.8285,P<0.05）。结论 轻型TBI后星形胶质细胞超急性期（3 h）即出现损害和胶质反应,神经元则在急性期（24 h）至亚急性期（72 h）出现明显损害,星形胶质细胞缺失程度可以反应神经元损伤程度。     

Protective role of melatonin in domoic acid-induced neuronal damage in the hippocampus of adult rats

Domoic acid (DA), a kainite-receptor agonist and potent inducer of neurotoxicity, has been administered intravenously in adult rats in the present study (0.75 mg/kg body weight) to demonstrate neuronal degeneration followed by glial activation and their involvement with inducible nitric oxide synthase (iNOS) in the hippocampus. An equal volume of normal saline was administered in control rats. The pineal hormone melatonin, which protects the neurons efficiently against excitotoxicity mediated by sensitive glutamate receptor, was administered intraperitoneally (10 mg/kg body weight), 20 min before, immediately after, and 1 h and 2 h after the DA administration, to demonstrate its role in therapeutic strategy. Histopathological analysis (Nissl staining) demonstrated extensive neuronal damage in the pyramidal neurons of CA1, CA3 subfields and hilus of the dentate gyrus (DG) in the hippocampus at 5 days after DA administration. Sparsely distributed glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes were observed in the hippocampus at 4-24 h after DA administration and in the control rats. Astrogliosis was evidenced by increased GFAP immunoreactivity in the areas of severe neuronal degeneration at 5 days after DA administration. Along with this, microglial cells exhibited an intense immunoreaction with OX-42, indicating upregulation of complement type 3 receptors (CR3). Ultrastructural study revealed swollen or shrunken degenerating neurons in the CA1, CA3 subfields and hilus of the DG and hypertrophied astrocytes showing accumulation of intermediate filament bundles in the cytoplasm were observed after administration of DA. Although no significant change could be observed in the mRNA level of iNOS expression between the DA-treated rats and controls at 4-24 h and at 5-day time intervals, double immunofluorescense revealed co-expression of induced iNOS with GFAP immunoreactive astrocytes, but not in the microglial cells, and iNOS expression in the neurons of the hippocampal subfields at 5 days after DA administration. Expression of iNOS was not observed in the hippocampus of control rats. DA-induced neuronal death, glial activation, and iNOS protein expression were attenuated significantly by melatonin treatment and were comparable to the control groups. The results of the present study suggest that melatonin holds potential for the treatment of pathologies associated with DA-induced brain damage. It is speculated that astrogliosis and induction of iNOS protein expression in the neurons and astrocytes of the hippocampus may be in response to DA-induced neuronal degeneration.     

脑损伤后N-乙酰天冬氨酰谷氨酸肽酶抑制的神经保护

目的 探索颅脑损伤后N-乙酰天冬氨酰谷氨酸(NAAG)肽酶抑制的神经保护效应.方法 将20只SD大鼠随机分成4组后制作中度液压颅脑损伤模型.伤后3个时间点(即时,8 h,16 h)分别经腹腔注射新型NAAG肽酶抑制剂ZJ-43(各组剂量:0,50,100,150 mg/kg).伤后24 h处死动物取脑组织行Fluoro-Jade B组织荧光染色以及GFAP免疫组化染色.通过立体细胞计数着重观察大鼠海马CA2/3区退变神经元及正常胶质细胞的数量变化.结果 ZJ-43能显著减少颅脑损伤后的神经元退变(P＜0.01),其中小剂量处理组(50 mg/kg)保护作用最佳,还能同时显著减少儋颅脑损伤后的胶质细胞丢失(P＜0.05).结论 阻断NAAG肽酶活性能有效放大内源性递质NAAG的神经保护作用,从而阻断谷氨酸兴奋毒性的病理进程,有望成为治疗颅脑损伤的新策略.     

Astrocyte targeted overexpression of Hsp72 or SOD2 reduces neuronal vulnerability to forebrain ischemia

Brief forebrain ischemia is a model of the delayed hippocampal neuronal loss seen in patients following cardiac arrest and resuscitation. Previous studies demonstrated that selective dysfunction of hippocampal CA1 subregion astrocytes occurs hours to days before delayed neuronal death. In this study we tested the strategy of directing protection to astrocytes to protect neighboring neurons from forebrain ischemia. Two well‐studied protective proteins, heat shock protein 72 (Hsp72) or superoxide dismutase 2 (SOD2), were genetically targeted for expression in astrocytes using the astrocyte‐specific human glial fibrillary acidic protein (GFAP) promoter. The expression constructs were injected stereotacticly immediately above the hippocampal CA1 region on one side of the rat brain two days prior to forebrain ischemia. Cell type specific expression was confirmed by double label immunohistochemistry. When the expression constructs were injected two days before transient forebrain ischemia, the loss of CA1 hippocampal neurons observed seven days later was significantly reduced on the injected side compared with controls. This neuroprotection was associated with significantly better preservation of astrocyte glutamate transporter‐1 immunoreactivity at 5‐h reperfusion and reduced oxidative stress. Improving the resistance of astrocytes to ischemic stress by targeting either the cytosolic or mitochondrial compartment was thus associated with preservation of CA1 neurons following forebrain ischemia. Targeting astrocytes is a promising strategy for neuronal preservation following cardiac arrest and resuscitation. © 2010 Wiley‐Liss, Inc.     

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.     

Dose-dependent neuronal injury after traumatic brain injury

The Fluoro-Jade (FJ) stain reliably identifies degenerating neurons after multiple mechanisms of brain injury. We modified the FJ staining protocol to quickly stain frozen hippocampal rat brain sections and to permit systematic counts of stained, injured neurons at 4 and 24 h after mild, moderate or severe fluid percussion traumatic brain injury (TBI). In adjacent sections, laser capture microdissection was used to collect uninjured (FJ negative) CA3 hippocampal neurons to assess the effect of injury severity on mRNA levels of selected genes. Rats were anesthetized, intubated, mechanically ventilated and randomized to sham, mild (1.2 atm), moderate (2.0 atm) or severe (2.3 atm) TBI. Four or 24 h post-TBI, ten frozen sections (10 microm thick, every 15th section) were collected from the hippocampus of each rat, stained with FJ and counterstained with cresyl violet. Fluoro-Jade-positive neurons were counted in hippocampal subfields CA1, CA3 and the dentate gyrus/dentate hilus. At both 4 and 24 h post-TBI, numbers of FJ-positive neurons in all hippocampal regions increased dose-dependently in mildly and moderately injured rats but were not significantly more numerous after severe injury. Although analysis of variance demonstrated no overall difference in expression of mRNA levels for heat shock protein 70, bcl-2, caspase 3, caspase 9 and interleukin-1beta in uninjured CA3 neurons at all injury levels, post hoc analysis suggested that TBI induces increases in neuroprotective gene expression that offset concomitant increases in deleterious gene expression.     

Fluoro-Jade B staining as useful tool to identify activated microglia and astrocytes in a mouse transgenic model of Alzheimer's disease

Fluoro-Jade B is known as a high affinity fluorescent marker for the localization of neuronal degeneration during acute neuronal distress. However, one study suggested that fluoro-Jade B stains reactive astroglia in the primate cerebral cortex. In this study, we analyzed the staining of fluoro-Jade B alone or combined with specific markers for detection of glial fibrillary acidic protein (GFAP) or activated CD68 microglia in the double APP(SL)/PS1 KI transgenic mice of Alzheimer's disease (AD), which display a massive neuronal loss in the CA1 region of the hippocampus. Our results showed that fluoro-Jade B did not stain normal and degenerating neurons in this double mouse transgenic model. Fluoro-Jade B was co-localized with Abeta in the core of amyloid deposits and in glia-like cells expressing Abeta. Furthermore, fluoro-Jade B was co-localized with CD68/macrosialin, a specific marker of activated microglia, and with GFAP for astrocytes in APP(SL)/PS1 KI transgenic mice of AD. Taken together, these findings showed that fluoro-Jade B can be used to label activated microglia and astrocytes which are abundant in the brain of these AD transgenic mice. It could stain degenerating neurons as a result of acute insult while it could label activated microglia and astrocytes during a chronic neuronal degenerative process such as AD for example.     

Morphological transformations of cells immunopositive for GFAP, TrkA or p75 in the CA1 hippocampal area following transient global ischemia in the rat. A quantitative study

Transient global ischemia induces intensive neuronal degeneration in the hippocampal CA1 pyramidal layer, accompanied by reactive transformation of glial cells. Previously, we have shown using the double immunostaining method that the NGF receptors (NGFR) p75 and TrkA are expressed mainly on subpopulations of GFAP+ astrocytes, and this expression increases progressively after ischemia. In the presented study, we analyzed quantitatively the morphological transformations of cells immunopositive for GFAP or NGF receptors in the stratum radiatum of the CA1 hippocampal area in different survival periods after ischemia, evoked by 10-min cardiac arrest in adult rats. In control brains, NGF receptors were expressed only on small cells with poorly ramified processes. After ischemia, the NGFR+ cells increased in size and morphological complexity (measured using fractal analysis). However, even 2 weeks after ischemia these cells did not reach the size and value of the fractal dimension typical of the largest GFAP+ astrocytes. Moreover, the reaction of NGFR+ cells was significantly delayed in comparison with the total astrocyte population. The obtained results suggest that NGF receptors are expressed mainly by immature astrocytes and ischemia induces the maturation of these cells.     

Kainate-induced zinc translocation from presynaptic terminals causes neuronal and astroglial cell death and mRNA loss of BDNF receptors in the hippocampal formation and amygdala

To evaluate the potential role of endogenous zinc in the pathophysiology of epilepsy, we injected kainic acid into the medial septum, which evokes seizure activity and delayed hippocampal degeneration. Different approaches were used. In the hippocampus, we found a movement of zinc from the synaptic compartment to CA1 pyramidal neurons and astrocytes after kainate. The same was true in the amygdala. We found that in those areas showing intense zinc bleaching there was also a loss of reactive astrocytes, which supports the view that release of synaptic zinc induces astrocytic cell death. We have also tested whether the kainate-induced zinc movement from the synaptic compartment to neuronal or glial cells alters the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, trkB. There was a prominent loss of expression of trkB mRNA in areas that coincided precisely with those displaying astrocyte loss and zinc bleaching. In the amygdala, these events were accompanied by a high upregulation of BDNF mRNA. To demonstrate further a role of synaptic zinc in hippocampal pathology, we used two different approaches. We first injected different doses of zinc chloride in the CA1 area. At lower doses (0.1-10 nmol), zinc chloride selectively induced apoptosis in CA1 pyramidal neurons and dentate granular neurons. In a second approach, we found that hippocampal zinc chelation was effective in protecting CA1 pyramidal neurons against kainate-induced cell death.     

脑创伤后bcl—2蛋白的神经保护作用

目的 探讨液压脑损伤后凋亡抑制基因bcl—2的变化规律及bcl—2基因在创伤性脑损伤后细胞凋亡中的作用。方法 应用免疫组化观察大鼠中型液压脑损伤伤前及伤后6h、12h、1d、3d、7dbcl—2蛋白表达情况,应用TUNEIL和电镜观察伤后细胞死亡的形态。结果 免疫反应阳性细胞主要位于伤侧大脑半球皮质、皮层下白质、海马CAl、CA3及齿状回的神经元和神经胶质细胞,以海马CA3区最为显。在高倍镜下,表达Bcl—2蛋白的神经细胞胞核形态正常,很少见到凋亡或坏死的形态特征。伤后早期(6h),打击侧海马CA3区Bcl—2蛋白表达显下降;Bcl—2早期改变出现在伤后6h,比细胞凋亡提前表现;伤后l—3h,Bcl—2的表达下降相对缓慢。结论 bcl—2蛋白在抑制脑创伤后细胞凋亡中起重要作用,bcl—2可能是一种可诱导的神经保护因子。     

Neuroprotection in the rat lateral fluid percussion model of traumatic brain injury by SNX-185, an N-type voltage-gated calcium channel blocker

Overload of intracellular calcium ([Ca(2+)](i)) following traumatic brain injury (TBI) has been implicated in the pathogenesis of neuronal injury and death. Voltage-gated calcium channels (VGCCs) provide one of the major sources of Ca(2+) entry into cells. Therefore, the potential neuroprotective activity of SNX-185, a specific N-type VGCC blocker, was tested in rats using the lateral fluid percussion (LFP) model of TBI. SNX-185 (50, 100, or 200 pmol) or vehicle was injected 5 min after injury into the CA2-3 subregion of the hippocampus ipsilateral to TBI. Acute neuronal degeneration was visualized in brain sections 24 h postinjury using the histofluorescent marker Fluoro-Jade (FJ), and the number of surviving neurons in the CA2-3 subregion of the hippocampus 42 days after injury was determined stereologically. Behavioral outcome after TBI and drug treatment was assessed in the beam walk test and Morris water maze. Direct injection of SNX-185 into the CA2-3 region of the hippocampus reduced neuronal injury 24 h after TBI and increased neuronal survival at 42 days at each of the three drug concentrations. Behavioral outcome in both the beam walk and Morris water maze were also improved by SNX-185, with 100 and 200 pmol, but not 50 pmol SNX-185 providing neuroprotection. These data support previous studies demonstrating substantial neuroprotection after TBI by treatment with N-type VGCC blockers.     

Effects of tumor necrosis factor-alpha central administration on hippocampal damage in rat induced by amyloid beta-peptide (25-35)

Male Wistar rats received unilateral intrahippocampal injections of 3 nmol (3.18 microg) aggregated Abeta(25-35), intracerebroventricular bilateral injections of 0.5 microg human recombinant TNFalpha or both (Abeta(25-35) + TNFalpha-treated animals). Seven days after the surgery brain sections were stained with cresyl violet (Nissl), for fragmented DNA (TUNEL), glial fibrillar acidic protein (GFAP) and isolectin B4-reactive microglia. In addition, caspase-3 activity in brain regions was measured fluorometrically. The morphology of the hippocampus after the injection of Abeta(25-35) or both Abeta(25-35) and TNFalpha (but not TNFalpha alone) showed cell loss in the CA1 pyramidal cell layer. The extension of neuronal degeneration measured in the CA1 field was significantly larger in Abeta(25-35)-treated groups compared to the contralateral hemisphere of both vehicle-treated controls and animals injected with TNFalpha alone. TNFalpha augmented the Abeta(25-35)-induced damage, significantly increasing the extension of degenerating area. Administration of Abeta(25-35) caused reactive gliosis in the ipsilateral hemisphere as demonstrated by upregulation of GFAP expression and the presence of hypertrophic astrocytes in the hippocampus. This effect was much more prominent in the hippocampi of rats treated with Abeta(25-35) + TNFalpha but absent after administration of TNFalpha alone. In both Abeta(25-35)-treated groups, the damaged area of the hippocampal CA1 field and lateral band of dentate gyrus displayed many darkly stained round isolectin B4-positive phagocyte-like microglial cells. Sparse TUNEL-positive nuclei were found in the hippocampi of rats treated with Abeta(25-35) alone or together with TNFalpha, but not in the control brain sections or in brain sections of TNFalpha-injected animals. The activity of caspase-3 increased significantly in the ipsilateral hippocampus after the injection of Abeta(25-35). Surprisingly, administration of TNFalpha into the cerebral ventricles prevented this Abeta(25-35)-induced increase in hippocampal caspase-3 activity. The results are discussed from the perspective of dual (neuroprotective and neurodestructive) roles of TNF in the brain.     

Selective alterations in the cellular distribution of apolipoprotein E immunoreactivity following transient cerebral ischaemia in the rat

The aim of this study was to examine the cellular localization and alterations of apolipoprotein E (apoE) following a transient ischaemic insult using immunohistochemistry. Transient cerebral ischaemia was induced in Wistar rats by occlusion of both carotid arteries with hypotension followed by reperfusion for 4 h (n = 5), 24 h (n = 5) or 72 h (n = 6). In sham-operated animals (n=9), the carotids were not occluded. In this model, ischaemia for 15 min results in selective neuronal damage in the caudate nucleus and neocortex (24 h after reperfusion) and the hippocampal CA1 pyramidal cells (72 h after reperfusion) while there is minimal damage in other areas such as the CA3 hippocampal region. In sham animals, apoE immunoreactivity was confined to astrocytes and their processes. ApoE immunoreactivity was not altered at 4 h post-ischaemic reperfusion. At 24 h reperfusion, intense apoE staining of the cytoplasm of astrocytes and neuropil within the caudate and neocortex was observed and at 72 h reperfusion apoE stained neuronal cell bodies within these regions. Within the CA1 region at 24 h reperfusion, there was increased immunoreactivity of the cytoplasm of astrocytes and the neuropil was more intensely stained compared with sham animals. At 72 h reperfusion, intense apoE staining of pyramidal cell bodies and dendrites was consistently observed in the CA1 region of the hippocampus. In contrast, at 72 h reperfusion, apoE staining of astrocytic processes was dramatically reduced in the CA1 region although GFAP staining indicated their preservation. The results demonstrate that following an ischaemic insult apoE is localized to degenerating neurons and their processes. This may indicate an inherent protective response of cells to injury. Alternatively, the results are consistent with the hypothesis that apoE is synthesized and released by astrocytes and taken up by neurons following injury.     

Dexamethasone enhances upregulation of nerve growth factor mRNA expression in ischemic rat brain.

Nerve growth factor (NGF) is well-established as a trophic factor that plays a crucial role in neuroregeneration and plasticity after brain insults. Dexamethasone (DEX), a powerful glucocorticoid steroid, has long been used in the clinical management of neurological disorders. We examined the relationship between NGF and DEX after an ischemic insult to the brain. In situ hybridization was used to measure NGF mRNA expression in the rat hippocampus after 20 min of transient forebrain ischemia. Immunostaining for NGF protein was performed using the avidin-biotin peroxidase method. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was also used to study the astrocyte reaction in the hippocampal CA1 area. Ischemic brain from rats not treated with DEX had a 2 and 3 fold increase in NGF mRNA compared to sham-operated rats at 4 and 6 h after ischemia, respectively. The NGF mRNA expression returned to basal levels 12 h to 7 days post-ischemia. Treatment with DEX potentiated the ischemia-induced increase of NGF mRNA to 4 times that of sham-operated rats at 6 h following reperfusion and NGF protein expression was similarly elevated. Additionally, the number of GFAP positive astrocytes in the CA1 region in the ischemic rats was markedly increased. These data suggest that DEX may play a role in modulating NGF mRNA expression in the hippocampal neuronal response to brain ischemia.     

TAK1抑制剂5Z-7-oxozeaenol对大鼠脑外伤海马CA1区的神经保护作用研究

目的探讨转化生长因子β激活激酶(TAK1)选择性抑制剂5Z-7-oxozeaenol在创伤性颅脑损伤中对伤侧海马的保护作用及可能机制;方法 72只雄性Sprague-Dawley大鼠随机分为3组:假手术组,外伤+二甲基亚砜组,外伤+5Z-7-oxozeaenol组,按照改良的Feeney法制作大鼠颅脑外伤模型,外伤后10 min通过左侧侧脑室给药,24 h后取标本,Western blot检测TAK1和磷酸化TAK1的表达,并用凝胶迁移实验检测其下游核因子κB和活化蛋白-1的活性,采用原位末端脱氧核苷酸转移酶介导的生物素脱氧尿嘧啶核苷酸缺口末端标记法(TUNEL)检测海马CA1区神经元的凋亡情况,胶质纤维酸性蛋白(GFAP)免疫组化染色检测星形胶质细胞的表达;结果与溶剂组相比,治疗组伤侧海马TAK1和磷酸化TAK1的表达量、核因子κB和活化蛋白-1的DNA结合活性显著降低,同时海马CA1区TUNEL和GFAP阳性细胞明显减少(P<0.05)。结论 5Z-7-oxozeaenol通过抑制TAK1信号通路降低海马组织中NF-κB和AP-1的活性起到抗凋亡作用。     

Effect of beraprost sodium (BPS), a prostacyclin analog,and dizocilpine (MK-801) on repeated ischemia-induced chronic cortical atrophy in gerbils

We investigated the effect of beraprost sodium (BPS), a new prostacyclin analog, and dizocilpine (MK-801) on repeated ischemia-induced cerebral atrophy and chronic cortical neuronal loss in gerbils. The left common carotid artery of gerbils was repeatedly occluded (for 10, 7, 7, and 7 min) at intervals of 24 h. The thickness of the cerebral cortex of the ischemic hemisphere diminished with increasing time of reperfusion after an ischemic insult. The animals were given BPS (1–100 μg/kg, po) or MK-801 (3–300 μg/kg, sc) after the first ischemic insult, and then twice daily for 4 wk. Increases in the amount of neuronal loss and acidophilic neurons, and progressive atrophy were observed with increasing time of reperfusion in the cerebral cortex of the ischemic hemisphere. Cortical sections revealed no astrocytes positive for glial fibrillary acidic protein (GFAP), whereas the hippocampal CA1 area showed neuronal loss accompanied by GFAP-positive astrocytes. In control animals at 4 wk survival, the area ratio neurons ratio (number of neurons in ischemic cortex/number of neurons in opposite cortex) were 89.8±3.0% and 74.6±3.4%, respectively. BPS was found to inhibit atrophy and chronic cortical neuronal loss in the ischemic hemisphere in a dose-dependent manner, whereas MK-801 showed no inhibitory effects at any dose tested. These results may suggest that the nature of neuronal degeneration differs between the cortical and hippocampal areas, that cortical neuronal degeneration might not involve glutamtate pathways with NMDA receptors in this model, and that prostacyclin could play an essential role in prevention of ischemia-induced progressive neuronal loss.     

Neurotrophic effects of basic and acidic fibroblast growth factors are not mediated through glial cells

Basic and acidic fibroblast growth factors (bFGF, aFGF) increase the survival of fetal hippocampal pyramidal neurons in serum-free cultures. bFGF is also a mitogen for astrocytes either in highly purified glial cultures or as a contaminant in neuronal cultures. The possibility that bFGF enhances neuronal survival indirectly through stimulating glial proliferation is unlikely. In the presence of 1 ng/ml bFGF, the total number of contaminating astrocytes (as defined by immunohistochemical staining for glial fibrillary acidic protein (GFAP] was increased to 4.3% vs 0.9% in control hippocampal cultures. aFGF did not significantly increase astrocyte number while supporting neuronal survival. Two other agents which stimulated equal or greater astrocytic proliferation, epidermal growth factor (EGF) and 10% serum, did not support neurons, and bFGF still significantly increased neuronal survival in their presence. When glial proliferation was inhibited by aphidicolin, contamination decreased to 0.1% in controls and 1.0% with 1 ng/ml bFGF, yet the neurons remained responsive to FGF. Cultures lacking any detectable GFAP-positive cells were identified, and even in the absence of glial cells, aFGF and bFGF increased neuronal survival. Because there is no significant correlation between the neuronal response and astrocyte number, it appears that bFGF and aFGF can directly support neuronal survival.     

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.