The Neuron-Astrocyte-Microglia Triad in Normal Brain Ageing and in a Model of Neuroinflammation in the Rat Hippocampus

Ageing is accompanied by a decline in cognitive functions; along with a variety of neurobiological changes. The association between inflammation and ageing is based on complex molecular and cellular changes that we are only just beginning to understand. The hippocampus is one of the structures more closely related to electrophysiological, structural and morphological changes during ageing. In the present study we examined the effect of normal ageing and LPS-induced inflammation on astroglia-neuron interaction in the rat hippocampus of adult, normal aged and LPS-treated adult rats. Astrocytes were smaller, with thicker and shorter branches and less numerous in CA1 Str. radiatum of aged rats in comparison to adult and LPS-treated rats. Astrocyte branches infiltrated apoptotic neurons of aged and LPS-treated rats. Cellular debris, which were more numerous in CA1 of aged and LPS-treated rats, could be found apposed to astrocytes processes and were phagocytated by reactive microglia. Reactive microglia were present in the CA1 Str. Radiatum, often in association with apoptotic cells. Significant differences were found in the fraction of reactive microglia which was 40% of total in adult, 33% in aged and 50% in LPS-treated rats. Fractalkine (CX3CL1) increased significantly in hippocampus homogenates of aged and LPS-treated rats. The number of CA1 neurons decreased in aged rats. In the hippocampus of aged and LPS-treated rats astrocytes and microglia may help clearing apoptotic cellular debris possibly through CX3CL1 signalling. Our results indicate that astrocytes and microglia in the hippocampus of aged and LPS-infused rats possibly participate in the clearance of cellular debris associated with programmed cell death. The actions of astrocytes may represent either protective mechanisms to control inflammatory processes and the spread of further cellular damage to neighboring tissue, or they may contribute to neuronal damage in pathological conditions.     

Effect of Oral Resveratrol on the BDNF Gene Expression in the Hippocampus of the Rat Brain

Resveratrol is a plant polyphenolic compound. Evidence indicates that resveratrol has beneficial effects against aging and neurodegenerative diseases. The goal of our study was in vivo examination of the effects of resveratrol on the abundance of mRNA encoding Brain Derived Neurotrophic Factor (BDNF) in the hippocampus of rat brain. Rats were administrated orally by different doses (2.5–20 mg/kg bwt) of resveratrol for 3, 10 and 30 days. Saline was used as control and 10% ethanol in saline was used as vehicle for resveratrol. Measurement of BDNF mRNA by Real-time RT–PCR showed that levels of the mRNA for BDNF were significantly and dose dependently elevated in the hippocampal tissues of rats. The findings suggest that the neuroprotective effects of resveratrol may be at least partly due to its inducing effects on the expression levels of the BDNF mRNA.     

Modulation of RAGE Isoforms Expression in the Brain and Plasma of Rats Exposed to Transient Focal Cerebral Ischemia

Activation of RAGE (receptor for advanced glycation endproducts) and of its subtypes may play a role in neuronal damage and neuroinflammation associated with brain ischemia, though the underlying mechanisms remain unclear. In this study, we have examined by Western blotting the expression of RAGE isoforms in the cerebral cortex and striatum of Wistar rats subjected to transient (1 or 2 h) middle cerebral artery occlusion (tMCAo). The findings show that the full-length RAGE (~50 kDa) and its isoforms in the 26-43 kDa range are significantly decreased in the ischemic cortex, but not in the striatum, after 1 and 2 h tMCAo when compared to the sham group. By contrast, in the striatum, ischemia-reperfusion injury caused a significant increase of full-length RAGE and its isoforms in the 72-100 kDa range. We also investigated the soluble form of RAGE, which was significantly decreased in the plasma of rats subjected to transient or permanent MCAo. In conclusion, the present data demonstrate that regional brain expression of RAGE is differentially affected by tMCAo in rat. These modifications are accompanied by a decrease in the plasma levels of soluble RAGE, thereby suggesting a potential role for soluble RAGE as a peripheral biomarker of focal ischemia.     

Xanthine and Uric Acid Levels in Rat Brain Following Focal Ischemia

Changes of the xanthine and uric acid (UA) levels in rat forebrain following focal cerebral ischemia were studied by reversed-phase HPLC with electrochemical detection. Focal ischemia was induced by occluding the left middle cerebral artery in the rat. The xanthine level in the normal group was 11.50 nmol/g tissue. In the ischemic group, the xanthine concentration in the ischemic hemisphere progressively increased after occlusion and reached a maximum value of 59.42 nmol/g tissue 4 h after operation. The UA level in the normal group was 2.20 nmol/g tissue, whereas in the ischemic group the UA concentration in the ischemic hemisphere gradually increased after occlusion, reaching a value of 38.53 nmol/g tissue 24 h after ischemia. The concentration of UA remained elevated in the ischemic hemisphere until 48 h after occlusion, and reached a maximum value of 38.98 nmol/g tissue. The xanthine and UA levels in the contralateral hemisphere remained unchanged. The xanthine and UA concentrations in the sham-operated group did not show a significant increase after operation. The time course of xanthine and UA levels suggests that in ischemic forebrain UA is formed from xanthine as a product of purine metabolism.     

Increased Expression of Neurotrophin 4 Following Focal Cerebral Ischemia in Adult Rat Brain with Treadmill Exercise

Neurotrophin 4 (NT-4) belongs to the family of neurotrophic factors, and it interacts with the tyrosine kinase B (trkB) receptor. NT-4 has neuroprotective effects following cerebral ischemia. Its role might be similar to brain-derived neurotrophic factor (BDNF), because both interact with trkB. Exercise also improves neural function by increasing neurotrophic factors. However, expression profiles of NT-4 in the brain during exercise are unknown. Here, we assessed the expressions of NT-4 and its receptor, trkB, following cerebral ischemia and hypothesized that exercise changes the expressions of NT-4 and trkB. Results showed that in a permanent middle cerebral artery occlusion rat model, ischemia decreased NT-4 and trkB expression. Immunohistochemistry showed their immunoreactivities around the region of the ischemic area. Treadmill exercise changed the expression of NT-4, which increased in the contralateral hemisphere in rats with ischemic injury. TrkB also showed similar patterns to its neurotophins. The change in NT-4 suggested that exercise might have primed NT4 production so that further injury causes slightly greater increases in NT4 compared with non-exercise controls.     

Human-Derived Physiological Heat Shock Protein 27 Complex Protects Brain after Focal Cerebral Ischemia in Mice

Although challenging, neuroprotective therapies for ischemic stroke remain an interesting strategy for countering ischemic injury and suppressing brain tissue damage. Among potential neuroprotective molecules, heat shock protein 27 (HSP27) is a strong cell death suppressor. To assess the neuroprotective effects of HSP27 in a mouse model of transient middle cerebral artery occlusion, we purified a “physiological” HSP27 (hHSP27) from normal human lymphocytes. hHSP27 differed from recombinant HSP27 in that it formed dimeric, tetrameric, and multimeric complexes, was phosphorylated, and contained small amounts of αβ-crystallin and HSP20. Mice received intravenous injections of hHSP27 following focal cerebral ischemia. Infarct volume, neurological deficit scores, physiological parameters, and immunohistochemical analyses were evaluated 24 h after reperfusion. Intravenous injections of hHSP27 1 h after reperfusion significantly reduced infarct size and improved neurological deficits. Injected hHSP27 was localized in neurons on the ischemic side of the brain. hHSP27 suppressed neuronal cell death resulting from cytochrome c-mediated caspase activation, oxidative stress, and inflammatory responses. Recombinant HSP27 (rHSP27), which was artificially expressed and purified from Escherichia coli, and dephosphorylated hHSP27 did not have brain protective effects, suggesting that the phosphorylation of hHSP27 may be important for neuroprotection after ischemic insults. The present study suggests that hHSP27 with posttranslational modifications provided neuroprotection against ischemia/reperfusion injury and that the protection was mediated through the inhibition of apoptosis, oxidative stress, and inflammation. Intravenously injected human HSP27 should be explored for the treatment of acute ischemic strokes.     

Plasticity Response in the Contralesional Hemisphere after Subtle Neurotrauma: Gene Expression Profiling after Partial Deafferentation of the Hippocampus

Neurotrauma or focal brain ischemia are known to trigger molecular and structural responses in the uninjured hemisphere. These responses may have implications for tissue repair processes as well as for the recovery of function. To determine whether the plasticity response in the uninjured hemisphere occurs even after a subtle trauma, we subjected mice to a partial unilateral deafferentation of the hippocampus induced by stereotactically performed entorhinal cortex lesion (ECL). The expression of selected genes was assessed by quantitative real-time PCR in the hippocampal tissue at the injured side and the contralesional side at day 4 and 14 after injury. We observed that expression of genes coding for synaptotagmin 1, ezrin, thrombospondin 4, and C1q proteins, that have all been implicated in the synapse formation, re-arrangement and plasticity, were upregulated both in the injured and the contralesional hippocampus, implying a plasticity response in the uninjured hemisphere. Several of the genes, the expression of which was altered in response to ECL, are known to be expressed in astrocytes. To test whether astrocyte activation plays a role in the observed plasticity response to ECL, we took advantage of mice deficient in two intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and vimentin (GFAP^−/−Vim^−/−) and exhibiting attenuated astrocyte activation and reactive gliosis. The absence of GFAP and vimentin reduced the ECL-induced upregulation of thrombospondin 4, indicating that this response to ECL depends on astrocyte activation and reactive gliosis. We conclude that even a very limited focal neurotrauma triggers a distinct response at the contralesional side, which at least to some extent depends on astrocyte activation.     

Mitochondrial Calcium Transport and Mitochondrial Dysfunction After Global Brain Ischemia in Rat Hippocampus

Here we report effect of ischemia-reperfusion on mitochondrial Ca²⁺ uptake and activity of complexes I and IV in rat hippocampus. By performing 4-vessel occlusion model of global brain ischemia, we observed that 15 min ischemia led to significant decrease of mitochondrial capacity to accumulate Ca²⁺ to 80.8% of control whereas rate of Ca²⁺ uptake was not significantly changed. Reperfusion did not significantly change mitochondrial Ca²⁺ transport. Ischemia induced progressive inhibition of complex I, affecting final electron transfer to decylubiquinone. Minimal activity of complex I was observed 24 h after ischemia (63% of control). Inhibition of complex IV activity to 80.6% of control was observed 1 h after ischemia. To explain the discrepancy between impact of ischemia on rate of Ca²⁺ uptake and activities of both complexes, we performed titration experiments to study relationship between inhibition of particular complex and generation of mitochondrial transmembrane potential (ΔΨ_m). Generation of a threshold curves showed that complex I and IV activities must be decreased by approximately 40, and 60%, respectively, before significant decline in ΔΨ_m was documented. Thus, mitochondrial Ca²⁺ uptake was not significantly affected by ischemia-reperfusion, apparently due to excess capacity of the complexes I and IV. Inhibition of complex I is favourable of reactive oxygen species (ROS) generation. Maximal oxidative modification of membrane proteins was documented 1 h after ischemia. Although enhanced formation of ROS might contribute to neuronal injury, depressed activities of complex I and IV together with unaltered rate of Ca²⁺ uptake are conditions favourable of initiation of other cell degenerative pathways like opening of mitochondrial permeability transition pore or apoptosis initiation, and might represent important mechanism of ischemic damage to neurones.     

Focal Experimental Injury Leads to Widespread Gene Expression and Histologic Changes in Equine Flexor Tendons

It is not known how extensively a localised flexor tendon injury affects the entire tendon. This study examined the extent of and relationship between histopathologic and gene expression changes in equine superficial digital flexor tendon after a surgical injury. One forelimb tendon was hemi-transected in six horses, and in three other horses, one tendon underwent a sham operation. After euthanasia at six weeks, transected and control (sham and non-operated contralateral) tendons were regionally sampled (medial and lateral halves each divided into six 3cm regions) for histologic (scoring and immunohistochemistry) and gene expression (real time PCR) analysis of extracellular matrix changes. The histopathology score was significantly higher in transected tendons compared to control tendons in all regions except for the most distal (P ≤ 0.03) with no differences between overstressed (medial) and stress-deprived (lateral) tendon halves. Proteoglycan scores were increased by transection in all but the most proximal region (P < 0.02), with increased immunostaining for aggrecan, biglycan and versican. After correcting for location within the tendon, gene expression for aggrecan, versican, biglycan, lumican, collagen types I, II and III, MMP14 and TIMP1 was increased in transected tendons compared with control tendons (P < 0.02) and decreased for ADAMTS4, MMP3 and TIMP3 (P < 0.001). Aggrecan, biglycan, fibromodulin, and collagen types I and III expression positively correlated with all histopathology scores (P < 0.001), whereas lumican, ADAMTS4 and MMP14 expression positively correlated only with collagen fiber malalignment (P < 0.001). In summary, histologic and associated gene expression changes were significant and widespread six weeks after injury to the equine SDFT, suggesting rapid and active development of tendinopathy throughout the entire length of the tendon. These extensive changes distant to the focal injury may contribute to poor functional outcomes and re-injury in clinical cases. Our data suggest that successful treatments of focal injuries will need to address pathology in the entire tendon, and that better methods to monitor the development and resolution of tendinopathy are required.     

Human Microglia Transplanted in Rat Focal Ischemia Brain Induce Neuroprotection and Behavioral Improvement

Background and Purpose

Microglia are resident immunocompenent and phagocytic cells of central nervous system (CNS), which produce various cytokines and growth factors in response to injury and thereby regulate disease pathology. The purpose of this study is to investigate the effects of microglial transplantation on focal cerebral ischemia model in rat.

Methods

Transient middle cerebral artery occlusion (MCAO) in rats was induced by the intraluminal filament technique. HMO6 cells, human microglial cell line, were transplanted intravenously at 48 hours after MCAO. Functional tests were performed and the infarct volume was measured at 7 and 14 days after MCAO. Migration and cell survival of transplanted microglial cells and host glial reaction in the brain were studied by immunohistochemistry. Gene expression of neurotrophic factors, cytokines and chemokines in transplanted cells and host rat glial cells was determined by laser capture microdissection (LCM) and quantitative real time-PCR.

Results

HMO6 human microglial cells transplantion group demonstrated significant functional recovery compared with control group. At 7 and 14 days after MCAO, infarct volume was significantly reduced in the HMO group. In the HMO6 group, number of apoptotic cells was time-dependently reduced in the infarct core and penumbra. In addition, number of host rat microglia/macrophages and reactive astrocytes was significantly decreased at 7 and 14 days after MCAO in the penumbra. Gene expression of various neurotrophic factors (GDNF, BDNF, VEGF and BMP7) and anti-inflammatory cytokines (IL4 and IL5) was up-regulated in transplanted HMO6 cells of brain tissue compared with those in culture. The expression of GDNF and VEGF in astrocytes in penumbra was significantly up-regulated in the HMO6 group.

Conclusions

Our results indicate that transplantation of HMO6 human microglial cells reduces ischemic deficits and apoptotic events in stroke animals. The results were mediated by modulation of gliosis and neuroinflammation, and neuroprotection provided by neurotrophic factors of endogenous and transplanted cells-origin.     

局灶缺血性脑卒中大鼠侧脑室下带Shh信号通路成分的动态表达

通过研究Sonic hedgehog(Shh)信号通路成分在局灶缺血性脑卒中大鼠侧脑室下带(subventricular zone,SVZ)的动态表达,初步探讨该通路在局灶性缺血性脑卒中后神经再生的调控作用.将84只健康成年雄性SD大鼠随机分为正常组(n=12)、假手术组(n=12)、缺血6、12、24 h和3、7 d,共7组(n=12).采用线栓法制备大鼠右侧大脑中动脉阻断(middle cerebral artery occlussion,MCAO)模型.分别应用逆转录聚合酶链反应(RT-PCR)、免疫组化、免疫印迹法检测局灶脑缺血大鼠侧脑室下带Shh、Gli1 mRNA和蛋白变化.与正常组比较,Shh、Gli1mRNA和蛋白在假手术组表达变化不明显(P>0.05),模型组6 h表达增高(P<0.01),24 h达峰值(P<0.01),3 d时接近正常水平(P>0.05),7 d表达又升高(P<0.01).缺血性脑卒中可以上调Shh信号通路成分在SVZ区的表达,提示Shh信号通路可能参与卒中后神经再生机制的调控.     

Electroacupuncture Prevents Cognitive Impairments by Regulating the Early Changes after Brain Irradiation in Rats

Cognitive impairments severely affect the quality of life of patients who undergo brain irradiation, and there are no effective preventive strategies. In this study, we examined the therapeutic potential of electroacupuncture (EA) administered immediately after brain irradiation in rats. We detected changes in cognitive function, neurogenesis, and synaptic density at different time points after irradiation, but found that EA could protect the blood-brain barrier (BBB), inhibit neuroinflammatory cytokine expression, upregulate angiogenic cytokine expression, and modulate the levels of neurotransmitter receptors and neuropeptides in the early phase. Moreover, EA protected spatial memory and recognition in the delayed phase. At the cellular/molecular level, the preventative effect of EA on cognitive dysfunction was not dependent on hippocampal neurogenesis; rather, it was related to synaptophysin expression. Our results suggest that EA applied immediately after brain irradiation can prevent cognitive impairments by protecting against the early changes induced by irradiation and may be a novel approach for preventing or ameliorating cognitive impairments in patients with brain tumors who require radiotherapy.