ABSTRACT
Aim To investigate the dynamic time-course changes in neuronal cytoskeleton after acute ischemia and reperfusion in rats. Methods Reperfusion was performedin rats by blocking the middle cerebralarteryfor 90 min, then therats wereobserved and collected at different time points. The brain damage wasobserved by Nissl staining,and neurobehavioural function was evaluated with neurological deficit score and forelimb placement test. The cellular changes in the alternations of cytoskeletal elements including microtubule associated protein 2 (MAP2) and neurofilament heavy chain (NF-H) were observed by immunohistochemistry staining and Western blot. Impaired axons, dendrites and cytoskeletal alternations were detected by electron microscope. Results Brain damage and neurobehavioural function were gradually aggravated with the prolongation of reperfusion. Brain damage appeared earlier and more severe in striatum than in cortex. Moreover, decreased MAP2-related and increased NF-H-related immunoreactive intensities were found in the ischemic areas. Impaired cytoskeletal arrangement and reduced dense were indicated. Damaged cytoskeletal components such as microtubules and neurofilament arrangement, decreased axonal filament density, and swelled dendrites were observed after cerebral ischemia reperfusion by ultrastructural observations. Conclusions Different brain regions have diverse tolerance to ischemia-reperfusion injury. Major elements of neuronal cytoskeleton show dynamic responses to ischemia and reperfusion, which may further contribute to brain damage and neurological impairment following MCAO and reperfusion.
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Ischemic stroke is the second leading cause of human death and the third reason of disability. Meanwhile, the incidence is rising year after year worldwide. Ischemic stroke could cause ischemia-reperfusion injury after blood recanalization treat-ment, but the mechanism of ischemia-reperfusion injury is still not very clear, so it is necessary to build a preclinical model with specific characteristics. Up to now, animal experiments have been still complicated, and the culture of brain slices has some limitations. The cell model in vitro has become a simplified and valuable tool widely used by researchers. The paper systematically summarizes the common type of nerve cells, and further analyzes establishment methods and principle, relevant research progress on the in vitro model of ischemia-reperfusion, in order to provide reference for rationally selecting hypoxia and reoxygenation model for basic research on cerebral ischemia and reperfusion and drug screening.
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Aim To observe cellular damage and astrocyte activation at different time points of cerebral ischemia and reperfusion. Methods The middle cerebral artery of male SpragueDawley rats was occluded for 90 min followed by different time points of reperfusion. Eighty-five SPF male SD rats were randomly divided into control group (Sham), IR3, 6, 12, 24 and IR48h (MCAO followed by 48 h of reperfusion) group. Cerebral ischemia and reperfusion injury was observed by HE staining, and the structure of astrocytes was estimated with transmission electron microscopy (TEM). GFAP expression was detected by immunofluorescence staining and Western blot. Results Cerebral ischemia following by different time points of reperfusion led to different degrees of cellular damage, which was the most serious at 24 h of reperfusion. TEM showed destruction of astrocytes structure, swollen organelles and broken mitochondrial ridge. After cerebral ischemia-reperfusion, the expression levels of GFAP were significant up-regulated in the ischemic penumbra cortex and the highest was at 48 h of reperfusion, indicating astrocytes were activated. In addition, the results showed the gradual decrease in GFAP expression in the infarct core. Conclusions After cerebral ischemia-reperfusion, cellular damage is aggravated, and astrocytes are gradually activated in the ischemic penumbra. With the extension of reperfusion time, the boundaries of infarct area and ischemic area are gradually clear, and scarring may occur.
ABSTRACT
Stroke is the second leading cause of death in the world, of which about 60 % - 80 % are ischemic stroke. Ischemic stroke will inevitably cause the damage of neurons in the core area. With the increase of ischemic time, other neurons in the ischemic penumbra will also die due to the loss of " signal connection", and further lead to body dysfunction. In view of the complexity of neuronal death mechanism after ischemic stroke, understanding the action principle of death mechanism can better save ischemic penumbra neurons. This review mainly expounds several main mechanisms and potential therapeutic targets of neuronal death after ischemic stroke, so as to provide basis and help for the improvement of action mechanism research and drug development.
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This study aims to explore the effect of Xiaoxuming Decoction on synaptic plasticity in rats with acute cerebral ischemia-reperfusion. A rat model of cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion(MCAO). Rats were randomly assigned into a sham group, a MCAO group, and a Xiaoxuming Decoction(60 g·kg~(-1)·d~(-1)) group. The Longa score was rated to assess the neurological function of rats with cerebral ischemia for 1.5 h and reperfusion for 24 h. The 2,3,5-triphenyltetrazolium chloride(TTC) staining and hematoxylin-eosin(HE) staining were employed to observe the cerebral infarction and the pathological changes of brain tissue after cerebral ischemia, respectively. Transmission electron microscopy was employed to detect the structural changes of neurons and synapses in the ischemic penumbra, and immunofluorescence, Western blot to determine the expression of synaptophysin(SYN), neuronal nuclei(NEUN), and postsynaptic density 95(PSD95) in the ischemic penumbra. The experimental results showed that the modeling increased the Longa score and led to cerebral infarction after 24 h of ischemia-reperfusion. Compared with the model group, Xiaoxuming Decoction intervention significantly decreased the Longa score and reduced the formation of cerebral infarction area. The modeling led to the shrinking and vacuolar changes of nuclei in the brain tissue, disordered cell arrangement, and severe cortical ischemia-reperfusion injury, while the pathological damage in the Xiaoxuming Decoction group was mild. The modeling blurred the synaptic boundaries and broadened the synaptic gap, while such changes were recovered in the Xiaoxuming Decoction group. The modeling decreased the fluorescence intensity of NEUN and SYN, while the intensity in Xiaoxuming Decoction group was significantly higher than that in the model group. The expression of SYN and PSD95 in the ischemic penumbra was down-regulated in the model group, while such down-regulation can be alleviated by Xiaoxuming Decoction. In summary, Xiaoxuming Decoction may improve the synaptic plasticity of ischemic penumbra during acute cerebral ischemia-reperfusion by up-regulating the expression of SYN and PSD95.
Subject(s)
Rats , Animals , Rats, Sprague-Dawley , Brain Ischemia/drug therapy , Reperfusion Injury/metabolism , Infarction, Middle Cerebral Artery , Neuronal Plasticity , ReperfusionABSTRACT
This study investigated the effect of Xiaoxuming Decoction(XXMD) on the activation of astrocytes after cerebral ischemia/reperfusion(I/R) injury. The model of cerebral IR injury was established using the middle cerebral artery occlusion method. Fluorocitrate(FC), an inhibitor of astrocyte activation, was applied to inhibit astrocyte activation. Rats were randomly divided into a sham group, a model group, a XXMD group, a XXMD+FC group, and a XXMD+Vehicle group. Neurobehavioral changes at 24 hours after cerebral IR injury, cerebral infarction, histopathological changes observed through HE staining, submicroscopic structure of astrocytes observed through transmission electron microscopy, fluorescence intensity of glial fibrillary acidic protein(GFAP) and thrombospondin 1(TSP1) measured through immunofluorescence, and expression of GFAP and TSP1 in brain tissue measured through Western blot were evaluated in rats from each group. The experimental results showed that neurobehavioral scores and cerebral infarct area significantly increased in the model group. The XXMD group, the XXMD+FC group, and the XXMD+Vehicle group all alleviated neurobehavioral changes in rats. The pathological changes in the brain were evident in the model group, while the XXMD group, the XXMD+FC group, and the XXMD+Vehicle group exhibited milder cerebral IR injury in rats. The submicroscopic structure of astrocytes in the model group showed significant swelling, whereas the XXMD group, the XXMD+FC group, and XXMD+Vehicle group protected the submicroscopic structure of astrocytes. The fluorescence intensity and protein expression of GFAP and TSP1 increased in the model group compared with those in the sham group. However, the XXMD group, the XXMD+FC group, and XXMD+Vehicle group all down-regulated the expression of GFAP and TSP1. The combination of XXMD and FC showed a more pronounced effect. These results indicate that XXMD can improve cerebral IR injury, possibly by inhibiting astrocyte activation and down-regulating the expression of GFAP and TSP1.
Subject(s)
Rats , Animals , Astrocytes , Brain Ischemia/metabolism , Brain , Reperfusion Injury/metabolism , Infarction, Middle Cerebral ArteryABSTRACT
Advances in mechanism and application of the heating effect in breeding of microorganism are reviewed in this paper. Heat produces mutagenesis effect and screening effect. Heating mutagenesis effect is occurred through the substitution of G-C base pair induced by heat, and heating screening effect produces higher forward mutation rate induced by other mutagens.
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Based on the strain of Micromonospora carbonacea JXNU-1 with board-spectrum antimicrobial activity, the technology for the isolation and purification of antibiotic from the fermentation broth of the Micromonospora carbonacea, and its physical-chemical properties were studied. The results showed that, the antibiotic was stable under the condition of high temperature and alkali, but not in acid solution. After the pretreatment of centrifugation and filtration to remove the cells and lipids, the antibiotic was absorbed to negative exchange resin, and the impurity was excluded when 2 mol/L NaCl was used as primary eluent. The antibiotic could be eluted with 20% alcohol as eluent, and the eluting speed of the antibiotic was greatly accelerated as 2 mol/L NaCl was added into 20% alcohol as final eluent. Aqueous solution of the antibiotic was yielded from the alcohol-salt eluant by decompression concentration to wipe off alcohol and by dialysis to exclude salt. One active component was detected in antibiotic solution by paper chromatography, and theHPLC purity was over 99%. As the antibiotic shows positive color-forming reaction to Molish reagents, Benedict’s reagents and Diohenvlamine reagents, combined with the characteristics of absorption spectra, it is deduced that the antibiotic belongs to nucleoside antibiotics.
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A rare strain of actinomycetes, with broad-spectrum antimicrobial activity, was isolated from the soil samples from the farmland in the area of Yaohu lake in Nanchang. The information about the taxonomic identification, such as the morphology, physiological properties, cell components and 16S rRNA gene se-quences, suggested that the rare strain of actinomycetes was identified as Micromonospora carbonacea.