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Objective To investigate the relationship between metabolite levels and the time of cerebral ischemia-reperfusion in the left cortex of rats, so as to explore the molecular mechanism of cortical metabolic disorders induced by ischemia-reperfusion injury. Methods Stroke models of focal cerebral ischemia in rats were established by middle cerebral artery occlusion (MCAO). Then a nuclear magnetic resonance (NMR)-based metabolome analytical approach was carried out to analyze the metabolite levels in the left cortex of MCAO rats at different time points (3, 6, and 24 h) after reperfusion. Results Some changes of metabolic pathways, such as energy deficiency, glycolysis aggravation, and neurotransmitter disorders, were observed in the left cortex of MCAO rats at 3 h after reperfusion. All the above-mentioned disorders were alleviated by the autoregulation at 6 h after reperfusion. However, the forementioned metabolic disturbances became severe after 24-hour reperfusion. Conclusion Our results suggest that different extents of metabolic disturbance appears in the cortex at different time points after reperfusion, which might help to understand the molecular mechanism of cerebral ischemia-reperfusion injury, providing reference for regulating metabolic disorders at different time points after stroke in clinic.
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Objective To observe the effect of electro-acupuncture (EA) intervention on expression of Slit2 and SrGAP1 in rats with focal cerebral infarction and to study the underlying mechanism of EA intervention on neuronal plasticity after cerebral ischemia. Methods Sixty male Sprague Dawley rats were randomly divided into Model group(n=30) and EA group (n=30); and according to elctro-apunture treatment duration, both 2 groups were further randomized into 3 subgroups:0 d(n=10),7 d(n=10), and 14 d (n=10).The middle cerebral arteries of the model group and EA group were embolized for 1.5 hours by suture. Nissl staining, immunofluorescence method and Western blotting analysis were sued to observe the histology and morphology change around the cerebral infarction and to assay the expression of Slit2 and SrGAP1 in the cerebral cortex on the ischemic side on day 0, 7, and 14. Results In comparison with the model group, the neurological score of EA group had no obvious difference on day 0 (P>0.05) and was significantly lower on day 7 and day 14 (P0.05); but on day 7 and day 14, the fluorescence intensities and grey levels of EA group were significantly higher than those of the model group(P<0.05). In the model group the above values on day 0 were significantly lower than those on day 7 (P<0.05), and those on day 7 were significantly higher than those on day 14 (P<0.05). In the EA group the fluorescence intensities and grey levels on day 0 were significantly lower than those on day 7 (P<0.05), and those on day 7 were significantly higher than those on day 14 (P<0.05). Conclusion Slit2 and SrGAp1 are lowly expressed following focal cerebral infarction on day 0, and EA intervention for one and two weeks can promote their expression and prolong the duration of high expression, subsequently accelerating axonal regeneration and repair, which may be a mechanism of EA therapy for neurological function recovery following cerebral infarction.
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Objective To explore the effect of cyclopamine (Cyc) on cerebral ischemia-reperfusion injury in rats. Methods Sixty male Sprague-Dawley (SD) rats were randomly divided into 3 groups (n=20): sham operation group, cerebral ischemia-reperfusion group (Con group) and cerebral ischemia-reperfusion+Cyc group (Cyc group). Cyc (10 mg/kg) or absolute ethyl alcohol was intraperitoneally injected in animals 3 h after cerebral ischemia for 7 d. Neurological deficit was assessed by Longa scale on day 1 and 14 after cerebral ischemia. At 24 h after cerebral ischemia, the cerebral water content, cerebral infaction area, pathological changes and cell apoptosis were evaluated by dry-wet method, 2,3,5-triphenyltetrazolium (TTC) staining, H-E staining and TUNEL method, respectively. Immunochemical method was used to examine the protein expression of NeuN and caspase-3 at 24 h after ischemia and glial fibrillary acidic protein (GFAP) expression on day 14 after ischemia. Results Neurological deficit score, cerebral water content, cerebral infaction area, TUNEL positive cell counting, protein levels of caspase-3 and GFAP in Cyc and Con groups were significantly higher than those in sham operation group (P<0.05), and the above parameters in Cyc group were also significantly higher than those in Con group (P<0.05). NeuN protein expressions in Cyc and Con groups were significantly lower than those in sham operation group (P<0.05), and NeuN protein in Cyc group was also significantly lower than that in Con group (P<0.05). Cellular and interstitial edema, neurocyte deformation, pyknosis and necrocytosis were more severe in Cyc group than in Con group. Conclusion Cyclopamine can aggravate rat cerebral ischemia-reperfusion injury.
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Objective To investigate the effect of transplantation with the three-dimensional spheroid-cultured mesenchymal stem cells (MSCs) on the expression of Nogo-A and NgR in rats with cerebral ischemia-reperfusion injury. Methods The experimental animals were randomly divided into Sham group, Vehicle group and MSCs treated group. The model of focal ischemia-reperfusion in rats was induced by intraluminal middle cerebral artery (MCA) occlusion with a nylon monofilament suture in Vehicle group and MSCs treated group. The fishing line was unpluged for reperfusion 2 h after ischemia and MSCs were transplanted in MSCs treated group one day later. Equivalent medium solution was given to the Vehicle group 1 d later. On the 1st day, 3rd day, and 7th day after transplantation, the neuromotor function of the animals was detected. The brain tissue of rats was harvested for RT-PCR detection of Nogo-A and NgR mRNA expression in the brain tissue of rats, and Western blotting analysis was used to detect the expression of Nogo-A and NgR protein. Results Compared with the Vehicle group, the neuromotor function was significantly improved in MSCs treated group on the 7th day; and the expressions of Nogo-A and NgR mRNA and protein were significantly down-regulated in MSCs treated group on the 1st day, 3rd day, and 7th day after transplantation (P<0.05). Conclusion Transplantation of the three-dimensional spheroid-cultured MSCs can improve the neuromotor function following cerebral ischemia/reperfusion injury, and its mechanism may be associated with down-regulation of Nogo-A and NgR in the brain tissue.
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Objective To investigate the protective effects of glutathione (GSH) against hepatic ischemia/reperfusion (I/R) injury and the related mechanism. Methods SD rats were randomized into Sham , I/R and GSH groups with 20 rats in each group. Rat models of segmental (70~Y) warm hepatic ischemia were established in I/R and GSH groups. GSH was injected through the femoral vein at the dose of 5 mg/kg 5 min before ischemia. At 24 h after reperfusion , liver injury was evaluated by serological and histological indices. Liver cell apoptoses were evaluated by TUNEL staining. The GSH/oxidized glutathione (GSSG) ratios of tissue level were compared between different groups. Liver mitochondria were collected and the mitochondrial calcium capacity (CRC) was evaluated. Results The serum aspartate transaminanse (AST) and alanine aminotransferase (ALT) levels in GSH group were significantly decreased 6 h after reperfusion compared with I/R group(P< 0. 05). 24 h after reperfusion , the liver injury was alleviated and the number of apoptosis cells was significantly decreased in GSH group compared with I/R group (P <0. 05). The GSH/GSSG ratio of tissue level in GSH group was significantly increased 6 h after reperfusion compared with I/R group (P<0. 05). Liver mitochondrial CRC in GSH group was significantly increased 6 h after reperfusion compared with I/R group (P<0. 05). Conclusion GSH preconditioning can protect liver from hepatic I/R injury, which is possibly by inhibiting oxidative response and subsequent inhibition of mitochondrial permeability transition.
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Objective To investigate the neuroprotective effects of low dose intravenous minocycline against ischemia reperfusion injury in rats after focal cerebral ischemia reperfusion and to explore the possible mechanism. Methods Seventy-two rats were randomly divided into sham-operated group (S group), cerebral ischemia/reperfusion group (I/R group), and minocycline intervention group (I/R+MC group). Focal cerebral ischemia was induced by filament medial cerebral artery occlusion method. Minocycline (3 mg/kg) in saline was administered intravenously via the caudal vein twice a day for 14 days in the I/R + MC group. At 2 days after ischemia reperfusion, the infarct volume was evaluated using TTC staining, the permeability of blood-brain barrier was assessed by Evan’s blue (EB) dye extravasation, and the expressions of high mobility group box-1 protein (HMGB1) and Iba1, a marker of activated microglia, were analyzed by using Western blotting analysis. The neurological function recovery was evaluated using the modified neurological severity score (mNSS) at 2d, 7d, and 14d after ischemia/reperfusion. Results Two days after cerebral ischemia reperfusion, the brain infarction volume and the extravasations of EB were significantly increased and the expressions of HMGB1 and Iba1 were significantly up-regulated in I/R groupcompared with sham group (P < 0.05). Compared with I/R group, minocycline at 3 mg/kg via the caudal vein significantly reduced the infarct volume in ischemic brain(P < 0.05), extravasation of EB and expressions of HMGBl and Ibal (P < 0.05). Additionally, the rats in I/R+MC group exhibited a significantly decreased neurological severity score compared with I/R group (P < 0.05). Conclusion Minocycline exerts a neuroprotective effect in rats with cerebral ischemia reperfusion injury, which may be related to decrease of infarct size, inhibition of EB extravasation and microglia activation.
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Objective To explore the effect of the intestinal ischemia/reperfusion injury on paracrine patterns of BM-MSCs in order to provide evidence for BMSCs repair.Methods BMSCs were isolated,cultured,characterized,and stained.The rat intestine ischemia/reperfusion model (IRI) was established.Intestinal epithelial mucosal extracts were obtained either from IRI rat or normal rat.Those two type extracts were added into the culture medium,and BM-MSCs were cultured with the two conditioned medium respectively.The culture medium was collected at 0 h and 24 h after culturing with intestinal epithelial mucosal extracts.The levels of TGF-ot,VEGF,FGF and IGF-1 in the medium were assayed by ELISA.Results The contents of all the growth factors detected in cultured medium with intestinal extracts from IRI rats were significantly higher than in regular rats (P ＜ 0.05).The levels of TGF-α,VEGF in culture medium containing IRI epithelium extracts were significantly higher than that in the medium containing normal intestinal epithelium extracts (P ＜ 0.05)Conclusion BM-MSCs can secret TGF-α,VEGF,FGF,and IGF-1 in the presence of intestinal extracts from IRI rat in the culture.These factors might stimulate the differentiation and proliferation of intestinal epithelial cells through paracrine mechanism.
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Objective To investigate the protective effects of simvastatin on cobalt choride ( CoCl2 ) -induced hypoxia and reoxygenation injury on alveolar type Ⅱ cells and the underlying mechanisms.Methods CoCl2 was used to establish the hypoxia and reoxygenation injury model on AT Ⅱ cells.Blank,control and variant doses simvastatin-treated groups ( 5,10,20,30,50,100 μ mol/L) were designed in the present study.The proliferation of AT Ⅱ cells was evaluated by Cell Counting Kit-8 ( CCK-8 ) assay.The percentage of apoptotic cells was assessed by flow cytometry AV/PI double-staining.The protein levels of surfactant protein-C (SP-C) and proliferating cell nuclear antigen (PCNA) in AT Ⅱ cells was determined by Western blot.Results As compared with the control group,pretreatment with low dose (5 - 20 μmol/L),but not high dose simvastatin (50 - 100 μmol/L) markedly reduced A549 cells apoptosis,and increased their proliferation and the protein levels of SPC and PCNAin vitro.The protective effect could be reversed in vitro by L-mevalonate,a simvastatin competitive inhibitor,which indicated that the inhibition of mevalorate pathway was involved in the simvastatin induced AT Ⅱ cells function restoration.Condusion Low doses simvastatin reversed CoCl2-induced hypoxia and reoxygenation injury of AT Ⅱ cells.The inhibition of mevalonate pathway contributed to simvastatin induced AT Ⅱ cells function restoration.
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Objective: To investigate the protective effect of astilbin on warm ischemia/reperfusion-induced liver injury and to study the related mechanism. Methods: C57BL/6 mice were randomly divided into four groups (n=8): sham-operated group (Sham), model control group (I/R), low dose astilbin treatment group (10 mg/kg) and high dose astilbin (40 mg/kg) treatment group. Mice in the two treatment groups were intraperitoneally injected with astilbin 24 hours and one hour before ischemia. Then 70% hepatic ischemia/reperfusion model (the left and middle hepatic lobe) was established. Mice in the I/R model control group and the sham operation group were administered with the same volume of normal saline. The blood sample and liver tissue samples were collected 90 min after ischemia and 6 h after reperfusion. Serum ALT activity was detected as an indicator of liver function damage and the content of MPO in liver tissues were detected by ELISA. The pathological changes of the liver were observed. The expression of IL-10 in liver tissues was detected by Western blotting and the expression of IL- 10 mRNA was detected by semi-quantitative RT-PCR. Results: Astilbin treatment can effectively lower the serum ALT level and MPO level in the liver tissues in some I/R mice. It could also improve the pathological manifestations of the liver. Compared with the I/R model control group, IL-10 protein levels gradually increased in the two treatment groups, which was consistent with the result of RT-PCR (low dose group P<0.05; high dose group, P<0.01). Conclusion: Astilbin can effectively reduce the inflammatory response after liver warm ischemia-reperfusion induced injury, effectively improve the mouse liver function and pathological damage, which might be related to the upregulation of IL-10 expression in the liver tissues.
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Objective: To study whether the neuroprotective effect of tibolone during cerebral ischemia injury is associated with its anti-apoptosis effect. Methods: A total of 30 healthy adult female Sprague Dawley rats were evenly randomized into sham-operated group, ovariectomized group, and tibolone group(0.25 mg/[kg·d]). Brain ischemia model was induced by left middle cerebral artery occlusion(MCAO) for 2 hours followed by 24 hours reperfusion. All animals were killed and the cerebral infarction volumes were determined by 2,3,5-triphenyltetrazlium chloride (TTC) staining and image analysis. Apoptotic cells were labeled by Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL). The expression of Bcl-2, Bax, and caspase-3 was analyzed by immunohistochemical SP method. Results: Compared with the sham-operated group(P<0.05), cerebral infarction volume was significantly reduced, TUNEL positive cells was significantly decreased(P<0.01), Bcl-2 positive cells were significantly increased, and Bax(P<0.01), caspase-3 postive cells were significantly reduced compared with tibolone treated groups (P<0.01). Conclusion: Tibolone can inhibit the neuron apoptosis following cerebral ischemia injury in OVX rats, displaying a neuroprotective effect.
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The effects of hepatic ischemia/reperfusion (I/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on storeoperated calcium current (Isoc) in isolated rat hepatocytes after hepaticischemia/reperfusion injuries were studied. Hepatic ischemia and reperfusion injury model was established and whole cell patch-clamp techniques were used to investigate the effects of 2-APB on Isoc. The results showed that ischemia/reperfusion injuries could significantly reduce hepatocellular viability and further increase Isoc in hepatocytes and 2-APB (20, 40, 60, 80, 100 μmol/L) produced a concentration-dependent decrease of Isoc with IC50 value of 64.63±10.56 μmol/L (n= 8). It was concluded that ischemia/reperfusion injuries could reduce hepatocellular viability, probably through increased Isoc in hepatocytes and 2-APB had a protective effect on ischemia/reperfusion-induced liver injury, probably though inhibiting Isoc.
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AIM: Apply the newly invented method of suppression subtractive hybridization to scan the mitochondria genes expression of hippocampus neurons in post ischemia/reperfusion rats. METHODS: Decapitate the rats suffering 10 min of whole brain ischemia and 24 h of reperfusion in Pulsinelli’s 4 vessels occlusion ischemia/reperfusion model. Dissociated hippocampus and isolated mRNA. After reversed transcription, use SSH to subtract the common sequences, then use suppressed PCR to amplify the differentially expressed sequences. After cloning and DNA sequencing, the dot hybridization was used to finally identify the differential gene expression. RESULTS: In the 78 positive clones acquired from cloning, 7 clones represent differential gene expression after confirmed by clone PCR, dot blot and DNA sequencing. The DNA sequencing data indicate these clones are ESTs of Cytochrome c oxidase subunit and NADH-ubiquinone oxidoreductase chain 4 gene. CONCLUSION: Cytochrome C oxidase mRNA level changs during brain ischemia /reperfusion. The differential expression of NADH-ubiquinone oxidoreductase chain 4, one of those 13 subunits of subcomplex ?, suggests that subcomplex ? may have important function associated with pathophysiologic changs in reperfusion injury.