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Aim To investigate the neuroprotective effect of picroside Ⅱ(PIC)on cyto C/caspase-9/caspase-3 signal pathway following ischemia/reperfusion(I/R)injury in rats.Methods Atractyloside(Atr)was selected as negative control,cyclosporin A(CsA)was selected as positive control,and PIC was selected as the treatment medicine.The I/R model was made by inserting a monofilament suture into internal carotid artery for 2 h,and then reperfused for 24 h.The cerebral infarction volume was detected by TTC staining,and the expression of cyto C,caspase-9 and caspase-3 were determined by immunohistochemical assay and Western blot.Results In model group,the cerebral infarct volume was obviously large;the expression of cyto C,caspase-9 and caspase-3 was increased significantly more than that in sham group(P<0.05).In PIC group,the cerebral infarct volume was significantly improved;the expression of cyto C,caspase-9 and caspase-3 was significantly decreased than that in model group(P<0.05).In Atr+PIC group,the rat infarction volume was reduced,and the expression of cyto C,caspase-9 and caspase-3 was significantly decreased than that in Atr group(P<0.05).Conclusion The mechanism of PIC inhibiting neuron apoptosis in focal cerebral I/R rats might be through down-regulating the expression of cyto C,caspase-9 and caspase-3.
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Objective To explore the neuroprotective effect and mechanism of picroside II on ERK1/2 signal transduction pathway after cerebral ischemia injury in rats.Methods The focal cerebral is-chemic models were established by inserting a monofilament threads into middle cerebral artery occlusion (MCAO) in 100 Wistar rats and treated by injecting picroside II (20 mg/kg) intraperitoneally.The neu-robehavioral function was evaluated by modified neurological severity score points ( mNSS) test.The cerebral infarct volume was measured by tetrazolium chloride ( TTC) staining.The apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL) assay.The expression of pERK1/2 in cortex was determined by the immunohistochemistry ( IHC) and Western Blot ( WB) .Results mNSS test showed that severe neurological dysfunction was found in model and LPS groups,and the scores of mNSS were significantly increased;meanwhile the scores of mNSS in treatment group and U0126 group were signifi-cantly lower than that in model and LPS groups (P<0.05).TUNEL assay showed that the apoptotic cell inde-xes (ACI) in different groups were (0.06±0.02),(0.27±0.03),(0.07±0.02),(0.26±0.03)and(0.09± 0.05) ,and the ACI in treatment and U0126 groups was obviously lower than that in model and LPS groups (P<0.05) .With IHC and WB,pERK1/2 level in model group was the highest,which was slightly higher than that of LPS group,and pERK1/2 expression in treatment and U0126 groups was significantly decreased com-pared with that in model and LPS groups (P<0.05) .Conclusion The activation of ERK1/2 by cerebral is-chemia could induce the cell apoptosis.Picroside II might reduce cell apoptosis by inhibiting the activation of ERK1/2 in ischemic brain injury.
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Objective To investigate the effect ofpicroside Ⅱ on mitochondria cytochrome C (CytC) expression and its significance in rats after ischemia/reperfusion.Methods Ninety-six Wistar rats were randomly divided into sham-operated group,model group,picroside Ⅱ group,Cyclosporin A (CsA,specific antgonist of CytC) group,CsA+picroside Ⅱ group,atractyloside (Atr,selective agonist of CytC) group,Atr+picroside Ⅱ group and DMSO group (n=12);the middle cerebral artery occlusion/reperfusion models referring to Longa's method with medications were adopted,which were established by inserting a monofilament suture into the internal carotid artery for 2 h and then reperfusion for 24 h.After 24 h of ischemia/reperfusion,modified neurological severity scale (mNSS) scores were observed,contents of reactive oxygen species (ROS) in brain tissues were measured by enzyme-linked immunosorbent assay (ELISA),morphology of brain tissues was observed by hematoxylin-eosin staining,ultrastructures ofmitochondria were observed by transmission electron microscopy,apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL),and CytC expression was determined by immunohistochemical assay and Western blotting.Results As compared with the sham-operated group,the model group had significantly increased mNSS scores,ROS contents,number ofapoptotic cells and CytC expression (P＜0.05),and the mitochondria structure was seriously destroyed.The picroside Ⅱ group had obviously decreased mNSS scores,ROS contents,number of apoptotic cells and CytC expression,and the morphology of brain tissue was improved and the mitochondria damage was reduced as compared with the model group,with significant differences (P＜0.05).The Atr+picroside Ⅱ group had significantly decreased mNSS scores,ROS contents,number of apoptotic cells and CytC expression (P＜0.05),and the mitochondria damage in the Atr+picroside Ⅱ group was reduced as compared with that in the Atr group with significant difference (P＜0.05).Conclusion The mechanism of picroside Ⅱ protecting against focal cerebral ischemia reperfusion might attribute to decrease of ROS contents,protection of mitochondria structure and down-regulation of CytC expression in middle cerebral artery occlusion/reperfusion rats.
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Objective To explore the regulating mechanism ofneuregulin1β (NRG1β) on extracellular signal-regulated kinase 5 (ERK5) signaling pathway in rats with cerebral ischemia reperfusion injury.Methods Fifty male Wistar rats were divided randomly into sham-operated group,model group,treatment group,inhibitor group,and inhibitor combined with treatment group (n=10).Focal cerebral ischemic models were established by inserting a monofilament thread to achieve middle cerebral artery occlusion (MCAO).The rats were injected 5 μL (2 μg/kg) NRGlβ to the internal carotid artery.This inhibitor BIX02189 was injected into the internal carotid artery before ischemia.The neurobehavioral functions were evaluated by modified neurological severity scale (mNSS).The apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick-end labeling,and the expressions of phosphorylated (p-) mitogen activated proteins kinase kinase 5 (MEKK5),ERK5 and myocyte enhancer-binding factor 2C (MEF2C) were determined by immunohistochemical assay and Western blotting.Results The rats in the model group appeared neurobehavioral dysfunction,the number of apoptotic cells in the cortex was increased,and the expressions of p-MEKK5,p-ERK5 and p-MEF2C showed compensable enhancement,which were significantly different as compared with those in the sham-operated group (P＜0.05).As compared with those in the model group and inhibitor combined with treatment group,the expressions of p-MEKK5,p-ERK5 and p-MEF2C were further significantly enhanced,the number of apoptotic cells was significantly decreased and the neurobehavioral functions were significantly improved in treatment group (P＜0.05).As compared with those in the model group and inhibitor combined with treatment group,the number of apoptotic cells was significantly increased,and the expressions ofp-MEKK5,p-ERK5 and p-MEF2C were significantly decreased in the inhibitor group (P＜0.05).Conclusion NRG1β could play a neuroprotective role by activating the MEKK5-ERK5-MEF2C signaling pathway and further up-regulating the expressions of p-MEKK5,p-ERK5 and p-MEF2C to inhibit the inflammation induced by cerebral ischemia reperfusion injury in rats.
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AIM: To verify the neuroprotective effect and optimize the therapeutic dose and time window of picroside Ⅱon cerebral ischemic injury in rats .METHODS:The forebrain ischemia model was established by the method of bilateral common carotid artery occlusion ( BCCAO ) .The successful model rats were randomly divided into 16 groups according to orthogonal design and treated by intraperitoneal injection of picroside Ⅱat different ischemic time poinis and different doses .The changes of the nerve fiber myelin were observed by fast green staining .The immunohistochemical assay and Western blotting were used to quantitatively and qualitatively determine the expression of myelin basic protein (MBP). The mRNA level of MBP in the brain tissues was tested by reverse transcription polymerase chain reaction (RT-PCR).RE-SULTS:Picroside Ⅱ increased the expression of MBP and decreased demyelination after cerebral ischemic injury .The best therapeutic time window and dose were:(1) ischemia for 2.0 h with picrosideⅡat dose of 10 mg/kg according to the results of fast green staining;(2) ischemia for 2.0 h with the dose of 10 mg/kg according to the results of immunohisto-chemical assay;(3) ischemia for 2.0 h with the dose of 10 mg/kg according to the analysis of Western blotting;(4) is-chemia for 1.5 h with the dose of 20 mg/kg according to the detection of RT-PCR.CONCLUSION:Given the principle of the lowest therapeutic dose with the longest time window , the optimized therapeutic dose and time window for rat cerebral ischemic injury is intraperitoneal injection of picroside Ⅱat the doses of 10~20 mg/kg and the time window of ischemia for 1.5~2.0 h.
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ObjectiveTo investigate the effect of neuregulin1β (NRG1β) on the learning memory abilities and the neuronal apoptosis and the expressions of nuclear factor kappa B (NFκB) in experimental Alzheimer's disease model in rats induced with beta-amyloid protein1-40 (Aβ1-40) injection.To explore the mechanisms of NRG in improving the capabilities of learning and memory.MethodsThirty adult healthy male wistar rats were randomly divided into control group (n =10),model group (n =10) and treated group (n =10).Alzheimer's disease models were established by stereotactically injecting Aβ1-40 into the left lateral ventricle,and treated by injecting NRG1β(0.3 μg · kg-1 ) into the right lateral ventricle.The learning and memory abilities of rats were evaluated with Y-electric maze before the experiment and 7 days after making Alzheimer's disease models and 14 days after treatment.HE staining was used to observe the structure of hippocampal neurons.The neuronal apoptosis of hippocampus was investigated by TUNEL assay.The expressions of NFκB in hippocampal neurons were determined with immunohistochemistry technique.ResultsCompared with control group (57.50 ± 1.58,7.20 ±1.03 ),the model group rats ( 59.50 ± 2.79,7.50 ± 1.08 ) showed low cognitive ability ( t =20.36,5.28,P ＜0.05 ),the hippocampal pyramidal cells of rats in the model group were sparse and disturbed pyramidal cells,noticeable neuron loss.The number of neuronal apoptosis and the expressions of NFκB increased significantly than those in control group (P＜0.05).Compared with model group (79.10 ±4.12,4.40 ±0.69),NRG1β strikingly improved cognitive ability ( 67.70 ± 4.90,5.80 ± 0.63 ) and normal cell structure ( t =5.63,4.69,P ＜ 0.05 ).The expressions of NFκB (25.90 ± 6.67 ) reduced while the number of neuronal apoptosis ( 23.50 ± 3.89 ) decreased markablely than those ( 41.10 ±7.95,29.30 ± 7.24) in model group(t =4.63,2.23,P ＜ 0.05).ConclusionNRG1β might decrease the neuronal apoptosis by inhibiting NFκB expressions,so that to improve the learning and memory abilities of experimental dementia rats.
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Objective To modify a classic two-vessel occlusion (2VO) modeling method in order to decrease the systematic errors in the behavioral experiments such as Morris water maze.Methods Thirty-two adult male Wistar rats were randomly allocated into classic 2VO model,modified model,sham operation and sham ligation groups (n =8 in each group).Only the bilateral common carotid arteries were ligated in the classic 2VO model group; the common carotid arteries were clipped intermittently,and the origins of pterygopalatine arteries of the internal carotid arteries were high selectively ligated in the modified model group; the common carotid arteries were only ligated intermittently in the sham ligation group; and only the common carotid arteries and the upper segment of pterygopalatine artery branches were separated in the sham operation group.The rat behavior was evaluated using the pupillary light reflex,Morris water maze and eight-arm radial maze.HE staining was used to observe the histological changes.Results The Morris water maze escape latency (F =72.169 - 163.102,all P ＜ 0.001) and the number of reference memory errors of eight-arm radial maze (F =33.515-74.726,all P ＜0.001) in the modified model and the classic 2VO model groups were longer and higher than those in the sham operation group.The pupillary light reflex of the rats was lost in the classic 2VO model group and the pupillary light reflex of the rats was normal in other groups.The reaching platform time in the classic 2VO model group was significantly longer than that in the modified model and sham operation groups (P ＜0.001).The percentage of target quadrant dwell time was also decreased significantly (at day 7 after procedure:F =13.770,P ＜0.001 ; at day 90 after procedure:F =14.780,P ＜0.001).HE staining showed pathological changes such as the cells decrease in hippocampal CA1 region and leukoaraiosis in the modified model and the classic 2VO model groups.In addition,there were more vacuole-like changes in the rat optic nerve region in the classic 2VO model group,while there were no such changes in the modified model group.Conclusions Establishing vascular dementia model with permanent occlusion of bilateral internal carotid arteries after intermittent occlusion of bilateral carotid arteries could avoid severe visual impairment in rats.In the Morris water maze and eight-arm maze test,the modified model rats showed significant decrease in learning and memory abilities and had hippocampal damage.
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Objective To investigate the hypoglycemic effects of Laminaria japonica (L. japonica) on diabetic model induced by alloxan in rats. Methods Sixty healthy female rats were used to establish diabetic models by injecting alloxan peritoneally, and L.japonica was applied as raw materials for potential marine drugs.The levels of fasting blood glucose (FBG) were detected by automatic blood glucose device. Enzyme linkedimmunoabsorbant assay was applied to determine the insulin level in serum. The shape and structure of isletcells were observed with histopathological staining, and the expression of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in islet cells were detected by immunohistochemical technique. Results After the treatment, the levels of FBG of L.japonica treated group B [(9.37±1.70) mmol/LandC (9.18±1.65 ) mmol/L, F= 32.81, q=6.35～11.72, P<0.05 ] reduced, while the serum levels of insulin in treated group A, Band C (0.0378±0.0026, 0.0378±0.0027, 0.0367±0.0035) increased(F= 11.40, q=4.28～8.47, P<0.05) significantly than those of diabetic model group (0.0456 ±0.0057) . The shape and structure of islet cells improved with the up-expressing SOD(t=4.73～4.76, P<0.05)and down-expressing iNOS (t=4.81～5.30, P<0.05) in L.japonica treated group B and C than those in diabetic model group. Conclusion L.japonica might decrease the serum level of FBG through promoting the islet cell recovery by an anti-oxide effect.
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Aim To explore the effect of picrodideⅡ on the expressions of Caspase-3 and poly ADP-ribose polymerase (PARP) in brain tissue following cerebral ischemic reperfusion injury in rats.Methods The middle cerebral artery occlusion reperfusion models were established with intraluminal thread methods in rats. PicrodideⅡ (10 mg·kg~(-1)) and salvianic acid A sodium (10 mg·kg~(-1)) were injected from tail vein for treatment. The neurological function was evaluated with Bederson's test and the cerebral infarction volume was observed with tetrazolium chloride (TTC) staining.The brain structure was observed by hematoxylin-eosin (HE) staining and the apoptosis was counted by TUNEL immunofluorescence assay. The expressions of Caspase-3 and PARP were detected with immunohistochemical and enzyme linked immunosorbent assay.Results After ischemia 2 h and reperfusion 22 h, the rats showed neurological function deficit and cerebral infarction in ischemic hemisphere. The expressions of Caspase-3 and PARP and the number of apoptotic cells in brain tissue increased compared with those in the sham operative group (P <0.05). In picroside and salvianic acid A sodium groups, the Bederson's scores and cerebral infarction volume, the expressions of Caspase-3 and PARP and the number of apoptosis cells were lower than those in the negative control group (P <0.05). While there was no significant difference in five indexes metioned above between picroside group and salvianic acid A sodium group (P >0.05).Conclusion PicrosideⅡ might reduce the expressions of Caspase-3 and PARP to inhibit the neuronal apoptosis induced by cerebral ischemia reperfusion injury and improve the neurological function of rats.
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Aim To study the interfering effects of picrosideⅡ on the expressions of nuclear transcription factor kappaB(NF-κB)and inhibitor of NF-κB(I-κB)after cerebral ischemic reperfusion in rats.Methods Intraluminal thread methods were applied to establish the middle cerebral artery occlusion reperfusion models in rats.PicrosideⅡ(10 mg·kg~(-1))and salvianic acid A sodium(10 mg·kg~(-1))were injected from the tail vein for treatment.TUNEL positive cells were counted by immunofluorescence assay.The expressions of NF-κB and I-κB were determined by immunohistochemical assay,and the concentration of NF-κB and I-κB in brain tissue was determined by ELISA.Results The exprssions of NF-κB and I-κB were weakly and the apoptotic cells were scattering at cortex,striatum and hippocampus in the sham operative group.In the negative control group,the number of TUNEL positive cells and the expressions of NF-κB and I-κB increased,the absorption(A)values and the concentration were significantly higher than those in the sham operative group(P<0.05).While in the positive control and picroside groups,the expressions(A values)and concentration of NF-κB and I-κB and the number of TUNEL positive cells were significantly lower than those in the negative control group(P<0.05).There was no significant difference between the positive control group and picroside group(P>0.05).Conclusion Picroside Ⅱ might downregulate the expressions of NF-κB and I-κB to inhibit neuronal apoptosis induced by inflammation after cerebral ischemia reperfusion injury in rats.
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Objective To investigate the neuroprotective effects of picrodideⅡ on cerebral ischemic reperfusion injury in rats. Methods Intraluminal thread methods were applied to establish the left middle cerebral artery occlusion reperfusion models (MCAO/R) in rats. PicrodideⅡ (10mg/kg) and salvianic acid A sodium (10mg/kg) were injected from tail vein for treatment. The neurological behavioral function was evaluated with Bederson's test. The cerebral infarction volume was observed with tetrazolium chloride (TTC) staining. The structure of cells was observed with histopathology. The apoptosis positive cells were counted by terminal deoxynucleotidyl transferase midiated dUTP nick end labeling (TUNEL). Results The neurological behavioral malfunction appeared in all rats with MCAO/R. The infarction focus showed in the ischemic hemisphere following cerebral ischemia reperfusion injury. In the picrodideⅡ and salvianic acid A sodium treatment groups, the number of apoptosis positive cells decreased and the cerebral infarction volume reduced, while the neurological behavioral function was significantly improved than those in the model control group (P<0.05). The cerebral infarction volume in the picrodideⅡ group was smaller than that in the salvianic acid A sodium group (P<0.05).Conclusion PicrodideⅡ might reduce cerebral infarction volume and improve the neurological behavioral function through inhibiting the neuronal apoptosis induced by ischemia reperfusion injury.
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Objective To investigate the effect of a Chinese herbs mixture(Monkshood,ginger and licorice)on blood pressure(BP)and its possible mechanism in renovascular hypertensive rats.Methods 2K1C hypertensive rats received placebo(n=8)or Sinitung(n=8)by gavage for 2 weeks.BP was measured by tail-cuff.Plasma angiotensin Ⅱ(Ang Ⅱ)and calcium gene related peptide(CGRP)were examined by histochemical assay.Results Sinitang treatment significantly decreased BP(116.2?8.3 mm Hg vs placebo:131.6?14.2 mm Hg,P
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AIM: To investigate the intervention effect of astragaloside on adhesion of polymorphonuclear neutrophils (PMN) and vascular endothelial cells and on expression of nuclear factor-?B (NF-?B) in human umbilical vein endothelial cells (hUVECs) injured by hypoxia/reoxygenation due to ischemia/reperfusion. METHODS: The experiment was performed at the Cell Bioengineering Laboratory of Institute of Cerebrovascular Diseases, Affiliated Hospital of Medical College, Qingdao University from September 2005 to May 2006. ①Neonatal umbilical cords were offered by Department of Gynecology and Obstetrics in Affiliated Hospital of Medical College of Qingdao University, with informed consent of puerperant and their families. The experiment was accorded with ethical standard of Helsinki declaration. ②The hUVECs were cultured to the future passage. After the third passage, hUVECs were randomly divided into three groups. Cells in a control group were cultured under normal conditions; Cells in a hypoxia/reoxygenation group were cultured under in closed container with hypoxia for 1 hour, and then under normal conditions for 1 hour; The hUVECs in a astragaloside group were pretreatment by different doses of astragaloside (20, 40, 80 mg/L). After 12 hours, hUVECs suffered from hypoxia/reoxygenation. ③The concentration of intercellular adhesion molecule (ICAM)-1 and content of malondialdehyde (MDA) in supernatant were detected by enzyme linked immunosorbent assay (ELISA) and thiobarbituric acid method. Expression of NF-?B of hUVECs was analyzed by immunohistochemistry. PMN adhesion to hUVECs was measured by rose Bengal staining. RESULTS: ①Compared to the control group, the content of MDA remarkably increased in hypoxia/reoxygenation group (P
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BACKGROUND: It has been demonstrated that insulin-like growth factor-1 (IGF-1) is a kind of neurotrophic factor and protects from cerebral ischemia/reperfusion injury, the expression of IGF-1 is associated with the attack of ischemic stroke. The effects of IGF-1 and its receptor (IGF-1R) on neurobehavioral function are to be further studied.OBJECTIVE: To observe the effects of IGF-1 and IGF-1R on neurobehavioral function in rat models of cerebral ischemia/reperfusion injury.DESIGN: A randomized controlled observation.SETTING: Institute of Cerebrovascular Diseases, Affiliated Hospital of Qingdao University Medical College.MATERIALS: The experiments were carried out in Shandong Key Laboratory for Prevention and Treatment of Brain diseases. Twenty-eight healthy adult Wistar rats of clean degree, weighing 220-260 g, were provided by the experimental animal center of Shandong University.METHODS: The rats were randomly divided into experimental group (n =24) and sham-operated group (n =4). The middle cerebral artery occlusion/reperfusion (MCAO/R) models were established by inserting a thread through left external-internal carotid arteries. The sham-operated rats were given the same treatments except inserting thread. ①Neurologic deficit test: The rats in the experimental group were assessed according to Bederson standard after 1-hour ischemia and 6, 12-hour, 1, 3, 7 and 14-day reperfusion respectively. The sham-operated rats were assessed at corresponding time points; Without neurologic deficit was marked as 0 point; flexion of anterior claws as 1 point; unable to act against the pushing from the contralateral side as 2 points; circling while walking as 3 points; shaking as 4 points;unconscious mind as 5 points. ② Sample collection and treatment: The samples in the experimental group were collected after 1-hour ischemia and 6, 12-hour, 1, 3, 7 and 14-day reperfusion, and those in the sham-operated group ere collected at 24 hours postoperatively. The rats were anesthetized, brain samples were got at about 5 mm posterior to optic chiasma after brains were removed completely, then serial coronal sections (5 μm) were prepared, and 1 from 10 sections was stuck to the cover glasses treated with poly-L-lysine. ③ Morphological observation of neurons: The neurons in brain were observed by toluidine blue staining. ④ Detection of IGF-1 and IGF-1R: The expressions of IGF-1 and IGF-1R in cortex and striatum were detected with immunohistochemical technique, 4 fields were randomly selected to count the positive cells under high-power microscope (×400).MAIN OUTCOME MEASURES: ① The neurologic deficit; ② Morphological changes of neurons in brain; ③ Expressions of IGF-1 and IGF-1R in cortex and striatum.RESULTS: All the 28 rats were involved in the analysis of results. ① The neurologic deficit: The scores of neurologic deficit were (1.50±058) and (1.50±0.78) in rats after 7 and 14-day reperfusion, which were lower than that in rats after 6-hour reperfusion [(3.00±0.00), P ＜ 0.05]. ② Morphological changes of neurons in brain: The neurons in ischemic area appeared as paryopyknosis and became irregular in shape, there were obvious gaps around the cells, also deeply stained as purplish blue, nucleolus disappeared, and there were many scattered cellular fragments. ③ Expressions of IGF-1 and IGF-1R in cortex and striatum: The numbers of IGF-1 positive cells in cortex were (8.75±2.06), (11.13±1.14),(19.75±3.18), (17.38±3.11 ) and (11.23±2.28) respectively in rats after 6, 12-hours and 1, 3, 7-day reperfusion, which all were higher than that in sham-operated rats [(3.88±1.46), P ＜ 0.05], the numbers of IGF-1 positive cells in striatum were(8.25±2.21), (11.34±2.21), (18.23±2.64), (18.56±2.34) and (11.31±2.14) respectively in rats after 6, 12 hours and 1, 3, 7days reperfusion , which were also higher than that in sham-operated rats [(4.12±2.24), P ＜ 0.05]. The numbers of IGF-1R positive cells in cortex were (7.63±1.50), (10.50±2.34), (15.55±3.12), (15.37±3.01), (8.86±2.75) respectively in rats after 6, 12-hours and 1,3,7-day reperfusion, which all were higher than that in sham-operated rats [(4.13±1.81), P ＜0.05]. Those in striatum were (8.33±2.31), (10.24±2.09), (14.72±2.17), (14.24±2.77), (8.38±2.05), which were also higher than that in sham-operated rats [(3.76±2.35), P ＜ 0.05].CONCLUSION: The neurological function is damaged after cerebral ischemia/reperfusion, but it has a trend of self-recovery. The expressions of IGF-1 and IGF-1R are mainly distributed in cortex and striatum. Higher expressions of IGF-1 and IGF-1R maintain during 12 hours to 7 days after reperfusion and have a peak value at 1-3 days, which suggests that early expression of IGF-1 and IGF-1R are certain related to the recovery of neurological function.
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BACKGROUND: Brain injury can induce the increased expression of basic fibroblast growth factor (bFGF) in brain,whereas FGFR is a very important player in the cell proliferation and differentiation, angiogenesis, skeletogeny, etc.OBJECTIVE: To observe the effect of bFGF and its receptor on neuronal apoptosis following cerebral ischemia/reperfusion injury in rats.DESIGN: A randomized grouping design and animal experiment.SETTING: Institute of Cerebrovascular Disease, Affiliated Hospital of Qingdao University Medical College.MATERIALS: Twenty-eight healthy adult Wistar rats of clean degree, weighing 220-260 g, were provided by the experimental animal center of Shandong University. Rabbit-anti-rat bFGF and fibroblast growth factor receptor-1(FGFR-1) monoclonal antibodies were provided by Wuhan Boster Biological Technology, Co.,Ltd.METHODS: The experiment was carried out in Shandong Key Laboratory for Prevention and Treatment of Brain diseases.① The rats were randomly divided into experimental group (n =24) and sham-operated group (n =4). Models of middle cerebral artery occlusion/reperfusion (MCAO/R) were established by thread occlusion via left external-internal carotid arteries, and 4 rats in the experimental group were sampled at 1-hour ischemia/6, 12-hour, 1, 3, 7 and 14-day reperfusion respectively. The rats in the sham-operated group were given the same treatment without inserting thread.After anesthesia, the brain was removed completely by cutting head, then the brain tissue at about 5 mm posterior to optic chiasma was cut down, then serial coronal sections (5 μm) were prepared. ② The brain tissues were stained with ematoxylin-eosin (HE), and the forms of neurons were observed under microscope. ③ TdT-mediated dUTP-biotin nick end labeling (TUNEL) method: there were buffy granules in nucleus which was positively stained (apoptosis). Four fields were randomly selected from cortex and striatum to count positive cells under high-power microscope (×400). ④ The sections were stained with rabbit-anti-rat bFGF and FGFR-1 monoclonal antibodies, 4 fields were randomly selected from cortex and striatum to count positive cells under high-power microscope (×400).MAIN OUTCOME MEASURES: Apoptosis and the expressions of bFGF and FGFR-1.RESULTS: All the 28 rats were involved in the analysis of results. ① In the experimental group, the neurons in the ischemic sites were obviously decreased, some neurons appeared as paryopyknosis and became irregular, also deeply stained as purplish blue, nucleolus disappeared, and there were many scattered cellular fragments. ② In the sham-operated group, there were a few apoptotic neurons in the brain tissue, and the apoptotic neurons were obviously increased after ischemia, which mainly observed in cortexes and striatums of frontal and paritetal lobes. In the experimental group, apoptotic cells in cortexes began to increase gradually at 6 hours, and there were more cells at 12hours and 3 days, which reached the peak value at 1 day, and began to decrease at 3 day, but there were still more apoptotic cells at 14 days than in the sham-operated group. The number of apoptotic neurons and the changing trend in striatums were generally the same as those in cortexes (P ＞ 0.05). ③ In the sham-operated group, there were weak bFGF expression in the neurons of brain tissue, but there were fewer lightly stained positive cells. After cerebral ischemia, the bFGF expressions were increased, mainly observed in cortexes and striatums. The bFGF expression appeared at 6 hours after cerebral ischemia/reperfusion, and the number was increased gradually and deeply stained as the time of reperfusion prolonged (Figure 3), it reached the peak value at 1-3 days, and then weakened gradually, but it was still higher than in the sham-operated group at 14 days [(5.01 ±1.71), (5.21 ± 1.62) cells/visual field; (2.03± 1.73),(2.46± 1.38) cells/visual field, P ＜ 0.05]. ④ In the sham-operated group, lightly stained FGFR-1 positive cells could be observed in brain tissue. At 6 hours after cerebral ischemia/reperfusion, the FGFR positive cells began to increased in cortexes and striatums, which were the most at 1-3 days, and gradually decreased after 3 days, and the number was still a little more than that in the sham-operated group at 14 days [(5.01± 1.41), (5.20± 1.33) cells/visual field; (2.25±1.67),(2.32± 1.61 ) cells/visual field].CONCLUSION: After cerebral ischemia/reperfusion, the expressions of endogenous bFGF and FGFR-1 may be activated in cortex and striatum, then inhibit the neuronal apoptosis, and play its neuroprotective role.
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BACKGROUND: Most animal experiments of transgene are derived from mice; therefore, it is necessary to establish a focal cerebral ischemiareperfusion model and significant to prevent and cure ischemic cerebrovascular diseases.OBJECTIVE: To establish a convenient and reliable model with middle cerebral artery occlusion reperfusion (MCAO/R) in mice.DESIGN: Randomized controlled animal study.SETTING: Institute of Cerebrovascular disease, Affiliated Hospital of Medical College of Qingdao University.MATERIALS: Twenty healthy BALB/c mice, of both genders, weighing 25-30 g, of SPF grade, were divided into sham operation group (n=5), ischemia group (n=10) and 22-hour reperfusion group (n=5) on the basis of digital table. In addition, according to digital table, 130 healthy male Kunming mice were divided into sham operation group (n=10), 24-hour ischemia group (n=30), 2-hour ischemia/22-hour, 46-hour and 70-hour reperfusion groups with 30 in each group; meanwhile, 30 female mice were divided into sham operation group, 24-hour ischemia group and 2-hour ischemia/22-hour reperfusion group with 10 in each group. All Kunming mice were weighing 25-30 g and of SPF grade.METHODS: The experiment was carried out in the Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University from July 2005 to March 2006. The 6-0 suture with silica gel covered at an end was sent from the left external carotid artery (ECA) into internal carotid artery (ICA) till arriving at the initiation of middle cerebral artery (MCA) to block the blood stream in it, then drawing the suture from ICA 2 hours after occlusion to accomplish reperfusion. Mice were cut off their heads in sham operation group at 24 hours after operation, in ischemia group at 24 hours after blocking blood stream and in reperfusion group at 24, 48 and 72 hours after operation. Reliability of models was evaluated with neurology score and tetrazolium chloride stain. Longa standard scores: neurology score ≥ 1 point was regarded as successful models;coronal sections of brain tissue were stained with tetrazolium chloride, and the white region was regarded as infarcted volume.MAIN OUTCOME MEASURES: Neurology score and infarcted volume after staining of triphenyltetrazolium chloride in brain tissue.RESULTS: All mice were involved in the final analysis. ① Successful rate was 20% of BALB/c mice, 66.7%-73.3% of male Kunming mice and 40%-50% of female Kunming mice. ② Brain sections of BALB/c mice in sham operation group were orange at both sides of cortex and infarction focus was not observed. A big infarcted volume was observed on brain sections of mice in ischemia group, and infarcted volume counted for 50%-70% as homonymy hemisphere on optochiasmatic coronal sections. The condition of Kunming mice was similar to that of BALB/c mice, but infarcted volume counted for 40%-65%. In addition, condition in ischemiareperfusion group was similar to that in ischemia group. A big infarcted volume was observed on brain sections, and infarcted volume counted for 50%-75% as homonymy hemisphere on optochiasmatic coronal sections.The condition of Kunming mice was similar to that of BALB/c mice, but infarcted volume counted for 40%-65%.CONCLUSION: The model with MCAO/R in mice characterizes by relatively smaller trauma, and the ischemic region is stable; therefore, it can be used to accurate timing control of ischemia/reperfusion. This model is an ideal one for researching pathophysiological changes, prognosis and therapy in cerebrovascular disease.
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BACKGROUND: A middle cerebral artery occlusion and reperfusion(MCAO/R) model in rats with suture has been widely used in the researches of acute focal ischemic cerebral infarction, while the model in rabbits by the same method is relatively rare. Magnetic resonance diffusion weighted imaging (MR DWI) has been paid close attention recently for its sharp sensitivity of cerebral ischemia.OBJECTIVE: To establish rabbit models of MCAO/R by intraluminal thread, and study the characteristics of MR DWI after cerebral ischemia and reperfusion.DESIGN: Random controlled animal experiment.SETTING: Institute of Cerebrovascular Diseases, Affiliated Hospital of Qingdao University Medical College.MATERIALS: The experiment was accomplished at the Key Laboratory of Brain Diseases Prevention and Cure of Shandong Province from March to June in 2005. A total of 103 adult healthy New Zealand rabbits of either sex, 10-12 weeks old and 1.8-3.3 kg weight were provided by the Experimental Animal Center of Shandong Agricultural Academy (SCX20040013).They were bred at quiet, sanitary and dry conditions.METHODS: Animal groups: 103 rabbits were divided randomly into group A (n=53) and group B (n=50). The rabbits in group A were treated with suture of 0.51-0.55 mm as the diameter of thread, while group B was reassigned into B1 (0.46-0.50 mm), B2 (0.51-0.55 mm) and B3 (0.56-0.60 mm).The successful MCAO/R models in 57 cases were randomly divided into permanent ischemia group (n=30, ischemia 1, 3, 6, 12, 24 andl 48 hours, 5ones at each time point) and ischemic reperfusion group (n=27, reperfusion 0, 2 and 5 hours, 5 ones at each time point; reperfusion 11, 23 and 47hours, 4 ones at each time point). Another 10 rabbits receiving sham operations were regarded as contrasts for permanent ischemia group and ischemia reperfusion group, with 5 ones in each.MAIN OUTCOME MEASURES: The changes of hyperintensity area on DWI and apparent diffusion coefficient (ADC) were measured in permanent ischemia group and ischemic reperfusion group.RESULTS: The data of 57 successful model rabbits were involved in the result analysis.①The successful rate in group A (26 cases, 49.1%) was significantly lower than that in group B (31 cases, 62.0%).②In ischemia group:The hyperintensity area on DWI with declined ADC appeared at ischemia 1 hour. The hyperintensity areas on DWI at different times increased gradually from ischemia 1 hour and unchanged within 24 hours. The mean ADC at different times declined at first and then gradually increased.③In reperfusion group: Comparing with ischemia 1 hour, the hyperintensity area on DWI reduced while ADC increased at reperfusion 2 hours and 5 hours, and enlarged with ADC high at reperfusion 11 hours, then continued to enlarge with ADC reduced significantly at 23 hours and 47 hours.CONCLUSION: The diameter of thread tip and the inserting distance of thread are main factors for establishing successful MCAO/R models. The hyperintensity area on DWI and the decreasing ADC after acute cerebral ischemia can be improved by early reperfusion, but the secondary decreasing ADC may be induced by continuously reperfusion.
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BACKGROUND: It is significant to establish a kind of effective, conve nient and reliable animal model of hypertension. At present, dogs, rats and rabbits are usually used to establish hypertensive models at home and abroad, and the renal artery stenosis induced hypertensive models are ex tensively used to research hypertension and its complication for human be ings because they are convenient and reliable, and there are many methods to establish them, but the effects are to be evaluated. OBJECTIVE: To establish convenient and reliable animal models of ex perimental renal artery stenosis induced hypertension. DESIGN: A randomized grouping design and animal experiment. SETTING: Institute of Cerebrovascular diseases, Medical College Hospital of Qingdao University. MATERIALS: The experiments were carried out in Shandong Key Labora tory for Prevention and treatment of Brain Disease from September 2005 to February 2006. Eighty-one healthy Wistar rats divided into 7 groups accord ing to the method of random number table: unilateral renal artery stenosis group (n=18), bilateral renal artery stenosis group (n=17), unilateral renal artery ligation group (n=15), bilateral renal artery ligation group (n=15), uni lateral renal artery stenosis sham-operated group (n=6), bilateral renal artery stenosis sham-operated group (n=4) and normal control group (n=6). METHODS: Unilateral renal artery stenosis group: Right renal artery was clamped with miniature silver clip, and left kidney was resected after 12 days. Bilateral renal artery stenosis group: Right renal artery was clamped with miniature silver clip, and the same treatment was given to the left side after 12 days. Unilateral renal artery ligation group: Right renal artery was ligated with filament, and left kidney was resected after 12 days. Bilateral renal artery ligation group: Right renal artery was ligated with filament, and the same treatment was given to the left side after 12 days. Unilateral renal artery stenosis sham-operated group: Right kidney was exposed, and returned to the original place without any treatment, and left kidney was resected af ter 12 days. Bilateral renal artery stenosis sham-operated group: Right kid ney was exposed, and returned to the original place without any treatment, and the same treatment was given to the left side after 12 days. Normal con trol group: The rats were not given any treatment. The blood pressure and heart rate were determined with RBP-2 hemomanometer for rats. MAIN OUTCOME MEASURES: The successful rate of model estab lishment, blood pressure and heart rate were observed. RESULTS: Totally 81 rats were used, and 61 of them died, all were in volved in the analysis of results without deletion. ① Blood pressures in the unilateral and bilateral renal artery stenosis groups and bilateral renal artery ligation group were obviously higher than those in the normal control group and bilateral renal artery stenosis sham-operated group [(138.0 ±36.5), (154.2±11.6), (160.5±0.7), (101.3±17.6), (108.3±5.7) mm Hg]. ② The changes of heart rate in the renal artery stenosis group were unstable, and the heart rates in the unilateral and bilateral renal artery stenosis groups, bilateral renal artery ligation group, normal control group and bilat eral renal artery stenosis sham-operated group were (367.5±47.2), (420.2 ±47.8), (386.0±4.2), (390.3±42.4), (417.3±27.5) beats per minute, respec tively. ③ The survival rates in the renal artery stenosis groups (22%, 29%) were significantly higher than those in the renal artery ligation groups (0,12%), and it was the highest in the unilateral renal artery stenosis group.CONCLUSION: The method of clamping bilateral renal arteries can establish stable rat models of hypertension induced by renal artery stenosis.
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Objective To study the gene expressions of nestin and stem cell factor(SCF)in neurons after ischemia-reperfusion injury in rat brain. Methods Thirty-six adult female rats were subject to left middle cerebral artery occlusion for 1.5h and different hours of reperfusion. In site hybridization was used to examine the expression of nestin and SCF mRNA in the rats subjected to 2h, 6h, 12h, 24h, 2d, 3d, 7d, 14d of reperfusion and sham-operation group (n=4). Results (1) Nestin expression in cortex, striatum and extraventricular zone was weak in the sham-operation group, and increased markedly in the ischemic hemisphere compared with sham-operation group except of reperfusion 2h in cortex, 2h, 6h in striatum, 2h, 6h and 14d in extraventricular zone. (2)SCF expression in cortex, striatum and extraventricular zone was weak in the sham-operation group, and increased markedly in the ischemic hemisphere compared with sham-operation group except of reperfusion 2h, 6h, 12h in cortex, 2h, 6h in striatum, 2h and 14d in extraventricular zone. Conclusion It is suggested that SCF expression might enhance the proliferation of neural stem cells following ischemia-reperfusion in rats.
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Objective To observe the effects of expressions of neurocyte adhesion molecule (NCAM) and growth associated protein-43 (GAP-43) in neurological function recovery following cerebral ischemia-reperfusion in rats. Methods The model of focal ischemia-reperfusion in SD rat was induced by intraluminal middle cerebral artery (MCA) occlusion with a nylon monofilament suture. In situ hybridization (ISH) was performed to examine the expression of NCAM mRNA and GAP-43 mRNA at 2,12 h and 1,2,3,7,14 d after reperfusion and in sham-operated controls. Results There was no functional deficit and little expression of NCAM mRNA and GAP-43 mRNA in brain cells in rats of the sham-operated group. In the experimental group, NCAM mRNA expression was observed after reperfusion for 2 h in cortex and striatum and peaked at 12 h and was still higher at 7 d. The neurological function improved at reperfusion of 3 d~14 d compared to reperfusion 2 h. In the ischemic cortex and striatum, GAP-43 mRNA expression demonstrated "double-peak" at 12 h and 2 d after reperfusion, then decreased gradually to the level of sham-operated group at 14 d. Conclusion The increasing NCAM expression might be an important factor of the neural reparation and GAP-43 might enhance the neurological functional recovery with ischemic brain injury in rat.