Effects of spermine on myocardial ischemia/ reperfusion injury in rats

AIM: To observe the effect of exogenous spermine (low concentration) on myocardial ischemia/reperfusion injury in rats.METHODS: 40 Wistar rats were randomly divided into 4 groups: sham- operation group (Sham), ischemic reperfusion group (I/R), spermine group (Sp) and natural saline group (NS). The model of ischemic/reperfusion injury was established by ligating rat coronary artery. In Sp group, spermine (0.5 mmol/L, 2 mL/kg) was injected slowly into rat vein. During the process, we recorded the electrocardiogram and the LV functional parameters, assayed the levels of SOD, LDH, NO and MDA in serum, and examined the ultrastructure of the myocardium. RESULTS: In I/R group, the incidence of arrhythmia was 90%, myocardial ultrastructure was injured seriously, values of LVSP and ±dp/dtmax decreased, levels of LDH, NO and MDA increased while SOD activity decreased (P<0.05 or P<0.01, compared with Sham group). Compared with I/R and NS group, all those indexes in Sp group changed significantly (P<0.05 or P<0.01). CONCLUSION: Exogenous spermine alleviates myocardial ischemia/ reperfusion injury in rats. The mechanism may be related to its antioxidant effect and relieving the injury caused by oxygen free radical.     

Ischemic postconditioning reduces myocardial damage in rats suffering from limb ischemia-reperfusion

AIM: To investigate the protective effect of limb ischemic postconditioning on the myocardial damage in the rats suffering from limb ischemia-reperfusion (LIR). METHODS: Wistar rats were randomly divided into control group (C group), ischemia-reperfusion group (IR group) and ischemic post-conditioning group (IR+IPostC group). For conducting ischemic postconditioning, the rats in IR+IPostC group underwent 5 min of ischemia and 5 min of reperfusion on their hind limbs repeatedly after 4 h of ischemia, and then, 4 h of reperfusion was applied. The activity of superoxide dismutase (SOD), xanthine oxidase (XOD) and myeloperoxidase (MPO) was measured. The levels of malonaldehyde (MDA) in plasma and myocardial tissues, the levels of creatine kinase (CK), creatine kinase MB (CK-MB), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase (α-HBDH) and myocardial troponin I (cTnI) were also detected. The changes of ultrastructure in the myocardium were observed under electron microscope. RESULTS: Compared with C group,the levels of CK-MB, AST, LDH,α-HBDH and cTnI were all increased in IR and IR+IPostC groups. The levels of MDA and XOD also increased (P<0.05), but the activity of SOD decreased (P<0.05). However, compared with group IR, the levels of CK-MB, AST, LDH, α-HBDH and cTnI decreased (P<0.05) in IR+IPostC group.The levels of MDA and XOD also decreased (P<0.05), but the activity of SOD increased (P<0.05). Under electron microscope, the cardiac myofibrils arranged neatly, light and dark bands were clear, the mitochondrial cristae arranged closely and neatly, and the mitochondrial matrix densification was observed in C group. However, the cardiac fiber arrangement was disordered or disappeared, stromal edema was obvious, most or all mitochondrial cristae and membrane became fusion or disappeared, mitochondrial vacuolization and decrease in glycogen were obvious in IR group. In IR+IPostC group, the pathological changes mentioned above were attenuated somewhat than those in IR group. CONCLUSION: Ischemic postconditioning protects rat myocardium under limb ischemia-reperfusion.     

Inhibitory effect of ginkgo-dipyridamole injection on ischemia/reperfusion injury in rat hearts in vitro

AIM：To investigate the effect of ginkgo-dipyridamole injection (GD) on ischemia/reperfusion (I/R) injury in rat hearts in vitro and its possible mechanism. METHODS：Forty male Sprague-Dawley rats were randomly divided into 5 groups (n=8): normal control (NC) group, I/R group, ischemic preconditioning (IPC)+I/R group, GD+I/R group and GD+LaCl3+I/R group. Cardiac function indexes, including heart rate (HR), left ventricular systolic pressure (LVSP) and the maximal rise/fall rate of left ventricular pressure (±dp/dtmax), were detected at 5 time points, including stabilizing point, 30 min after ischemia, and 5, 30 and 60 min after reperfusion. The activity of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent at the five time points was assayed. The concentration of Ca2+ and the content of α-ketoglutarate dehydrogenase (α-OGDH) in myocardial mitochondria were determined at the end of the whole experiment. RESULTS：Compared with I/R group, the cardiac function indexes in IPC+I/R and GD+I/R groups were improved at the reperfusion period (P<0.05), the activity of LDH and CK in coronary effluent and the concentration of Ca2+ in mitochondria were significant reduced (P<0.01), and the content of α-OGDH was increased (P<0.05). However, the protective effect of GD was inhibited by LaCl3 (P<0.05). CONCLUSION：GD protects rat hearts against I/R injury by inhibiting calcium overload and improving mitochondrial enzyme activity to stabilize mitochondrial energy metabolism.     

Expression of dopamine receptor in rat anoxia-reoxygenation injured cardiac tissue

AIM: To investigate the expression of dopamine receptor (DR) in rat cardiac tissue and the relationship with the anoxia-reoxygenation injury (ARI).METHODS: The rat cardiac ARI model in vitro was rebuilt on Langendorf device.RT-PCR and Western blotting were used to detect the mRNA and protein expression of DR1and DR2.The activity of LDH in coronary effluent volume was assayed with ultraviolet spectrophotometer.The ultrastructure of cardiac tissue was determined by transmission electron microscope.RESULTS: The mRNA and protein expressions of DR1and DR2 in rat cardiac tissue increased at anoxia 40 min (P<0.05)，markedly increased at reperfusion 1 h (P<0.01),reached the highest level at 2 h,but lightly decreased at 3 h and 4 h.The activity of LDH increased and the myocardial ultrastructure injured seriously after reperfusion.CONCLUSION: The expressions of DR1 and DR2 in rat cardiac tissue increased firstly then decreased during anoxia-reoxygenation processes.The two kinds of receptors may take part in the cardiac anoxia-reoxygenation injury.     

Effects of diazoxide preconditioning on mitochondrial respiratory function and enzyme activity in rats

AIM: To investigate the effects of the selective mitochondrial ATP-sensitive K+ channels opener diazoxide on mitochondrial respiratory function and enzyme activity in isolated rat myocardium under ischemia/reperfusion.METHODS: Observation was made on rat hearts perfused with Langendorff apparatus.72 Sprague-Dawley (SD) rats were randomly divided into 4 groups: normal group (NOR),ischemia reperfusion (IR),diazoxide group (DIA) and 5-hydroxydecanoate (5-HD) antagonized diazoxide group (5HD-DIA).Hearts isolated from SD rats were mounted on a Langendorff apparatus and started with a 20 min perfusion for equilibration.NOR went on perfusion for another 100 min after equilibration.IR underwent 40 min global ischemia and followed by 30 min reperfusion after 30 min stabilization.DIA was administered with K-H solution containing diazoxide at concentration of 50 μmol/L for 10 min before ischemia and reperfusion.5HD-DIA was infused with 100 μmol/L 5-HD (a specific mitochondrial ATP sensitive K+ channel blocker) and the same procedure was carried out as DIA group.Hearts were taken down to extract mitochondrial at the end-equation,before ischemia and at the end-reperfusion for determination of mitochondrial respiratory function and the enzyme activity of mitochondria.RESULTS: At the end of reperfusion,mitochondrial respiratory function (mitochondrial respiratory control rate,P/O ratio and state 3 respiration) and mitochondrial enzyme activity (NADH oxidase,succinate oxidase and cytochrome C oxidase) in DIA group were better than those in IR group and 5HD-DIA group (P<0.05),but worse than those NOR group (P<0.01).No significant difference in all parameters was observed between IR and 5HD-DIA (P>0.05).CONCLUSION: Preconditioning with mitochondrial ATP sensitive potassium channel opener,diazoxide,protects rat heart mitochondria against ischemia-reperfusion injury.The mechanisms are involved in the safeguarding of respiratory function and activity of enzymes of respiratory chain.     

Role of heme oxygenase-1 in the ischemic preconditioning of isolated rat heart

AIM: To investigate the influence of ischemic preconditioning on heart function, the activities of lactate dehydrogenase (LDH), malondialdehyde (MDA), and heme oxygenase-1 (HO-1) after ischemia/reperfusion in isolated rat heart. METHODS: The model of Langendorff was used in isolated rat heart perfusion. Ischemic preconditioning protocol: stopping perfusion for 5 minutes and reperfusion for 5 minutes, repeating three times. Ischemia protocol: stopping perfusion for 40 minutes and reperfusion for 20 minutes. Indexes of heart function were recorded in control M8, ischemia and reperfusion group (IR), and ischemic preconditioning group (IPC). The content of LDH of coronary effluent was measured. Moreover, the content of MDA and activity of HO-1 in myocardium were also measured. RESULTS: The recovery percentage of heart function in IPC group was significantly higher than that in IR group (P<0.01) and the activity of heme oxygenase-1 also increased significantly (P<0.05). CONCLUSION: The contents of LDH and MDA significantly decreased in IPC group compared with IR group. The increase in heme oxygenase-1 activity might be involved in the protective effect of ischemic preconditioning on ischemic/reperfused rat heart.     

Effects of adiponectin on expression of connexin 43 in rat myocardium during ischemia-reperfusion

AIM: To observe the effects of adiponectin(APN) on the expression of connexin 43 (Cx43) in rat myocardium during ischemia-induced arrhythmias. METHODS: The SD rats were randomly divided into 4 groups (n=12): sham operation group (SM group), ischemia and reperfusion group (I/R group), I/R+adiponectin(APN1) group: pre-ischemia with 3.5 μg/kg of APN; I/R+APN2 group: post-ischemia with 3.5 μg/kg of APN. The incidence of ventricular arrhythmias and ventricular arrhythmia score (VAS) were determined. The expression of Cx43 in the ischemic myocardium was studied by the techniques of immunohistochemistry and RT-PCR. The levels of malondialdehyde(MDA) and superoxide dismutase(SOD) were measured by the methods of xanthine oxidase and thiobarbituric acid. The expression of endothelial nitric oxide synthase (eNOS) at mRNA and protein levels was determined by RT-PCR and Western blotting,respectively.The morphological changes of the myocardial tissues were observed under electronic microscope. RESULTS: The VAS and concentration of MDA increased obviously and the activity of SOD was decreased in I/R group as compared with SM group (P<0.01). The expression of Cx43 was evidently decreased and the distribution of Cx43 in the myocardium was disturbed. The expression of eNOS at mRNA and protein levels was decreased in I/R group (P<0.05). The ultrastructure of ventricular myocardium was abnormal in I/R group. Compared with I/R group, APN obviously decreased the VAS caused by ischemia and reperfusion (P<0.01) no matter the drug was given before or after ischemia. APN increased the activity of SOD, inhibited the MDA content in serum, and resulted in normal distribution of Cx43 and increased the expression of Cx43 and eNOS. Compared with I/R group, the changes of heart ultrastructure attenuated greatly in APN group, but didn't recover to normal state. CONCLUSION: Adiponectin antagonizes the arrhythmias during myocardial ischemia and reperfusion via inhibiting oxidative stress and regulating Cx43.     

Changes of cardiac energy metabolism and structure during ischemia/reperfusion injury of liver in rats

AIM: To investigate the effect and mechanism of liver ischemia/reperfusion (I/R) injury on the changes of cardiac energy metabolism and structure.METHODS: 48 healthy Wistar male rats were randomly divided into 6 groups as follows (n=8 in each group): control group (CTL), simply ischemia for 30 min without reperfusion(I group); reperfusion following ischemia for 30 min (I/R group); 2 h reperfusion following ischemia for 30 min (I/R 2 h group); 4 h reperfusion following ischemia for 30 min (I/R 4 h group) and 6 h reperfusion following ischemia for 30 min (I/R 6 h group). The level of serum endotoxin was measured. The levels of insulin and insulin antibody in heart were detected by radioimmunoassay. The contents of MDA, MPO and lactic acid in heart were also determined. RESULTS: During the process of liver I/R injury, the level of endotoxin increased in I group and I/R group and declined gradually for long time during reperfusion, but was still longer than that in CTL group (P<0.05). The level of MDA obviously increased in I group and in all reperfusion groups compared to CTL (P<0.05). The obvious significant differences among I/R 2 h group, I/R 4 h group, I/R 6 h group with CTL were observed (P<0.05). The activities of MPO obviously increased in all reperfusion groups, there was also an obvious significant difference compared with CTL and I group (P<0.05). The level of lactic acid obviously increased with prolongation in reperfusion (P<0.05), but decreased in I/R 6 h group (P<0.05). The level of insulin decreased in I/R 4 h group and I/R 6 h group (P<0.05). No difference of insulin antibody was observed among all groups (P>0.05). CONCLUSION: During the process of liver I/R injury, endotoxin is absorbed from intestine and impairment of liver detoxication leads to endotoxemia, which might play a role in the changes of the energy metabolism and structure in heart.     

Effect of intervention for mast cell function before reperfusion on intestinal ischemia-reperfusion-induced early liver injury in rats

AIM：To explore the effect of intervention for mast cell function before reperfusion on intestinal ischemia-reperfusion (IR)-induced early liver injury. METHODS：Adult SD rats (n=35) were randomized into 5 groups with 7 rats each: sham operation group (S group), IR group, cromolyn sodium treatment group (IR+C group, 25 mg/kg), ketotifen treatment group (IR+K group, 1 mg/kg), compound 48/80 treatment group (IR+CP group, 0.75 mg/kg). IR was induced by superior mesenteric artery occlusion for 75 min followed by 4 h of reperfusion. The agents were intravenously administered 5 min before reperfusion. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and histamine, and the liver levels of lactate dehydrogenase (LDH), tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), malondialdehyde (MDA) and superoxide dismutase (SOD) were assessed. The liver histopathologic changes were also evaluated. RESULTS：IR resulted in severe liver injury as demonstrated by great increases in injury scores, concomitant significant increases in serum levels of AST, ALT and histamine, and liver levels of LDH, TNF-α, IL-8, and MDA, accompanied by reduced SOD activity (all P<0.05 vs S group). Treatment with cromolyn sodium or ketotifen markedly alleviated IR-mediated liver injury as confirmed by significant reduction of the above biomedical changes, whereas compound 48/80 further aggravated liver injury by dramatically enhancing the biomedical changes (all P<0.05 vs IR group). CONCLUSION：Inhibition of mast cell function before reperfusion may reduce early liver injury induced by intestinal ischemia reperfusion. Histamine, oxidative stress and inflammatory response may provide promising effects on it.     

Changes of gut mucosa antioxidant system and liver,renal function in rats after intestinal ischemia-reperfusion injury

AIM: To investigate the changes of the gut mucosa antioxidant system and liver, renal functions during rat intestinal ischemia-reperfusion injury. METHODS: 30 male Wistar rats underwent 45 min of intestinal ischemia by clamping the superior mesenteric artery followed by reperfusion. The levels of malondialdehyde (MDA), glutathione (GSH), the activities of antioxidant enzymes in the gut mucosa including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), glutathione S- transferase (GST) activity and serum ALT, AST, BUN, Cr were assayed at 2, 4, 8, 12 and 24 h after reperfusion. RESULTS: The levels of MDA and GSH in the gut mucosa increased and decreased significantly at 2 h of reperfusion, respectively (P<0.05). MDA was still lower than sham at 24 h of reperfusion (P<0.05), while GSH decreased to 40% of sham at 4 h of reperfusion (P<0.01) but returned to the level of control at 12 h. The activities of CAT, SOD and GSH-Px did not show significant changes in rat after intestinal ischemia reperfusion. GST decreased 39% at 2 h of reperfusion compared with the sham group and decreased to 56% of sham at 4 h (P<0.05), but returned to the level of control at 12 h after reperfusion. Serum ALT, AST, BUN and Cr increased significantly at 2 h of reperfusion (P<0.05) and increased 208%, 100%, 103%, 41% compared with control at 4 h of reperfusion (P<0.01). However, at 24 h of reperfusion, they returned to normal. CONCLUSION: Intestinal ischemia/reperfusion diminishes GSH level and GST activity, increases MDA level and causes liver and renal reversible damages.     

Effects of human urotensin II on ischemia/reperfusion injury in isolated perfused rat hearts

AIM: To investigate the effects of human urotensin II (hUII) on ischemia/reperfusion (I/R) injury in isolated rat hearts. METHODS: In the ischemia/reperfusion (I/R) model of isolated perfused rat hearts, the effects of hUII pretreatment on cardiac function was monitored with cardiac function software of MFL Lab200. ATP, total calcium, and malondialdehyde (MDA) content in myocardium were detected. The coronary perfusion flow (CPF) and lactate dehydrogenase (LDH) activity in coronary effluent were measured during reperfusion. RESULTS: In the hUII pretreated group, the release of LDH from myocardium was lower [(78.3±18.1)U/L] than I/R group [(109.3±23.9) U/L, P< 0.05], with decreased contents of MDA and calcium in myocardium (decreased by 24% and 27%, respectively, P< 0.05) and an increased myocardial ATP content [(3.8±0.4)μmol/g dw vs (2.2±0.4)μmol/g dw, P< 0.05)]. At the same time, hUII pretreatment increased CPF [(5.4±0.7) mL/min vs (3.8±0.8) mL/min in I/R group, P< 0.05], reduced left ventricular end-diastolic pressure (LVEDP) by 20% ( P< 0.05) with increased±d p /d t max [(217±38) kPa/s and (119±18) kPa/s vs (173±29) kPa/s and (82±25) kPa/s in I/R groups, respectively, P< 0.05]. hUII pretreatment also increased natrite/natrate (NO₂^-/NO₃^-) content in coronary effluent [(52.2±12.0)μmol/L vs (32.1±10.2)μmol/L in I/R group, P< 0.05)]. CONCLUSION: hUII pretreatment attenuated I/R injury in isolated perfused rat hearts. The protective mechanism might be associated with NO-mediated coronary vasodilation.     

全国设施蔬菜生产可持续发展学术研讨会在沈阳召开

会议由中国园艺学会主办 ,中国农业工程学会设施园艺工程专业委员会协办 ,沈阳农业大学园艺系承办 ,于 2 0 0 0年 4月 19日至 2 2日在沈阳农业大学召开。来自我国 13个省、自治区、直辖市的与会代表共 78人 ,会议论文 4 3篇 ,由沈阳农业大学学报专刊发表。经承办单位和与会者努力 ,会议圆满成功。开幕式由中国园艺学会副理事长李树德研究员主持 ,辽宁省科技厅、农业厅、沈阳市副食品局、沈阳农业大学的领导到会祝贺并发表讲话。会议就确定的主题进行了大会发言 ,小组讨论及现场考察。代表们欣喜地看到 ,近 2 0年来我国蔬菜设施生产迅猛发展 …     

Effects of Astragalus injection on neuronal apoptosis and expression of JNK3 in rat hippocampus after cerebral ischemia reperfusion

AIM:To investigate the effects of Astragalus injection on neuronal apoptosis and expression of c-Jun N-terminal kinase 3(JNK3) in the rat hippocampus after cerebral ischemia reperfusion. METHODS:The rat model of cerebral ischemia reperfusion was set up by a four-vessel occlusion method. The SD rats were randomly divided into 4 groups:sham operation group, cerebral ischemia reperfusion group(model group), cerebral ischemia reperfusion+Astragalus injection group(Astragalus injection group) and cerebral ischemia reperfusion+vehicle group(vehicle group). The rats in model group, Astragalus injection group and vehicle group after transient global cerebral ischemia(30 min) were then divided into 7 subgroups according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. The apoptosis of the neuron in the hippocampus was measured by the method of TUNEL staining. The expression of JNK3 at mRNA and protein levels was determined by real-time PCR and Western blotting,respectively. RESULTS:Compared with sham operation group, the number of apoptotic neurons increased in model group(P<0.05). Compared with model group, the number of apoptotic neurons decreased obviously in Astragalus injection group(P<0.05). Compared with sham operation group, the expression of JNK3 at mRNA and protein levels in the hippocampus increased obviously in model group at all time points except 120 h(P<0.05). Compared with model group, the expression of JNK3 at mRNA and protein levels in the hippocampus decreased obviously in Astragalus injection group at all time points except 120 h(P<0.05). CONCLUSION:Astragalus injection decreases neuronal apoptosis in rat hippocampus after cerebral ischemia reperfusion by inhibiting the expression of JNK3 at mRNA and protein levels.     

Relationship between the intracellular calcium concentration changes and left ventricular hypertrophy and function in the spontaneously hypertensive rats

AIM:To elucidate the relationship between the intracellular calcium concentration changes and left ventricular hypertrophy and function in the spontaneously hypertensive rats (SHR).METHODS:Intracellular free calcium concentrations were measured by Fura 2 methodology and left ventricular function quantitated by cardiac catheterization in 20 SHR aged 10, 22, and 34 weeks and 20 age-matched Wistar-kyoto (WKY) rats.RESULTS:(1) The systolic blood pressure(SBP), intracellular calcium concentrations and left ventricular mass / body weight index (LVM/BW) were significantly higher in all three age groups of SHR than the corresponding groups of WKY; (2) Compared with age-matched WKY groups, the peak left ventricular pressure descending rate(-dp／dt_max) decreased while left ventricular relaxation time constant (τ)increased significantly in SHR aged 22 and 34 weeks. The peak left ventricular pressure ascending rate(dp／dt_max) and the left ventricular contractility index were significantly increased only in the 34 weeks SHR; (3) Intracellular calcium concentrations showed a positive correlation with LVM/BW,SBP,-dp／dt_max and τ(r=0.47-0.83,P<0.01)and a negative correlation with dp／dt_max and the left ventricular contractility index (r=-0.46,P<0.05 and r=-0.81, P<0.01).CONCLUSION:Intracellular calcium overload is one of the potential mechanisms in the induction of left ventricular hypertrophy as well as of systolic and diastolic dysfunction.     

Role of nitric oxide in the development of glomerular ischemia reperfusion injury in rats

AIM:To investigate the effects of nitric oxide on ultrastructure and anionic sites of glomerular in renal ischemia reperfusion injured (I-RI) rats. METHODS:Animals were divided randomly into five groups:(1) sham group (n=6);(2) I-RI group (n=6), 0.3 mL normal saline was injected via venae lingualis 20 min before ischemia;(3) SNP+I-RI group (n=6), 2.5 μg/kg sodium nitroprusside (SNP) was injected via venae lingualis 20 min before ischemia;(4) AG+I-RI group (n=6), 10 mg/kg aminoguanidine (AG) was injected via venae lingualis 20 min before ischemia;(5) L-NNA+I-RI group (n=6), 10 mg/kg Nω-nitro-L-arginine (L-NNA) was injected via venae lingualis 20 min before ischemia. Anionic sites of glomerular were studied with a cationic probe-polyethyleneimine (PEI) and ultrastructure was observed under electron microscope in renal I-RI rats. RESULTS:(1) Ultrastructure of glomerular was normal and anionic sites (AS) was located clearly in lamina rare externa of GBM in sham rats. The PEI particles arranged regularly in line (19.3±1.7/1 000 nm) under electronic microscope. Obvious foot processes derangement and effacement were observed and the AS number in GBM of I-RI group was fewer (16.6±1.0/1 000 nm, P<0.05) and the particle was smaller than that in sham group. (2) Compared with I-RI group, the foot process effacement was aggravated in SNP+I-RI group and L-NNA+I-RI group. SNP caused the numbers of anionic sites reduced after renal I-RI (11.7±3.2/1 000 nm, P<0.05), and the electronic density of the PEI granule was also reduced. AG lead a increase in anionic site number (17.8±1.0/1 000 nm, P<0.05), but still fewer than that in sham group (P<0.05). The numbers of anionic sites was not changed in L-NNA+I-RI group (14.7±0.9/1 000 nm, P>0.05). CONCLUSION:Foot process effacement and reduction of anionic sites were present in glomerular filtration membrane in renal I-RI rats. NO aggravated those injuries, indicating that NO plays a role in the ultrastructure damages of glomerular filtration membrane in I-RI rats.     

Establishment and evaluation of rat heart ischemia-reperfusion injury model in vivo

AIM: To establish and evaluate a rat model of heart ischemia-reperfusion injury in vivo. METHODS: Seventy-two male Sprague-Dawley rats weighing(250±50)g were randomly divided into sham operation group(sham), ischemia-reperfusion group(I/R) and normal group. The animals were anesthetized and heparinized. Myocardial ischemia-reperfusion was induced by ligating the left anterior descending coronary artery with "U-shape tube" for 35 min followed by 120 min or 240 min reperfusion in vivo. The heart infarct size was measured by triphenyltetrazolium chloride(TTC) staining. The myocardial cell apoptotic index was determined by the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling(TUNEL). Immunohistochemical method was used to detect the expression of Bcl-2 and Bax in rat ischemia myocardium. The blood level of MB isoenzyme of creatine kinase(CK-MB),cardiac troponin I(cTnI),nitric oxide(NO),malondialdehyde(MDA), total superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px) were detected after reperfusion for 2 h and 4 h. RESULTS: Compared with normal group and sham group, there were obvious changes of ST-T segment and Q wave in the electrocardiogram of I/R group. The blood level of CK-MB, cTnI, NO, MDA and GSH-Px in I/R group increased(P<0.05,P<0.01) after reperfusion for 2 h and 4 h, and the blood level of T-SOD in I/R group after reperfusion for 2 h and 4 h also increased(P<0.05). The heart infarct size in I/R group was the largest as compared to other groups. Among these groups, the apoptotic index of I/R group was the highest and the Bcl-2/Bax ratio in I/R group decreased(P<0.01).CONCLUSION: The rat model of heart ischemia-reperfusion injury in vivo can be successfully established with the "U-shape tube". There are obviously changes of heart infarct size, blood level of CK-MB, cTnI, NO, MDA, T-SOD and GSH-Px, myocardial apoptotic index and Bcl-2/Bax ratio between I/R rats and control animals.     

Effects of ketotifen administered after intestinal ischemia reperfusion on the survival rate in rats

AIM: To investigate the effect of antihistaminic, ketotifen, administered for three days after intestinal ischemia reperfusion (I/R), on survival rates in SD rats. METHODS: The intestinal I/R model of SD rats was set up with clamping the superior mesenteric artery for 75 min. 72 SD rats were divided into 3 groups: group S for sham group, group M for model group, group K for ketotifen treatment (model + ketotifen 1mg/kg), each group was divided into 2 sub-group: 3rd d and 7th d (eg: group S3, S7, M3, M7, K3, K7; n=12 per sub-group). Ketotifen was administered via caudal vein 5 min before reperfusion in group K, while normal saline was administered in groups S and M and was kept on administering once a day for 3 days after reperfusion. The survival rates were recorded at 3 d or 7 d after operation. The morphological changes of intestinal mucosal and mast cells were observed under light and electron microscopes in survival rats. The concentration of histamine was also determined in the intestinal samples. RESULTS: The survival rate at 3rd d in group M and K was lower than that in group S (P<0.05 or P<0.01). No significant difference of the 3rd d survival rate between group K and M was observed (P>0.05). The survival rate at 7th d in group M7 was lower than that in group S7 and K7 (P<0.05), no significant difference between group K7 and group S7 was found (P>0.05). Light and electron microscope examinations showed slight damage of intestinal mucosal in group M and K in survival rats, while the result in group S was almost normal. Mast cell degranulation was obvious in group M7, partly in group K while none was found in group S. There was no significant difference in Chiu’s score, the expression of tryptase and the number of IMMC among the groups in the survival rats (P>0.05). The concentration of histamine in the intestine in group K7 was lower than that in group S7 and M7. CONCLUSION: Ketotifen administered after intestinal ischemia reperfusion increases the survival rate in rats. The mucosal mast cell degranulation and release of histamine may be adverse to the prognosis of intestinal ischemia reperfusion injury in rats.     

Correlation of peripheral leukocyte apoptosis insufficiency and intestinal injury following mesenteric ischemia/reperfusion in rats

AIM: To investigate the role of peripheral blood leukocytes and polymorphonuclear neutrophils (PMNs) on intestinal injury following mesenteric ischemia/reperfusion (IR) in rats. METHODS: Twenty adult, male Sprague-Dawley rats, weighing 200-230 g, were randomly divided into two groups. The control group (CON) consisting of 10 rats was subjected to laparotomy and separation of superior mesenteric artery (SMA) only. The ischemia/reperfusion (IR) group consisting of 10 rats, was subjected to laparotomy, followed by occlusion of the superior mesenteric artery (SMA) by an atraumatic microvascular clamp for 30 min. At the end of ischemic period in IR, the microvascular clamp was removed and the intestinal segment was reperfused for 60 min. The pathological changes of the ileal mucosal tissue were evaluated. The apoptosis of intestinal mucosal epithelial cells was examined by terminal deoxylnucleotidy-l transferase mediated-dUTP nick end-labeling (TUNEL). The enzymatic activity of casapse-3 in mucosal cells was determined using a colorimetric assay. The percentages of apoptotic peripheral blood leukocytes and PMNs were measured by flow cytometry using Annexin-V/PI double staining assay. The numbers of peripheral blood leukocytes in each animal was measured at baseline, 30 min of ischemia, and 30 min and 60 min of reperfusion. RESULTS: (1) Compared to CON group animals, the most severe mucosal injury was observed in IR group under optical microscope. (2) The number of apoptotic mucosal epithelia cells and enzymatic activity of caspase-3 were significantly higher in IR than those in CON group (P<0.05). (3) The percentages of apoptotic peripheral blood leukocytes and PMNs were significantly lower in IR group than those in CON group (P<0.05). The number of peripheral blood leukocytes in IR group was increased obviously after SMA closed for 30 min (P<0.05), and was higher following reperfusion (P<0.05), and significantly higher than that in CON group (P<0.05). (4) A negative correlation between the enzymatic activity of caspase-3 in the ileal mucosal tissue and the percentage of apoptotic peripheral blood leukocytes (r=-0.764, P<0.05), and of PMNs (r=-0.845, P<0.05) was found. A negative correlation between the apoptosis index of mucosal epithelial cells and the apoptotic percentage of PMNs (r=-0.638, P<0.05) was also observed. CONCLUSION: Apoptosis insufficiency and higher numbers of peripheral blood leukocytes are correlated with the mucosal cells injury following mesenteric ischemia/reperfusion (IR) in rats.     

Role of mitochondrial KATP channels in cardioprotection of hyperpolarized cardioplegia

AIM:To study the protective effect of hyperpolarized cardioplegic arrest on reperfused rat heart performance and to investigate the role of mitochondrial ATP-sensitive K+ channels (mitoKATP) opening in the protection of hyperpolarized cardioplegia against ischemia/reperfusion damage. METHODS:Forty Sprague-Dawley rats were randomized into five groups (n=8 in each group): control group (Con); depolarized arrest group (D); hyperpolarized arrest group (H); depolarized cardioplegia with 5-hydroxydecanoate (5-HD) group (5HD+D); hyperpolarized cardioplegia with 5-HD group (5HD+H). The rat hearts were quickly removed to Langendorff apparatus. The heart perfusion was performed for 20 min with 37 ℃ Krebs-Henseleit buffer balanced with gas mixture (O2∶〖KG-*2〗CO2=95%∶〖KG-*2〗5%) at 5.8 kPa perfusion pressure, then cardial arrest was induced by different cardioplegic solution. Hearts were subjected to ischemia at 37 ℃ for 40 min followed by 30 min reperfusion. (1) The hemodynamics was detected at recovery after 30 min reperfusion. (2) Before ischemia and at the end-reperfusion, tissue was harvested for mitochondrial isolation and ultrastructure was observed by transmission electron microscopy (TEM). (3) Production of reactive oxygen species (ROS) was also determined at different time points. RESULTS:(1) Compared with end-equilibration, 30 min reperfusion caused significant differences in left ventricular developed pressure (LADP), left ventricular end-diastolic pressure (LVEDP), double product (DP), heart rate (HR), coronary flow (CF) (P<0.01). TEM showed that the ultrastructures of myocardial and mitochondrial were damaged remarkably. (2) When H group was compared with D, 5HD+H and Con group, significant differences were found in LVDP, LVEDP, DP, HR and CF (P<0.01). TEM showed that the myocardial and mitochondrial ultrastructures were improved remarkably. (3) The rate of ROS generating was lower in group H than that in other four groups at end-reperfusion (P<0.01). CONCLUSION:(1) Of the four cardioplegias, hyperpolarized cardioplegia is superior to improve myocardial performance, attenuates myocardial and mitochondrial injury, and reduces rate of ROS generating. (2) Mitochondrial preservation is one of mechanisms of myocardial protection in hyperpolarized cardioplegia, opening of mitoKATP enhances cardioprotection through decreasing ROS generating, providing better energe supply for reperfused myocardium.     

Effects of soybean isflavones on mitochondrial damage and neuronal apoptosis induced by cerebral ischemia/reperfusion

AIM: To study the effects of soybean isoflavones on mitochondrial ultrastructure, neuronal apoptosis and expression of cytochrome C, caspase-9 and caspase-3 in the rats with cerebral ischemia/reperfusion.METHODS: Adult healthy SD rats (n=60) were randomly divided into 3 groups: sham group, ischemia/reperfusion injury (I/R) group and soybean isoflavone (SI) pretreatment group. Soybean isoflavones (120 mg·kg-1·d-1) were fed by gastric lavage for 21 d. The global ischemia/reperfusion model of the rats was established by blocking 3 vessels, and then reperfused for 1 h after 1 h of ischemia. The morphological change of the cerebral cortex cells was observed under light microscope. The mitochondrial ultrastructure of the cerebral cortex cells was determined by transmission electron microscope. The apoptotic rate of the cerebral cortex cells was detected by flow cytometry. The expression of cytochrome C, caspase-9 and caspase-3 in the cerebral cortex cells was determined by semi-quantitative RT-PCR and immunohistochemical techniques.RESULTS: Disintegration of mitochondria membrane and disappearance of the mitochondrial cristae were seen in I/R group. Compared with I/R group, the change of ultrastructure of mitochondria was significantly improved by soybean isoflavone pretreatment, and the neuronal apoptotic rate was also significantly decreased (P<0.01). The mRNA expression and protein content of cytochrome C, caspase-9 and caspase-3 in I/R group were obviously higher than those in sham group (P<0.01). Compared with I/R group, the mRNA expression and protein content of cytochrome C, caspase-9 and caspase-3 in SI group were significantly decreased (P<0.01).CONCLUSION: Soybean isoflavones attenuate cerebral ischemia/reperfusion injury by stabilizing the structure of mitochondria, preventing cytochrome C release to the cytoplasm, inhibiting the activation of caspase-9 and caspase-3 and decreasing cell apoptosis．