Objective To investigate the protective effect of ischemic preconditioning (IP) on ischemicreperfusion injury of rat liver graft. MethodsMale Sprague Dawley rats were used as donors and recipients of orthotopic liver transplantation,the period of cold preservation and anhepatic phase were 100 min and 25 min respectively.Sixtyfour rats were randomly divided into 2 groups (n=32),control group: donor livers were flushed through the portal veins with physiological saline solution containing heparin only before harvested; IP group: before donor livers were harvested,the portal veins and hepatic arteries of them were interrupted for 10 min,and reflow was initiated for another 10 min,then did as control group.One half of each group were used to investigate 1 week survival rate of recipients,and another half of each group were used to take sample of blood and hepatic tissue after 2 hours of reperfusion of liver graft. ResultsOne week survival rate,amount of bile,serum NO and activity of antioxidase were higher in IP group than those in control group(P＜0.05),meanwhile,serum ALT,AST,LDH,TNF and superoxide in hepatic tissue were lower in IP group than those in control group (P＜0.05),and histological findings in IP group showed less injury than those in control group. Conclusion IP could increase production of serum NO,reduce the level of serum TNF and protect rat liver graft from ischemicreperfusion injury.
【Abstract】Objective To study the mechanisms of enhancing effect of mild hypothermia (MH) to ischemic preconditioning (IP) on hepatic ischemiareperfusion (I-R) injury. Methods To observe the content of the marker enzymes of liver damage (ALT,AST,LDH) and malondialdehyde (MDA), and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPX), total antioxidase (TAX) in inferior vena cava blood above liver in nonischemic control group (n=6), I-R group (n=6), IP group (n=6) and mild hypothermic ischemic preconditioning (MHIP) group (n=6). Results After I-R the content of ALT,AST, LDH and MDA were significantly elevated (P＜0.01), SOD,CAT,GSH-PX,ACT activities were declined obviously (P＜0.01). The content of ALT,AST,LDH and MDA were significantly lower in IP group than those in I-R group, and in MHIP group than those in IP group (P＜0.01,P＜0.05), and the content of SOD, CAT,GSH-PX, ACT activities were significantly higher in IP group than those in I-R group, and in MHIP group than those in IP group (P＜0.01,P＜0.05). Conclusion Ischemic preconditioning may enhance the oxidation-resistance of liver, and reduce the oxygen free radical injury to liver after ischemia-reperfusion. Mild hypothermia may enhance the protective effect of IP on hepatic ischemiareperfusion injury.
Abstract： Objective To observe the expression changes of microRNA 1 （miRNA-1） and microRNA 21（miRNA-21） after ischemic preconditioning （IPC）, ischemic postconditioning （IPO） and remote ischemic preconditioning （RIPC）in an ischemia-reperfusion rat heart model in vitro, as well as the expression of their target protein heat shock protein 70 （HSP70） and programmed cell death 4 （PDCD4）, and evaluate whether miRNA are involved in endogenous cardio-protective mechanism. Methods The Langendorff-perfused Sprague-Dawley rat hearts were randomly assigned into one of the four groups, control group （CON group, n=12）, ischemia preconditioning group （IPC group， n=12） , ischemia postconditioning group （IPO group， n=12） and remote ischemia preconditioning group （RIPC group，n=12）. Cardiac function was digitalized and analyzed. The expression of HSP70, PDCD4, B-cell lymphoma/leukemia-2 （Bcl-2） and Bax was detected by Western blotting. The expression of miRNA-1 and miRNA-21 was detected by real-time reverse transcriotion-polymerase chain reaction （RT-PCR）. Assessment of cardiac infarct size and myocardial apoptosis was determined using triphenyltetrazolium chloride （TTC） assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） assay respectively. Results The expressions of miRNA-1 and miRNA-21 were up-regulated in IPC group, but the expression of miRNA-1 was down-regulated in RIPC group and IPO group （P＜0.05）. The expressionsof PDCD4, HSP70 and Bax were down-regulated in ‘conditioning’ groups compared with CON group （P＜0.05）. The expression of Bcl-2 was not statistically different among the four groups. The infarct size and the myocardial apoptosis in ‘conditioning’ hearts were significantly decreased compared with CON group （P＜0.05）. Conclusion The expressions of the miRNA-1 and miRNA-21 are different in IPC, RIPC and IPO groups, and their target proteins are not inversely correlated with the miRNAs in all the ‘conditioning’ groups.
Abstract： Objective To study the changes of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) expression of isolated rat hearts after diazoxide preconditioning (DPC), and to explore the possible mechanism of cAMP signaling pathway in myocardial protection by DPC. Methods Isolated working heart Langendorff perfusion models of 40 Wistar rats were set up and were divided randomly into four groups. For the ischemia reperfusion injury(I/R) group (n=10), 30 min of equilibrium perfusion was followed by a 60 min reperfusion of KrebsHenseleit (K-H) fluid. The DPC group (n=10) had a 10 min equilibrium perfusion and two cycles of 5 min of 100 μmol/L diazoxide perfusion followed by a 5 min diazoxidefree period before the 30 min ischemia and the 60 min reperfusion of K-H fluid. The blank control group (control group, n=10) and the Dimethyl Sulphoxide(DMSO) group (n=10) were perfused with the same treatment as in the DPC group except that diazoxide was replaced by natriichloridum and DMSO respectively. The activity of creatine kinase (CK) in coronary outflow, the activity of malonyldialdehyde (MDA) and superoxide dismutase (SOD) in myocardium were detected. And the scope of myocardial infarction and the concentrations of myocardial cAMP and PKA were also assessed. Results Compared with the I/R group, the level of MDA for the DPC group decreased significantly (8.28±2.04 nmol/mg vs. 15.52±2.18 nmol/mg, q=11.761,Plt;0.05), the level of SOD increased significantly (621.39±86.23 U/mg vs. 477.48±65.20 U/mg, q=5.598,Plt;0.05). After a 30 min reperfusion, compared with the I/R group, the content of CK decreased significantly (82.55±10.08 U/L vs. 101.64±19.24 U/L, q=5.598, Plt;0.05) and the infarct size reduced significantly (5.63%±9.23% vs.17.58%±5.76%, q=6.176,Plt;0.05) in the DPC group. The cAMP concentration in the DPC group was much higher than that in the I/R group (0.64±0.07 pmol/g vs. 0.34±0.05 pmol/g, q=14.738,Plt;0.05), and PKA concentration was also much higher than that in the I/R group ［17.13±1.57 pmol/(L·min·mg) vs. 12.85±2.01 pmol/(L·min·mg), Plt;0.05］. However, there were no significant differences between the I/R group, DMSO group and the control group in the above indexs (Pgt;0.05). Conclusion DPC significantly improves the releasing of cAMP and PKA, decreases oxygen free radicals, and relieves myocardial ischemia reperfusion injury. The cAMP signaling pathway may be involved in triggering the process of myocardial protection mechanisms of DPC.
Objective To investigate the effect of ischemic preconditioning(IPC) on myocardial ischemic reperfusion injury(I/R) of elderly rats. Methods Fiftysix Wistar rats, of which there were 28 aged from 21-23months(elderly rat) and 28 aged from 4-5months(young rat), were used to build isolated heart perfusion Langendorff model. The rats were divided into 7 groups with random number table（8 in each group）: adult control group, adult I/R group, adult IPC group, elderly control group, elderly I/R group, elderly IPC group and elderly enhanced IPC group. The control group underwent a 90-min perfusion without any intervention; the I/R group underwent a 30-min equilibration period, then a 30-min ischemia and a 30-min reperfusion; the IPC group underwent a 10-min equilibration period, then a 5-min ischemia for twice and a 5-min reperfusion, after that a 30-min ischemia and [CM(158-3mm]a 30-min reperfusion; the enhanced IPC group underwent a 10-min equilibration period, then a 5-min ischemia for 4-times and a 5-min reperfusion, after that a 30-min ischemia and a 30-min reperfusion. The recovery rates of cardiac output(CO), left ventricular developed pressure (LVDP), the maximum rising and descending rate of left ventricular pressure (±dp/dtmax) after a 30-min reperfusion were compared among groups. The activity of creatine kinase (CK) in coronary outflow, the level of malonyldialdehyde (MDA) and superoxide dismutase (SOD) before ischemia and after a 30min reperfusion were detected. The myocardial infarction areas were compared among groups. Results After a 30min reperfusion, compared with adult I/R group, in adult IPC group CK reduced significantly(89.48±18.72 U/L vs. 115.76±16.72 U/L，q=6.061，Plt;0.01)，the level of MDA decreased significantly（9.53±3.44 nmol/ml vs. 16.84±2.29 nmol/ml，q=7.732，Plt;0.01)，the level of SOD increased significantly（584.7±122.62 U/ml vs. 429.46±85.24 U/ml，q=4.754，Plt;0.01)，the recovery rates of CO,LVDP,+dp/dtmax and -dp/dtmax increased ignificantly(78.69%±9.68% vs. 65.10%±8.63%，83.61%±8.46% vs. 67.23±8.68%，81.68±8.68% vs. 67.89%±6.89%，89.79%±7.78% vs. 66.79%±8.46%，Plt;0.01), the myocardial infarction areas reduced significantly (5.25%±4.33% vs. 14.75%±8.02%,q=7.458，Plt;0.01)。There was no statistical significance between elderly IPC group and elderly I/R group in the above indexes（Pgt;0.05）.However, There was statistical significances between elderly enhanced IPC group and I/R group. CK reduced significantly (88.60±28.32 U/L vs. 105.76±9.64 U/L，q=5.620，Plt;0.01)，the level of MDA decreased significantly（8.38±3.36 nmol/ml vs. 16.80±3.06 nmol/ml，q=7.500，Plt;0.01)，the level of SOD increased significantly（558.87±78.66 U/ml vs. 433.75±86.65 U/ml，q=7.335，Plt;0.01)，the recovery rates of CO,LVDP,+dp/dtmax and -dp/dtmax increased significantly (77.99%±10.02% vs. 66.26%±9.78%，85.59%±6.67% vs. 73.90%±6.66%，83.87%±9.98% vs. 68.90%±8.68%，86.01%±766% vs. 70.39%±7.98%，Plt;0.01)， the myocardial infarction areas reduced significantly (795%±6.32% vs. 1568%±10.36%,q=8.680， Plt;0.01). 〖WTHZ〗Conclusion The protective effect of IPC on I/R elderly rat hearts has weakened. The enhanced IPC is able to regain the protective effect of IPC on elderly rat hearts.
Objective To investigate the effect of peroxisome proliferatoractivated receptor-γ coactivator-1α(PGC-1α) on early ischemic preconditioning (IPC) which may act as an important role in early IPC. Methods Building isolated working rat heart Langendorff model, thirty Wistar rats were divided randomly into three groups. Control group(CON group,n=10): a 120-min perfusion was performed without any intervension; ischemia and reperfusion group(I/R group,n=10): a 30-min equilibration period perfusion, a 30-min ischemia and a 60-min reperfusion were performed.; IPC group (n=10): a 10-min equilibration period perfusion was performed, then was elicited by two cycles of 5-min of ischemia interspersed with 5-min reperfusion prior to 30-min ischemia and a 60-min reperfusion. Frozen sections of myocardium at cardiac apex were made and immunohistochemical staining was used to detect expression and the intergrated optical density average (IODA) of PGC-1α. Ultrathin sections were made and the mitochondria under each specimen was evaluated according to Flameng score. Results PGC-1α expression in IPC group (IODA 10.94±5.23) was significantly higher than that in I/R group (IODA 3.88±1.72) and that in CON group (IODA 3.39±2.46; P=0.009, 0.007). The mitochondria changes in I/R group were significant edema and severe damage; but there were not so severe in CON group and IPC group.Flameng score of IPC group (0.44±0.13) and CON group (0.88±0.22) were lower than that in I/R group(1.78±0.14;P=0.003, 0.014) respectively. Conclusion IPC can protect myocytes mitochondria from ischemia and reperfusion.The cardioprotection may be related with the activation and the high expression of PGC-1α, which may act as one of the most important endogenous defence factors of the heart.
Objective To investigate whether single cycle ischemic preconditioning （IP） improves the myocardial preservation in patients undergoing cardiac valve replacement. Methods From August 2002 to April 2006, 85 patients who had chronic heart valve disease and required cardiac valve replacement were randomly divided into two groups. IP group, 47 allocated to receive IP and arrested with 4 C St. Thomas＇ Hospital cardioplegic solution during cardiopulmonary bypass（CPB）, preconditioning was accomplished by using single cycle of 2 minutes occlusion of aorta followed by 3 minutes of reperfusion before cross-clamping. Control group, 38 allocated to receive 4 C St. Thomas＇ Hospital cardioplegic solution alone. Myocardial protective effects were assessed by determinations of creatinine kinase-MB isoenzyme （CK-MB） and cardiac troponin I（cTnI）, ST-T changes, ventricular arrhythmias and other clinical data in ICU. Results Serum CK-MB and cTnI concentrations were increased postoperatively in two groups. At 24, 48 and 72h after operation, values of CK-MB in IP group was significantly lower than that in control group （P〈0.05）, cTnI at 24 and 48h after operation also less in IP group （P〈0.05）. The duration for patients needed for antiarrhythmic drugs in IP group was lower than that in control group （P〈0.05）. Compared with control group, fewer inotropic drugs were used in IP group. As a result, ICU stay time in IP group was shorter than that in control group （P〈0.05）. Conclusion IP enhances the myocardial protective effect when it was used with hypothermic hyper kalemic cardioplegic solution in patients undergoing cardiac valve replacement, IP significantly reduces the postoperative increase of CK-MB, cTnI and plessens the severity of postoperative ventricular arrhythmias.
Objective To investigate the effects and mechanism of doxorubicin preconditioning in providing ischemic tolerance for rats abdomen island flaps. Methods Twenty-four healthy adult Sprague Dawley rats, 12 males and 12 females, were randomly divided into 3 groups (n=8): control group (group A), ischemic preconditioning group (group B), and doxorubicin preconditioning group (group C). After the abdomen island flap (6 cm × 3 cm in size) based on the superficial inferior epigastric neurovascular bundle was prepared, group A had no further treatment; group B was given a 10-minute ischemia followed by a 10-minute reperfusion for 4 times; and group C was given pretreatment with doxorubicin (1 mg/kg) by injection of the inferior epigastric vein. After 24 hours, the inferior epigastric vessels were blocked by vascular clamp for 4 hours, followed by reperfusion 2 hours to prepare ischemia/reperfusion (I/R) injury model. The rat survival was observed after operation; at 0, 8, 12, 24, and 30 hours after I/R injury, the malonyldiadehyde (MDA) and superoxide dismutase (SOD) levels were measured. At 7 days after I/R injury, the survival rate of flap were calculated and the flaps were harvested for histological observation. Results During experiment, 5 rats died (1 rat in groups A and B respectively, 3 rats in group C) and were added. The survival rates of the flap in group A (10.10% ± 0.43%) was lower than those in group B (91.63% ± 1.76%) and in group C (92.75% ± 1.48%) at 7 days after I/R injury, showing significant differences (P lt; 0.05), and there was no significant difference between groups B and C (t=0.29, P=0.77). Significant difference was found in MDA level and SOD level between group A and groups B, C after 8 hours (P lt; 0.05), and there was no significant difference between groups B and C (P gt; 0.05). Histological observation showed that inflammatory cells infiltration was more obvious and hyperplasia of fibers was weaker in group A than in groups B and C. Conclusion Doxorubicin preconditioning can provide ischemic tolerance for rats abdomen island flaps and protect flaps from the I/R injury. The possible mechanism may be related to that doxorubicin can induce endogenous protections.
Objective To determine whether the different durations and times of the ischemic preconditioning affect the effectiveness of the ischemic preconditioning. Methods Ninety male Wistar rats were randomly divided into the control group and the eight preconditioned groups of 10 rats each. A transverse rectus abdominis musculocutaneous flap (TRAM) was elevated in each rat. The flaps were preconditioned by clamping the pedicle and reperfusing for 5 or 10 minutes per cycle. This was repeated for one or two cycles. The controls were simply perfused for 30 minutes. Each flap was then subjected to 4 hours of the global ischemia. Three rats in each group were killed for anestimate of the water content in the muscle and for observation on the muscularstructure under microscope. The flap surface survival areas of the other rats were calculated on the 7th postoperative day by the computerized video planimetry. Results The water content in the muscle was evidently reduced. The mean survival area of the flap in every preconditioned group increased by2-3 times compared with that of the controls(P＜0.001). The different proceduresof the ischemic preconditioning produced different protective effects. Conclusion The ischemic preconditioning is an available means to alleviate an ischemiareperfusion injury to the transverse rectus abdominis musculocutaneous flap in rats. The effect of the ischemic preconditioning is affected by the duration and time of the ischemic preconditioning.
Objective To study the protective effects of ischemic preconditioning(IP) duration against ischemic reperfusion injury of skeletal muscle. Methods Thirty-six Wister rats were made amputation-like models, which underwent temporary amputation at the level of the femur, excluding the femoral vessels. They were divided into 6 groups(n=6) according to different treatments before ischemiareperfusion: group A(4 hours of ischemiareperfusion); groups B, C, D, E(5, 10,15, 20 minutes of ischemia and 5, 10, 15, 20 minutes of reperfusion respectively, for 3 cycles, 4 hours ischemiareperfusion ); group F (no ischemia-reperfusion). The malondialdehyde(MDA), the extent of edema and necrosis of skeletal muscle were measured to observe protective effects of different ischemic preconditioning duration. Results Five minutes of ischemic preconditioning（IP5）could protect skeletal muscle of ischaemia against necrosis and the survival area of the muscle was 82.47%.The effects of IP10 and IP 15 were significantly superior to that of IP5 and the survival areas of the muscle were 89.03% and 89.49%. The effect of IP20(78.27%) was significantly inferior to that IP5. IP5 could reduce edema of skeletal muscle, the effect of IP10 was significantly superior to that of IP5. IP5, IP 10,and IP 15 could decrease the level of MDA, but IP20 did not decrease it. Conclusion The trend of protective effect of IP on ischemia-reperfusion injury of themuscle in rats first rise to the peak and then go down,10minutes ofIPis optimal.