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Objective To evaluate the individualized fluid therapy in the elderly patients with coronary heart disease undergoing gastrointestinal surgery.Methods From January 2009 to December 2012,80 cases of coronary heart disease patients (aged 65-80 years) undergoing gastrointestinal surgery were divided into test group and control group by random digits table with 40 cases each.Traditional fluid therapy was used in control group in the intraoperative and postoperative period.Individualized fluid therapy was used in test group in the intraoperative and postoperative transferred to the intensive care unit (ICU) during the period of 24 h:in cardiac index (CI),stroke output index,stroke variation degree,under the guidance of indicators such as capacity titration type treatment.Hemodynamic index,fluid intake,incidence of cardiac adverse events and recovery of gastrointestinal function were compared in two groups into the operating room (T1),after anesthesia induction (T2),the operation started (T3),intraoperative 1 h (T4),the end of surgery (T5),transferred to the ICU 6 h (T6),transferred to the ICU 12 h (T7) and transferred to the ICU 24 h (T8).Results Compared with T1,two groups of patients with mean arterial pressure (MAP),central venous pressure (CVP),CI and stroke volume (SV) were lower than those at T2 [test group:(68.1 ±6.1) mm Hg(1 mm Hg =0.133 kPa) vs.(84.4 ±5.2) mm Hg,(5.5 ±0.8) cm H2O(1 cm H2O =0.098 kPa) vs.(6.2 ± 1.0) cm H2O,(2.8 ± 1.6) L/(min·m2) vs.(3.3 ± 0.8) L/(min·m2),(65.7 ± 4.5) ml vs.(74.3 ± 7.5) ml;control group:(65.4 ± 7.3) mm Hg vs.(85.1 ± 6.6) mm Hg,(4.6 ± 0.8) cm H2O vs.(6.4± 1.1) cm H2O,(2.7 ±0.7) L/(min·m2) vs.(3.3 ±0.6) L/(min·m2),(60.6 ± 7.6) ml vs.(73.8 ±7.5)ml],stroke variation degree (SVV) was significantly increased [test group:(15.9 ±5.1)% vs.(12.1 ±5.9)%; control group:(15.8 ± 9.4)% vs.(12.6 ± 8.4)%],there was significant difference (P < 0.05).Compared with the same time of control group,MAP was higher at T3,CI was higher at T4 and T5,SV was higher at T2-T7,there was significant difference (P< 0.05).The total transfusion amount,crystal usage and urine in intraoperative and transferred to the ICU 24 h in test group were less than those in control group,while colloid usage was more than that in control group,there was significant difference (P < 0.05).The incidence of cardiac adverse events between two groups had no significant difference (P =0.232).The postoperative ICU stay time,exhaust time,defecation time,into the liquid diets time and hospital stay in test group were less than those in control group [(37 ± 13) h vs.(55 ± 25) h,(72 ± 12) h vs.(99 ± 13) h,(92 ± 16) h vs.(113 ± 16) h,(4.0 ±0.8) d vs.(4.9 ± 1.9) d,(17 ±4) d vs.(27 ±5) d],there was significant difference (P < 0.05 or < 0.01).Conclusion In the elderly patients with coronary heart disease undergoing gastrointestinal surgery,individualized fluid therapy can effectively decrease adverse cardiac events,improve postoperative gastrointestinal function,and reduce length of hospital stay.
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ObjectlveTo evaluate the role of cannabinoid receptor 2 (CB2 receptor) in microglial injury induced by glutamate.MethodsMicroglia cells were randomly divided into 4 grups:control group (group C),microglial injury group ( group Ⅰ),specific CB2 receptor agonist AM 1241 group ( group AM1241 ) and specific CB2 receptor antagonist AM630 group (group AM630).In group C,the cells were cultured routinely for 26 h.In group Ⅰ,the cells were incubated in the culture medium containing glutamate 10 mmol/L for 24 h.In group AM1241,the cells were incubated in the culture medium containing AM1241 2 μmol/L for 2 h,and then in the culture medium containing glutamate 10 mmol/L for 24 h.In group AM630,the cells were incubated in the culture medium containing AM630 2 μmol/L for 2 h,and then in the culture medium containing glutamate 10 mmol/L for 24 h.The cell viability and release of LDH were measured.Microglial morphology was observed under microscope.Results Compared with group C,the cell viability was significantly decreased,and the release of LDH was significantly increased in groups Ⅰ,AM1241 and AM630 (P < 0.05).Compared with group Ⅰ,the cell viability was significantly increased,and the release of LDH was significantly decreased in group AM1241 ( P < 0.05).There was no significant difference in the cell viability and the release of LDH between groups 1 and AM630 ( P > 0.05).Conclusion Glutamate induces microglial injury through inhibiting the function of CB2 receptor.
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Objective To investigate the effect of methylene blue(MB)on liver ischemia-reperfusion (I/R) injury in rabbits.Methods Twenty-four healthy New Zealand white rabbits of both sexes welshins 2.0-2.3 kg were randomly divided into 3 groups (n=8 each):group Ⅰ sham operation(group s);group Ⅱ I/R and group Ⅲ methylene blue (group MB).The animals were anesthetized with intravenous 2% pentobarbital 30 mg/kg.Liver I/R was produced by occlusion of hepatic blood flow for 40 min followed by 60 min repeffusion.In group MB methylene blue 5 mg/kg was injected iv at 20 min before liver ischemia.Femoral artery was carmulated for MAP monitoring and blood sampling.MAP and HR were recorded immediately before(T1,baseline)and at 20 and 40 min of ischemia (T2,3) and 1,5,30,60 min(T4-7)of repedusion.Blood samples were collected at T1,T5,T6 and T7 for measurement of seruln TNF-α and IL-6 concentrations.Plasma AST and ALT activities were measured at T1,T6 and T7.Liver specimens were obtained at the end of experiment for determination of SOD activity and MDA content.Results In group I/R MAP was significantly decreased at T4-7 during reperfusion and HR at T7 as compared with the baseline at T1;while in group MB no significant change in MAP and HR Was observed during ischemia and reperfusion as compared with the baseline.The gerum TNF-α and IL-6 concentrations and the plasma ALT and AST activities were significantly increased during reperfusion as compared with the baseline immediately before ischemia in group I/R and MB and were significantly lower in group MB than in group. I/R. The SOD activity was significantly higher while MDA content was significantly lower in group MB than in group I/R. Microscopic examination showed that liver damage was less severe in group MB than in group I/R. Conclusion The administration of MB can maintain hemodynamic stability and attenuate liver I/R injury in rabbits.
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Objective To investigate the role of Notch signaling pathway in cerebral ischemic tolenmce induced by clectroacupuncture (EA) preconditioning.Methods Fifty-two adult male SD rats weighing 280-320 g were randomly divided into 2 groups(n=each):control group(group C)and electroacupuncture preconditionig group(group EA).Group C received no treatment.Group EA received EA at the Baihui acupoint (GV20) for 30 min a day for 5 days.Twenty-four hours after the last preconditionig,focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 120 min,followed by 72 h of reperfusion.Notch intracellular domain(NICD)expression was determined by Western blot and expression of Notch1,Notch4,Jag1,and Hes1 mRNA by real-time PCR immediately before iachemia and 24 and 72 h of reperfusion.The neurological deficit was scored at 72 h of reperfusion.The infarct volumes were then determined after evaluation of the neurological deficit score .Results There was no significant difference in Hesl mRNA and NICD expression immediately before ischemia between group EA and C(P>O.05).NICD expression was up-regulated at 24 and 72 h of reperfusion in both groups, and Hesl mRNA expression at 72 h of reperfusion in group C and at 24 h of reperfusion in group EA was up-regulated compared with those immediately before ischemia (P < 0.05). Hes1 mRNA and NICD expression was up-regulated in group C, while down-regulated in group EA at 72 h of reperfusion compared with those at 24 h of reperfusion ( P < 0.05 ). Compared with group C, the expression of Notchl,Notch4 and Jag1 mRNA was up-regulated immediately before ischemia, and Hes1 mRNA and NICD expression was up-regulated at 24 h of reperfusion while down-regulated at 72 h of reperfusion in group EA( P < 0.05). EA preconditioning significantly reduced infarct volumes and increased neurological deficit scores at 72 h of reperfusion (P < 0.05 ). Conclusion Notch signaling pathway may participate in cerebral ischemic tolerance induced by EA preconditioning.
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Objective To investigate the effects of propofol on intracellular free Ca2+ concentration and nitric oxide synthase (NOS) activity in PC12 cells exposed to bupivacaine. Methods The PC12 cells were provided by Shanghai Cell Biology Research Institute, Chinese Academy of Sciences and cultured in DMEM liquid culture medium. The cultured PC12 cells were seeded in 36 well plates and randomly assigned to one of 4 groups (n=9 wells each): group Ⅰ control (C);group Ⅱ propofol (P);group Ⅲ bupivacaine (B) and group Ⅳ propofol + bupivacaine (PB). In control group D-Hank solution was added. The cells were exposed to propofol 2 mmol/L and bupivacaine 0.09 mmol/L in group P and B respectively. In group PB the cells were incubated with propofol 2 mmol/L and bupivacaine 0.09 mmol/L simultaneously. After being incubated for 6 and 24 h the apoptosis in BC12 cells was assessed by flow cytometry. Apoptotic rate was calculated. NOS activity and intracellular free Ca2+ coneentration in PC12 cells were determined. Results Bupivacaine significantly increased the apoptotic rate of PC12 cells, the intracellular free Ca2+ concentration and NOS activity in PC12 cells in group B as compared with control group. Propofol significantly decreased the toxic effects of bupivacaine on PC12 cells in group PB compared with group B. Conclusion Bupivacaine is toxic to PC12 cells by increasing apoptosis, intracellular Ca+ concentration and NOS activity in the cells. The toxic effects can be prevented to some extent by concomitant administration of propofol.
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The standardized training for residents is the key point of medical education and clinical level enhancement.The future clinicians not only must have the solid specialized knowledge,but also should have the rich humanities and the social sciences knowledge,good interpersonal communication ability,as well as lofty occupational ethics.Therefore,how to train residents to be rich in both the spirit of science and humanity and the knowledge of science and humanity,has become an important topic in the clinical medicine education.
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Objective To explore the anesthetic management during high intensity focused ultrasound(HIFU) for liver cancer. Methods A total of 21 patients underwent combined intravenous and inhaled general anesthesia.The anesthesia was induced by the administration of propofol,midazolam,fentanyl,and vecuronium.Endotracheal intubation was performed for mechanical ventilation.The anesthesia was maintained with inhalation of isoflurane or enflurane combined with additional administration of fentanyl,propofol,and vecuronium.Optimal analgesia and muscular relaxation condition were maintained and the hemodynamic parameters and body temperature were kept stable during the operation.Results All the 21 patients were successfully treated with HIFU under general anesthesia.A deepen anesthesia was required to depress stimulus responses while HIFU treatment in 2 patients.The duration of operation was 245~423 min(329?48 min),the time to recovery from the anesthesia was 7~18 min(11?3 min),and the time to extubation was 9~24 min(15?4 min).A small amount of pleural effusion was observed after operation in 1 patient and spontaneously disappeared after 2 weeks.Conclusions Combined intravenous and inhaled general anesthesia is a safe and effective anesthetic method for HIFU treatment for liver cancer.
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BACKGROUND:Repeated hyperbaric oxygenation (HBO) preconditioning induces tolerance against ischemia in spinal cord. The confirmation of this phenomenon paves a new way in clinic to prevent and treat complications of spinal ischemia following thoracoepigastric aorta surgery. Probing into its mechanism of is chemic tolerance has provided basis for clinical application.OBJECTIVE: To investigate the changes of mitochondrial structure and ATPase activity during spinal cord ischemic tolerance induced by repeated HBO preconditioning in rabbits.DESIGN: Randomized and controlled trial.SETTING: Department of Anesthesiology, Xijing Hospital of Fourth Military Medical University of Chinese PLA.MATERIALS: The experiment was conducted in the hyperbaric oxygen chamber of the Department of Air Medicine; Experimental Animal Center of the Department of Anesthesiology, Xijing Hospital of Fourth Military Medical University of Chinese PLA, between March and June 2004. Totally 50 male New Zealand rabbits were selected.METHODS: Fifty male New Zealand rabbits were randomly divided into two groups: simple ischemia control group and HBO group with 25 rabbits in each group: 0.25 Mpa, 100% O2, one hour per day for 5 days. Spinal ischemia model was prepared 24 hours after the last preconditioning.Spinal cord ischemia (20 minutes)/reperfusion model was prepared by blocking the infrsrenal aorta. The motor function of hind-limbs was scored at 6 hours, 24 hours and 48 hours after reperfusion. The mitochondrial ATPase activity was measured and compared between two groups before ischemia, at 6 hours, 24 hours and 48 hours after reperfusion.MAIN OUTCOME MEASURES: ① The score of motor nerve function of the hind-limbs; ② The activity of mitochondrial ATPase; ③ Pathological evaluation.RESULTS: Totally 50 rabbits were involved for result analysis, and finally 25 rats in each group entered statistical analysis with no loss in the midway. ① The score of motor nerve function of the hind-limbs in HBO group at 6 hours, 24 hours and 48 hours after HBO preconditioning was significantly higher than that in control group (P < 0.01). ② The activity of mitochondrial ATPase: the mitochondrial Na+, K+-ATPase activity and Ca2+, Mg2+-ATPase activity at each time point of the animals in the two groups after is chemia were significantly decreased; Na+, K+-ATPase activity in HBO preconditioning group at 6 hours, 24 hours and 48 hours of ischemia was significantly higher than that in control group (P < 0.01); Ca2+, Mg2+-ATPase activity at 6 hours and 24 hours after ischemia was significantly higher than that in control group (P < 0.01). ③ Pathological evaluation: The structure of mitochondria in control group was seriously damaged while it was found almost normal in spinal cord in HBO group animals at 48 hours after reperfusion.CONCLUSION: The mechanism of ischemic tolerance induced by hyperbaric oxygenation pretreatment may partly be related to the attenuation of mitochondria ATPase activity decrease after ischemia, thereby protecting the function of mitochondria.
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BACKGROUND: How to choose traditional Chinese remedy to treat cerebrovascular disease, not only improving the brain blood supply but also not affecting the blood pressure and heart rate, has been a promising research.OBJECTIVE: To study the role of yangyintongnao granule on average blood pressure and heart rate in anaesthetized dog.DESIGN: Complete grouping design and randomized controlled study based on hybrid dog.SETTING: Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University of Chinese PLA and Department of Pharmacology, Faculty of Preclinical Medicine, Fourth Military Medical University of Chinese PLA MATERIALS: The experiment had finished by Cardiovascular Laboratory of Physiology Department in the Fourth Military Medical University of Chinese PLA from March to June 2003. Totally 23 healthy hybrid dogs in either sex were selected. These dogs were divided randomly into 4 groups: high dose group of yangyintongnao( n = 8), moderate dose group of yangyintongnao ( n = 6), low dose group of yangyintongnao( n = 5) and saline group( n = 4).METHODS: Anesthetic dogs in high, moderate and low dose group of yangyintongnao granule were given different doses of yangyintongnao granule: 2 g/kg, 1 g/kg and 0. 5 g/kg respectively. All doses of drug were calculated based on the body mass of dogs, and the drugs were dissolved in 100 mL saline and given through the gastric canal. Dogs in saline group were perfused with equal saline. Aortic average blood pressure was measured by femoral artery intubation via a piezometric transducer. Heart rate was obtained from R-R intervals of a standard Ⅱ lead of electrocardiograph(ECG). The mean blood pressure and heart rate were recorded 0. 5, 1, 1.5, 2.0,3.0, 4. 0, 5.0, 6. 0 hours after treatment.MEAN OUTCOME MEASURES: The changes of blood pressure and heart rate at different time before and after medication.RESULTS: Totally 29 dogs were brought into the final analysis. Blood pressure: The mean blood pressure was reduced -5.4% to -6. 2% respectively in high dose group and moderate dose group after treatment. But in low dose group the average blood pressure sometimes increased sometimes decreased, mainly decreased. It increased by 6.6% ( P > 0. 05) and decreased by -4. 1% ( P > 0.05) at maximum. The average blood pressure in saline group changed by - 9.6% ( P > 0.05). Heart rate: The heart rate in high and medium dose group gradually reduced as time went on. It reduced by - 4. 4%, - 12.2% and - 9.5% respectively in high, moderate and low doses group. The change in each group was not statistically significant as the same in saline group( P > 0.05).CONCLUSION: Yangyintongnao granule has no significant influence on average blood pressure and heart rate.
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<p><b>OBJECTIVE</b>To investigate whether pretreatment with repeated electroacupuncture (EA) at the Baihui acupoint could induce ischemic tolerance against transient focal cerebral ischemic injury in rats.</p><p><b>METHODS</b>Thirty male Sprague-Dawley (SD) rats were randomly divided into 3 groups (n=10 for each): the control group consisted of animals receiving no treatment, the isoflurane (ISO) group had animals that inhaled 1.5% isoflurane for 30 min a day for 5 days, and animals in the EA group received electroacupuncture at the Baihui acupoint for 30 min a day for 5 days under 1.5% isoflurane anesthesia. Twenty-four hours after the last treatment, the middle cerebral artery was occluded with No. 3 nylon monofilament for 120 min. The neurological outcomes were evaluated 24 h after reperfusion. The infarct volumes were then assessed using 2% triphenyltetrazolium chloride staining after the neurological outcome evaluation.</p><p><b>RESULTS</b>The neurological deficit score (NDS) of the EA group [1 (0-2)] was lower than that of the ISO group [2 (1-3)] and the control group [2 (1-4)], P < 0.05. The infarct volume of the EA group (38.3 +/- 25.4 mm(3)) was significantly smaller than that of the control group (220.5 +/- 66.0 mm(3)) and the ISO group (168.6 +/- 57.6 mm(3)) 24 h after reperfusion.</p><p><b>CONCLUSION</b>Electroacupuncture at the Baihui acupoint 30 min a day for 5 days significantly reduces neurological injury induced by transient middle cerebral artery occlusion.</p>
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Animals , Male , Rats , Electroacupuncture , Ischemic Attack, Transient , Therapeutics , Rats, Sprague-DawleyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of injecting Shenfu, an extract of traditional Chinese herbal medicines, on the central nervous system (CNS) and the cardiac toxicity of bupivacaine in rats.</p><p><b>METHODS</b>Sixteen male Sprague-Dawley rats, weighing form 280 to 320 g, were randomly assigned to two groups (n = 8 in each group). Animals in the control group received a saline injection 10 ml/kg while animals in the Shenfu group received an injection of Shenfu 10 ml/kg intraperitoneally 30 minutes before intravenous infusion of bupivacaine. Lead II of an electrocardiogram (EEG) was continuously monitored after 3 needles were inserted into the skin of both forelimbs and the left hind-leg of each rat. The femoral artery was cannulated for measurement of arterial blood pressure and blood sampling. The femoral vein was cannulated for the infusion of bupivacaine. After baseline measurement (arterial blood pressure, heart rate and arterial blood gas), 0.5% bupivacaine was infused intravenously at a rate of 2 mg.kg(-1).min(-1) to all animals until asystole occurred. The time of bupivacaine-induced convulsions, arrhythmia and asystole were determined. The dose of bupivacaine was then calculated at the corresponding time point.</p><p><b>RESULTS</b>The doses of bupivacaine that induced convulsions, arrhythmia and cardiac arrest were remarkably larger in Shenfu injection-treated animals than in saline-treated rats [convulsions, (10.5 +/- 1.9) mg/kg vs (7.2 +/- 1.5) mg/kg; arrhythmia (10.5 +/- 2.0) mg/kg vs (7.2 +/- 1.9) mg/kg; asystole, (32.8 +/- 8.5) mg/kg vs (25.0 +/- 5.0) mg/kg; P = 0.006, 0.009 and 0.044, respectively].</p><p><b>CONCLUSION</b>The Shenfu injection is able to reduce the toxicity of bupivacaine to CNS and cardiac system in rats.</p>
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Animals , Male , Rats , Anesthetics, Local , Toxicity , Bupivacaine , Toxicity , Drugs, Chinese Herbal , Pharmacology , Electrocardiography , Heart Rate , Injections, Intraperitoneal , Random Allocation , Rats, Sprague-DawleyABSTRACT
Objective To evaluate the anesthesia and perioperative treatment for simultaneous liver-kidney-pancreas transplantation. Methods The preoperative preparation included improvement of the hepatic function, alleviation of uremia and control of blood glucose level. General anesthesia was employed and maintained with isoflurane combined with intermittent intravenous administration of midazolam, fentanyl and vecuronium. Dopamine and low dose of epinephrine were used to maintain arterial blood pressure if necessary. The parameters of blood coagulation, the indexes of hepatic and renal function, blood glucose and amylase levels in blood and urine were surveyed regularly. The treatment was adjusted according to the results of tests mentioned above. Results The circulation was stable and blood gas was normal in the course of surgery. The concentration of blood glucose was higher at the end of the operation than that of pre-operation. Normal hepatic and pancreatic function was achieved about a week after operation, while the renal function showed no satisfactory improvement. The patient was given hemodialysis until the second transplantation of kidney. Three days later, the renal function recovered to normal. Up to the present, all the grafted organs showed good function. Conclusion Appropriate preoperative preparation, optimal anesthetic procedure and management, perfect protection of function of multiple organs, and maintenance of stable hemodynamics and homeostasis were the key points of successful anesthesia and management for simultaneous liver-kidney-pancreas transplantation.
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Objective To observe the influences of different analgesic methods on blood sugar, stress hormones and cytokinesMethods According to different postoperative analgesic methods, 30 patients immediately after elective cholecystectomy, were randomly divided into four groups: normal saline was infused epidurally in group C as control; analgesia via epidural infusion of morphine 2 mg in group EM; patient-controlled analgesia via epidural or intravenous fentanyl in group PCEFA or group PCIFABlood sugar, cortisol, insulin, interlukin-2 (IL-2), interlukin-6 (IL-6) and tumor necrosis factor-?(TNF-?) levels were determined during the perioperative periodResults Postoperative visual analog score was higher in group C than that in group EM, group PCEFA or group PCIFA (P005)Conclusions Postoperative application of PCEFA can much more help to maintain the stability of neuroimmunoendocrinal system
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Objective To investigate the effect of tetramethylpyrazine(TMP), a traditional Chinese herbal medicine, on the neural injury caused by spinal cord ischemia and reperfusion in rabbit.Methods Twenty-two male New Zealand white rabbits were anesthetized with isoflurane. Spinal cord ischemia was induced by 20min by infra-renal aortic occlusion. Animals were randomly allocated to 3 groups. Group C received no pharmacologic intervention. Group P and T received 30 mg?kg -1 TMP infused iv at a constant rate over 30min before aortic crossclamping(prevention) and after reperfusion(therapeutic) respectively. Neurologic deficit was assessed at 4, 8, 12, 24 and 48h after reperfusion using neurologic dificit score(NDS 4 = normal, 0 = paraplegia) . The animals were sacrificed at 48h after reperfusion and spinal cords (L 5-7) were removed immediately for histopathologic study.Results All animals survived the experiment. The NDSs at each observation interval were significantly higher in group P and T than those in group C (P
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Objective To explore the neuroprotective effect of repeated preconditioning with cannabinoid receptor agonist WIN 55,212-2 on focal cerebral ischemia-reperfusion injury in rats.Methods Focal cerebral ischemia was induced by middle cerebral artery occlusion(MCAO)for 120min.Fifty male SD rats were randomly assigned to five groups(10 each):rats in control group and dimethyl sulphoxide group(DMSO group)were intraperitoneally administered 0.3ml normal saline and 0.3ml DMSO once a day for 5 days.Rats in WIN 55,212-2 preconditioning groups(including WIN1,WIN3 and WIN5 group)received intraperitoneal injection of 1mg/kg WIN 55,212-2(dissolved with 0.3ml DMSO)once a day for 1d,3d and 5d,respectively.All animals underwent MCAO operation 24h after last pretreatment to reproduce temporal(120min)focal cerebral ischemia model.The neurological function score(NFS)was evaluated at 24,48 and 72h after reperfusion.Brain infarct was identified with 2% 2,3,5-triphenyltetrazolium chloride(TTC)staining 72h after reperfusion,and the brain infarct volume was expressed as percentage of normal cerebral hemisphere volume.Results The NFSs of rats in WIN 55,212-2 preconditioning groups(WIN1,WIN3 and WIN5 group)were significantly higher,and the infarct volumes were significantly smaller than that in control group and DMSO group at 24h,48h and 72h after reperfusion(P0.05).Conclusion The neuroprotective effect of repeated preconditioning with cannabinoid receptor agonist WIN 55,212-2 may be enhanced by increased time of pretreatment.