| The central nervous system cerebrovascular disorder is a common serious disease.Thrombosis may lead to ischemia or rupture of blood vessels,often resulting in neurological deficits.It is the most common fatal neurological disease,it is also the leading cause of chronic disability,with many survivors leaving a lifelong irreversible neurological dysfuction,such as hemiplegia,aphasia,ataxia,etc..Cerebral thrombosis,cardiogenic embolism or atherosclerotic plaques in the cerebrovascular disease cause ischemic cerebral apoplexy to account for about 80% of cerebrovascular diseases.Currently,the only drug approved by the FDA for ischemic stroke is recombinaint tissue plasminogen activiator,which is used on patients presented with ischemic stroke within 3 hours of symptom onset to dissolve blood clots and restore blood supply.Thrombolytic therapy is time-limited,and achieving thrombolytic and blood circulation will increase the risk of intracerebral hemorrhage.It may initiate reperfusion injury when blood supply returns to ischemic tissue.There are no effective treatments for reducing the neurological deficits caused by reperfusion injury and ischemia.Therefore,it is promising to find drugs that can reduce ischemia reperfusion injury and promote the recovery of nerve function.12-O-tetradecanoylphorbol-13-acetate(TPA)is is a potent Protein Kinase C(PKC)activator.PKC is a family of protein kinase enzymes that consists of at least12 isoenzymes formed by serine or threonine amino acid.It involves in the necessary signal networks in the brain and cellular signal transduction pathways.It also plays an important role in the regulation of cell function and development of various diseases,including cell growth,differentiation,apoptosis,transformation,synaptic function,memory,behavior,cognitive and reperfusion injury.It was reported that TPA 200ug/kg at a single time was given at 60 min or 30 min later after the focal cerebral ischemia reperfusion in rats.After 24 hours,it could reduce the degree of brain edema and brain injury.However,the dose of TPA 200 ug/kg is far beyond the acceptable safe dose in the first phase.Therefore,this experiment was conducted to investigate the protective effects of TPA with the clinical safe dose on cerebral infarction,edema and the neurological function recovery after focal cerebral ischemia reperfusion in rats.Objective:To explore whether TPA can improve cerebral infarction and brain edema caused by focal cerebral ischemia reperfusion injury in rats and its protective mechanism.Methods:1 Protective effect of TPA after 72 h of focal cerebral ischemia reperfusion injury in rats.66 male SD rats with weight 250-280 g were randomly divided into 6 groups.There are sham group,model group and four TPA groups injected with differrent doses of TPA(12.5,25,50,100 ?g/kg).We established a middle cerebral artery occlusion model by suture method to keep focal cerebral tissue ischemic for 2 hours.And we gave a tail vein injection of TPA one hour later after ischemia,then we administered once per 24 hours for 3 days.The sham group and model group were injected with the same concentration of TPA as the control solvent.We performed a behavioral score evaluation at 24 hours after the surgery to exclude failure models,and we did it again 72 hour later after reperfusion.Then the rats were narcotized with inhaled sevoflurane,and the brians of the rats were taken out of their heads to get stained with TTC(chlorinated triphenyl four azole nitrogen).Subsequently we processed and calculated the volume ratio of cerebral infarction and cerebral edema with Photoshop.2 Effects of TPA on nerve recovery after 10 days of focal cerebral ischemia reperfusion injury in rats.20 Sprague-Dawley rats were randomly divided into model group and TPA group(25 ug/kg).We established a middle cerebral artery occlusion model by suture method to keep focal cerebral tissue ischemic for 2 hours.And we gave a tail vein injection of TPA one hour later after ischemia,.then we administered once per 24 hours for 3 days.Two days apart,the we gave the injection of TPA again for 5consecutive days.At the same tine,we gave the intraperitoneal injection of5-bromine-2–deoxyuridine at the dose of 50 ug/kg.The model group were injected with the same concentration of TPA as the control solvent.We performed the behavioral scores evaluation at 24 hours after the surgery to exclude failure models and recorded the weights every day.And We performed the comprehensive neurological function scores(balance beam walking and plane behavior)on 2nd,4th,6th,8th and 10 th days.On the 10 th day,the brains were collected.And the brain tissues were stained with HE to observe the pathological changes of the brains.The expression of Neu N was detected by immunohistochemistry and the expression of GFAP,BDNF and Brd U-DCX was detected by immunofluorescence.Results:1 The protective effect of TPA on focal cerebral ischemia-reperfusion injury in rats after 72 hours1.1.The results of behavioral scores : After 72 hours of cerebral ischemia-reperfusion in rats,the behavioral score on the plane of the model group was 3.44 ± 0.73,The behavioral scores of four TPA groups(12.5,25,50,100 ?g/kg) at 72 hours after cerebral ischemia-reperfusion were 3.50 ± 0.71,1.80 ± 1.00,2.33 ±1.11 and 2.10 ± 1.52.Compared with the model group,the behavioral scores of three TPA groups(25,50,100 ?g/kg)decreased significantly(P < 0.01,P < 0.01,P < 0.01),It indicates that TPA can improve the behavior disorder caused by injury.1.2.The results of TCC staining: After 72 hours of cerebral ischemia-reperfusion in rats,the brain infarct volume of model group was 41.95%,the brain infarct volume of TPA groups(12.5,25,50,100 ?g/kg)were 45.74%,22.68%,26.91% and 26.15%,.Compared with the model group,the percentage of brain infarct volume of three TPA groups(25,50,100 ?g/kg)decreased significantly(P < 0.01,P < 0.01,P < 0.01);The brain edema volume of model group was 15.82%,the brain edema volume of TPA groups(12.5,25,50,100 ?g/kg)were 6.35%,1.98%,7.58% and 8.41%.Compared with the model group,the percentage of brain edema volume of four TPA groups decreased significantly(P < 0.01,P < 0.01,P < 0.01,P < 0.01).2 Effects of TPA on neuroplasticity after focal cerebral ischemia-reperfusion injury in rats after 10 days2.1.General living conditions of SD rats:After the cerebral ischemia-reperfusion,the rats tended to eat less and lose weight,the initial weights of sham group were266.7 ± 4.85 g,then reduced to lowest level 242.6 ± 8.77 g three days later,and then gradually recover to the initial weights on the 10 th day.The initial weights of model group were 266.4 ± 3.14 g,then reduced to lowest level 212.1 ± 19.58 g 5 days later after reperfusion.The initial weights of TPA group were 266.8 ± 5.35 g,then reduced to 225.0 ± 21.56 g 5 days later after reperfusion.There was no significant difference in the weights of TPA group and model group,but both of them were less than the weights of sham group(P < 0.05).2.2.The results of comprehensive neurological function scores: On the 2nd,4th,6th,8th,and 10 th days,the scores of the model group were 6.83 ± 3.90,5.67 ± 3.76,2.17 ± 3.18 and 1.00 ± 1.67,the scores of TPA group were 6.29 ± 2.56,3.43 ± 2.13,1.71 ± 1.29,0.86 ± 1.32,and 0.57 ± 0.91.Compared with the model group,the comprehensive neurological function scores of the TPA groups significantly decreased on the forth day(P < 0.05).The results suggested that the neurological function could recover faster after treatment of TPA.2.3.The results of HE staining:The normal brain tissue of sham group had no injury area,in which the nerve fibers and the nucleus arrange evenly.The model group had larger cerebral infarction area,in which we could find the formation of vacuolar tissue,sparse necrotic brain tissue,pycnosis of nucleus,staining deepened,nucleus dissolved and disappeared in infarction cerebral infarction area of model group.The cerebral infarction area of TPA group is relatively smaller,part of which was full of of macrophages or was infiltrated by aggregated inflammatory cells.2.4.The results of Neu N immunohistochemistry :The number of positive neurons in normal brain tissue of sham group accounted for 67.08%.The number of positive neurons in infarct penumbra of model group and TPA group accounted for29.01% and 61.64%.Compared with the model group,there were more positive neurons in infarct penumbra of TPA group(P < 0.05).The result indicated that the treatment of TPA could significantly reduce the mortality rate of neurons.2.5.The results of GFAP immunofluorescent staining: The expression of GFAP in infarcted brain tissue of model group and TPA group were 32.65 ± 3.59 and 48.14± 4.30,both of which were higher than in contralateral hemisphere(20.66 ± 3.60,39.87 ± 1.24).And the expression of GFAP in the ipsilateral and contralateral infarcted brain tissue of TPA 25 ?g/kg group were higher than that of model group(P< 0.01,P < 0.01).This result suggested that the infarct development and TPA treatment could promote the activation of astrocytes.2.6.The results of BDNF immunofluorescent staining:The expression of BDNF in the ipsilateral and contralateral infarcted brain tissue of TPA group were 37.60 ±4.19 and 31.82 ± 2.49,both of which were significantly higher than that of the model group(23.06 ± 3.54,27.13 ± 1.08),(P < 0.01,P < 0.01).The result showed that TPA could increase the expression of brain BDNF and provide neurotrophy.2.7.The double staining results of Brd U-DCX immunofluorescence: In the penumbra of the side of cerebral infarction,the expression of Brd U-DCX in the penumbra of the side of cerebral infarction of model group and the TPA group were14.75 ± 3.43,and 27.88 ± 5.19.The expression of Brd U-DCX of TPA group was significantly higher than model group(P < 0.01);In TPA group,the expression of Brd U-DCX in the cerebral cortex,subventricular zone and striatum were 41.26 ±7.68?52.98 ± 18.75 and 20.20 ± 5.52.The expression of Brd U-DCX in the cerebral cortex and subventricular zone were significantly higher than that in striatum(P <0.05,P < 0.05).This result indicated that TPA could promote the differentiation of positive newborn neurons.Conclusion:Rats with focal cerebral ischemia-reperfusion injury in the process of TPA treatment could alleviate ischemia-reperfusion injury,may be related to reducing the death of neurons and the promoting the produce of brain derived neurotrophic factor. |
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