| Objective:The high morality after cardiac arrest and cardiopulmonary resuscitation is mainly due to the global cerebral ischemia reperfusion injury.The blood brain barrier injury and brain edema are important pathological changes of cerebral ischemia reperfusion injury.The damage of blood-brain barrier can aggravate the secondary brain injury after ischemia.Therefore,reducing BBB damage and brain edema is an important part of reducing brain injury.The treatment of therapeutic hypothermia is a safe and effective treatment method after recovery,low temperature can play a role in brain protection through a variety of mechanisms.However,many experimental studies have confirmed that hypothermia alone is difficult to achieve the desired effect of cerebral resuscitation in clinical practice.Therefore,seeking a combined approach has become a research direction to improve the success rate of cerebral resuscitation.Hydrogen sulfide has been referred as a neuromodulator and neuroprotectant in the central nervous system.Therefore,this study will test the hypothesis whether the use of hydrogen sulfide in combination with therapeutic hypothermia would improve survival and neurological outcome after cardiac arrest and resuscitation in rats,furthermore,investigate whether hydrogen sulfide combined with hypothermia would protect BBB structure,decrease blood-brain barrier permeability and brain edema induced by cardiac arrest and resuscitation,and examine the possible mechanisms for the effects.Methods:The experiment will be divided into two parts.In the first part,we investigated the effects of hydrogen sulfide and(or)therapeutic hypothermia on the survival rate and neurological function of rats after cardiopulmonary resuscitation.Male Sprague Dawley(SD)rats were randomly divided into five groups:(1)Sham operation group(Sham group,n=5):rats received only anesthesia,oral tracheal intubation and femoral artery,vein catheterization and other operations;(2)The cardiac arrest model group(CA+Normothermia+Saline group,n=15):rats were subjected to 4 minutes cardiac arrest induced by transoesophageal cardiac pacing and then resuscitated successfully.(3)Hydrogen sulfide treatment group(CA+Normothermia+NaHS group,n=15):After cardiac arrest and resuscitation,rats were treated with hydrogen sulfide;(4)The therapeutic hypothermia treatment group(CA+Hypothermia+Saline group,n=15):After cardiac arrest and resuscitation,rats were treated with low temperature;(5)Hydrogen sulfide combined therapeutic hypothermia treatment group(CA+Hypothermia+NaHS group,n= 15):After cardiac arrest and resuscitation,rats were treated with hydrogen sulfide and low temperature.The dosage regimen of hydrogen sulfide:NaHS(Sigma-Aldrich,St.Louis,MO,USA)was freshly diluted in normal saline to the desired concentration(0.3 mg/ml)before administration.The NaHS was infused intravenously with an initial loading dose of 0.5 mg/kg at the start of CPR,followed by a maintenance infusion of NaHS(1.5 mg·kg-1·h-1)until 6 hours after ROSC.In hypothermic groups,we initiated cooling after ROSC by applying alcohol and ice bags to the body surface under anesthesia.Rectal temperature was reduced to 34℃ within 15 minutes of initiating reperfusion.Hypothermia was maintained for 6 hours by exposing the rat to ice bag or a heat lamp,and the distance between the rats and the lamp was adjusted to maintain the target rectal temperature.The 7-days survival rate after resuscitation was recorded,and the neurologic outcome was evaluated by tape removal test.The second part discusses the hydrogen sulfide and(or)therapeutic hypothermia on the permeability of blood brain barrier brain edema,blood brain barrier ultrastructure and the expression of tight junction protein occludin,matrix metalloproteinase-9(Matrix metalloproteinase-9 MMP-9)expression after resuscitation.The grouping and treatment were the same as experiment one,25 rats in each group.The brain water content and permeability of blood brain barrier were detected at 24 h after resuscitation;the expression of tight junction protein occludin in blood brain barrier was detected by western blotting;The ultrastructural changes of blood-brain barrier were detected by transmission electron microscopy(TEM),the change of MMP-9(matrix metalloproteinase-9)expression was detected by immunohistochemistry.Gelatin zymography was used to detect the changes of MMP-9 activity.Results:Compared with group Sham,the 7-days survival rate was significantly decreased,the time consumption for tape removal test was significantly increased,the cerebral content and the permeability of blood brain barrier were significantly increased,the number of MMP-9 positive cell was significantly increased,the ultrastructural damage of BBB was obvious and the expression of occludin was significantly decreased in the other four groups(P<0.05).Compared with group CA+Normothermia+Saline,the 7-days survival rate was significantly increased,the time consumption for tape removal test was significantly decreased,the cerebral content and the permeability of blood brain barrier were significantly decreased,the Ultrastructural damage of blood-brain barrier was reduced,the number of MMP-9 positive cell was significantly decreased,the activity of MMP-9 was significantly decreased,and the expression of occludin was significantly increased in the CA+Normothemia+NaHS group,CA+Hypothermia+Saline group and CA+Hypothermia+NaHS group(P<0.05).Among them,the changes in the CA+Hypothermia+NaHS group were the most obvious,and the differences were statistically significant(P<0.05).Conclusions:The effect of H2S combined with therapeutic hypothermia on brain injury in rats with cardiac arrest and resuscitation was better than that of alone.The mechanism is related to inhibiting the expression and activity of MMP-9,and reducing occludin degradation and decreasing the permeability of blood brain barrier. |
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