Study On Brain Injury Following Deep Hypothermic Circulatory Arrest In Animal Experiments

Part One Neuroprotective Effect of Deep Hypothermic Circulatory Arrest with Low Priming Volume:Study in a Rabbit ModelObjective:The aim of the study was to investigate the possible neuroprotective effects of a low priming volume following deep hypothermic circulatory arrest (DHCA) by setting up a model of cardiopulmonary bypass(CPB) and DHCA associated with cerebral microdialysis in rabbits.Method:Rabbits were randomized into three groups:DHCA with low priming volume(Group L,n=7),DHCA with high priming volume(Group H,n=7),and sham-operated goup(Group S,n=5).The priming volume of Groups L and H were 75mi and 210ml,respectively.The rabbits were simultaneously placed on CPB and brain microdialysis,cooled to 16 to 18℃with DHCA for 60 minutes.After weaning from CPB,the animals were monitored and observed up to 2 hours of recovery.The microdialysis was continuous from consciousness to recovery from CPB. Physiological parameters were regularly recorded.The extracellular levels of glutamate,glucose,lactate,and pyruvate in the hippocampus were collected by microdialysis and measured by HPLC and a microdialysis analyzer.The brain tissue sections,in parietal cortex and hippocampus(CA1),for morphological studies were stained with hematoxylin and eosin and TUNEL,simultaneously,examined by electron microscope.Brain damage was evaluated by these three means.Statistical analysis was performed with the SPSS 13.0.All data were expressed as mean+ standard deviation(SD).Different groups were compared by one-way analysis of variance(ANOVA).Repeated measures ANOVA or Kruskal-Willis analysis were used for comparisons between relevant time-points and the baseline in the same group. A p value of less than 0.05 was considered statistically significant.Results:In order to keep the mean arterial pressure and acid-base balance within defined physiological ranges,more doses of dopamine and sodium bicarbonate were administered in Group H than in Group L(P＜0.05).The ratios of lactate/glucose and lactate/pyruvate in Group H increased significantly compared with those in Group L from the beginning of weaning from CPB(P＜0.05).The levels of extracellular glutamate in the two DHCA groups increased significantly(p＜0.05).After weaning from CPB,the glutamate values in Group H remained at higher levels compared with those in Group L(P＜0.05).The percentage of injured neurons,TUNEL positive staining,and the mitochondria score of the hippocampus CA1 in Group H were significantly higher than in Group L(P＜0.05).Conclusions:A low priming volume during DHCA could have a neuroprotective effect compared with a high priming volume. Part Two Molecular Events in Neuronal Injury after Deep Hypothermic Circulatory Arrest:Study in a Rabbit ModelObjective:Although deep hypothermic circulatory arrest has been known to induce neuronal injury,the molecular mechanism of this damage has not been identified.We studied the key molecular mediators through cellular energy failure, excitotoxicity,and overactivation of poly(adenosine diphosphate-ribose) polymerase 1(PARP-1) in brain tissues of a rabbit model of deep hypothermic circulatory arrest similar to clinical settings.Method:We established 2 models of cardiopulmonary bypass(n=11) and deep hypothermic circulatory arrest(n=11) associated with cerebral microdialysis in rabbits.Deep hypothermic circulatory arrest lasted for 60 minutes.The measurements of glucose,lactate,pyruvate,and glutamate collected by means of microdialysis were quantified by using a microdialysis analyzer and high-performance liquid chromatography.The overactivation of PARP-1 was assessed by detecting immunostaining of poly(adenosine diphosphate-ribose)(PAR).Histologic studies were used to identify neuronal morphologic changes,and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining(TUNEL) and PARP-1 Western blotting were used to identify apoptotic cells and early apoptotic signals.Results:Deep hypothermic circulatory arrest significantly increased the lactate/pyruvate and lactate/glucose ratios and the glutamate value,whereas cardiopulmonary bypass did not(P＜0.05).Deep hypothermic circulatory arrest significantly increased the numbers of PAR-positive and apoptotic neurons compared with cardiopulmonary bypass(P＜0.05).The cleavage of PARP-1 was only found in the deep hypothermic circulatory arrest group.More injured neurons were found in the deep hypothermic circulatory arrest group(histologic scores,P＜0.05).Conclusions:This study demonstrated that deep hypothermic circulatory arrest results in a series of molecular events consisting of cellular energy failure, excitotoxicity,overactivation of PARP-1,and necrosis and/or apoptosis in neuronal injury.