The Clinical Analysis And Experimental Study Of The Related Mechanisms Of Cerebral Lesions Induced By Extertional Heatstroke

Background and objective:The occurrence rate of heatstroke(HS) increased significantly with global warming and the increasing in the intensity and frequency of heat waves assault. Without appropriate treatment, HS could develop into severe multiple organ dysfunction, with mortality rate as high as10-50%. Cerebral lesions presents at early stage of HS, and coma, spasm and delirium are the most common manifestations.20～30%of survivors could have permanent central nervous system impairment.The exact pathophysiological mechanisms of cerebral lesions due to HS are still unclear. Rat and baboon models subjected to moderate whole body hyperthermia showed that plenty of apoptosis in the spleen, lymph tissue and gut. Studies in neurons also found that heat stress directly induced plenty of apoptosis. It is proved the reactive oxygen species (ROS) induced by heat stress increased explosively and induce apoptosis. However, the intermediate mechanism of heat stress-induced burst out of reactive oxygen species how to regulate neuronal apoptosis is not well understood. Besides, nuclear factor-κB (NF-κB) involve in the regulation of multiple apoptosis gene transciption. Kim and his coworkers found that the cerebral cortex and hippocampal neuron contained with high NF-κB activity. Whether NF-κB participates in the high lever oxidative stress reaction and resulted neuronal apoptosis need further exploration.Based on these studies, the clinical data of patients with exertional HS in recent10years in our hospital were retrospective analysized and summarized. By investigating the cerebral lesion features, imaging performances and other clinical manifestations, the law of cerebral lesions under exertional HS was analyzed and clinical strategies were provided. In addition, through the establishment of HS mice and heat stress neuron model, the correlation between heat and neuronal apoptosis and related signal pathways were discussed to reveal pathophysiology mechanisms of brain damage in HS and provide theoretical evidence for prevention and novel treatment.Part1The clinical analysis of the characteristics of cerebral lesions induced by extertional heatstrokeMethodsAll cases of EHS patients were retrospectively included in ICU department of Guangzhou General Hospital of Guangzhou Military Command from May2004to Sep2013, and were divided into survival group and death group. The survivors were further divided into curing subgroup and sequela subgroup based on the cerebral sequela. The demographic characteristics, highest body temperature, cooling time under38.5℃, cerebral lesion manifestations, cranial MRI or CT scan results, brain dysfunction when discharged, vital organ dysfunction at early stage and within72h at admission were recorded.SPSS17.0were used to process the clinical data. Kruskal-Wallis H analysis was used to analyze the count data, while Levene variance homogeneity test was applied to compare measuring data. The survival rate at30days were calculated by Kaplan-Meier analysis and survival curves was displayed by log-rank test. P<0.05was considered to have a statistical significance. Multi-factor COX regression analysis were adopted to screen the risk factors.Results 126cases of patients with exertional HS were included, with19cases of death and107cases of survivors, and15cases of cerebral sequela when discharged. At admission, the highest temperature were similar in3groups (P>0.05). The rate of cooling delaying (2-4hours) and severe cooling delaying (>4hours) were highest in the death group, followed by sequela subgroup. There was significant difference in the cooling rate in3groups (P<0.05), and different cooling rate correlated with survival rate in30days (P<0.05)There was no difference in the primary conscious disturbance after EHS occurred in3groups, as long as the survival rate in mild, moderate and severe conscious disturbance.95patients regained consciousness in24h, with18cases in2-5days, and13cases deteriorated. The duration of conscious disturbance was of statistical difference in curing subgroup, sequela subgroup and death group (P<0.05).The hospital30days survival rates were of statistical difference between the duration of conscious disturbance exceed24hours and the duration of conscious disturbance less than24hours(P<0.05).19cases were detected with cerebral lesions through cranial MRI/CT, with9cases of death group manifesting as diffused lesion, including7cases of diffused brain swelling,1cases of cerebral infarction and hernia,1cases of cerebellar and brain stem infarction. Limited intracranial bleeding were developed in6cases.4cases found punctate and platelike abnormal focal signal, with high signal in T2-weighted imaging, FLAIR sequence and DWI-weighted imaging. The abnormal punctate focal signal located in the white matter around the frontal horn of lateral ventricle and corona radiate. The abnormal platelike focal signal located in the hippocampus, parahippocampal gyrus and insular lobe.28cases of patients (22.2%) complicated with thrombopenia in24h after admission, with14cases of death group. The survival rate was much lower in the patients with thrombopenia at early stage than those without (P<0.05).26cases of patients complicated with disseminated intravascular coagulation (DIC) in72h after admission, with14cases of death group, the survival rate decreased signifcantly in the patients with DIC at early stage than those without (P<0.05).53cases of patients (42.1%) complicated with acute liver failure in72h after admission, with8cases of death group, the survival rate was similar in the patients with acute liver failure at early stage and those without (P>0.05).22cases of patients (17.5%) complicated with acute lung failure in72h after admission, with9cases of death group. The survival rate was lower in the patients with acute lung failure at early stage and those without (P<0.05).24cases of patients (19%) complicated with shock in72h after admission, with12cases of death group. The survival rate was much lower in the patients with shock at early stage and those without (P<0.05).108cases of patients (85.7%) complicated with rhabdomyolysis in72h after admission, with19cases of death group. The survival rate was similar in the patients with rhabdomyolysis and those without (P>0.05)Multi-factor COX analysis showed that shock(RR3.219;95%CI:1.097～9.444; P=0.033), primary conscious disturbance duration>24h(RR5.247;95%CI:1.419～18.473; P=0.027) and cooling time(RR3.241;95%CI:1.407～7.466; P=0.006)were risk factors of mortality in30days.ConclusionThere was no significant difference of the degree of primary conscious disturbance in patients with EHS, most patients recover within24hours after onset. The duration of primary consciousness exceed24hours was the risk factor in30days survival rates in patients of EHS.Small number of EHS patients could present cerebral lesions such as diffused lesions like diffused brain swelling or extensive cerebral infarction, intracranial bleeding and punctate or platelike focal degeneration. The abnormal punctate focal lesions located in the white matter around the frontal horn of lateral ventricle and corona radiate, while the platelike focal signal located in the hippocampus, parahippocampal gyrus and insular lobe. ALL of these parts were the sensitive damage parts of brain damage in heat stroke.Shock and the cooling time were also the risk factors in30days survival rate in EHS patients.Part2Experimental study of heat-induced apoptosis in cortical neuronsMethods1. Animal experiments906-8weeks male specific pathogen-free BALB/c mice, bodyweight18-22g, were randomly assigned to control group and heat stress group,30mice in the control group and60mice in the heat stress group. Control animals were always placed in the room temperature, then put the heat stress group mice into the biological oxygen tank which temperature of (35.5±0.5)℃,humidity(60±5)%, the rectal temperature measured once every30min, when the rectal temperature reached41.5±, the rectal temperature measured once every10min, when the rectal temperature reached42±stop heat stress and rewarming at room temperature. Heat stress group were randomly assigned to rewarming0h,3h,6h,12h, each group with12. In half of each group cerebral cortex were stained by HE, then observe the pathological changes under optical microscope and conduct the nerve pathological injury evaluation. The expression of caspase-3in the cerebral cortex were detected by immunohistochemical staining. The expression of p-NF-kB65and active caspase-3in the cerebral cortex were detected by Western blot The levels of ROS in the cerebral cortex were detected by fluorescence spectrophotometer.Ultrastructural changes in the cerebral cortex in the other half of each group were observed under transmission electron microscopy.2. Neuron experimentsThe day before experiment, the neurons in accordance with the density of5×104/hole in the cell culture dish, and continue to incubate for24h. Control group were incubated at37℃,5%CO2, While heat stress group of neurons were incubated at39℃、41℃、43℃for2h,then all the cells were further incubated at37℃for12h. Cell viability of neurons in each group were analyzed by CCK-8(heat stress group of neurons were incubated at39℃、41℃、43℃、45℃for2h).Neuronal apoptosis was analyzed by flow cy tometry using Annexin V-FITC/PI staining and Hoechst33258fluorescent nuclear staining.The expression of p-NF-kB65and active caspase-3in the neurons in each group were detected by Western blot. The amounts of ROS in the neurons in each group were assayed by DCFH staining. In order to observe the role of NF-KB in heat stress-induced neurons apoptosis, both control group and heat stress group were pretreatment the NF-KB’inhibitor PTDC (20μl,30min)before heat stress, to explore the effect of PTDC in heat stress-induced neurons apoptosis and analysis it in heat stress-induced the expression of active caspase-3. In order to proof the key role of ROS in heat stress-induced neurons apoptosis, both control group and heat stress group were pretreatment the ROS’inhibitor MnTMPyP (10μmol/Ld,1h) before heat stress, to explore the effect of MnTMPyP in heat stress-induced neurons apoptosis and the expression of p-NF-kB65and active caspase-3.3. Statistical analysisGraphpad Prism and SPSS17.0were used to analyze the data, measurement data were reported as the mean±standard deviation(x±5). Analysis of one-way ANOVA was used to evaluate the data among groups. P<0.05were accepted as significant.Result1. Animal experiments1.1Cerebral cortex in each group were observed pathology change under optical microscope:Both control group and heat stress re warming Oh group, The structure of cortical neurons was normal, the neurons were in good arrangement and the form was complete. Heat stress rewarming3h group:same neurons arrangement was scattered, cells were stained with shrinkage, cell nucleus contracted and was stained deeply. Heat stress rewarming6h and12h group:most neurons arrangement was scattered, cells nuclear chromatin condensation stained and cell nucleus contracted and was stained deeply. The nerve pathological injury evaluation was low both in control group and heat stress rewarming Oh group, the difference was no significant. With the extension of rewarming time, the nerve pathological injury evaluation was increased in heat stress rewarming6h and12h group,it was significant difference compared with the control group (P<0.05).1.2Ultrastructural changes in the cerebral cortex in each group observation under transmission electron microscopy:There was no apoptosis both in control group and heat stress rewarming Oh group, the structure of cortical neurons was normal and clear, the nuclear membrane is complete, the nuclear chromatin evenly. There was plenty of apoptosis both in heat stress rewarming6h group and heat stress rewarming12h group, the structure of cortical neurons was not normal and clear, the nuclear chromatin condensation stained and were gathered surrounding the nuclear membrane as the massive porphyritic, cytoplasm concentrated, nuclear membrane was shrinkage.1.3The expression of caspase-3in the cerebral cortex in each group were detected by immunohistochemical staining:it was no significant difference both in control group and heat stress rewarming Oh group. The expression of caspase-3began to appear in heat stress rewarming3h group, significantly increased in heat stress rewarming6h group and continues to express in heat stress rewarming12h group (P<0.05)1.4The expression of p-NF-KB65and active caspase-3in the cerebral cortex in each group were detected by Western blot:it was no expression of active caspase-3in control group, it began to appear in heat stress rewarming Oh group. With the extension of rewarming time, the expression of active caspase-3was significant increased in heat stress rewarming3h,6h,12h group (P<0.05)1.5The levels of ROS in the cerebral cortex of each group were detected by fluorescence spectrophotometer:Compare with the control group, the levels of ROS was significant increased in heat stress rewarming0h,3h,6h,12h group (P<0.05) The levels of ROS was peaked in heat stress rewarming3h group. With the extension of rewarming time, the levels of ROS was declined in heat stress rewarming6h,12h group.2. Neuron experiments2.1Cell viability of neurons in each group were analyzed by CCK-8:Compare with the control group, the cell viability of neurons was no significant difference in39℃. With increasing of heat stress temperature (41℃-45℃), the cell viability of neurons showed a progressive decline, it was significant difference compared with the control group (P<0.05).2.2Neuronal apoptosis was analyzed by Hoechst33258fluorescent nuclear staining:Compare with the control group, the neuronal apoptosis was no significant difference in39℃.With increasing of heat stress temperature (41℃-43℃),there were more positive nuclei stained. Neuronal apoptosis was analyzed by Annexin V-FITC/PI staining, also find that the neuronal apoptosis was no significant difference in39℃compare with the control group. With increasing of heat stress temperature (41℃-43℃), the neuronal apoptosis was significant increased, apoptosis rate was10.19%in41℃, apoptosis rate rose sharply to43.02%in43℃, it was significant difference compared with the control group (P<0.01).2.3The expression of p-NF-κB65and active caspase-3in the neurons in each group were detected by Western blot:Compare with the control group, the expression of p-NF-κB65and active caspase-3was no significant difference in39℃. With increasing of heat stress temperature (41℃-43℃), the expression of p-NF-kB65and active caspase-3was significant increased, the trend of its expression is consistent with the trend of neuronal apoptosis.2.4The amounts of ROS in the neurons in each group were assayed by DCFH staining:Compare with the control group, the amounts of ROS was no significant difference in39℃.With increasing of heat stress temperature (41℃-43℃), the amounts of ROS was significant increased. Compare with the control group, it was increased nearly7times and15times in41℃and43℃respectively.2.5The intervention of NF-κB’ inhibitor PDTC:Compare with the heat stress43℃group (41.67%), the neuronal apoptosis was significant decreased in the PDTC pretreatment+heat stress43℃group (1120%,P<0.01).Western blot analysis found that the PDTC pretreatment significantly inhibited the expression of active caspase-3(P<0.01)2.6The intervention of ROS’ inhibitor MnTMPyP:Compare with the heat stress43℃group (47.42%), the neuronal apoptosis was significant decreased in the MnTMPyP pretreatment+heat stress43℃group (18.45%,P<0.01).Western blot analysis found that the MnTMPyP pretreatment significantly inhibited the expression of p-NF-KB65and active caspase-3(P<0.01)Conclusion1. Heat stress can induce the neuronal apoptosis and the occurrence of neuronal apoptosis is related to the strength of heat stress of a certain range.2. Heat stress-induced burst out of reactive oxygen species as an upstream signal to mediate neuronal apoptosis through NF-κB pathway.3. Neuronal apoptosis may be regulate to the subsequent development of brain damage and may be one of the pathophysiology mechanisms of permanent decrements in neurologic function in heat stroke survivors.SummaryIt was found that the primary conscious disturbance presented similar in patients with EHS, and most patients could regain consciousness in24h. The primary conscious disturbance>24h,shock and cooling time were risk factors of mortality in30days. Part of EHS patients could present cerebral lesions such as diffused lesions like diffused brain swelling or widespread cerebral infarction, intracranial bleeding and punctate or platelike focal degeneration. The abnormal punctate focal lesions located in the white matter around the frontal horn of lateral ventricle and corona radiate, while the platelike focal signal located in the hippocampus, parahippocampal gyrus and insular lobe. ALL of these parts were the sensitive damage parts of brain damage in heat stroke.It is indicated that heat stress could induce explosive ROS increase in neurons as upstream signal to mediate neuronal apoptosis through NF-κB pathway in our establishment of heat-stress neuron and mouse model. The occurrence of neuronal apoptosis is related to the strength of heat stress of a certain range.