Mechanisms of selective head cooling for resuscitating damaged neurons during post-ischemic reperfusion

Objective To evaluate the efficacy and the mechanism of application of selective head cooling on neuronal morphological damage during postischemic reperfusion in a rabbit model.Methods 168 New Zealand rabbits were randomized into three groups. Group Ⅰ [n=24, (38±0.5)℃, non-ischemic control]; Group Ⅱ [n=72, (38±0.5)℃, normothermic reperfusion]; Group Ⅲ [n=72, (28±0.5)℃, selective head cooling, initiated at the beginning of reperfusion). Animals in three subgroups (n=24, each) of Group Ⅱ and Group Ⅲ had reperfused lasting for 30, 180 and 360 min respectively. Using computerized image analysis technique on morphological changes of nucleus, the degree of neuronal damage in 12 regions were differentiated into type A (normal), type B (mild damaged), type C (severely damaged) and type D (necrotic). Fourteen biochemical parameters in brain tissues were measured.[KH*2/5D]Results As compared with Group Ⅰ, the counts of type A neuron decreased progressively, and those of type B, C and D increased significantly in Group Ⅱ during reperfusion (P【0.01). In Group Ⅱ, vasoactive intestinal peptide, b-endorphine, prostacyclin, T 3 and Na +, K +-ATPase were correlated with the changes of type A; b-endorphine and thromboxane with type B; glucose and vasopressin with type C; Na +, K +-ATPase, glutamic acid, T 3 and vasoactive intestinal peptide with type D (P【0.05). As compared with Group Ⅱ, the counts of type A increased, and those of type C and D significantly decreased in Group Ⅲ (P【0.01). In Group Ⅲ, Ca 2+ , Mg 2+ -ATPase were correlated with the changes of type A, C and D (P【0.01). Conclusion Selective head cooling for sex hours during postischemic reperfusion does improve neuronal morphological outcomes in terms of morphological changes.

Inhibiting effect of moderate hypothermia on cell apoptosis after diffuse brain injury in rats

To explore the variant processes of cell apoptosis and the inhibiting effect of moderate hypothermia on cell apoptosis after diffuse brain injury. Methods: Models of diffuse brain injury were induced by the trauma device reported by Marmarou. 1 A total of 128 Wistar rats were divided into 4 groups: the uninjured group (Group A, n=8), the severely injured group (Group B, n=60), the mildly injured group (Group C, n=30) and the mild hypothermia group (Group D, n=30). In Group D, the severely injured rats were treated with moderate hypothermia to keep the rectal temperature at 32℃ (standard deviation for 0.1 ℃) for 6 hours. Then the morphosis, the characteristics and the quantity of apoptotic cells in the cerebral cortex and in the hippocampus regions after different severities of craniocerebral injuries were observed and compared under an electronic microscope, with terminal deoxynucleotidyl nick end labeling (TUNEL) in DNA fragmentation and with agarose gel electrophoresis. Results: TUNEL showed apoptotic cells increased according to the injury severity, and they peaked at 48 hours after injury and then declined. In Group C, apoptosis was located in the CA 2 and CA 3 areas of the hippocampus. And in Group B, apoptosis increased evidently, and located in the whole hippocampus and in the frontal and parietal cortex regions. The hypothermia treated rats had some apoptotic cells, too. However, even at 24, 48 and 72 hours after injury there were significantly fewer apoptotic cells in the cortex and in the hippocampus in Group D than that in the non treated groups. Electron microscopy showed that the apoptotic cells were round and shrunken in morphology and the nuclei were round and condensed at 24 and 48 hours after injury. And the apoptosis at 48 hours was more severe than that at 24 hours. The hypothermia treated rats had no apoptotic cells. Gel electrophoresis showed that characteristic DNA “ladders” were observed in the cortex and in the hippocampus at 48 hours after severe injury. But there was no DNA “ladder” at other time points in the severely injured group, in the mildly injured group and in the hypothermia treated group. Conclusions: It suggests that apoptosis occurs after diffuse brain injury and apoptotic cells increase with the injury severity. Moderate hypothermia has a specific inhibiting effect on cell apoptosis after diffuse brain injury in rats.

Thrombocytopenia after therapeutic hypothermia in severe traumatic brain injury

To investigate the clinical characteristics and significance of thrombocytopenia after therapeutic hypothermia in severe traumatic brain injury （TBI）. Methods： Ninety-six .inpatients with severe brain injury were randomized into three groups： SBC （ selective brain cooling ） group （ n =24）, MSH （ mild systemic hypothermia ） group （ n = 30）, and control （normothermia） group （n=42）. The platelet counts and prognosis were retrospectively analyzed. Results： Thrombocytopenia was present in 18 （75 % ）, 23 （ 77 % ） and 15 （36 % ） patients in SBC group, MSH group and control group, respectively （P 〈0.01 ）. Thrombocytopenia, in which the minimum platelet count was seen 3 days after hypothermia, showed no significant difference between SBC and MSH group （P 〉 0.05 ）. Most platelet counts （37 cases, 90% ） in hypothermia group were returned to normal level after 1 to 2 days of natural rewarming. The platelet count in SBC group reduced by 16%, 27% and 29% at day 1, 3 and 5 respectively compared with the baseline value. Good recovery （ GOS score 4-5 ） rate of thrombocytopenia 1 year after injury for hypothermia group （ 17 cases, 37 % ） was significantly lower than that of control group （ P 〈 0. 01 ）. Conclnsions： Therapeutic hypothermia increases the incidence of thrombocytopenia in severe TBI, and patients with thrombocytopenia after therapeutic hypothermia are associated with unfavorable neurological prognosis.

Effect of mild hypothermia on glucose metabolism and glycerol of brain tissue in patients with severe traumatic brain injury

Objective： To study the effect of mild hypothermia on glucose metabolism and glycerol of brain tissue in patients with severe traumatic brain injury （STBI） using clinical microdialysis. Methods：Thirty-one patients with STBI （ GCS ≤ 8 ） were randomly divided into hypothermic group （Group A ） and control group（ Group B）. Microdialysis catheters were inserted into the cerebral cortex of perilesional and normal brain tissue. All samples were analyzed using CMA mlcrodialysis analyzer. Results. In comparison with the control group, lactate/glucose ratio （ L/G ）, lactate/pyruvate ratio （ L/P ） and glycerol（Gly） in perilensional tissue were significantly decreased; L/P in normal brain tissue was significantly decreased. In control group, L/G, L/P and Gly in perilensional tissue were higher than that in normal brain tissue. In the hypothermic group, L/P in perileusional tissue was higher than that in relative normal brain. Conclusions： Mild hypothermia protects brain tissues by decreasing L/G, L/P and Gly in perilensional tissue and L/P in ＂ normal brain＂ tissues. The energy crisis and membrane phospholipid degradation in perilensional tissue are easier to happen after traumatic brain injury, and mild hypothermia protects brain better in perilensional tissue than in normal brain tissue.

Changes of evoked potentials and evaluation of mild hypothermia for treatment of severe brain injury

To observe the changes of evoked potentials after severe brain injury and the effect of mild hypothermia on acute severe brain injury. Methods: A total of 44 patients with severe closed head injury (GCS 3 8, admitted within 10 hours from injury) admitted from May 1998 to March 1999 were selected for this study. All patients were admitted into the intensive care unit and divided into 2 groups, Group A (GCS 3 5) and Group B (GCS 6 8). Patients were also randomly assigned to either normothermia or hypothermia subgroups. Patients in the hypothermia group were cooled to 32 34℃. Median nerve short latency somatosensory evoked potentials (SLSEP) and brain stem auditory evoked potentials (BAEP) were recorded before cooling and 4, 24, 48, 72, 96 and 120 hours, respectively after cooling and temperature resuming. SLSEP and BAEP were measured at the same time in the normothermia group (control group). The changes of evoked potentials (EP) were analyzed by statistical methods. Results: In the Group B, N 20 amplitudes in SLSEP and I/V amplitudes in BAEP after mild hypothermia treatment in the hypothermia group differed significantly from those in the control group (P< 0.05 ). However, in the Group A, no significant difference in all parameters was found. Conclusions: These results demonstrate that mild hypothermia treatment (32 34℃) in the Group B has a significant neuroelectrophysiological effect on severe brain injury. Nevertheless, the effect of mild hypothermia in the Group A is not apparent and needs further studying.

Effects of acupuncture plus mild hypothermia on apoptosis-related factors in rats with cerebral ischemia-reperfusion

Objective： To investigate the effect of acupuncture plus mild hypothermia on neurological function impairment score, cerebral infarct size and apoptosis-related factors in cerebral ischemia reperfusion injury（CIRI） rats. Methods： Sixty healthy male Sprague-Dawley（SD） rats were routinely reared for 1 week. Ten rats were randomly selected as the sham operation group and 10 rats as the blank control group, while the remaining 40 rats were subjected to preparing the middle cerebral artery occlusion（MCAO） model by modified filament occlusion method. The 40 MCAO rats were further randomly divided into a model group, an acupuncture group, a mild hypothermia group and an acupuncture plus mild hypothermia group, with 10 rats in each group. Rats in the sham operation group, the blank control group and the model group did not accept treatment except binding; rats in the acupuncture group received acupuncture treatment; rats in the mild hypothermia group received mild hypothermia treatment; rats in the acupuncture plus mild hypothermia group received acupuncture and mild hypothermia treatment. 72 h after the treatment, neurological function impairment score was performed; the infarct area ratio was determined by 2,3,5-tripheyl tetrazolium chloride（TTC） staining; apoptosis of brain cells was observed by TUNEL method; the expressions of Bcl-2, Bax and Caspase-3 were detected by immunohistochemistry.Results： Compared with the blank control group and the sham operation group, the neurological function impairment score, cerebral infarct area ratio, apoptosis, and the expressions of Bax and Caspase-3 in the model group were significantly increased, while the expression of Bcl-2 was significantly decreased, and there were significant between-group differences（all P〈0.05）. After the treatment, there were statistically significant differences among the treatment groups in the neurological function impairment score, cerebral infarct area ratio and apoptosis in the ischemic side of rats, as well as the expressions of Bcl-2, Bax and Caspase-3（all P〈0.05）, and from the figures, tables and statistical analysis, it was found that a better tendency in the acupuncture plus mild hypothermia group than the acupuncture group or mild hypothermia group. Conclusion： Acupuncture plus mild hypothermia can protect the brain cells by improving neurological function impairment, decreasing cerebral infarct area ratio, reducing the number of apoptotic cells in the ischemic area and regulating the expressions of apoptosis related proteins to inhibit apoptosis.

Exploring the effect of acupuncture plus mild hypothermia on miRNA-204 and its target gene expressions in CIRI rat brain tissues based on MAPK signal pathway

Objective:To explore the protective mechanism of acupuncture plus mild hypothermia for cerebral ischemia-reperfusion injury(CIRI)by observing the effects of acupuncture plus mild hypothermia on miRNA-204 and its target gene expressions in CIRI rat brain tissues.Methods:Sixty Sprague-Dawley rats were divided into a blank control group,a sham operation group,a model group,an acupuncture group,a mild hypothermia group and an acup un cture plus mild hypothermia group accordi ng to the random nu mbertable method(n=10).Except for the blank c on trol group an dthe sham operati on group,rats in the other 4 groups received CIRI modeling.After the model was successfully established,rats in the blank control group were bred routinely for 72 h without any interventions;rats in the sham operation group and the model group were bred routinely for 72 h,and only received bin ding without other interve ntions after surgery;rats in the acup un cture group were bred routinely for 72 h,and received acupuncture at Dazhui(GV 14),Baihui(GV 20)and Shuigou(GV 26)after binding;rats in the mild hypothermia group were bred routinely for 72 h,and received mild hypothermia intervention for 72 h after binding;rats in the acupuncture plus mild hypothermia group were bred routinely for 72 h,followed by receiving acupuncture as in the acup uncture group and mild hypothermia therapy as in the mild hypothermia group after bin ding.The n eurological impairme nt score,cerebral infarcti on area ratio,the expressions of miRNA-204 and its target genes in eluding Map3k8,Ntrk2 and Ppp3rl in the ischemic hippocampus of each group were observed after 72 h of intervention.Results:Before intervention,compared with the bgnk control group and the sham operation group,the neurological impairment scores and the infarction area ratios of the modelled rats were statistically significa ntly increased(all P<Q.Ql)f indicating that the model was successful.After intervention,compared with the model group,the neurological impairment scores of the three in tervention groups were sign ifica ntly reduced(all P<0.01);compared with the acupuncture group and the mild hypothermia group,the infarction area ratio in the acupuncture plus mild hypothermia group was signtiicantly reduced(both P<0.01);compared with the model group,the three intervention groups showed significant inhibition of miRNA-204 expression in brain tissues(all P<0.05),which was most significant in the acupuncture plus mild hypothermia group(P<0.01);compared with the acupuncture group and the mild hypothermia group,the Map3k8 expression in the acupuncture plus mild hypothermia group was significantly increased(both P<0.01),but there were no sign ificant d iff ere nces in Ntrk2 and Ppp3rl expressions between groups(all P>0.05).Conclusion:Acupuncture,mild hypothermia,and acupuncture plus mild hypothermia reduced the neurological impairment score and the cerebral infarction area in CIRI rats,while acupuncture plus mild hypothermia showed the most significant effect.In regulating miRNA-204 target gene expressions,acupuncture plus mild hypothermia showed the same effect on Ntrk2 and Ppp3rl expressions,while better effect on Map3k8 expression compared with either acupuncture or hypothermia.

Effect of acupuncture plus mild hypothermia on MAPK/ERK pathway of brain tissues in rats with cerebral ischemia-reperfusion injury

Objective： To observe the protective effect of acupuncture plus mild hypothermia on brain tissues in rats with cerebral ischemia-reperfusion injury （CIRI）, and the influence on protein expression levels of phosphorylated Raf-1, MEK-2 and ERK3/2 in the mitogen-activated protein kinase （MAPK）/extracellular regulated protein kinases （ERK） pathway, and to explore the mechanism of acupuncture plus mild hypothermia therapy for the ischemic stroke. Methods： Ninety Sprague-Dawley （SD） rats were randomly divided into a blank control group, a sham operation group, a model group, an acupuncture group, a mild hypothermia group and an acupuncture plus mild hypothermia group, 15 rats in each group. Except the rats in the blank control group, the remaining rats were used to prepare the middle cerebral artery occlusion （MCAO） models according to the modified occlusion method using lines, while only the occlusion lines were inserted without blocking the brain arteries of rats in the sham operation group. When the vital signs of rats were stable, rats in the blank control group did not receive any intervention; rats in the sham operation group and the model group received fastening without treatment; rats in the acupuncture group, the mild hypothermia group, and the acupuncture plus mild hypothermia group were treated with the corresponding therapeutic methods. 72 h later, observed neurologic injury score, evaluated infarction area ratio by 2,3,5-tripheyl tetrazolium chloride （TTC） staining, determined apoptosis by TUNEL assay, and measured the phosphorylated Raf-1, MEK-2 and ERK3/2 protein expression levels in rat ischemic hippocampal tissues by Western blot assay. Results： Compared with the blank control group and the sham operation group, after modeling, the neurologic injury score, infarction area ratio and apoptotic cells were increased, and phosphorylated Raf-1, MEK-2 and ERK：1/2 protein expression levels were significantly increased in the model group; the differences were statistically significant （P〈0.05 or P〈0.01）. Compared with the model group, after acupuncture or mild hypothermia therapy, neurologic injury score and infarction area ratio were decreased; apoptotic cells and phosphowlated Raf-1, MEK-2 and ERK1/2 protein expression levels were significantly decreased; the differences were statistically significant （P〈0.05 or P〈0.01）. Compared with the acupuncture group, neurologic injury score and phosphorylated Raf-1, MEK-2 and ERK3/2 protein expression levels were decreased in the acupuncture plus mild hypothermia group; differences between the groups were statistically significant （P〈0.05 or P〈0.01）. Compared with the mild hypothermia group, phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels decreased in the acupuncture plus mild hypothermia group, and differences were statistically significant （P〈0.01）. Conclusion： Acupuncture or mild hypothermia therapy can improve neurologic injury, reduce infarction area and apoptosis, which brought about protective effect on the brain tissues, in the MCAO model. The protective effect of acupuncture plus mild hypothermia group is the strongest. The mechanism may involve the MAPK/ERK pathway, by reducing the phosphorylated Raf-l, MEK-2 and ERK：1/2 protein expression levels.