| BackgroundEach year more than 2 billion people around the world need surgery treatment. Most of them use the general anesthesia of inhaled anesthesia. Inhaled anesthetics has been safe, effective and indispensable tool for anesthesia in clinical. Isoflurane, sevoflurane and desflurane are commonly used inhaled anesthetics.With many excellent features, isoflurane is widely used as mainstream in today's clinical anesthetics. But studies of recent years have shown that the postoperative cognitive dysfunction seems have relationship with isoflurane. Further experiments found that isoflurane has neurotoxicity and can lead to neuronal apoptosis. However, the mechanisms are unclear. To determine whether the increase of reactive oxygen species is one of mechanisms of isoflurane neurotoxicity, we conducted the first part of the experiment. In this part we use C57BL/6J mice to study whether the increased of reactive oxygen species is one of mechanisms of isoflurane neurotoxicity.Sevoflurane is a relatively new type of inhalation anesthetics. Compared with isoflurane, sevoflurane is much closer to the ideal inhaled anesthetics that people look forward to. And the significant difference with other fluorinated inhalation anesthetics is its lower blood /gas partition coefficient, which is lower than isoflurane. This will ensure the rapid induction of anesthesia and shortening recovery time. A large number of experiments show that inhaled anesthetics preconditioning have neuroprotective effect on cerebral ischemic injury in normal rats. Sevoflurane is the most widely used inhaled anesthetics for pretreatment.Now nearly 100 million diabetic patients in China, and the incidence of diabetes up to 9.7%. Concomitant cardiovascular and cerebrovascular disease is the main reason for disability or death in diabetic patients. Cerebral infarction in diabetic patients was 2-4 times of non-diabetic patients. Diabetes mellitus with cerebral infarction recurrence had a significantly higher morbidity and mortality than in normals. So the prevention work of diabetic cerebrovascular disease is a top priority to prolong life and improve quality of life undoubtedly. The second part of the experiment examined whether sevoflurane preconditioning had neuroprotective effects on focal cerebral ischemia-reperfusion injury in STZ (streptozocin)-induced T1DM (type 1 diabetes mellitus) rats. The results showed that sevoflurane pretreatment on T1DM rats did not have neuroprotective effects, which laid a foundation for looking for other control measures and mechanisms study of later. Objective To make sure if isoflurane can induce cellular apoptosis, that is to say to make sure if isoflurane can induced neurotoxic effect. Methods Sixteen male C57BL/6J mice were randomly divided into isoflurane treatment (Iso, n=8) and control (Con,n=8) groups. Mice in Iso group were exposed to three sessions of 1.4% isoflurane, 2h for each session. Mice in Con group were exposed 100% oxygen for 6h and 20min. Isoflurane was delivered by 100% oxygen. The changes of cell morphology in prefrontal cortex were observed by HE staining, and the expressions of caspase-3 were observed by immunocytochemical staining. Results 1. Blood gas analysis: The results suggest that isoflurane or pure oxygen explosure can induce metabolism or respiration in mice no significant changes. 2. HE staining: Compared with the Con group, cell morphology of the mice in the Iso group was changed: the cells gradually shrink, small nuclei, irregular cell morphology, different sizes, some crescent-shaped chromatin, margination at the nuclear envelope. 3. The expressions of caspase-3: the number of caspase-3 positive cells in Iso group was increased significantly than in the Con group (P<0.05). Conclusion The cellular apoptosis in the forebrain of mice existed when were exposed to three sessions of 1.4% isoflurane, 2h for each session, that is to say isoflurane can induce neurotoxic effect. Objective To investigate the changes of reactive oxygen species when isoflurane induced neurotoxicity and reactive oxygen species whether involved in isoflurane induced neurotoxic effect in the mice. Methods Forty male C57BL/6J mice were randomly divided into isoflurane treatment (Iso, n=12) group,control (Con, n=12) group,dimethylthiourea treatment (DMTU, n=8) group and imethylthiourea plus isoflurane treatment (DMTU+Iso, n=8) group. Mice in Iso group and DMTU+Iso group were exposed to three sessions of 1.4% isoflurane, 2h for each session. Mice in the Con group and DMTU group were exposed 100% oxygen for 6h and 20min. Thirty min before oxygen inhalation in DMTU group and first isoflurane inhalation in DMTU+Iso group, 50mg/kg of radical scavenger DMTU were intraperitoneally injected to the mice in the two groups. Isoflurane was delivered by 100% oxygen. The activities of SOD and content of MDA were measured. The changes of caspase-3 expression in the prefrontal cortex were observed by immunocytochemical staining. Results The measurement of the activities of SOD and content of MDA in brains: There was no significant difference in the activities of SOD between the Iso and Con groups (P>0.05), but MDA content in Iso group was significantly higher than that in Con group (P<0.05). Compared with the DMTU group, the number of caspase-3 positive cells in DMTU+Iso group was not obviously changed (P>0.05). Both were little. Conclusions The reactive oxygen species was changed when isoflurane induced neurotoxicity, which pointed out ROS may be involved in isoflurane induced neurotoxicity. The free radical scavenger-DMTU can inhibit isoflurane induced neurotoxicity in the forebrain of mice partly, which is to say the increase of ROS is one of mechanisms of isoflurane induced neurotoxicity. Objective To establish an appropriate animal model of type I diabetes, that is T1DM rat model, which is ready for the study follow-up. Methods 90 healthy male SD rats, the weight of which was 300-320g, were randomly divided into two parts: One part was used for inducing T1DM rats by STZ(n = 55); The other part was used as control group(n = 35). The mothod of making T1DM rats model was to inject STZ (65mg/kg) solution intraperitoneally 12h after fasting normal SD rats. Random blood glucose was measured at 3d, 1w, 2w, 4w, 8w after STZ injected. Random blood glucose greater than 16.7mM was the inclusion criteria[1]. Rats in control group were made by injecting the same concentration of citric acid solution, in the same location, at the same time and using the same batch of rats as the rats in T1DM gpoup for 8 weeks. Blood glucose was measured by the rat tail vein blood glucose meter. Result After testing we had got 48 T1DM rats and 35 control rats. Conclusion Obtained appropriate diabetic rats by this method and laid the foundation for follow-up.Experiment 2 The effect of sevoflurane pretreatment on T1DM rats with focal cerebral ischemia-reperfusion injury.Objective To study whether 2% sevoflurane pretreatment has neuroprotective effect on T1DM rats. Methods The rats in T1DM and control groups were randomly divided into 3 groups respectively, they were MCAO,Sevo+MCAO and Sevo+Sham groups, 11 rats in each group. DM (diabetes mellitus) was represented for the diabetic rats, control was represented for rats with normal blood, MCAO was represented for implementation of MCAO (middle cerebral artery occulision) operation, Sevo was represented for implementation of sevoflurane pretreatment 24h before MCAO or sham surgery, Sham was represented for sham surgery. For pretreatment, the concentration of sevoflurane should be 2%, delivered by 100% oxygen, inhalation time is 1h/d, continuous 5d, and each time interval between the 24h. Rats without pretreatment were exposed to the air directly. 24h after MCAO surgery, neurological score was measured by Garcia scoring, then infarct volume was measured by TTC ( 2,3,5-triphenyl tetrazolium chloride) staining and apoptotic cells was measured by TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay) staining. Results Neurological score of results: DM-MCAO group and DM-Sevo+MCAO group were statistically significant different (P>0.05), control-MCAO group and the control-Sevo+MCAO group were statistically significant different(P<0.05), DM-MCAO group and the control-MCAO group were statistically significant different(P<0.05); TTC staining and TUNEL staining results were consistent with the results of neurological score, slightly.Conclusion Sevoflurane preconditioning does not have neuroprotective effect on T1DM rats with focal cerebral ischemia-reperfusion injury. ConclusionsThe first part of this experiment confirmed that the neurotoxicity of isoflurane in C57BL/6J mice and on the basis of which revealed the role of ROS in isoflurane neurotoxicity; In the second part of the experiment, we made out the T1DM rats induced by STZ successfully firstly, and then observed the effect of sevoflurane pretreatment on ischemic injury of brain in T1DM rats.1.The mice were exposed to 1.4% isoflurane 2h, repeated three times, can cause the expression of caspase-3 was increased, suggesting that it can produce neurotoxicity.2. Isoflurane inhalation leaded to higher content of oxygen free radicals in forebrain cells. Oxygen free radical scavengers reverse isoflurane induced apoptosis in mouse brain cells partially, which suggested that the increase of oxygen free radical is one of mechanisms of isoflurane induced neurotoxicity in mice.3. Sevoflurane preconditioning does not have neuroprotective effect on T1DM rats with focal cerebral ischemia-reperfusion injury. |
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