Effect Of Isoflurane, Individually Or Combined With Midazolam, On The Expression Of Caspase-3of Neural Cells In The7-day-old Rats

ObjectiveTo establish a model of applying inhalation anesthesia for neonatal rats and evaluate thereliability of this model, and to investigate the effect of isoflurane, individully or combinedwith midazolam on the expression of Caspase-3of neural cells in the7-day-old rats.MethodsConnetcted the anaesthesia mechine equipped with a calibrated vaporizer of isoflurane,to the domestic air-tight chamber with an air-scoop and an air-out to form anesthesia pipeline.97seven-day-old male and female Sprague Dawley rats were used for the experiments.1. Modle and evaluate:35seven-day-old SD rats were divided into seven randomlyselected groups （n=5each）. Experiment groups: according to the differentconcentrations of the isoflurane in the air stream, the mixture gas were divided into sixgroups as follows:0.125%(groupⅠ)，0.25%(groupⅡ)，0.5%(group Ⅲ)，1%(groupⅣ)，1.5%(group Ⅴ),2%(groupⅥ)； control groups(group Ⅶ): air stream composedonly of70%nitrogen mixed30%oxygen. The experimental rats were exposed toisoflurane in the air-tight chamber, with the control rats who only supplied with themixed30%oxygen, together placed in a constant temperature incubator, setting up at 37℃. Isoflurane was continuously delivered into chamber through inlet with an airstream (70%nitrogen+30%oxygen) containing desired anesthetic concentration usinga calibrated vaporizer. The mixture gas samples in chamber of experiment groups weretaken at5min,10min,15min,30min,90min,180min and360min after isofluraneintervention. Isoflurane concentrations in the chamber were measure by gaschromatograph. The samples of both the experiment groups and the control group weretaken at the end of anesthesia for blood gas analysis. The other20seven-day-old SD ratswere divided into two randomly selected groups(n=10), control group and1.5%isoflurane group, to establish the isoflurane inhalation model according as mentionedabove, and using immunohistochemistry to detect the expression of Active Caspase-3on the area of hippcampal.2. The last42seven-day-old SD rats were randomly divided into three groups (n=14each)as follows: control group(C): receiving intraperitoneal(i.p.) injection of0.9%saline for6h of mock anesthesia; isoflurane group(I): receiving1.5%isoflurane exposure for6hanesthesia; midazolam combined isoflurane group(MI): receiving intraperitoneal(i.p)injection of9mg/kg midazolam, then immediately inspiring1.5%isoflurane for6hanesthesia. As above, at the end of anesthesia, four rats of each group were taken tocollect100μl arterial blood from the left cardiac ventricle to measure the values of pH,PaO2, PaCO2, BE, HCO3-and SaO2. After2h of the end of anesthesia, the expression ofCaspase-3mRNA of the cortex and hippocampus were detected by Real-time PCR; andthe expression of Active Caspase-3were detected by immunohistochemistry.Results1. Whereafter the isoflurane concentrations in these groups became linearly dependented,the linear dependence equation of them is Y=1.5472X－0.0575(r=0.9993).2. Compared with the group Ⅶ, the values of pH, PaO2, PaCO2, BE, HCO3-and SaO2ofthe groupⅠ-Ⅵ had no difference at the end of anesthesia time point (P＞0.05).Compared with the groups C, the values of pH, PaO2, PaCO2, BE, HCO3-and SaO2ofgroups I and MI had no significant difference at the end of anesthesia time point(P＞0.05). 3. Compared with group C, the expression of Caspase-3mRNA was up-regulated ingroups I and MI in cortical and hippocampal areas(P＜0.05).4. Compared with group C, the Active Caspase-3immono-stain positive cells weresignificantly increased in the group I and MI in the cortical, hippocampal and thalamicregions (P＜0.05); moreover, the number of Active Caspase-3postive cells wassignificantly increased in group MI (P＜0.001). Compared with group I, the number ofActive Caspase-3postive cells was in hippocampal and thalami regions(P＜0.05).ConclusionThe model for evaluating the effects of isoflurane on neonatal rats is successfullyestablished by an anaesthesia apparatus, a calibrated vaporizer of isoflurane, a domesticair-tight chamber with an air-scoop and an air-out. Exposured to isoflurane, the expression ofCaspase-3elevate in seven-day-old SD rat brain; combined with midazolam can trigger evenmore expression of Caspase-3, which was result to the activation of Caspase-3, but theconcrete mechanism need more researches to explore.