| Background and purposeRecent studies have indicated that anesthetic agents are toxic to developing brains and that this injury results in a long-term impairment of cognitive function. Pregnant women, newborns, and infants are often exposed to various anesthetic agents during childbirth or for surgical procedures. The administration of anesthetic agents usually occurs during an important period of brain growth, the brain growth spurt period, ranging from the last3months of pregnancy until approximately2yr after birth (in humans) or during the first2weeks after birth (in rodents) Therefore, it is important to study the mechanisms underlying these agents’deleterious effects on the developing nervous system during this critical period.Although the widely recognized neurotoxic effect of anesthesia is usually neonatal brain cell death, recent studies suggest that cell death after certain anesthetic agent, i.e. isoflurane, by itself is not sufficient to cause neurocognitive dysfunction. The developing brain has a high degree of plasticity. In postnatal day7(P7) rats, cell birth is a very common event. Neurogenesis occurs during development and persists in adulthood in the subventricular zone and the hippocampal dentate gyrus (DG). The peak of DG neuron expansion occurs around P7. Appropriate neurogenesis in DG is crucial in hippocampal-dependent episodic learning and memory. Any intervention that affects this process could result in impaired hippocampal learningSevoflurane (2,2,2-tri fl uoro-1-[tri fl uoromethyl]ethyl fl uoromethyl ether) is one of the most frequently used volatile anesthetics during surgery and cesarean delivery because of its low blood gas partition coefficient and low pungency. It is especially useful for infants and children because of its properties of rapid induction and recovery together with less irritation to the airway It has been recently shown that neonatal exposure to sevoflurane induces learning disabilities. In this study, we tested the hypothesis that sevoflurane induces acute brain cell death and decreases progenitor proliferation in P7rats, and that the impaired neurogenesis in DG causes delayed onset of neurocognitive deficit in these animals.MethodsPostnatal day7(P7) male Sprague Dawley rats were anesthetized in groups of6at1MAC as determined by tail clamping. In the pilot study, we established that sevoflurane concentrations between3%and5%maintained suffient depth of anaesthesia, as determined by50%mice lack of reaction to a painful stimulus, that is tail clamping Animals were closely monitored during the experiment and the concentration of sevoflurane was adjusted between3and5vol%to maintain normal skin color and adequate respiratory efforts. Control rats were placed in the anesthesia box for4h without exposure to anesthetic agent but otherwise identical conditions as animals in the sevoflurane group. The rats were divided into three groups according to the time of injection. Group1was used to see how sevoflurane affected the proliferation of progenitors labeled just before anesthesia. Rats in group1were given300mg/kg BrdU i. p.(intraperitoneally) four hours before anesthesia. Group2targeted the proliferation of progenitors that are in the S-phase of the cell cycle during the time of the anesthesia. In this group rats were injected300mg/kg BrdU i.p. just before anesthesia. Group3was used to see if it still had effect after4d,50mg/kg BrdU was injected i. p. to the rats twice with the interval of12hours. The brains were removed12hours after last injection. BrdU stain was done in all groups while Fluoro-Jade stain was done in group2and3. DG was the area that we were interested in. PT-PCR was used to detect the expression of Ki67. Changes of cleaved-caspase-3was detected by western blot. Morris water mazing was used to see if the neurocognitive function was impaired after2weeks of anesthesia and repeated6weeks after anesthesia.Result:Sevoflurane caused enhanced FJ staining upon anesthesia in P7rat pups right after anesthsia (FJ positive cells per50X50μm2:75.4±2.0[sevo] vs.67.3±1.8[control]; p<0.05, n=6), and this effect remained significant at4days after initial drug administration (FJ positive cells per50×50μm2:74.9±1.0[sevo] vs.61.6±1.5[control]; p<0.05, n=6). Caspase-3immunoblotting showed visible increases in the protein levels of cleaved caspase-3after sevoflurane anesthesia when compared with the control condition. DG Ki-67mRNA level was reduced by approximately70%in sevoflurane-treated rats as compared to control rats. The number of BrdU+cells per DG was significantly less in sevoflurane-treated group as compared to control (BrdU+cell/DG:control39430vs. sevoflurane22480; p<0.05, n=6) in group2.4days after initial sevoflurane administration, the proliferation was still found to be inhibited in the sevoflurane-treated group ((BrdU+cells/DG:control28480vs. sevoflurane14120; p<0.05, n=6)Both the sevoflurane group and the control group learned at a steady but slow rate when spatial reference memory was tested in the MWM on2weeks after4h of sevoflurane on P7. Rats that had received sevofl urane at P7spent significantly more time searching for the platform in the target quadrant during the cued trial. At the end of the cued trial the two groups performed the same. There was no difference for platform-site crossovers between the two groups on probe trial.Discussion and conclusion:In this study, we report for the first time that sevoflurane induced deficit in hippocampal-dependent learning and memory in neonatal rats. The impairment on the CNS by sevoflurane is twofold:acute neuronal damage on cellular level and a delayed-onset of neurocognitive malfunction. The spatial learning ability of the rats was enhanced according to the MWM6weeks after anesthesia while the long term memory was impaired significantly. The neurogenesis was inhibited by sevoflurane according to the decline of BrdU+cells and Ki67expression in DG. The FJb+cells were significantly increased and cleaved-caspase-3was high which suggested apoptotic neurodegeneration by sevoflurane. In conclusion we suggested that sevoflurane exposure in developing brain would cause a decline in neurogenesis, an increase in apoptotic neurodegeneration and these effects led to subtle, progressive deficit in hippocampal-dependent long term memory. |
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