| Background and purposePostoperative cognitive dysfunction (POCD) defined as patients without mental disorders before operation suffered cognitive decline, impaired memory, declined language comprehension and social integration ability a few days or several weeks after anesthesia and operation. This phenomenon usually occurres in the old people.In recent years, the high incidence of POCD attracts widespread attention, but the exact factors and mechanisms are still unknown. Previous studies showed that aging was the exact risk factors of POCD, At the same time, increasing evidences indicated that anesthetics and operation were closely correlated with the occurrence of POCD. The previous study suggested that the damage of hippocampal cholinergic neurons was the morphological basis of the decline of cognitive function. Choline acetyltransferase (ChAT), a rate-limiting enzyme, is always regarded as the special symbol of the cholinergic neurons. Study on the central nervous system showed that the reduction in the number of neurons due to apoptosis might be the basis of cognitive decline in various neurodegenerative diseases including Alzheimer's disease (AD). With the increasing study of apoptosis, it has gradually been realized that mitochondria may be the central regulation of apoptosis and the release of mitochondrial Cytochrome C (Cyt.C) into the cytoplasm is the main feature of mitochondrial apoptosis pathway. Based on the theories mentioned above, we speculated that, on the basis of central cholinergic system degeneration, isoflurane or operation might induce neuronal apoptosis through the classical mitochondrial apoptosis pathway and caused the damage of cholinergic nervous system and then triggered POCD. this was the main content of this study. In the present study, we adopted Morris water maze test, immunohistochemistry and reverse transcription polymerase chain reaction for observation the changes of choline acetyltransferase of hippocampal neurons and Cyt.C of cytoplasm expression and cognitive function in elder rats to explore the exact factors and the possible mechanism of POCD. MethodsOne hundred and three aged (20 months old) male Sprague-Dawley(SD) rats weighing 500-600g were randomly divided into 4 groups: group C inhaled 30% O2 for 2 h(control,n=16); group I1 was anesthetized with 0.75% isoflurane in 30% O2 for 2 h(0.75% isoflurane,n=29); group I2 was anesthetized with 1.5% isoflurane in 30% O2 for 2 h(1.5% isoflurane,n=29); operation group O underwent abodominal operation under 1.5% isoflurane (in 30% O2) for 2 h(operation,n=29). In the course of anesthesia and surgery, arterial oxygen saturation was maintained between 95% and 100% and body temperature was kept 37.8℃～38.7℃.Five rats were randomly selected from each group and arterial blood samples (0.3ml) were obtained at 0.5,1 and 2 h for blood gas analysis by ABL700 blood gas analyzer.1. Effects of isoflurane and surgical operation on cognitive function in aged rats. Cognitive function was assessed by Morris water maze test at 24 h after anesthesia or operation.2. Effects of isoflurane and surgical operation on the expression of ChAT in hippocampus in aged rats24 h after anesthesia or operation, the animals were anesthetized with 10% chloral hydrate intraperitoneally. Then the animals were killed and their brains were immediately removed for the detection of the expression of ChAT in hippocampus by RT-PCR and immunohistochemistry.3. Effects of isoflurane and surgical operation on the expression of Cyt.C in hippocampus cytoplasm in aged rats24 h after anesthesia or operation, the animals were anesthetized with 10% chloral hydrate intraperitoneally. Then the animals were killed and their brains were immediately removed for the detection of the expression of Cyt.C in hippocampus cytoplasm by Western blot and immunohistochemistry.Result1. General state of experimental animalsDuring the period of anesthesia and operation, the vital signs of rats were stable; Lips were rosy, as well as the end part of the body; No limb movement, hypotension, respiratory depression and hypoxia signs were found. There weren't significant difference between the groups in age, body weight, swimming speed, blood oxygen saturation and blood gas analysis (P>0.05).2. Change of cognitive function in aged rat2.1 The results of place navigation test showed that the escape latency of rats became shorter with the increase of the training days, which showed that the learning and memory were gaining gradually. Learning and memory curve was shift right in group I1, group I2 and group O, and turned to be stable at the 4th day but at the 3rd day in group C. At day 2, 3, 4 and 5 after treatment, the escape latency in group I1, group I2 and group O were observed significantly longer compared with group C(P<0.05). The escape latency at day 4 and 5 was significantly longer in group O than that in group I2(P<0.05).No significant differences was found between group I1 and group I2(P>0.05).2.2 The results of spatial probe test showed that the frequency of crossing the original platform and the time consumption of staying at the original platform quadrant was lower in group I1, group I2 and group C compared with those in group C(P<0.05). The time consumption of staying at the original platform quadrant at day 4 and 5 was significantly lower in group O than that in group I2(P<0.05).There weren't significant differences between group I1 and group I2.3. Change of ChAT expressions in hippocampusCompared with group C, the mRNA and protein expression levels of ChAT in hippocampus in aged rats was significantly lower in group I1, group I2 and group O(P<0.05). No significant difference was found between group I1 and group I2(P>0.05). The mRNA and protein expressions of ChAT was significantly lower in group O than group I2(P<0.05).4. Change of Cyt.C expressions in hippocampal cytoplasmCompared with group C, the protein expression levels of Cyt.C were significantly increased in group I1, group I2 and group O(P<0.05). At the same time, The protein expression level of Cyt.C was lower in group I1 compared with group I2(P<0.05). The protein expression level of Cyt.C was higher in group O than that in group I2(P<0.05).Conclusion1. Isoflurane reduced cognitive function in aged rats in the early period after anesthesia and operation, and operation further increased this damage.2. Isoflurane or operation resulted in the reduced expression of ChAT and the increased expression of cytosolic Cyt.C in neurons in hippocampal. Operation aggravated these changes in the expressions of ChAT and Cyt.C. This abnormal change of the expression levels of ChAT and Cyt.C was consistent with the cognitive decline in rats. This suggests that anesthesia or operation based on degeneration of the central cholinergic system induced apoptosis in hippocampal neurons and damaged hippocampal cholinergic nervous system, and in turn leaded to cognitive dysfunction in aged rats. This was the possible mechanisms of POCD.
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