| Implications and objectiveStudies demonstrate lasting cognitive impairment in elderly persons after anesthesia such as isoflurane and surgery; it can be summarize into post-operative delirium (PD) and postoperative cognitive dysfunction (POCD). PD usually occurs 1～3 days after surgery and anesthesia ,and persist for several hours to several days or even weeks; while POCD generally happens one week after operation, some studies indicate that it is most serious in 2～5 days after surgery and anesthesia. Its symptoms include mental confusion, anxiety, personality change, memory impairment, etc, in severe situation, it may be deteriorated to the senile dementia, however, the mechanism thereof is not known, at present, aging is considered as one of the risk factors. The researches have indicated that the central cholinergic nervous system is closely related to study and memory. Studies showed that activity of ChAT,the marker enzyme of cholinergic system, is an important cause leading to dysfunction in study and memory, besides, numerous studies also have proved that the transmission system of acetylcholine may be the important target for many general anesthetics. It is therefore inferred that the mechanism for cognitive disfunction of aged people after surgery and anesthesia may be related to the anesthetic's inhibition of central cholinergic system and to the function of central cholinergic system declining along with the aging. In consideration of these, our study adopts Morris Water Maze to test the influence of isoflurane on spatial memory of aged and young rats in two and three days after anesthesia, and observe the changes of ChAT protein and its mRNA level in hippocampus, to illuminate the relationship of isoflurane and CHAT for mechanism of cognitive disfunction in aged people after isoflurane anesthesia and surgery.Materials and methods1. Test animal and main materials We select healthy female SD rats (provided by The Third Military Medical University) including the aged group: 14 months old with a weight of 400～420g; young group: 3 months with a weight of 190～210g.Adopt Morris Water Maze (Stoelting Company, USA), Dager Fabius anesthesia machine (Germany), HYUNDA anesthetic gas monitor, isoflurane (Shanghai Yapei Pharmaceutical Co, Ltd.), RNAsimple Total RNA Kit (Tian'gen Biotech Co., Ltd.), RT-PCR Kit (TOYOBO company), PCR amplification primer (Shanghai Sangon Biological Engineering Technology & Service Co., Ltd.), SYBR quantitative PCR kit (TOYOBO company), 2000bp DNA Ladder Marker (Tian'gen Biotech Co., Ltd.), DEPC (Sigma company, USA), Agarose (Spain Biowest company), the first antibody: anti-Rat -CHAT(SANTA CRUZ company), goat anti-GAPDH monoclonal antibody (SANTA CRUZ company); the second antibody: horseradish peroxidase (HRP) marked goat anti-rat IgG (H+L) (Beijing Zhong Shan Golden Bridge Biological Technology Co., Ltd.), horseradish peroxidase marked rabbit anti-goat IgG (H+L) (Beijing Zhong Shan Golden Bridge Biological Technology Co., Ltd.), ABI Prism7500 fluorescence quantitative PCR system (American ABI company), PCR system 9700 amplifier (American ABI company), nucleic acid protein analyzer (German Beckman Coupe company), Trans-blot (Beijing Liuyi Instrument Factory), American Bio-Rad enzyme-labeled instrument, American Bio-Rad electrophoresis tank (MINI PROTEAN IITM), American Bio-Rad electrophoresis apparatus (3000XI), American Bio-Rad gel image analysis system, etc.2. Methods2.1 Changes on spatial memory of aged and young rats in 2, 3 days after isoflurane anesthesia2.1.1 Test procedure of Morris Water Maze:①Place navigation test (PNT): put rats into the water maze to swim freely for 2 minutes to familiar with the environment in the afternoon before the day of test. The test is carried out in two phases: the first phase starts before isoflurane anesthesia of rats and lasts for 4 and a half days, it is done at a regular time of each day in the morning and afternoon, including 10 time intervals, 4 times of training are implemented in each of the interval. The platform is located in the second quadrant, on testing, lightly put rats into the water facing the wall from midpoint of pool wall of the four quadrants, and their entry into the water is randomly from different quadrants in four times of each time interval. Rats swim a while and climb up the platform and stay at the platform for 5 seconds, it is deemed that they find the platform; if they fail to find the platform in 120 seconds, the tester lead them to the platform and they stay 20 seconds, the escape latency is considered as 120s.The second phase is carried out in 2, 3 days after isoflurane anesthesia. Image collection system and analysis system automatically keep a record of and store the movement track of rats, the escape latency of finding the platform, swimming time, speed and distance, searching strategy for looking for the platform, etc.②Spatial probe test (SPT): after the place navigation test, make the rats leave the platform, choose the midpoint of the quadrant relative to the platform quadrant, record the strategy of their searching the platform and the ratio of swimming distance of platform quadrant to the total distance.2.1.2 Isoflurane anesthesia of aged and young ratsAged and young rats are divided into the isoflurane anesthesia group (n =24 aged and 24 young) and the control group (n =16 aged and 16 young).For the anesthesia group, take two hours of 1.2% isoflurane anesthesia and monitor the concentration of isoflurane gas, such vital signs as average arterial pressure, heart rate, pulse oxygen saturation, blood gas, etc of the rats, and make them naturally regain consciousness after anesthesia. After completion of anesthesia, divide each group of animals into two parts, decapitate one part and take out hippocampus tissue after 2, 3 days of anesthesia, test the protein and transcription level expression of CHAT in hippocampus of aged and young rats by real-time PCR and Western method; and test the learning memory and special location ability of the other part after 2, 3 days of anesthesia using Morris Water Maze.2.2. Influence of isoflurane on CHAT in hippocampus of aged and young ratsIn 2, 3 days after isoflurane anesthesia, decapitate each group of rats and take out hippocampus tissue, divide them equally into two parts after PBS rinsing and store them at -80℃.Test the protein and transcription level expression of CHAT in hippocampus of aged and young rats by real-time PCR and Western blot method. 2.2.1 Test CHATmRNA relative expression level in hippocampus of aged and young rats by real-time PCRMethod: take hippocampus tissue to extract the total RNA as per steps in operating instructions of RNAsimple Total RNA Kit, calculate the obtained RNA content and purity at 260nm and 280nm absorbance, reversely transcribe cDNA the first strand synthesis, and store the end product at -20℃.Design of the primer: take a specificity analysis for gene order blast of GAPDH and CHAT, and design the primer with Primer5. CHAT upstream primer: 5'-GCCCGAGACCTGTGCAAAGA-3'; downstream primer: 5'-CCACAGCTT CCGCAAACTCC-3'; the product is 248bp long.GAPDH upstream primer: 5'-ACCCATCACCATCTTCCAGGAG-3'; downstream primer: 5'-GAAGGGGCGG AGATGATGAC -3'; the product is 159bp long. The two pairs of primers are synthesized by Shanghai Bioengineering Co., Ltd. Take GAPDH as the internal standard, measure the relative expression level of CHAT in hippocampus for the control group and the isoflurane anesthesia group with the real-time PCR amplifier and test the specificity of amplified product.2.2.2 Test the protein level expression of CHAT in hippocampus of aged and young rats by Western methodMethod: put the hippocampus tissue into liquid nitrogen to grind to tiny powder and have a mechanical homogenate in the cracking liquid, centrifugate 15 minutes at 4℃, 12000 r/min. Measure the protein concentration by BCA method and store it at -70℃in frozen condition. Prepare 10%SDS polyacrylamide gel, denature the sample at 100℃, then add equivalent 50ug protein sample for each aperture, transfer to cellulose nitrate membrane in wet state after electrophoresis. Oscillate and close the 5% skimmed milk power TBS buffer solution at room temperature for 2 hours, then wash the membrane, wash the TBST three times, 10min for each of time. Incubate the first antibody at 4℃; put it on the shaker for a night. Transfer it to room temperature shaking state for 30 minutes to restore the room temperature. Wash the membrane three times, 10 min for each of time, add the second antibody horseradish peroxidase, seal the bag, put it in shaking state for 2 hours, wash the membrane three times, 10 min for each of time. Prepare A, B luminous solution in 1:1 proportion, collect images with Bio-Rad gel imaging analysis system, analyze the optical density of strips, calculate the ratio of CHAT optical density to GAPDH optical density for the two groups, and judge the change of CHAT protein expression level from the ratio.Results1. The spatial memory of aged and young rats in 2, 3 days after isoflurane anesthesia Compared with the control group, in 2, 3 days after isoflurane anesthesia, the escape latency for anesthesia group of aged and young rats rises, the ratio of memory test platform quadrant path to the total path drops, it is more distinct for the anesthesia group of aged group (P < 0.05).The vital signs of isoflurane anesthesia group of both the aged and young rats keep stable at 1.2% isoflurane for 2 hours, no such symptom as low blood pressure, breath inhibition or hypoxia.2. Change of CHAT mRNA expression levelThe result of real-time PCR: dissociation curve of both GAPDH and CHAT presents a sharp single peak, indicating that the specificity is high for amplified product of CHAT and GAPDH genes.2 days after isoflurane anesthesia: the CHATmRNA relative expression level in the hippocampus for the aged isoflurane anesthesia group is 76.9% lower than that for the control group, the level for the young group is 46.6% lower than that for the control group. 3 days after isoflurane anesthesia: the CHATmRNA relative expression level in the hippocampus for the aged isoflurane anesthesia group is 57.7% lower than that for the control group; the level for the young group is 46.8% lower than that for the control group. Therefore, it is shown that 2, 3 days after isoflurane anesthesia, the CHATmRNA expression level in hippocampus is lower for the anesthesia group than for the control group; the CHATmRNA reduces more significantly in the aged group than the young group.3. Change of CHAT protein expression levelThe results of Western blot indicate that compared with the control group, the CHAT protein expression level reduces in hippocampus of both the aged and young rats after isoflurane anesthesia, and there is a more significant difference in the aged group (P < 0.05). ConclusionsFor the spatial memory ability of aged and young rats at early time of isoflurane anesthesia, the CHAT expression level in the hippocampus reduces compared with the control group, which is more distinct for the aged group than the young group.The results suggest that the isoflurane anesthesia may have an influence on the central cholinergic nervous system of both the aged and the young, and have a greater influence on the aged ones, thus, it is easy to change for cognitive function of the aged people after the isoflurane anesthesia. The reducing of CHAT expression level in the hippocampus may be one of the important reasons for cognitive disfunction of the aged at the early time of isoflurane anesthesia.
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