Combination Ketamine With Midazolam To Effects Of NR2B Subunit Expression On The Hippocampus CA1, CA3 In The Fear Rats

ObjectiveEveryone expresses fear to the operation, regardless of the adults or the children. The present study observed the expression of NR2B subunit in the hippocampus CA1 ,CA3 of the fear rats, by setting up the classical Pavlov' s fear conditioning model. Then administered drugs and compared the expression of NR2B subunit in the hippocampus CA1, CA3 to the former to elucidate the mechanism of acquisition of conditioned fear and to provide evidences to explain how to antagonize fear.Experimental Material1. Experimental animals : 80 Male Wistar rats obtained from China Medical University Laboratory Animal Center, aging 5~6 weeks, weighing 150 ~ 200g.2. Main equipments: DSC - 60 Analyizer( Japan);High - performace Liquid Chromatography ( HPLC ) (Japan);pHS - 3C acidic agent (China);U -MCB Olympus Photographic system (Japan);BX60 Olympus microscopic camera ( Japan );METAMORPH/COOL SNAP + X/AX70 microscope ( fluorescence) photograph analyzing system (VIC/ROPER/OLYMPUS) US/JP ( Japan);CM1800 LEICA paraffin slicer (Germany);H - 600 Penetration electron microscope (Japan);YSD - 4G Pharmacological multipurpose stimulator (China);Low - frequency signal generator (China);Loudspeaker (China).3. Main reagent: Immunohistochemistry: first antibody: anti - NR2B, ( Wuhan Boster biological technique company );Immunohistochemistry dyeingkit (Wuhan Boster biological technique company) .4. Experimental medicine: CKQS ( Compound Ketamine Oral Solution) [Ketamine, KET 50mg/ml;Midazolam, MZ5mg/ml];KET 50mg/ml;MZ 5mg/ml.Experimental Method1. Copy fear conditioning model1. 1 Equipment: shock cage (40 X.30 x 30 cm) with organic glass and grid floor spacing 1. 2cm. Electric shock was provided by a YSD -4G Pharmacologic multipurpose stimulator. The Low — frequency signal generator is connected with a Loud - speaker. Repeating electric foot shock paired with tone shock to copy fear conditioned model.1. 2 Animal grouping:(T)Animal experiment: 80 rats were grouped to Train group (T group) and control group(C group) randomly ,T group has 60 rats, C group has 20rats. The rats were absolute diet 12h, but not refrained to water before experiments.(2)Experimental procedure: T group: put rats into shock cage, then the rats were presented with 20 tone - footshock pairings( tone, 1 kHz,75dB,5sec;foot-shock, 0. 5mA, 0. 5 sec) with 1 minute intertribal intervals averagely in 4 days. C group: only presented with tone shock but not electric foot shock.1. 3 Behavioural observationFreezing Behavior experiment: 48h after the last training, rats were placed to the shock case again and observed freezing behavior by only conditioned stimulus (tone shock) for 5 minutes.Monitoring targets:(1) Freezing Behavior: freezing is commonly identified as cessation of any movement except for respiratory movements.(2) Exploratory Behavior: The tendency to explore or investigate a novel environment.(3)activity: hind limb movement, grooming, standing.2. Combination Ketamine with Midazolam to effects of NR2B subunit ex-pression on the hippocampus CA1 ,CA3 in the fear rats2. 1 Grouping;80 male, healthy, clean Wistar rats were randomly divided into 4 groups, each group containing 20 rats. Then each group was divided into 5 time -points, each point containing 4 rats.A group: (control 1 group) only tone shock, Sodium Chloride 2u,l/g was administrated into stomach.F group: ( control 2 group) present with tone - footshock pairings, Sodium Chloride 2(xl/g was administrated into stomach.KET group: (experiment 1 group) present with paired shock, KET solution 2jxl/g was administrated into stomach.CKOS group: (experiment 2 group) present with paired shock, CKOS solution 2|xl/g was administrated into stomach.2. 2 Observation and examinationAt the time of 15 ,30,60,90,120min after drugs administration respectively, catheter was inserted to the aortic ventricle of heart. Then irrigate 0. 9% cold Sodium Chloride to general circulation. When color of liver gradually change from red to white ( 150ml) , fix all body organs with 4% paraformalde-hyde (120ml). Removing brain and abruptioing diencephalon and cerebellum after all body organs were fixed completely. The removal brain tissue were soaked in 4% paraformaldehyde for 72h 4t refrigerator, then placed in 4°C refrigerator.Observe the expression changes of NR2B in rats hippocampus CA1 NCA3 by immunehistochemistry staining after drugs administration. Microscope image analyzing system was used to analyze brain slices.Experimental results1. Behavioural changes of fear ratsContrast to control group, training group rats increased freezing response to 865% (P<0.001) , exploratory movement within the limited space decreased to 20% ( P <0.05) , and didnt performance any activity.2. Drugs interfere in expression of NR2B subunit in the hippocampusCAl,CA3 in the fear ratsContrast to control group, expression of NR2B subunit in the hippocampus CA1, CA3 of the fear rats increased without any drug administration before. NR2B expression began to decrease at 15min after administrating CKOS and decrease to the lowest at 60min point. Then, expression of NR2B gradually recovered at 120min, but was still higher than control group . Contrast to F group, NR2B expression decreased at 30min after administrating KET, and began to recover at 120min point. Expression of NR2B in hippocampus CA1 ,CA3 have decreased after administrating KET, but inhibitive level is lower than CKOS group.ConclusionContrast to control group, expression of NR2B subunit in the hippocampus CA1, CA3 of the fear rats which is not given pre - anesthetic drug increased. Ketamine, non - selected NMDA antagonis, Midazolam, GABAA excitomotory, eliminate fear and inhibit expression of NR2B subunit. The present experiments provide evidences to elucidate mechanism of fear.