动物造模,认知功能障碍,大麻素2型 z-bo 2022-03-17 410

　　Background: Postoperative cognitive dysfunction (POCD) is increasingly recognized as a common complication of major surgery. POCD refers to the relative decline of learning, memory, and executive function in short-term and long-term postoperative neuropsychological tests compared with preoperative. Mainly found in the peripheral immune system, cannabinoid receptor type 2 (regular) has anti-inflammatory and immunomodulatory effects. CB2R also appears in small amounts in the central nervous system. Significant up-regulation of CB2R expression under neuroinflammatory conditions has been observed in the brain, especially microglia. Neuroinflammation is generally considered to be involved in the pathogenesis and progression of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) ). In addition, the regular increase of CB2R expression in brain tissue has been reported in these neurodegenerative diseases. The neuroprotective effect of CB2R activation has been confirmed in animal models, especially in animal models of AD and stroke. In addition, CB2R plays an important role in controlling the chemotaxis of immune cells in the lesions induced by chemokines. The activation of CB2R is considered to inhibit the production of inflammatory mediators and promote the production of pro-survival factors by controlling the activity and toxicity of glial cells. CB2R is playing an increasing role in immune regulation and inflammation. How CB2R contributes to the inflammatory response and cognitive function of surgery is still lacking effective evidence. In this study, we evaluated CB2R expression changes and hippocampal and prefrontal cortex pro-inflammatory cytokines, IL-1β, TNF-α, and monocyte chemoattractant protein (MCP)-1 in orthopedic mouse models. We also investigated whether there are selective CB2R agonists and antagonists that can change the expression pattern of CB2R, neuroinflammation, learning and memory impairment. Our main hypothesis is that CB2R expression is related to neuroinflammatory response and cognitive impairment in surgical mice.

　　Method: Animals: Adult male C57BL/6 mice (4 months old, weighing 20-30 grams). Experimental design: The mice were randomly divided into control group, JWH133 group, AM630 group, anesthesia group (isoflurane anesthesia), surgery group (surgery under isoflurane anesthesia), surgery + JWH133 group (after surgery under isoflurane anesthesia) jwh133 treatment), surgery + AM630 group (AM630 treatment after surgery under isoflurane anesthesia). The mice received fear training 24 hours before training. The fear state test was performed 1, 3, and 7 days after the operation. Within each treatment group, group assessment was performed at each time point (n=6). The animals were sacrificed after 2 hours after the behavior assessment on 1, 3, and 7 days after operation. In addition, an open field test was conducted to evaluate the spontaneous activity of the mice.

　　Surgery model: In the previous study, mice underwent intramedullary fixation of tibial fractures under isoflurane anesthesia, and the anesthesia protocol was modified. Simply put, anesthesia includes 3% isoflurane induction, 100% oxygen, and 1.5% isoflurane to maintain anesthesia. Make a skin incision under the knee. The tibia is exposed, and a 0.3mm needle is inserted into its medullary cavity to achieve intramedullary fixation. The bone fractured in the middle. Finally, the wound is sutured after necessary debridement. The operation lasted about 10 minutes, and the total time of anesthesia was 20 minutes. The rectal temperature of the mice was kept at 37±0.5°C during the operation. After anesthesia, the mice were placed on a heated mat to recover. After recovery, he was sent back to his cage. In order to treat the pain caused by the skin incision, 2% lidocaine solution is used locally before the incision, and 1% tetracaine hydrochloride is applied to the wound, twice a day for 3 days.

　　Behavior test: Open field test: Perform an open field test for 15 minutes before each test phase of the training phase and fear training. Each mouse gently enters an open indoor center under dim light and is allowed to move freely for 5 minutes. Connect to any maze animal tracking system software through the camera, and calculate the motion parameters according to the software. The total distance was used to determine the spontaneous activity of the mice. The indoor floor was cleaned with 75% ethanol to avoid olfactory signals after each test.

　　Fear test: using previously published Fear Condition Test (FCT) parameters. This test includes the training period before the operation and the test period 1, 3, and 7 days after the operation. The day before the operation, the mice received fear training to build long-term memory. Each mouse was placed in an air-conditioned room for 120s, followed by six pairs of conditional-unconditional stimulation, and an additional 60s in the air-conditioned room. A pair of conditional-unconditional stimulation consists of 20 seconds, 70 dB sine wave tone (conditional stimulation), 2s interval, and receives a 2S, 0.7mA electric shock (unconditioned stimulation). Conditional unconditional stimuli are randomly spaced from 45-60S. FCT reflects the hippocampus dependent memory, and the pitch test evaluates hippocampal independent memory. In the environmental test, the mice were returned to the air-conditioned room for 5 minutes without a tone or electric shock. Perform a 2 hour tone test. The mouse was placed in a new room with a different shape for 5 minutes, during which the tone was transmitted for 3 minutes. The percentage of freezing time (defined as the time that the mouse has no movement except breathing) is recorded by the maze animal tracking system software.

　　Immunofluorescence detection of CD11b: from the control group, the operation group, the operation+JWH133 group, the operation+AM630 group, the mice were sacrificed 1, 3, and 7 days after anesthesia or operation, respectively. Immunofluorescence was used in the hippocampal CA1 area and the medial prefrontal cortex. Staining evaluates the expression of microglia marker CD11b. After the mice were anesthetized with isoflurane, they were first perfused with cold normal saline and then perfused with 4% formalin. The brain was quickly dissected and fixed at 4 degrees. The hippocampal CA1 area and the medial prefrontal cortex were continuously sectioned. After washing, the sections were incubated with FITC-labeled goat anti-mouse IgG secondary antibody for 2 hours at room temperature. After washing, observe the immunostained sections with a fluorescence microscope. .

　　Result: Spontaneous activity: The baseline spontaneous activity of mice was assessed for 15 minutes before FCT training. The effects of anesthesia, surgery, and postoperative drug treatment on voluntary activities were evaluated before the FCT test phase 1, 3, and 7 days after surgery. The effects of each drug were also evaluated in mice that did not undergo surgery. The total distance in the 5-minute open field experiment was used to assess spontaneous activity. The data shows that the baseline spontaneous activity of the mice in each group is equal, and the above treatment does not affect the spontaneous activity.

　　Fear experiment: There is no significant difference in the freezing time of each stage of fear training, indicating that the baseline learning and memory abilities of each group are the same. The FCT test assesses hippocampal-dependent memory. Compared with the control group, JWH133, AM630 or isoflurane anesthesia did not significantly change the freezing time of mice at any time point. Compared with the control group, surgery under isoflurane anesthesia reduced the freezing time 1, 3 and 7 days after the operation. The postoperative administration of JWH133 reduces the freezing time of 3 and 7 days after operation. The administration of AM630 only reduced the freezing time of 7 days after operation.

　　The expression of pro-inflammatory factors in the hippocampus: immunoblotting method determined the expression of IL-1β, TNF-α and MCP-1 in the hippocampus of mice. Compared with the control group, the expressions of IL-1β, TNF-α and MCP-1 in the hippocampus increased significantly at 1, 3, and 7 days after surgery. Compared with mice in the surgery group, postoperative JWH133 treatment reduced the expression of hippocampal IL-1β at 3 and 7 days after surgery. The expression of TNF-α and MCP-1 in hippocampus tissue decreased on the 1, 3 and 7 days after operation. Postoperative AM630 administration increased the expression of IL-1β and TNF-α in the hippocampus on the 3rd and 7th day after surgery.

　　The expression of pro-inflammatory factors in the prefrontal cortex: 1, 3, 7 days after surgery, the expression of IL-1β, TNF-α and MCP-1 in the prefrontal cortex was significantly higher than that of the control group. Compared with the level of the operation group, JWH133 treatment reduced the expression of IL-1β, TNF-α and MCP-1α after surgery. AM630 treatment increased the expression of IL-1β, TNF-α and MCP-1α after prefrontal cortex surgery.

　　Hippocampal CB2R expression: Compared with the control group, the hippocampal CB2R expression of mice in the operation group increased significantly. JWH133 treatment reduced CB2R expression at 3 and 7 days after surgery, and AM630 increased CB2R expression at 3 and 7 days after surgery.

　　The expression of CB2R in the prefrontal cortex: The expression of CB2R in the prefrontal cortex of mice in the operation group was increased. The expression of CD11b in the hippocampal CA1 area: 1, 3, and 7 days after the operation, the expression of the microglia marker CD11b in the hippocampus CA1 area of the mouse operation group was increased compared with the control group. JWH133 treatment reduced CD11b expression at 1, 3, and 7 days after surgery, and AM630 increased CD11b expression at 7 days after surgery.

　　The expression of CD11b in the medial prefrontal cortex: The expression of the microglia marker CD11b in the medial prefrontal cortex of the mice in the operation group was up-regulated at 1, 3 days after surgery. JWH133 treatment reduced the expression of CD11b at 3 and 7 days after surgery, and AM630 increased the operation CD11b expression after 7 days.

　　Conclusion: CB2R activator has a potential protective effect on early postoperative memory. With jwh133 CB2R agonist treatment inhibited postoperative neuroinflammation and enhanced memory, while CB2R antagonist AM630 aggravated postoperative neuroinflammation and worsened memory. These results emphasize that CB2R is a potential target for the treatment of postoperative cognitive dysfunction, which needs further investigation.