Application of GM1 in protection of developmental-period dog nerve injury caused by propofol anesthesia

Abstract

The invention discloses application of GM1 in protection of nervous injury of developmental dogs caused by propofol anesthesia, and relates to the technical field of neuroprotective effect of GM1. The invention finds that the GM1 can relieve the hippocampus injury of a dog in the development period caused by propofol anesthesia and improve the cognitive function of the dog through anti-inflammatory, anti-pyroptosis and anti-oxidation effects, so that the neuroprotective effect is exerted; the GM1 can alleviate neuroinflammation, pyroptosis, oxidative stress and cognitive dysfunction of developmental dogs caused by propofol anesthesia by regulating a PI3K / Akt / NF-kappa B pathway and the expression of BDNF; 10 mg / kg of GM1 has a protective effect on nervous injury, caused by propofol anesthesia, of a developing-period dog, and has potential veterinary clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of neuroprotective effect of GM1, and in particular to application of GM1 in protecting nerve damage of developing dogs caused by propofol anesthesia. Background Art

[0002] Propofol is an anesthetic drug of the succinic acid fatty acid ester class, which is widely used clinically for anesthesia and pain management in perioperative and developmental animals. Its mechanism of action is to enhance GABAA receptors, regulate NMDA receptors and the inhibitory effects of the central nervous system (CNS), thereby producing anesthetic and sedative effects. However, clinical studies have found that because the brain of developing animals is still at a critical stage of development, long-term or repeated exposure to propofol can easily cause nerve damage in the brain of developing animals, causing great harm. At present, the mechanism of action of propofol-induced neurotoxicity in developing animals is still unclear, and there is still a lack of effective intervention measures for the above problems. Therefore, it is particularly important to develop protective agents against propofol neurotoxicity to resist nerve damage caused by anesthesia in developing animals.

[0003] Monosialoganglioside 1 (GM1) is one of the most important gangliosides, existing in the cell membranes of mammals, especially in the CNS, and has neurotrophic and protective effects. As a neuroprotectant, GM1 has the effects of promoting neuronal differentiation, inducing synaptic growth, reducing neuronal cell damage and inhibiting neuronal cell apoptosis. It has been widely used in the treatment of various neurological diseases caused by abnormal neurodevelopment or neurological damage. Summary of the invention

[0004] The present invention provides the use of GM1 in protecting the nerve damage of developing dogs caused by propofol anesthesia, aiming to explore the protective effect of GM1 on the nerve damage of developing dogs caused by propofol anesthesia and the possible mechanism of action.

[0005] To achieve the above object, the present invention provides the use of GM1 in protecting developing dogs from nerve damage caused by propofol anesthesia.

[0006] Preferably, protecting developing dogs from propofol anesthesia-induced neurological damage comprises: Ⅰ. Alleviate propofol anesthesia-induced hippocampal damage in developing dogs and improve their cognitive function; II. Alleviate neuroinflammation, pyroptosis, oxidative stress, and cognitive dysfunction in developing dogs caused by propofol anesthesia.

[0007] Preferably, reducing propofol anesthesia-induced hippocampal damage in developing dogs and improving cognitive function in dogs include anti-inflammatory, anti-pyroptotic and antioxidant effects.

[0008] Preferably, alleviating neuroinflammation, pyroptosis, oxidative stress and cognitive dysfunction in developing dogs caused by propofol anesthesia comprises: regulating the expression of PI3K / Akt / NF-κB pathway and BDNF.

[0009] A drug for protecting developing dogs from propofol-induced neurological damage: including GM1.

[0010] Preferably, the GM1 content is 10 mg / kg.

[0011] The application of GM1 of the present invention in protecting the nerve damage of developing dogs caused by propofol anesthesia has the following beneficial effects: (1) GM1 can alleviate propofol anesthesia-induced hippocampal damage in developing dogs and improve cognitive function through anti-inflammatory, anti-pyroptotic and antioxidant effects, thereby exerting a neuroprotective effect; (2) GM1 can reduce neuroinflammation, pyroptosis, oxidative stress, and cognitive dysfunction in developing dogs induced by propofol anesthesia by regulating the PI3K / Akt / NF-κB pathway and the expression of BDNF; (3) 10 mg / kg of GM1 has a protective effect on propofol anesthesia-induced nerve damage in developing dogs and has potential veterinary clinical application value.

[0012] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In the figures, * indicates statistical difference compared with the control group, * indicates P<0.05, ** indicates P<0.01, # indicates statistical difference compared with the PPF group, # indicates P<0.05, ## indicates P<0.01.

[0014] Figure 1 This is the result of respiratory rate monitoring; Figure 2 The trend of respiratory rate change; Figure 3 The results of biochemical index tests for dogs; A is total bilirubin; B is aspartate transferase; C is alanine aminotransferase; D is alkaline phosphatase; E is urea nitrogen; F is creatinine; Figure 4 The results of the test of inflammatory factors in the canine hippocampus, where A is IL-1β, B is IL-6, C is IL-10, D is IL-18, and E is TNF-α; Figure 5 This is the result of the detection of HSP-70 level in canine hippocampus; Figure 6 HE staining results for canine hippocampal pathological analysis; Figure 7 The semi-quantitative analysis results of the number of cells in the CA1, CA3 and DG regions of the canine hippocampus stained with HE; Figure 8 Nissl staining results for canine hippocampal histopathological analysis; Fig. 9 The semi-quantitative analysis results of the number of cells in the CA1, CA3 and DG regions of the canine hippocampus stained with Nissl staining; Fig.10 This is the observation picture of BDNF-positive cells; Fig.11 is the number of BDNF-positive cells; Fig.12 Immunofluorescence results of IBA1 in microglia; Fig.13 is the number of IBA1-positive cells; Fig.14 The results of GFAP immunofluorescence in astrocytes; Fig.15 is the number of GFAP-positive cells; Fig.16 The results of ultrastructural observation of hippocampal tissue are shown in Figure 2. Fig.17 The results of the oxidative stress index test of the canine hippocampus, where A is malondialdehyde; B is superoxide dismutase; C is trace reduced glutathione; D is catalase; E is myeloperoxidase; Fig.18 The changes of pyroptosis-related proteins in canine hippocampus tissue; Fig.19 The changes of PI3K / Akt / NF-κB signaling pathway proteins in canine hippocampus tissue; Fig. 20 The changes of brain-derived neurotrophic factor protein in canine hippocampus; Fig.21 Scoring results for each domain of cognitive dysfunction for the experimental dogs; Fig. 22 This is the total score result of canine cognitive dysfunction; Fig.23 To score the results for each domain of cognitive dysfunction in dogs in the clinic; Fig.24 This is the total score of canine cognitive dysfunction in clinical practice. DETAILED DESCRIPTION

[0015] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0016] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0017] Experimental animals Thirty-two healthy beagles (weight 2±0.1kg, half male and half female) aged one month were selected. All beagles were purchased from the Experimental Animal Center of Harbin Medical University. The dogs were clinically examined for health before the experiment. All beagles were kept in single cages in an animal room with a good environment, quiet and comfortable (temperature 20-24℃, humidity 40%-55%). They were walked twice a day at a fixed time. All dogs were adaptively raised for 7 days before the start of the experiment.

[0018] Twelve canine surgical cases were selected from the Animal Hospital of Northeast Agricultural University. Before the clinical trial, the animal owners agreed and signed the "Informed Consent Form", issued questionnaires, and strictly followed animal welfare standards. All the above animal experiments were approved by the Animal Ethics Committee of Northeast Agricultural University (NEAUEC2023-03-145), and all operations in the experiment were strictly carried out in accordance with the "Guidelines for Animal Experiments of Northeast Agricultural University".

[0019] All test reagents and test equipment used are commercially available products.

[0020] All data in the experiment were statistically analyzed using SPSS27.0 software. The experimental results were expressed as mean ± standard deviation (mean ± SD). Normal distribution data were analyzed using one-way ANOVA and Tukey's post hoc test; non-normal distribution data were analyzed using Dunnettst test. GraphpadPrism10 software was used for drawing. P < 0.05 was considered statistically significant.

[0021] Embodiment 1 (1) Experimental animal grouping Beagle dogs were divided into four groups (n=8 in each group) according to the random number table method, namely control group (CON group), propofol group (PPF group), propofol+monosialotetrahexosylganglioside group (PPF+GM1 group), and monosialotetrahexosylganglioside group (GM1 group).

[0022] (2) Preparation before anesthesia for dogs Before the experiment, the animals were fasted for 12 hours and deprived of water for 6 hours. Before anesthesia, the weight of each group of dogs was recorded. In the small animal operating room, after the dogs were fully adapted to the environment and calmed down, the physiological indicators of the dogs were tested and recorded, and a catheter was implanted in the cephalic vein of the forelimb. This process was performed by the same experimenter to reduce stress. The endotracheal tube was connected to the ventilator to establish an anesthesia route. The heating pad was turned on for warmth to avoid a drop in body temperature caused by anesthesia.

[0023] (3) Anesthesia and drug intervention Slowly push propofol from the cephalic vein of the dog's forelimb to induce anesthesia. After confirming that the dog's upper and lower jaws are relaxed and tension-free, and its swallowing reflex is significantly weakened or completely disappeared, immediately perform endotracheal intubation and ensure that the catheter is fixed in place. Connect the respiratory anesthesia machine and monitor, place the dog in the left lateral position, gently pull out the tongue to expose it to the outside, and then clamp the blood oxygen probe on the monitor on the tongue, ensuring that the infrared light emitting surface faces under the tongue to accurately monitor the blood oxygen level. After disinfection and lubrication, the temperature monitoring probe is inserted into the dog's rectum 2-3cm, and the non-invasive blood pressure measurement cuff is fixed above the elbow joint of the dog's left forelimb, while ensuring that the position of the cuff is consistent with the height level of the heart to facilitate accurate blood pressure measurement.

[0024] After testing various physiological indicators, propofol was continuously infused at a constant rate using an injection pump. From the time of intubation, the anesthesia process was recorded in detail. The dog's basic physiological indicators and anesthesia effect score were recorded every 10 minutes. Any reactions of the dog during the maintenance of anesthesia were carefully observed and recorded.

[0025] After the anesthesia lasted for 180 minutes, the dog was allowed to wake up naturally, and the time required for awakening was recorded. At the end of anesthesia, oxygen was continued to be supplied to the dog until the animal was able to respond to external stimuli. Only then could the endotracheal tube be carefully removed, and the awakening quality of the experimental dog was subsequently evaluated and scored.

[0026] When inducing anesthesia in dogs, the dose of propofol is 5.5 mg / kg, and the infusion is completed in 40-60 seconds. When maintaining anesthesia, the dose of propofol is 100 mg / kg / h, and it can be increased or decreased as appropriate according to the actual situation to achieve the conditions for surgical operation.

[0027] The GM1 dosage for dogs was determined based on the conversion coefficient between different animals: CON group: intravenous infusion of appropriate amount of normal saline; PPF group: continuous intravenous infusion of propofol at a constant rate for 3 hours; PPF+GM1 group: intravenous injection of 10 mg / kg GM1 30 minutes before propofol infusion, and continuous intravenous infusion of propofol at a constant rate for 3 hours; GM1 group: intravenous injection of 10 mg / kg GM1.

[0028] The experimental dogs reached the surgical anesthesia period 20 minutes after the injection of propofol. During this period, the sedation, analgesia and muscle relaxation effects were good, and this good anesthesia effect could be maintained until the end of the operation.

[0029] During the anesthesia maintenance period, the body temperature showed a downward trend over time, but it was within the normal range. With the anesthesia lasting 180 minutes, there was no significant difference in the changes of body temperature between groups and at each time point (P>0.05). This shows that propofol total intravenous anesthesia has little effect on the body temperature of medium-sized young dogs.

[0030] SpO2 mainly reflects the ability of blood to transport oxygen. Its changes are related to many factors such as the body's respiratory function, circulatory function, nervous system, etc. It can comprehensively evaluate the impact of anesthesia on the body.

[0031] During the entire monitoring process, the SpO2 of the two groups of anesthetized dogs fluctuated within the normal range, with no significant difference (P>0.05).

[0032] During anesthesia maintenance, respiratory rate (RR) monitoring results are as follows Figure 1 , Figure 2 shown.

[0033] Figure 1 The results showed that compared with the CON group, there were significant differences in RP values ​​at 120min, 150min, 160min, 170min and 180min in the PPF group (P<0.01); there were significant differences in RR values ​​at 70min, 130min and 140min (P<0.05); at 160min, there was a significant difference between the PPF+GM1 group and the CON group (P<0.05); at 160min, there was a very significant difference between the GM1 group and the PPF group (P<0.01); at 120min, 140min, 150min, 170min and 180min, there were significant differences between the GM1 group and the PPF group (P<0.05).

[0034] Figure 2 The results showed that the RR values ​​of the PPF group and the PPF+GM1 group showed an overall downward trend, which indicated that both groups had entered a state of deep anesthesia.

[0035] The results of blood oxygen saturation (SpO2) and respiratory rate (RR) showed that long-term infusion of propofol would cause a certain degree of depression on the respiratory system.

[0036] During the maintenance of anesthesia, at 0 min, the heart rate of the PPF+GM1 group was significantly higher than that of the CON group (P<0.05), which may be due to the fact that the heart rate of the experimental dogs changed too much after the injection of the drug. At 40 min, the heart rate of all experimental dogs was around 140 beats / min and remained stable until 180 min.

[0037] From before anesthesia to 180 minutes, the diastolic blood pressure values ​​of the experimental dogs in each group fluctuated within the normal range. Except for the significant increase in SBP in the PPF group compared with the COM group at 100 minutes (P<0.05), the systolic blood pressure values ​​of the other groups fluctuated within a small range. From before anesthesia to 180 minutes, the mean arterial pressure (MBP) values ​​of the experimental dogs in each group fluctuated within the normal range. The blood pressure fluctuations were relatively stable, and no adverse reactions such as hypertension or hypotension occurred during the operation.

[0038] During the anesthesia maintenance period, the end-tidal carbon dioxide (P ET CO2) showed a trend of first increasing and then decreasing. At each time point, the P ET There was no significant difference in CO2 (P>0.05).

[0039] Awakening quality monitoring After the end of anesthesia, the recovery time and recovery quality during the recovery period were evaluated and recorded according to Table 1.

[0040] Table 1 Awakening quality score table ;

[0041] The awakening period and awakening quality (n=8) are shown in Table 2. It can be seen that there was no significant difference in the awakening period between the two groups of dogs (P>0.05), and both recovered in about 15 minutes. There was no significant difference in the awakening quality score (P>0.05). The awakening quality of the experimental dogs in both groups was good.

[0042] Table 2 Recovery period and recovery quality (n=8) ;

[0043] (4) Sample collection and preservation Before and after anesthesia, blood was collected from each group of dogs and placed in EDTA-K2 blood collection tubes and blood collection tubes without anticoagulants. After standing at room temperature for 60 minutes, part of the blood was used for routine blood tests; the other part was centrifuged at 3000r / min for 15 minutes, and the upper layer of serum was stored in a -80℃ ultra-low temperature refrigerator for biochemical index detection.

[0044] After anesthesia in all groups, some dogs (n=3) were kept until adulthood (12 months old) for behavioral testing; the other dogs (n=5) were euthanized and hippocampal samples were collected for pathological histological analysis, detection of related indicators such as inflammatory factors, protein expression, oxidative stress, and transmission electron microscopy observation.

[0045] The blood routine related indicators were detected using a fully automatic blood cell analyzer, and the blood routine results measured before anesthesia were used as the basic values.

[0046] ① The results of routine blood tests of dogs are shown in Table 3. Compared with the CON group, the white blood cell count of the PPF group and the PPF+GM1 group increased after anesthesia, but the PPF+GM1 group was lower than the PPF group, and the differences among the groups were not significant (P>0.05), which indicates that propofol anesthesia may cause a certain degree of acute inflammation in young animals, and GM1 can alleviate this symptom. After anesthesia, the hemoglobin of the PPF group and the PPF+GM1 group increased significantly, and there were significant differences compared with the CON group and the GM1 group (P<0.05), which may be due to the blood concentration or physiological increase caused by the reduction of body fluids due to water deprivation before anesthesia, but the blood routine values ​​of each group were within the normal range.

[0047] Table 3 Results of routine blood tests in dogs ;

[0048] Note: * indicates statistical difference compared with the control group, * means P<0.05; ** means P<0.01; # indicates statistical difference compared with the PPF group, # means P<0.05, ## means P<0.01.

[0049] ② The levels of serum aspartate transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TBTL), urea nitrogen (UREA), and creatinine (CREA) were measured using an automatic biochemical analyzer, and the biochemical indicators measured before anesthesia were used as the baseline values.

[0050] The results of this test are Figure 3 As shown in the figure, only the ALP of the PPF+GM1 group was significantly increased compared with the CON group (P<0.05), which may be due to physiological reasons. There were no significant differences in the other indicators between each group, indicating that propofol anesthesia and GM1 injection had no significant effect on the liver function of dogs.

[0051] (5) Detection of inflammatory factor levels in canine hippocampus ELISA kits were used to detect the levels of IL-1β, IL-6, IL-18, IL-10 and TNF-α in canine hippocampal tissue. The operation was strictly in accordance with the instructions. The OD value was detected at a wavelength of 450nm and the concentration was calculated. The results were as follows Figure 4 shown.

[0052] Compared with the CON group, the expression of IL-1β in the PPF group was significantly increased (P<0.01). Compared with the PPF group, the expression of IL-1β in the PPF+GM1 group and the GM1 group was significantly decreased (P<0.05).

[0053] Compared with the CON group, the expression level of IL-6 in the PPF group was significantly increased (P<0.01). Compared with the PPF group, the expression level of IL-6 in the PPF+GM1 group was significantly decreased (P<0.05), and the expression level of IL-6 in the GM1 group was significantly decreased (P<0.01).

[0054] Compared with the CON group, the expression level of IL-10 in the PPF group was significantly decreased (P<0.01). Compared with the PPF group, the expression level of IL-10 in the PPF+GM1 group was significantly increased (P<0.05), and the expression level of IL-10 in the GM1 group was significantly increased (P<0.01).

[0055] Compared with the CON group, the expression of TNF-α in the PPF group was significantly increased (P<0.05). Compared with the PPF group, the expression of TNF-α in the PPF+GM1 group was significantly decreased (P<0.05), and the expression of TNF-α in the GM1 group was significantly decreased (P<0.01).

[0056] Compared with the CON group, the expression level of IL-18 in the PPF group was significantly increased (P<0.05). Compared with the PPF group, the expression level of IL-18 in the PPF+GM1 group was significantly decreased (P<0.05), and the expression level of IL-18 in the GM1 group was significantly decreased (P<0.01).

[0057] (6) Detection of HSP-70 levels in canine hippocampus The ELISA kit was used to detect the level of HSP-70 in canine hippocampal tissue. The operation was strictly in accordance with the instructions. The OD value was detected at a wavelength of 450 nm and the concentration was calculated. Figure 5 As shown in the figure, compared with the CON group, the expression level of HSP70 in the PPF group was significantly decreased (P<0.01), and compared with the PPF group, the expression levels of HSP70 in the PPF+GM1 group and the GM1 group were significantly increased (P<0.01).

[0058] (7) HE staining and Nissl staining were used to analyze the pathological histology of the hippocampus of dogs in each group.

[0059] ①HE staining results are as follows Figure 6 As shown, in different divisions of the hippocampal tissues of the CON group, PPF+GM1 group, and GM1 group, the pyramidal cell structures were clearly visible, maintaining integrity and neat arrangement; in the CA1 and CA3 regions of the PPF group, a relative increase in the intercellular spaces was observed, accompanied by a disordered arrangement of cells. In addition, the cells swelled, the nuclei shrank, and even the nucleolus disappeared. Some cells dissolved, and the number of cells in the DG region decreased, with condensed or disappeared nucleoli.

[0060] For semiquantitative analysis of cell number, nine fields of view (each with an area of ​​104 μm) were randomly selected in the CA1, CA3, and DG regions of the hippocampus.2 ), the analysis results are as follows Figure 7 shown.

[0061] Compared with the CON group, the number of neurons in the CA1 region of the PPF group was significantly decreased (P<0.01), and compared with the PPF group, the number of neurons in the PPF+GM1 group and GM1 group was significantly increased (P<0.01); compared with the CON group, the number of neurons in the CA3 region of the PPF group was significantly decreased (P<0.01), and compared with the PPF group, the number of neurons in the GM1 group was significantly increased (P<0.05); compared with the CON group, the number of neurons in the DG region of the PPF group was significantly decreased (P<0.01), and compared with the PPF group, the number of neurons in the PPF+GM1 group was significantly increased (P<0.05); compared with the PPF group, the number of neurons in the GM1 group was significantly increased (P<0.01).

[0062] The above results show that GM1 significantly reversed the propofol-induced decrease in the number of neurons in the CA1, CA3 and DG regions of the hippocampus of developing dogs, and GM1 can alleviate the morphological damage of the hippocampus caused by propofol.

[0063] ② Nissl staining results are as follows Figure 8 As shown, the neuronal cell structures in the CA1, CA3, and DG regions of the hippocampus in the CON, PPF, PPF+GM1, and GM1 groups were similar to the results of HE staining.

[0064] For semiquantitative analysis of cell number, nine fields of view (each with an area of ​​104 μm) were randomly selected in the CA1, CA3, and DG regions of the hippocampus. 2 ), the analysis results are as follows Fig. 9 shown.

[0065] Compared with the CON group, the number of neurons in the CA1 region of the PPF group was significantly decreased (P<0.05), and compared with the PPF group, the number of neurons in the PPF+GM1 group and the GM1 group was significantly increased (P<0.05); compared with the CON group, the number of neurons in the CA3 region of the PPF group was significantly decreased (P<0.01), and compared with the PPF group, the number of neurons in the PPF+GM1 group and the GM1 group was significantly increased (P<0.05, P<0.01); compared with the CON group, the number of neurons in the DG region of the PPF group was significantly decreased (P<0.01), and compared with the PPF group, the number of neurons in the PPF+GM1 group was significantly increased (P<0.05), and compared with the PPF group, the number of neurons in the GM1 group was significantly increased (P<0.01).

[0066] ③ The expression level of BDNF positive cells was analyzed by immunohistochemistry. Fig.10 As shown: The BDNF-positive cells in the hippocampus of the CON group, PPF+GM1 group, and GM1 group were arranged neatly and orderly, with high and dense cell density. The cytoplasm was stained dark brown, showing strong immunoreactivity. In contrast, although the BDNF-positive cells in the PPF group maintained a certain degree of neatness in arrangement, the number of cells decreased significantly and showed a scattered distribution. In addition, atrophy of neuronal cell bodies was observed, and the intensity of immunostaining was weakened, showing a light brown color, indicating that the expression level of BDNF was reduced.

[0067] The number of BDNF-positive cells was Fig.11 As shown, compared with the CON group, the number of BDNF-positive cells in the CA1 region of the PPF group was significantly decreased (P<0.01), and compared with the PPF group, the number of positive cells in the PPF+GM1 group and the GM1 group was significantly increased (P<0.01); compared with the CON group, the number of BDNF-positive cells in the CA3 region of the PPF group was significantly decreased (P<0.01), and compared with the PPF group, the number of positive cells in the PPF+GM1 group was significantly increased (P<0.05), and compared with the PPF group, the number of positive cells in the GM1 group was significantly increased (P<0.01); compared with the CON group, the number of BDNF-positive cells in the DG region of the PPF group was significantly decreased (P<0.01), and compared with the CON group, the number of positive cells in the DG region of the PPF+GM1 group was significantly decreased (P<0.05), and compared with the PPF group, the number of positive cells in the PPF+GM1 group was significantly increased (P<0.05), and compared with the PPF group, the number of positive cells in the GM1 group was significantly increased (P<0.01).

[0068] The above results show that the PPF+GM1 group can effectively increase the number of BDNF-positive cells in various regions of the hippocampus.

[0069] (8) Immunohistochemistry test The wax blocks were dewaxed, rehydrated, and antigen repaired at 98°C for 10 min, cooled to room temperature, and washed 2 to 3 times with PBS (5 min each time), and then washed with 3% H2O2 （ The slides were soaked in methanol solution, left at room temperature for 20 minutes, washed with PBS 2-3 times (5 minutes each time), blocked with horse serum, left at 37℃ for 1 hour, and the excess liquid was shaken off. 100μL of I antibody was added and left at 4℃ overnight. After staying at 94℃ overnight, the slides needed to be rewarmed at room temperature for 45 minutes, washed with PBS 3 times (5 minutes each time), 50-100μL of II antibody (0.05% tween-20) was added, left at room temperature for 1 hour, washed with PBS 3 times (5 minutes each time), and developed with DAB for 5-10 minutes. The slides were immediately observed under a microscope to grasp the degree of staining, rinsed with distilled water, counterstained with hematoxylin for 2 minutes, differentiated with hydrochloric acid and alcohol, and returned to blue with blueing solution, examined under a microscope, rinsed with tap water, dehydrated, transparent, and sealed.

[0070] (9) Immunofluorescence test Place the slices at 37°C overnight, wash with PBS buffer, repeat 3 times (5 minutes each time), and place pH 6.0 sodium citrate buffer in a microwave oven and heat it on high for 10 minutes. After heating, place the slices in warm pH 6.0 sodium citrate buffer, repair them on low heat in the microwave for 15 minutes, and then let them cool naturally to room temperature. Wash with PBS again for 3 minutes. Soak in PBS buffer with 1% Triton-X100 and permeabilize for 20 minutes. Wash with PBS 2 to 3 times (5 minutes each time).

[0071] Use a special oil pen to completely circle the specimen to prevent the added liquid from flowing out. Add 5% BSA (diluted with PBS buffer) to the specimen and incubate at 37℃ for 1h. Remove BSA, dilute the primary antibody with 5% BSA, add it to the specimen, and incubate at 4℃ overnight. Wash 3 times with 0.5% TBST (5min each time). Add the diluted secondary antibody to the tissue specimen and incubate at 37℃ for 1h. Wash 3 times with 0.5% TBST (5min each time). Add DAPI to the specimen and incubate at room temperature for 5min. Wash 3 times with 0.5% TBST (5min each time), and seal the slide with antifade agent.

[0072] ①IBA1 immunofluorescence results of microglia Microglia are macrophage-like cells and important immune effector cells in the central nervous system. Excessive activation of microglia can lead to neuronal damage, and their typical activation marker is IBA1.

[0073] Immunofluorescence staining was used to observe the expression of IBA1 positive cells. Fig.12 As shown, the expression of IBA1-positive cells in the CA1, CA1, and DG regions in the PPF group was significantly higher than that in the other three groups.

[0074] The number of IBA1-positive cells was Fig.13As shown, compared with the CON group, the number of microglia (IBA1) positive cells in the CA1 region of the PPF group was significantly increased (P<0.01), compared with the PPF group, the number of positive cells in the PPF+GM1 group was significantly decreased (P<0.05), and compared with the PPF group, the number of positive cells in the GM1 group was significantly decreased (P<0.01); compared with the CON group, the number of microglia (IBA1) positive cells in the CA3 region of the PPF group was significantly increased (P<0.01), compared with the CON group, the number of positive cells in the PPF+GM1 group was significantly increased (P<0.05), and compared with the PPF group, the number of positive cells in the PPF+GM1 group and GM1 group was significantly decreased (P<0.01); compared with the CON group, the number of microglia (IBA1) positive cells in the DG region of the PPF group and PPF+GM1 group was significantly increased (P<0.01), and compared with the PPF group, the number of positive cells in the PPF+GM1 group and GM1 group was significantly decreased (P<0.01).

[0075] ② Astrocyte GFAP immunofluorescence results Astrocytes are a major type of cell in the central nervous system and are key response cells to central nervous system damage. Their most prominent feature is that they can be activated in injury, inflammation, and central nervous system degenerative diseases, and their typical activation marker is GFAP.

[0076] Immunofluorescence was used to observe the expression of GFAP positive cells. Fig.14 It can be seen that the expression of GFAP positive cells in CA1 and CA3 regions of the PPF group was significantly higher than that of the other three groups. Fig.15 As shown, compared with the CON group, the number of GFAP-positive cells in the CA1 region of the PPF group was significantly increased (P<0.01), and compared with the PPF group, the number of positive cells in the PPF+GM1 group and GM1 group was significantly decreased (P<0.01); compared with the CON group, the number of GFAP-positive cells in the CA3 region of the PPF group was significantly increased (P<0.01), and compared with the PPF group, the number of positive cells in the PPF+GM1 group and GM1 group was significantly decreased (P<0.01); compared with the CON group, the number of GFAP-positive cells in the DG region of the PPF group was significantly increased (P<0.01), and compared with the PPF group, the number of positive cells in the PPF+GM1 group and GM1 group was significantly decreased (P<0.01).

[0077] (10) Ultrastructural observation of canine hippocampal tissue Use a scalpel to trim the hippocampus to a size less than 1 mm. 3Tiny lumps. Soak the trimmed tissue blocks in 5% glutaraldehyde solution for 12 hours, then wash them three times with 0.1M phosphate buffer for 15 minutes each time, transfer the tissue to 1% osmium acid fixative and continue to fix for 2 hours, and then wash them three times with 0.1M phosphate buffer for 15 minutes each time. Place the tissue in increasing concentrations of alcohol solutions in sequence: 50%, 70%, and 90% alcohol solutions for 8 minutes each, then place them in anhydrous ethanol I and II for 10 minutes each, and then place them in a mixture of 100% ethanol + 100% acetone for 10 minutes. All of the above steps are carried out at 4°C.

[0078] The tissue was finally soaked in 100% acetone at room temperature for 5 minutes. Then soaked in 100% acetone and embedding solution (1:1) for 40 minutes, then soaked in 100% acetone and embedding solution (1:2) for 2 hours, and soaked in 100% acetone and embedding solution (1:3) overnight; fixed in embedding solution for 2 hours, then carefully placed in the embedding plate and treated in an oven at 45°C for 12 hours, then heated to 60°C for 24 hours. The embedded tissue was sliced ​​using an ultrathin slicer, and the thickness was controlled within the range of 50-60nm. Then it was stained with uranyl acetate-lead citrate double staining method. Finally, it was observed with a transmission electron microscope and the image was saved.

[0079] The ultrastructural observation results of hippocampus tissue are as follows Fig.16 As shown, arrows of different colors are marked to point out specific ultrastructural changes: the black arrow highlights the damaged nuclear membrane, whose integrity is destroyed; the red arrow points to the vacuolated mitochondria, indicating the abnormality of their internal structure; the blue arrow reveals the disappearance of mitochondrial cristae and the initial dissolution process; and the yellow arrow marks the swelling reaction of mitochondria, in which the original cristae structure no longer exists.

[0080] The hippocampal cells in the CON, PPF+GM1, and GM1 groups all showed plump cytoplasm, with clear mitochondrial and endoplasmic reticulum structures of internal organelles, and the double-layer integrity of the nuclear membrane was maintained, and the chromatin distribution also appeared uniform and orderly. The mitochondrial cristae structure was clear and intact, and the endoplasmic reticulum structure was intact; in contrast, the PPF-treated group showed more severe cell damage, including impaired integrity of the nuclear membrane, reduced nuclear volume, and particularly significant mitochondrial damage, manifested as widespread mitochondrial expansion, vacuolization, and rupture and disappearance of cristae, and some mitochondria even completely disintegrated or could not be observed. The nuclear membrane of the PPF+GM1 group was intact, and although a few mitochondria were observed to be slightly swollen, the nuclear membrane remained intact.

[0081] (11) Detection of oxidative stress indicators in canine hippocampus 0.1 g of canine hippocampal tissue was taken and added with physiological saline to prepare a 10% tissue homogenate. The mixture was centrifuged at 2500 r / min for 10 min. The supernatant was taken and one part was used for BCA protein concentration determination. The other part was used to determine the levels of malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), trace reduced glutathione (GSH), and catalase (CAT) in strict accordance with the instructions of the oxidative stress kit. The results were as follows: Fig.17 shown.

[0082] ① Results of malondialdehyde (MDA) content determination: compared with the CON group, the MDA content in the PPF group was significantly increased (P<0.01); compared with the PPF group, the MDA content in the PPF+GM1 group was significantly decreased (P<0.05); compared with the PPF group, the MDA content in the GM1 group was significantly decreased (P<0.01).

[0083] ② Results of superoxide dismutase (SOD) content determination: Compared with the CON group, the SOD content in the PPF group was significantly decreased (P<0.01); compared with the PPF group, the SOD content in the PPF+GM1 group was significantly increased (P<0.05); compared with the PPF group, the SOD content in the GM1 group was significantly increased (P<0.01).

[0084] ③ Results of determination of micro-reduced glutathione (GSH) content: Compared with the CON group, the GSH content in the PPF group was significantly lower (P<0.05).

[0085] ④ Results of catalase CAT content determination: Compared with the CON group, the CAT content in the PPF group was significantly decreased (P<0.01), and the CAT content in the PPF+GM1 group was significantly decreased (P<0.05); compared with the PPF group, the CAT content in the GM1 group was significantly increased (P<0.01).

[0086] ⑤ Results of myeloperoxidase (MPO) content determination: Compared with the CON group, the MPO content in the PPF group was significantly increased (P<0.01); compared with the PPF group, the MPO content in the GM1 group was significantly decreased (P<0.01).

[0087] (12) Detection of related protein levels in canine hippocampal tissue Western Blot was used to detect the expression of PI3K, P-PI3K, Akt, P-Akt, P65, P-P65 and BDNF proteins as well as pyroptosis-related proteins (NLRP3, Caspase-1 and GSDMD) in canine hippocampal tissue.

[0088] ① Changes of pyroptosis-related proteins in canine hippocampal tissue like Fig.18As shown, compared with the CON group, the expressions of NLRP3, GSDMD and caspase-1 proteins in the PPF group were significantly increased after administration (P<0.01). Compared with the PPF group, the expressions of NLRP3 and caspase-1 proteins in the PPF+GM1 group were significantly decreased (P<0.01), and the expression of GSDMD protein was significantly decreased (P<0.05). Compared with the PPF group, the expressions of NLRP3 and caspase-1 proteins in the GM1 group were significantly decreased (P<0.01), and the expression of GSDMD protein was significantly decreased (P<0.05).

[0089] ② Changes of proteins in the PI3K / Akt / NF-κB signaling pathway in canine hippocampus tissue like Fig.19 As shown in the figure, there was no significant difference in the protein expression of PI3K, AKT and P65 among the groups. Compared with the CON group, the protein expression of P-PI3K and P-AKT in the hippocampus of the PPF group was significantly decreased (P<0.01), and the protein expression of P-P65 in the hippocampus was significantly increased (P<0.01); compared with the PPF group, the protein expression of P-PI3K and P-AKT in the hippocampus of the PPF+GM1 group was significantly increased (P<0.01), and the protein expression of P-P65 was significantly decreased (P<0.05); the protein expression of P-PI3K and P-AKT in the GM1 group was significantly increased (P<0.01), and the protein expression of P-P65 was significantly decreased (P<0.05).

[0090] By calculating the ratio of phosphorylated protein to non-phosphorylated protein (P-PI3K / PI3K, P-AKT / AKT and P-P65 / P65), it was found that compared with the CON group, the PPF group had significantly decreased P-PI3K / PI3K and P-AKT / AKT values ​​(P<0.01), and a significantly increased P-P65 / P65 value (P<0.05); compared with the PPF group, the PPF+GM1 group had significantly increased P-PI3K / PI3K and P-AKT / AKT values ​​(P<0.01), and no significant change in P-P65 / P65 value (P>0.05); compared with the PPF group, the P-PI3K / PI3K and P-AKT / AKT values ​​in the GM1 group were significantly increased (P<0.01), and a significantly decreased P-P65 / P65 value (P<0.05).

[0091] ③ Changes of brain-derived neurotrophic factor (BDNF) protein in canine hippocampus like Fig. 20 As shown in the figure, compared with the CON group, the BDNF protein expression in the PPF group was significantly decreased (P<0.01); compared with the PPF group, the protein expression in the PPF+GM1 group and the GM1 group was significantly increased (P<0.01).

[0092] (13) Canine cognitive function score Canine cognitive dysfunction is mainly manifested as a decline in learning, memory and spatial orientation, accompanied by changes in social interaction and sleep patterns. A double-blind test method was used to score the cognitive abilities of dogs in each group that continued to be raised to adulthood (over 12 months old) using the Canine Cognitive Dysfunction Rating Scale to ensure the objectivity and reliability of the evaluation results.

[0093] When using the Canine Cognitive Dysfunction Rating Scale for evaluation, the evaluators perform a double-blind test process to ensure the fairness and accuracy of the scoring. During this process, 17 items in four areas, including spatial orientation, social interaction, sleep-wake cycle, and house contamination, are comprehensively evaluated to test the cognitive ability of the test dogs. The scores for each area of ​​cognitive dysfunction in the test dogs are as follows: Fig.21 shown.

[0094] After 12 months of feeding of the experimental dogs anesthetized with propofol, it was found that compared with the CON group, the spatial orientation score of the PPF group was significantly increased (P<0.05); the social interaction, sleep-wake cycle, and house contamination scores were all increased, but the differences were not significant (P>0.05); compared with the PPF group, the spatial orientation scores of the PPF+GM1 group and the GM1 group were significantly reduced (P<0.01), and the house contamination score of the GM1 group was significantly reduced (P<0.05).

[0095] The total scores of cognitive dysfunction in the experimental dogs are shown in Table 4 and Fig. 22 As shown. Compared with the CON group, the PPF group was significantly higher (P<0.01); compared with the PPF group, the PPF+GM1 group and the GM1 group were significantly lower (P<0.01). The total score of cognitive dysfunction in the PPF group was 14.67 points, which was within the range of mild cognitive impairment (8-23 points), and the total scores of the other groups were within the normal range (0-7 points).

[0096] Table 4 Results of cognitive dysfunction scores in dogs (n=3)

[0097] The results showed that mild cognitive dysfunction was observed in developing dogs receiving propofol-maintained anesthesia as they grew into adulthood. After GM1 intervention, the scores of the four areas of spatial orientation, social interaction, sleep-wake cycle, and house contamination were all reduced, and the total score of cognitive dysfunction was significantly reduced (P<0.01). This shows that GM1 intervention can reduce the cognitive dysfunction score of dogs caused by propofol anesthesia and effectively alleviate the cognitive dysfunction of dogs caused by propofol anesthesia. It further verified that GM1 has a protective effect on neural damage caused by propofol anesthesia in developing dogs.

[0098] Embodiment 2 Clinical application trial To verify the neuroprotective effect of GM1 on propofol anesthesia in developing dogs and its effective intervention dose, 12 dogs (5 male dogs and 7 female dogs) who needed propofol anesthesia surgery or treatment were collected from the Animal Clinical Teaching Hospital of Northeast Agricultural University. The dogs were aged between 3 and 6 months old and divided into a control group (CON group, n=6) and a GM1 group (n=6).

[0099] After the consent of the owners, the GM1 group was given GM1 intervention 30 minutes before the propofol anesthesia surgery, with a dose of 10 mg / kg (the dose selection was calculated through previous animal experiments). A detailed questionnaire was designed and produced in combination with the Canine Cognitive Dysfunction Rating Scale. After the surgery, the questionnaire was distributed to the owners, and the dogs were followed up until adulthood (more than 12 months old). After the veterinarian screened the dogs for diseases, their cognitive abilities were assessed using the Canine Cognitive Dysfunction Rating Scale combined with the questionnaire.

[0100] Clinical application test results The dogs were followed up from the clinical development period to adulthood, and the cognitive function of each adult dog was comprehensively evaluated using the Canine Cognitive Impairment Rating Scale, combined with questionnaire feedback collected from the dog owners. This evaluation aims to assess the changes in cognitive function of dogs over a long period of time and to evaluate the long-term effectiveness and potential sustained effects of the GM1 intervention. The clinical manifestations of cognitive behavioral changes are divided into four stages (total score of four areas): normal score 0-7 points; mild cognitive impairment 8-23 points; moderate cognitive impairment 24-44 points; severe cognitive impairment 45-95 points.

[0101] The scores of each domain of canine cognitive dysfunction in clinical practice are shown in Table 5 and Fig.23 As shown in Table 5, the average scores of spatial orientation, social interaction, and sleep-wake cycle in the GM1 group were lower than those in the CON group. The average scores of house pollution in the two groups were the same, with no significant difference (P>0.05). Fig.24 As shown in the results, the total score of the CON group was 8.49, which just reached the score range of mild cognitive dysfunction (8-23 points), and the total score of the GM1 group was 6.49, which was in the normal score range (0-7 points), but the difference between the two groups was not significant (P>0.05). This result shows that when propofol anesthesia is used for surgery in clinical middle and developing dogs, intervention with 10mg / kg GM1 can reduce the cognitive dysfunction score of dogs in adulthood and reduce the risk of cognitive dysfunction in dogs in adulthood. It provides an important preliminary research basis for the potential value of GM1 in clinical applications.

[0102] Table 5 Results of clinical canine cognitive dysfunction scores (n=6) ;

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of GM1 in protecting developing dogs from nerve damage induced by propofol anesthesia.

2. The use according to claim 1, characterized in that: Protection against propofol anesthesia-induced neurological damage in developing dogs includes: Ⅰ. Alleviate propofol anesthesia-induced hippocampal damage in developing dogs and improve their cognitive function; II. Alleviate neuroinflammation, pyroptosis, oxidative stress, and cognitive dysfunction in developing dogs caused by propofol anesthesia.

3. The use according to claim 2, characterized in that: Alleviating propofol anesthesia-induced hippocampal damage and improving cognitive function in developing dogs includes anti-inflammatory, anti-pyroptotic, and antioxidant effects.

4. The use according to claim 2, characterized in that: Alleviation of propofol-induced neuroinflammation, pyroptosis, oxidative stress, and cognitive dysfunction in developing dogs, including regulation of the PI3K / Akt / NF-κB pathway and the expression of BDNF.

5. A drug for protecting dogs during their development from nerve damage caused by propofol anesthesia, characterized in that: Including GM1.

6. The drug according to claim 5, characterized in that: The GM1 content is 10 mg / kg.

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