Construction method of plateau traumatic craniocerebral injury mouse model

Abstract

A construction method of a mouse model of plateau traumatic severe craniocerebral injury (HTBI) comprises the following steps: feeding a mouse in a simulated plateau environment for 7-10 days, and carrying out controlled cortex impact (CCI) striking treatment, specifically, fixing the mouse after anesthesia craniotomy on a stereotactic instrument, and adopting a falling rod with the weight of 20g to freely fall from a position with the height of 15cm so as to vertically impact the dura mater of the mouse at the depth of 1.5 mm. The mouse plateau traumatic severe craniocerebral injury model is constructed by simulating vertical striking of the falling rod with specific weight and height in a plateau environment, so that the equipment acquisition cost is reduced, the survival rate of the injured mouse is increased (up to 60-70%), the construction success rate is high, and the experiment cost is greatly reduced; and a foundation is laid for improved variety breeding of experimental animals. The model can more accurately simulate the TBI pathological process in the plateau environment, the pathological difference of model reaction is small, the model has excellent consistency, and a reliable model is provided for researching a secondary injury mechanism of HTBI and researching and developing prevention and treatment drugs.

Description

Technical Field

[0001] The invention relates to the technical field of animal model construction, and in particular to a method for constructing a mouse model of plateau traumatic craniocerebral injury. Background Art

[0002] Traumatic brain injury (TBI) is a pathological change in brain tissue or neurological dysfunction caused by external mechanical force, and is an important cause of death and disability. There is currently no effective treatment. There are many animal models of TBI, with different injury modes and biomechanical mechanisms. They can be divided into impact injury models, non-impact acceleration injury models, and thin-grid blast wave injury models. Different models have their own advantages and disadvantages, and their application ranges are also different. Among them, impact injury is mainly focal injury, but the animal mortality rate is high during the modeling process, and the local brain compression time and force are often uneven. Controlled cortical impact (CCI) is mainly focal injury, with good modeling stability and controllability, which can avoid diffuse TBI and has potential advantages in studying the biomechanical and pathophysiological changes of the body after TBI.

[0003] Due to the harsh geographical environment and backward medical conditions in the plateau area, the treatment of plateau traumatic brain injury (HTBI) is limited and the mechanism is unclear. Existing HTBI animal models are mostly constructed according to low-altitude standards, resulting in high animal mortality and waste of resources. Animal models suitable for plateau TBI are urgently needed. Related studies have mostly focused on the construction and application of general TBI models, without fully considering the particularity of the plateau environment, and lack of targeted guidance on the construction of plateau TBI models. Secondly, the existing construction of controlled cortical impact injury often uses a controlled cortical impact instrument to construct craniocerebral trauma of different degrees of injury, and the construction equipment is costly. Summary of the invention

[0004] Based on the above existing technologies, the purpose of the present invention is to provide a method for constructing a controlled cortical impact injury (CCI) mouse model of severe traumatic brain injury at high altitude. This method can effectively construct a mouse model of traumatic brain injury at high altitude, effectively save the construction cost, and improve the success rate of model construction.

[0005] The purpose of the present invention is achieved through the following technical solutions: A method for constructing a mouse model of plateau traumatic severe craniocerebral injury, characterized by: raising mice in a simulated plateau environment for 7 to 10 days, anesthetizing the mice after craniotomy and fixing them on a stereotaxic apparatus, and performing CCI impact treatment, so as to establish a CCI plateau model that conforms to the characteristics of high altitude and takes into account cost-effectiveness.

[0006] Furthermore, the simulated plateau environment uses a low-pressure oxygen chamber to simulate an environment at an altitude of 4,000 meters. Specifically, the pressure in the oxygen chamber is set to 0.6 absolute atmospheres, and the temperature is controlled at 20±1°C.

[0007] Furthermore, the breeding is to feed the mice in a simulated plateau environment for 7 to 10 days, and the oxygen chamber is opened twice a day for 30 minutes each time to add granular food and water, and clean it to ensure that the basic living needs of the mice are met.

[0008] Furthermore, after the feeding was completed, blood was collected from the mouse orbits to test hemoglobin and red blood cell count. When the hemoglobin reached above 200 g / L and the red blood cell count reached 9.5×10 12 / L or above, CCI strike processing can be performed.

[0009] Furthermore, the craniotomy is performed by lifting the mouse scalp with tweezers, using a scalpel to longitudinally incise the skin along the midline of the head, using tissue scissors to separate the subcutaneous tissue to both sides along the edge of the incision, and bluntly separating the skin from the fascia and muscle tissue on the surface of the skull. After fixing the mouse head on a stereotaxic instrument, the skull drill is started so that the drill bit is perpendicular to the skull surface and slowly and evenly drilled into the skull, the blade of the expander is gently placed into the surgical incision, and the incision is gradually expanded to fully expose the skull.

[0010] Furthermore, the CCI impact treatment is performed by using a 20 g drop rod to freely drop from a height of 15 cm and vertically impact the mouse dura mater at a depth of 1.5 mm.

[0011] Furthermore, before CCI treatment, the mice underwent craniotomy. Specifically, the mice were fasted for 8 hours before the operation and anesthetized intraperitoneally with pentobarbital at a dosage of 50 mg / kg. After anesthesia, the mice were fixed on a stereotaxic apparatus, and a 2.5 mm craniotomy was performed 2.0 mm lateral to the right anterior bregma of the mice.

[0012] During the construction process, if the weight and height of the drop bar are improperly regulated, the local shear strain during the striking process will be uncontrollable, resulting in the damage function performance of the constructed model being affected, and the pathological differences of craniocerebral injuries are obvious, and even the constructed model will not reach severe craniocerebral injuries, but will belong to mild craniocerebral injuries, resulting in poor model consistency. In the present invention, the weight of the drop bar is determined to be 20g, and it is dropped and struck at 15cm. The pathological differences of the constructed model are small, the consistency of the model is high, and the striking standards for severe craniocerebral injuries are better standardized.

[0013] Most specifically, a method for constructing a mouse model of plateau traumatic severe craniocerebral injury is characterized by comprising the following steps: (1) A hypobaric oxygen chamber was used to simulate the plateau environment at an altitude of 4000 meters. The pressure in the oxygen chamber was set to 0.6 absolute atmospheres and the temperature was controlled at 20±1°C. The mice were placed in the hypobaric oxygen chamber for 7-10 days. The oxygen chamber was opened twice a day for 30 minutes each time to add pelleted food and water and clean it to ensure that the basic living needs of the mice were met. After the feeding period, blood was collected from the mouse eye sockets. When the hemoglobin level reached above 200 g / L and the red blood cell count reached 9.5×10 12 / When it is above L, the subsequent CCI strike processing is carried out; (2) The mice were fasted for 8 hours before the operation and anesthetized intraperitoneally with 50 mg / kg of pentobarbital. After anesthesia, the mice were fixed on a stereotaxic apparatus and a 2.5 mm craniotomy was performed 2.0 mm lateral to the right anterior bregma of the mice. (3) A 20 g drop rod was dropped freely from a height of 15 cm to vertically impact the mouse dura mater at a depth of 1.5 mm.

[0014] The present invention has the following technical effects: The present invention constructs a plateau traumatic severe craniocerebral injury model for mice by simulating vertical impact with a falling pole of specific weight and height in a plateau environment. This not only reduces the equipment purchase cost, but also improves the survival rate of craniocerebral injury in mice (the survival rate reaches 60-70%). The success rate of construction is high, which greatly reduces the experimental cost and lays a foundation for the breeding of fine varieties of experimental animals.

[0015] This model can more accurately simulate the pathological process of TBI in a plateau environment. The pathological differences in the model response are small and have excellent consistency, providing a reliable model for studying the secondary injury mechanism of HTBI and the development of preventive and therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Pictures of mouse breeding; A is a low-pressure oxygen chamber that simulates the plateau environment of 4,000 meters above sea level, and B is a live picture of mice after 7 days of breeding.

[0017] Figure 2 : Simulate the CCI impact injury process, divided into two heights of 15cm and 20cm, and the sampling and testing site after the impact.

[0018] Figure 3 : Picture of the mouse striking process in step (3), where A is the whole process of mouse striking injury and B is the sampling site.

[0019] Figure 4: Results of neurological function tests on the constructed mouse model; A: Morris water maze test; B: Modified neurological injury severity score (MNSS) score; C: Balance beam test; D: Number of platform crossings; E: Average speed during OFT; F: Escape latency; * P <0.05, ** P <0.01 or *** P <0.001, NS means: no significant difference.

[0020] Figure 5 : H&E staining of mice; A: Hematoxylin and eosin (H&E) staining image of mouse whole brain; B: Typical H&E staining image of damaged tissue, (Bar = 200 µm).

[0021] Figure 6 :Typical Nissl staining images of different mouse high models constructed (Bar = 5 µm).

[0022] Figure 7 :Prussian blue staining images of different mouse models constructed, (Bar = 20 µm).

[0023] Figure 8 :TUNEL assay results; A: TUNEL staining fluorescence intensity of each group; B: TUNEL fluorescence intensity statistics of each group (results are expressed as mean ± standard deviation, n=6 per group); C: Hemoglobin concentration of each group (results are expressed as mean ± standard deviation, low altitude 20cm: n=23, high altitude 20cm: n=35, high altitude 15cm: n=25. *** P <0.001, NS means: no significant difference).

[0024] Fig. 9 :Constructed Fe of different mouse models 2+ Concentration graph, results are expressed as mean ± SD, n = 6 per group. ** P <0.01, NS means: no significant difference.

[0025] Fig.10 : Diagram of brain tissue water content and inflammatory factor content in different mouse models, A: brain tissue water content in each group (results are expressed as mean ± standard deviation, n=6 for each group); B: TNF-α content in each group (results are expressed as mean ± standard deviation, n=6 for each group); C: IL-ß content in each group (results are expressed as mean ± standard deviation, n=6 for each group).

[0026] Fig.11 :Transmission electron microscopy (TEM) images of different mouse models constructed (Bar= 500 nm).

[0027] Fig.12 : Kaplan-Meier survival curves 7 days after CCI (*P<0.05, compared with the high altitude 20 cm group). DETAILED DESCRIPTION

[0028] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0029] Mouse strain and source in the present invention: Male C57BL / 6J mice aged 6-7 weeks and weighing 20.21±2.10 g were purchased from SPF Biotechnology Co., Ltd. (Beijing, China).

[0030] Ethical approval: All animal care and use procedures were performed in strict accordance with the ethical guidelines for the care and use of laboratory animals established by Chengdu University of Traditional Chinese Medicine (approval number 10.111 / 1348-0421.12984). The animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Chengdu University of Traditional Chinese Medicine.

[0031] Example 1 A method for constructing a mouse model of plateau traumatic severe craniocerebral injury comprises the following steps: (1) A hypobaric oxygen chamber model (Binglun Corp, Yantai, People's Republic of China) was used to simulate an altitude of 4000 meters. The pressure in the oxygen chamber was set to 0.6 absolute atmospheres (ATA) and the temperature was controlled at 20±1°C. Mice were divided into a high-altitude 20 cm group, a high-altitude 15 cm group, and a low-altitude 20 cm group. The mice in the high-altitude 20 cm group and the high-altitude 15 cm group were placed in the hypobaric oxygen chamber for 7 days; the mice in the low-altitude 20 cm group were raised under normal atmospheric pressure (400 meters above sea level) for 7 days; the oxygen chamber was opened twice a day for 30 minutes each time to add pelleted food and water and clean it to ensure that the basic living needs of the mice were met; after the end of the feeding, blood was collected from the orbits of the three groups of mice to test hemoglobin and red blood cell count. When the hemoglobin reached above 200 g / L and the red blood cell count reached 9.5×10 12 / When it is above L, subsequent CCI strikes can be made; (2) The mice were fasted for 8 hours before the operation and anesthetized intraperitoneally with 50 mg / kg of pentobarbital. After anesthesia, the mice were fixed on a stereotaxic apparatus and a 2.5 mm craniotomy was performed 2.0 mm lateral to the right anterior bregma of the mice. (3) For the high-altitude 15 cm group, a 20 g drop bar was freely dropped from a height of 15 cm to vertically impact the mouse dura mater at a depth of 1.5 mm. In addition, the drop height of the drop bar was 20 cm for both the high-altitude 20 cm group and the low-altitude 20 cm group. After hemostasis was confirmed, the incision was sutured and the mice were recovered in a 37°C incubator.

[0032] Figure 1 These are pictures of mouse breeding; A is a low-pressure oxygen chamber that simulates the plateau environment of 4,000 meters above sea level, and B is a live picture of mice after 7 days of breeding.

[0033] Figure 2 It simulates the CCI impact injury process and is divided into two heights of 15cm and 20cm, as well as the parts for sampling and testing after the impact.

[0034] Figure 3 This is a picture of the mouse striking process in step (3), where A is the entire process of mouse striking injury and B is the sampling site.

[0035] Model Validation: 1. Neurological function testing (1) Morris water maze test (MWM): The test consists of two parts: cued navigation and spatial exploration, which are used to evaluate the spatial memory ability of mice after TBI. During the test, the swimming path and residence time of the mice are recorded by an automatic video tracking system.

[0036] The results are as follows Figure 4 As shown in A, mice in the high-altitude 20 cm group showed more complex swimming paths and longer swimming distances than those in the high-altitude 15 cm and low-altitude 20 cm groups.

[0037] (2) Modified Neurological Severity Scale (MNSS) score: The test was performed on the third day after TBI. The mice were assessed for neurological deficits, such as ataxia and hemiplegia, and scored on a scale of 0 to 10. Higher scores indicated more severe neurological deficits.

[0038] The MNSS system was used to evaluate the neurological dysfunction in TBI mice. Figure 4 As shown in B, the experimental results showed that the MNSS score of the high-altitude 20cm group was significantly higher than that of the high-altitude 15cm and low-altitude 20cm groups.

[0039] (3) Balance beam test (BBT): The motor coordination ability of mice was assessed on day 7 after TBI by recording the number of falls and the ability to maintain balance when walking on a balance beam.

[0040] The results are as follows Figure 4As shown in C, the BBT results showed that the mice in the high altitude 20 cm group showed a significant increase in the number of foot slips compared with the high altitude 15 cm and low altitude 20 cm groups.

[0041] (4) Out-of-field test (OFT): On day 7 after TBI, mice were placed in the center area of ​​an open field box, and the total distance traveled and the time taken within 5 min were recorded, and the average speed was calculated to assess the spontaneous locomotor activity of the mice.

[0042] The results are as follows Figure 4 As shown in D, the number of platform crossings of mice in the high altitude 20 cm group was significantly reduced compared with the high altitude 15 cm and low altitude 20 cm groups. During OFT, the average speed of mice in the low altitude 20 cm group was (3.01±0.34) cm / s, that in the high altitude 20 cm group was (2.32±0.29) cm / s, and that in the high altitude 15 cm group was (3.10±0.37) cm / s. Figure 4 E. Among them, the average speed of mice in the high altitude 20cm group was significantly lower than that in the high altitude 15cm group and the low altitude 20cm group. In addition, the escape latency of mice in the low altitude 20cm group was (34.83±2.79) s, that in the high altitude 20cm group was (45.17±2.93) s, and that in the high altitude 15cm group was (35.83±5.04) s. The results showed that the escape latency of mice in the high altitude 20cm group was significantly longer than that in the high altitude 15cm group and the low altitude 20cm group. Figure 4 As shown in F.

[0043] 2. Histopathological examination (1) Hematoxylin-eosin (H&E) staining: The brain slices were subjected to a series of treatments including dewaxing, hydration, and staining, and microscopic imaging was used to observe the damage to the brain tissue, such as changes in cell morphology and inflammatory cell infiltration.

[0044] The results are as follows Figure 5 As shown in A, in the whole brain HE staining, the parietal cortex tissue structure of the mice in the 20 cm high altitude group was significantly damaged, characterized by continuous interruption of the cortex and a large damaged area. Figure 5 As shown in B, the damaged areas of mice in the high altitude 15 cm group and the low altitude 20 cm group were reduced, the damaged tissues showed less loose structure, less obvious infiltration of inflammatory cells, and extensive changes in brain tissue damage were mild.

[0045] (2) Nissl staining: Brain tissue sections were dewaxed with xylene, rehydrated through a series of 100%, 95%, and 75% ethanol, stained with toluidine blue for 3 min, and then rinsed twice with distilled water. Sections were dehydrated with a gradient of 75%, 95%, and 100% ethanol, cleared in xylene, and sealed with neutral resin before observation, imaging, and analysis under a microscope.

[0046] Nissl staining results Figure 6 As shown, neurons in the brain tissue of mice in the 20 cm high altitude falling pole impact group showed dark staining, nuclear condensation, vacuolar atrophy, and a decrease in the number of Nissl bodies in other cell morphologies. The damage in the 15 cm high altitude and 20 cm low altitude groups was significantly alleviated.

[0047] (3) Prussian blue staining: Paraffin-embedded brain tissue sections underwent a dewaxing and rehydration process and then were subjected to Perls staining with a Prussian blue staining kit (Solarbio, Beijing, China) in the dark. Iron deposition in brain tissues was examined under an optical microscope.

[0048] Prussian blue staining results Figure 7 As shown, the high altitude of 20 cm significantly increased the iron deposition in the brain tissue of TBI mice compared with the high altitude of 15 cm and low altitude of 15 cm groups, and the iron ion concentration was significantly increased.

[0049] (4) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay: TUNEL staining was performed using a cell death detection kit (Applygen, Beijing, China). The morphology and number of neurons, especially the changes in Nissl bodies, were observed to assess the degree of neuronal damage.

[0050] The results of TUNEL staining showed that the fluorescence intensity of the high altitude 20 cm group was significantly increased compared with the low altitude 20 cm and high altitude 15 cm groups, indicating an increase in apoptotic neurons ( Figure 8 AB).

[0051] The hemoglobin concentrations of each group were: low altitude 20cm group (129.01±11.19g / L), high altitude 20cm group (208.67±5.75g / L), high altitude 15cm group (206.83±6.11g / L). The hemoglobin concentrations of the high altitude 20cm and high altitude 15cm groups were similar, indicating that the high altitude 15cm impact model has a better ability to simulate the state of high altitude craniocerebral injury ( Figure 8 C).

[0052] 3. Iron content detection: To quantify Fe in brain tissue samples 2+ Content, using Fe 2+ Assay kit (Solarbio). The samples were lysed and centrifuged to collect the supernatant. According to the assay kit protocol, the specified iron detection reagent was introduced into the supernatant. The absorbance value of the sample was determined by spectrophotometry and compared with the known Fe 2+ The concentration of Fe in the sample was calculated by comparing it with the standard curve. 2+ content.

[0053] The results are as follows Fig. 9 As shown, it can be seen that the Fe 2+ The concentration was the highest, while the Fe 2+ The concentration levels are similar.

[0054] 4. Brain water content: Three days after TBI, rats (n=6 per group) were euthanized. The mouse brain was divided into left and right cerebral hemispheres, and the right cerebral hemisphere was weighed (wet weight). The right cerebral hemisphere was dried at 100°C for 24 hours and the dry weight was measured (established method: Tan et al., 2017). The percentage of water content = (wet weight - dry weight) / wet weight × 100%, and the water content of brain tissue was calculated by this formula to evaluate the degree of brain edema.

[0055] like Fig.10 As shown in A, the water content of brain tissue in the high altitude 20 cm group was significantly higher than that in the high altitude 15 cm and low altitude 20 cm groups, indicating more obvious cerebral edema.

[0056] (V) Enzyme-linked immunosorbent assay (ELISA): To detect the concentrations of TNF-α and IL-ß, ELISA kits (Esebio, Shanghai, China) were used according to the manufacturer's instructions, and the absorbance values ​​of TNF-α and IL-β in each well were measured at a wavelength of 450 nm. The sample concentrations were calculated according to the equations derived from the standard curves.

[0057] like Fig.10BC showed that the inflammatory factor TNF-α was (325.17±12.69) pg / mL in the low altitude 20cm group, (343±9.86) pg / mL in the high altitude 20cm group, and (324.67±11.17) pg / mL in the high altitude 15cm group; IL-β was (96.83±4.92) pg / mL in the low altitude 20cm group, (106.17±5.49) pg / mL in the high altitude 20cm group, and (97.33±5.24) pg / mL in the high altitude 15cm group. The results showed that the concentrations of TNF-α and IL-β in the high altitude 20cm group were significantly higher than those in the high altitude 15cm group and the low altitude 20cm group.

[0058] (vi) Transmission electron microscopy (TEM): Mouse brain tissues were immersed in 2.5% glutaraldehyde overnight at 4°C and then fixed with 1% osmium tetroxide for 1 hour. Subsequently, the samples were stained with 2% uranyl acetate and dehydrated with graded ethanol. After embedding in 100% resin overnight, the samples were cut into 100 nm sections and stained with 2% uranyl acetate and lead citrate. TEM images were acquired using a JEM-1400 Flash TEM (JEOL, Tokyo, Japan) and analyzed using Image J software.

[0059] The ultrastructure of mitochondria was examined by TEM. Fig.11 As shown. In the high altitude 20 cm group, mitochondria showed obvious structural abnormalities, including mitochondrial swelling, fragmentation, vacuolization, and increased cristae width. In addition, the reduction in mitochondrial length and increase in width indicated that the mitochondrial morphology was significantly distorted. In the low altitude 20 cm and high altitude 15 cm groups, slight mitochondrial swelling and synaptic structural abnormalities were seen. The ultrastructure of mitochondria showed a certain degree of damage, and the overall shape and structure were lighter than those of the high altitude 20 cm group.

[0060] 7. Survival rate analysis The number of mice that died in each group was counted daily, and Kaplan-Meier survival curves were plotted 7 days after TBI. All data analyses were performed by two observers in a blinded manner.

[0061] In the process of model construction, the main indicator for distinguishing whether it is severe craniocerebral injury is the mortality rate of the constructed craniocerebral injury model. When the mortality rate exceeds 30%, the craniocerebral injury model is judged to be severe craniocerebral injury. The Kaplan-Meier survival curve shows that the survival rate of the 20cm high altitude group is significantly lower than that of the 15cm high altitude group and the 20cm low altitude group (P<0.05) ( Fig.12). Experimental data show that after being raised for 7 days in a simulated 4000-meter plateau environment, the hemoglobin concentration of mice can be as high as 200g / L, which is in line with the characteristics of high altitude. However, if a 20cm impact height is selected in high-altitude areas, the mortality rate of mice will increase significantly, reaching more than 70%. Such a high mortality rate indicates that the model construction has failed, which not only causes a waste of funds, but also consumes a lot of manpower and time costs. The present invention finds that at a high altitude impact height of 15cm, the mortality rate is only between 30% and 40%, which is close to the judgment limit standard for severe craniocerebral injury, and can still have the same effect as a low-altitude 20cm impact (the control standard for severe craniocerebral injury). That is, while ensuring a high survival rate, a model of severe craniocerebral injury caused by plateau trauma in mice is constructed. The model of severe craniocerebral injury in mice constructed by this method has excellent consistency and higher practical value. It fully proves the significant advantages of the present invention in exploring traumatic severe craniocerebral injury in high-altitude mice.

[0062] During the construction process, we studied the effects of different feeding days on the final model. We found that when the feeding days were less than 7 days, the hemoglobin content and red blood cell content of mice could not reach the stable physiological state under plateau conditions (for example, after feeding for 3 days, the hemoglobin content was only 180g / L, and the number of red blood cells was far less than 9.5×10 12 / L), and after 7 to 10 days, the hemoglobin and other indicators of mice reached a peak and tended to stabilize. The same CCI treatment (falling bar weight 20g, hitting height 15cm) was performed on mice raised for 30 days. The constructed craniocerebral injury model was basically consistent with the craniocerebral injury model constructed by mice raised for 7 days, and the degree of damage to motor coordination (balance beam experiment) and cognitive function (water maze) was comparable. In addition, we also studied the influence of the selection of falling bar weight and hitting height on the construction of the model. The results showed that if the falling bar weight was too heavy and the hitting height was too high, the mortality rate of the model would increase. If the hitting height was too low, the pathological characteristics of clinical craniocerebral injury could not be simulated, resulting in the construction of a craniocerebral injury model that deviated from reality. However, when the falling bar weight was too small, the hitting force was controlled by adjusting the hitting height, the accuracy was not high, and the local shear strain was uncontrollable, which affected the injury function performance of the constructed model, and the pathological manifestations of the craniocerebral injury model showed obvious differences, resulting in poor model consistency, and even causing some of the constructed models to fail to reach severe, but belong to mild craniocerebral injury.

Claims

1. A method for constructing a mouse model of plateau traumatic severe craniocerebral injury, characterized in that: A CCI plateau model that conforms to the characteristics of high altitude and is cost-effective was established by raising mice in a simulated plateau environment for 7 to 10 days and then subjecting them to CCI impact treatment. The CCI impact treatment involved fixing the anesthetized mice after craniotomy on a stereotaxic apparatus and using a 20g drop rod to freely drop from a height of 15cm, vertically impacting the mouse dura mater at a depth of 1.5mm.

2. The method for constructing a mouse model of plateau traumatic severe craniocerebral injury as claimed in claim 1, characterized in that: The craniotomy is performed by lifting the mouse scalp with forceps, using a scalpel to longitudinally cut the skin along the midline of the head, using tissue scissors to separate the subcutaneous tissue to both sides along the edge of the incision, and bluntly separating the skin from the fascia and muscle tissue on the surface of the skull. After fixing the mouse head on a stereotaxic instrument, the skull drill is started so that the drill bit is perpendicular to the skull surface and slowly and evenly drilled into the skull, the blade of the expander is gently placed into the surgical incision, and the incision is gradually expanded to fully expose the skull.

3. The method for constructing a mouse model of plateau traumatic severe craniocerebral injury as claimed in claim 1, characterized in that: The simulated plateau environment uses a low-pressure oxygen chamber to simulate an environment at an altitude of 4,000 meters. The pressure in the oxygen chamber is set to 0.6 absolute atmospheric pressure, and the temperature is controlled at 20±1°C.

4. The method for constructing a mouse model of plateau traumatic severe craniocerebral injury according to any one of claims 1 to 3, characterized in that: The breeding is to feed the mice in a simulated plateau environment for 7 to 10 days, open the oxygen chamber twice a day, each time for 30 minutes, add granular food and water, and clean it to ensure that the basic living needs of the mice are met.

5. The method for constructing a mouse model of plateau traumatic severe craniocerebral injury according to any one of claims 1 to 4, characterized in that: After the feeding, blood was collected from the mouse orbits to test hemoglobin and red blood cell count. When the hemoglobin reached above 200 g / L and the red blood cell count reached 9.5×10 12 / L or above, CCI strike processing can be performed.

6. The method for constructing a mouse model of plateau traumatic severe craniocerebral injury as claimed in claim 5, characterized in that: Before the craniotomy, the mice were fasted for 8 hours and anesthetized intraperitoneally with pentobarbital at a dosage of 50 mg / kg. After anesthesia, the mice were fixed on a stereotaxic apparatus and a 2.5 mm craniotomy was performed 2.0 mm outside the right anterior bregma of the mice.

7. A method for constructing a mouse model of plateau traumatic severe craniocerebral injury, characterized in that: The steps include: (1) A hypobaric oxygen chamber was used to simulate the plateau environment at an altitude of 4000 meters. The pressure in the oxygen chamber was set to 0.6 absolute atmospheres and the temperature was controlled at 20±1°C. The mice were placed in the hypobaric oxygen chamber for 7-10 days. The oxygen chamber was opened twice a day for 30 minutes each time to add pelleted food and water and clean it to ensure that the basic living needs of the mice were met. After the feeding period, blood was collected from the mouse eye sockets. When the hemoglobin level reached above 200 g / L and the red blood cell count reached 9.5×10 12 / When it is above L, the subsequent CCI strike processing is carried out; (2) The mice were fasted for 8 hours before the operation and anesthetized intraperitoneally with 50 mg / kg of pentobarbital. After anesthesia, the mice were fixed on a stereotaxic apparatus and a 2.5 mm craniotomy was performed 2.0 mm lateral to the right anterior bregma of the mice. (3) A 20 g drop rod was dropped freely from a height of 15 cm to vertically impact the mouse dura mater at a depth of 1.5 mm.

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