Construction method of model for rat traumatic brain injury caused by explosive shock waves

Abstract

The invention belongs to the technical field of damage assessment, and particularly relates to a construction method of a rat traumatic brain injury model caused by explosive shock waves. By protecting other important visceral organs except the brain, interference of damage of visceral organs such as the lung to brain injury is avoided, the overall function stability of the body is maintained, the caused brain injury can more truly reflect the characteristics of explosive traumatic brain injury, and the credibility and repeatability of experimental data are improved. The construction method simulates loading of explosion shock wave parameters in a real environment, constructs an animal traumatic brain injury model through live ammunition explosion, is closer to an actual combat environment, and can truly reflect physical parameters related to explosion and a complex action process generated by interaction of the physical parameters. And a support is provided for developing molecular mechanisms of explosive traumatic brain injury, clinical diagnosis, screening of therapeutic drugs and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of damage assessment, and in particular relates to a method for constructing a rat traumatic brain injury model induced by explosion shock waves. Background Art

[0002] The shock waves generated by explosions cause extremely serious damage to the human body, among which brain damage is one of the important causes of casualties and disability. In the process of studying the relevant mechanisms and treatment methods of explosive traumatic brain injury, models are indispensable tools. Common models for explosive traumatic brain injury research include animal models, dummy models, digital simulation models, etc., each of which has its own advantages and disadvantages due to different research purposes, objects and applicable conditions. Animals are the most commonly used models in the study of explosive shock injuries, mainly based on the principle that the injury effects of animals and personnel are similar, and their organ structure, function, anatomical characteristics, injury threshold, etc. are close to those of humans. Since rats have certain similarities in physiological characteristics and nervous systems with humans, they are often used to establish brain injury models. At present, most explosive traumatic brain injury models are mainly based on laboratory biological shock tubes, improvised explosive devices, etc. Although their experimental parameters are controllable, repeatable and low-cost, they cannot simulate the complexity and destructiveness of shock waves under actual explosion conditions, and there is still a large gap with the damage data obtained in actual combat environments.

[0003] Free-field explosions can realistically simulate explosion damage in real environments and fully reproduce the pathological features of brain injury, which can provide a reliable animal model for the study of the mechanism and treatment of explosive brain injury. However, existing methods expose the whole body of rats to a free-field environment without protecting their chest and abdomen. The shock wave not only causes brain damage to the rats, but also damages the lungs. Lung damage interferes with the elucidation of the mechanism of brain injury. Therefore, establishing an animal model that simulates simple brain damage caused by explosion shock waves in a real environment is an urgent problem to be solved. Contents of the invention The purpose of the present invention is to provide a method for constructing a rat traumatic brain injury model induced by explosion shock waves, so as to overcome the above-mentioned technical problems existing in the prior art.

[0004] To this end, the technical solution provided by the present invention is as follows: A method for constructing a rat traumatic brain injury model induced by explosion shock wave comprises the following steps: Step 1) Laying out explosives; Step 2) Protecting the chest and abdomen of the rat; Step 3) Place the anesthetized and chest-abdomen-protected rats at a fixed distance from the explosion center, with their heads facing the explosion source; Step 4) Shock wave exposure: After debugging, the explosives were remotely detonated to obtain a rat model of explosive traumatic brain injury.

[0005] After the shock wave exposure, the injury of the acquired blast traumatic brain injury rat model was also evaluated, and the injury of the rats was detected by gross anatomy and histopathology.

[0006] Step 1) The explosive power of the laid explosives is 200-250 kg TNT equivalent.

[0007] Step 2) Protect the chest and abdomen of the rat. The specific process is as follows: After the rat is anesthetized, wrap the rat's trunk and limbs in gauze and fix them to the mouse board with tape. Place the rat in a prone position on the bottom wall of the protective box, insert the upper board, and expose the rat's head.

[0008] Step 3) The fixed distance from the explosion center point is 18-30m.

[0009] Step 3) also includes arranging an overpressure sensor on the same arc as the rat, and making the sensitive surface of the overpressure sensor level with the ground, and the electrical signal of the overpressure sensor is connected to the computer.

[0010] The gross anatomical manifestations of brain injury in rats were dilation of blood vessels and macular hemorrhage on the brain surface; the histopathological manifestations were blurred contours of cortical neurons, dark staining of the cytoplasm, and visible nuclear shrinkage and nuclear dissolution.

[0011] The gross anatomy of the rat lungs showed that the lung capsule was smooth, the alveoli of each lobe were well inflated, punctate hemorrhages were visible in some lobes, and no plaque or flaky hemorrhages or exudates were observed; histopathological manifestations showed that the alveolar epithelial cells had regular morphology and were closely arranged, without cell swelling, inflammatory cell infiltration, or pulmonary interstitial congestion.

[0012] The protection box is a rectangular parallelepiped, the mouse plate is placed in the protection box, a through hole is opened on the front side of the protection box to expose the rat's head, and an extension plate is provided on the lower bottom surface of the protection box toward the front side to support the rat's head.

[0013] The beneficial effects of the present invention are: The method for constructing a rat traumatic brain injury model induced by explosion shock waves provided by the present invention avoids the interference of damage to organs such as the lungs on brain injury by protecting other important organs other than the brain, maintains the overall functional stability of the body, and the resulting brain injury can more truly reflect the characteristics of explosion traumatic brain injury, thereby improving the credibility and repeatability of experimental data.

[0014] The construction method of the present invention simulates the loading of explosion shock wave parameters under real environment, and constructs an animal traumatic brain injury model through live ammunition explosion, which is closer to the actual combat environment and can truly reflect the complex action process of explosion-related physical parameters and their interactions, providing support for the molecular mechanism, clinical diagnosis and screening of therapeutic drugs of explosive traumatic brain injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic structural diagram of an embodiment of the rat chest and abdomen protection device of the present invention; Figure 2 is a diagram of the placement of rats and overpressure sensors in an embodiment of the present invention; Figure 3 is the shock wave overpressure curve; Figure 4 This is the anatomical diagram of the lungs of the unprotected group of rats in the chest and abdomen; Figure 5 This is the lung anatomy of the rat chest and abdomen protection group; Figure 6 It is a diagram of the anatomy of rat brain tissue; Figure 7 is the H&E staining of the lungs of the rat chest and abdomen unprotected group; Figure 8 This is the H&E staining picture of the lungs in the rat chest and abdomen protection group; Fig. 9 This is an H&E staining image of rat brain tissue.

[0016] In the figure: 1. protective box; 2. extension plate; 3. through hole; 4. upper plate; 5. lower plate. DETAILED DESCRIPTION

[0017] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0018] The exemplary embodiments of the present invention are now described with reference to the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided to disclose the present invention in detail and completely and to fully convey the scope of the present invention to those skilled in the art. The terms used in the exemplary embodiments shown in the accompanying drawings are not intended to limit the present invention. In the accompanying drawings, the same units / elements are marked with the same reference numerals.

[0019] Unless otherwise specified, the terms (including technical terms) used herein have the commonly understood meanings to those skilled in the art. In addition, it is understood that the terms defined in commonly used dictionaries should be understood to have the same meanings as those in the context of the relevant fields, and should not be understood as idealized or overly formal meanings.

[0020] Example 1 This embodiment provides a method for constructing a rat traumatic brain injury model induced by explosion shock wave, comprising the following steps: Step 1) Laying out explosives; Step 2) Protecting the chest and abdomen of the rat; Step 3) Place the anesthetized and chest-abdomen-protected rats at a fixed distance from the explosion center, with their heads facing the explosion source; Step 4) Shock wave exposure: After debugging, the explosives were remotely detonated to obtain a rat model of explosive traumatic brain injury.

[0021] The present invention avoids the interference of damage to organs such as the lungs on brain injury by protecting other important organs besides the brain, maintains the overall functional stability of the body, and the resulting brain injury can more truly reflect the characteristics of explosive traumatic brain injury, thereby improving the credibility and repeatability of experimental data.

[0022] Example 2 Based on Example 1, this example provides a method for constructing a rat traumatic brain injury model induced by explosion shock wave, comprising the following steps: Step 1) Lay out explosives; the explosive power is 200-250 kg TNT equivalent.

[0023] Step 2) Protect the chest and abdomen of the rat: Based on the rat chest and abdomen protection device. The protection device includes a protection box and a mouse board. After the rat is anesthetized, the rat's trunk and limbs are wrapped in gauze and fixed to the mouse board with tape. The rat is placed in a prone position on the bottom wall of the protection box, and the upper board is inserted to expose the rat's head.

[0024] like Figure 1 As shown, the protective box 1 is a six-sided rectangular parallelepiped, and the front side is composed of an upper plate 4 and a lower plate 5. The lower plate 5 is fixed to the lower bottom surface of the box body, and the upper plate 4 is not fixed and can be removed. The upper plate 4 is composed of five semicircular holes with the same radius, the same left and right spacing, and the center of the circle is equidistant from the upper wall. Two holes with the same radius and depth are set on the left and right sides of the bottom of the upper plate 4. Correspondingly, the lower plate 5 is composed of five semicircular holes with the same radius, the same left and right spacing, and the center of the circle is equidistant from the lower bottom surface (the upper and lower semicircular holes form a through hole 3), and the top of the lower plate 5 is provided with pins of equal length on the left and right sides, which are used to fit the two holes at the bottom of the upper plate 4.

[0025] The rear side is a box cover that can be opened and closed, and a metal buckle is set on the box cover to fasten the box cover. A handle is set on the upper bottom surface for carrying. There is an extension plate 2 from the lower bottom surface to the front side direction, which is used to support the rat head and ensure that all rat heads are on the same horizontal plane. The mouse board is a rectangular board that supports the rat body.

[0026] Step 3) Place rats: Place anesthetized rats with chest and abdomen protection at a fixed distance from the explosion center, with their heads facing the explosion source. At the same time, place overpressure sensors on the same arc as the rats, and make the sensitive surface of the overpressure sensors level with the ground; Figure 2 As shown; Among them, the fixed distance refers to 18-30 m away from the center of the explosion.

[0027] Step 4) Shock wave exposure: after debugging is completed, the explosive is remotely detonated to obtain a rat model of explosive traumatic brain injury, and the pressure data of the overpressure sensor is recorded at the same time to obtain the dose-effect relationship between rat brain injury and overpressure.

[0028] like Figure 3 As shown, the shock wave pressure is 0.06-0.14MPa. A is 18m away from the explosion center, B is 20m away from the explosion center, C is 25m away from the explosion center, and D is 30m away from the explosion center.

[0029] Example 3 Based on Example 1, this example provides a method for constructing a rat traumatic brain injury model caused by an explosion shock wave. After the shock wave exposure is completed, it also includes an injury assessment of the obtained explosion traumatic brain injury rat model, and the injury of the rat is detected by gross anatomy and histopathology methods.

[0030] Brain: Gross anatomy showed dilated blood vessels and patchy hemorrhage on the brain surface; histopathology showed blurred contours of cortical nerve cells, darkly stained cytoplasm, and nucleus shrinkage and nuclear dissolution. Lungs: The gross anatomy showed that the lung capsule was smooth, the alveoli of each lobe were well inflated, punctate hemorrhages were visible in some lobes, and no plaque or flaky hemorrhages or exudates were found. The histopathological manifestations showed that the alveolar epithelial cells had regular morphology and were closely arranged, without cell swelling, inflammatory cell infiltration, or pulmonary interstitial congestion.

[0031] Example 4 In order to further illustrate the effect of the present invention, this example conducted the following experiment using a control (Ctrl) group and an experimental group.

[0032] 1. Animal grouping 90 rats were weighed and numbered 1-90. The corresponding random numbers were generated using Excel software. The random numbers were arranged in ascending order and the rats were divided into two groups in order, a control (Ctrl) group (10 rats) and an experimental group (80 rats). The experimental group was further divided into two subgroups, a chest and abdomen protection group and a chest and abdomen unprotected group (40 rats in each). The experimental group was placed at different distances from the explosion center, with 10 rats in the protection group and 10 rats in the unprotected group at each point. The control group was placed in a shelter 2 km away from the explosion source.

[0033] Model making This model was constructed with the help of a warhead static explosion power test. The specific operation method is: select explosives with an explosion power of 250 kg TNT equivalent (here only 250 kg TNT equivalent is used as an example, but it does not mean that only under this explosion power can the traumatic brain injury effect be achieved) for deployment, and then randomly place 40 rats in the test group at 18 m, 20 m, 25 m, and 30 m away from the explosion center, with 20 rats at each point, of which 10 are protected in the chest and abdomen.

[0034] Before the experiment, the rats in the protection group were anesthetized by inhalation of isoflurane and then placed in a homemade chest and abdomen protection device ( Figure 1 ) (exposed head) and fixed to the ground. In the unprotected group, rats were fixed in a wire cage with their heads facing the explosion source and fixed to the ground. Overpressure sensors were set up at each position and transmitted to the terminal computer via cables. The power switch was turned on and overpressure data was recorded before the test. After the test, the anatomical and histopathological examinations of the rats were performed to evaluate whether the model was successful. The layout of rats and overpressure sensors is shown in the figure below. Figure 2 shown.

[0035] 3. General observation After the experiment, the casualties and mental state of the experimental animals were observed.

[0036] 4. Animal dissection Rats were anesthetized by intraperitoneal injection of Zota (50 mg / kg), the head skin was cut open and then cut along the cranial sutures. After the brain tissue was fully exposed, it was gently removed and placed in a culture dish filled with physiological saline to wash away blood stains. The brain damage was observed and recorded; Cut the skin from under the jaw along the xiphoid process upwards, bluntly peel off the skin and subcutaneous tissue to both sides, expose the thorax and cut it open along the middle, clamp the organ with tweezers, remove the lung tissue as a whole, place it in a culture dish filled with physiological saline, wash away the blood, and observe and record the lung injury. 5. Histopathological examination At the same time, the whole brain of the above rats was selected to prepare paraffin sections for histopathological analysis; wherein the specific method of histopathological section preparation is as follows: ① Sampling: Take a brain tissue block of about 3-4 mm fixed with 4% paraformaldehyde solution and place it in an embedding box; ② Dehydration and transparency: Use a fully automatic dehydrator for dehydration, the dehydration setting program is: 75% ethanol (1 h) → 85% ethanol (1 h) → 95% ethanol I (1 h) → 95% ethanol II (1 h) → 100% ethanol I (45 min) → 100% ethanol II (45 min) → 100% ethanol III (0.5 h); transparency setting program: xylene I (20 min) → xylene II (20 min); ③ Embedding: Use a paraffin embedding machine to embed the dehydrated specimens in paraffin, and trim the wax blocks for easy sectioning; ④ Sectioning: Use a rotary microtome to cut the specimen into 0.5 μm sections; ⑤ Spread the film: Use a clean brush in your left hand to gently pick up the wax film, and use ophthalmic forceps in your right hand to gently transfer the wax film to the water surface of the film spreader (water temperature 40~45℃) to float; ⑥ Drying: After the slices are fully unfolded, use a glass slide to pick up a good slice and place it on a slice drying machine to dry (65-70°C), and immediately write the sample number; ⑦ Staining: Use a fully automatic staining machine for staining, set the program: xylene I (5min) → xylene II (5min) → xylene III (5min) → 100% ethanol (1min) → 95% ethanol (1min) → 85% ethanol (1min) → 75% ethanol (1min) → water wash I (1min) → hematoxylin (6min) → water wash II (1min) → hydrochloric acid alcohol differentiation (4s) → water wash III (1min) → ammonia blueing (1min) → water wash IV (1min) → eosin (1.5min) → water wash V (1min) → 85% ethanol (20s) → 90% ethanol (1min) → 95% ethanol I (1min) → 95% ethanol II (1min) → 100% ethanol I (1min) → 100% ethanol II (1min) → xylene I (1min) → xylene II (1min); ⑧Seal the slides: Use an automatic sealer to seal the slides; ⑨Microscopic examination: Use an optical microscope to read the film, observe the pathological changes in the specimen in detail, make records, and take photos.

[0037] 6. Results 6.1 Symptoms and signs of animals after the test After the test, rats in the unprotected group at the locations 18 m and 20 m away from the explosion center died, with mortality rates of 30.0% (3 / 10) and 10.0% (1 / 10), respectively; all rats in the protected group survived, and the surviving rats' spontaneous activities were significantly reduced. All rats in other locations survived.

[0038] 6.2 Rat dissection The lungs of rats in the unprotected group showed varying degrees of ecchymosis and hemorrhage ( Figure 4 ), the lung hemorrhage of the dead rats involved the entire layer, intercostal indentations were visible, and the degree of bleeding was related to the distance. The lung capsule of the protection group was smooth, the alveoli of each lobe were well inflated, punctate hemorrhage was visible in some lobes, and no plaque or sheet hemorrhage was observed ( Figure 5 ); The brain showed varying degrees of cerebral surface vascular dilation, punctate and / or macular hemorrhage, and the degree of hemorrhage was related to the distance ( Figure 6 ).

[0039] 6.3 Pathology The alveolar septa of rats in the unprotected group were widened, and red blood cells and inflammatory cells infiltration were observed in the lung interstitium ( Figure 7 In the lung tissue of the rats in the protection group, the alveolar epithelial cells were regular in shape and arranged closely, and no cell swelling, inflammatory cell infiltration, or pulmonary interstitial congestion was observed ( Figure 8 ); H&E staining of rat cerebral cortex showed that the control group had intact nerve cell structure, clear outline, round or oval cells, obvious nuclei, and no nuclear shrinkage or dissolution; in the 18 m and 20 m exposure groups, a large number of nerve cells had blurred outlines, dark cytoplasm, and nuclear shrinkage or dissolution ( Fig. 9 ), in the 25m and 30m exposure groups, a small number of nerve cells were observed with blurred outlines, darkly stained cytoplasm, and shrunken or dissolved nuclei.

[0040] Through the above experiments, it can be known that the degree of brain injury bleeding is related to the distance, and the corresponding distance can be selected according to the needs. The lungs of the protection group were not damaged, which avoided the interference of damage to the lungs and other organs on brain injury, maintained the overall functional stability of the body, and the brain injury caused could more truly reflect the characteristics of blast traumatic brain injury, and improved the credibility and repeatability of the experimental data. The invention method can obtain a rat traumatic brain injury model induced by explosion shock waves that meets the requirements.

[0041] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. A method for constructing a rat traumatic brain injury model induced by explosion shock wave, characterized in that: The following steps are involved: Step 1) Laying out explosives; Step 2) Protecting the chest and abdomen of the rat; Step 3) Place the anesthetized and chest-abdomen-protected rats at a fixed distance from the explosion center, with their heads facing the explosion source; Step 4) Shock wave exposure: After debugging, the explosives were remotely detonated to obtain a rat model of explosive traumatic brain injury.

2. The method for constructing a rat traumatic brain injury model induced by explosion shock wave according to claim 1, characterized in that: After the shock wave exposure, the injury of the acquired blast traumatic brain injury rat model was also evaluated, and the injury of the rats was detected by gross anatomy and histopathology.

3. The method for constructing a rat traumatic brain injury model induced by explosion shock wave according to claim 1, characterized in that: Step 1) The explosive power of the laid explosives is 200-250 kg TNT equivalent.

4. The method for constructing a rat traumatic brain injury model induced by explosion shock wave according to claim 1, characterized in that: Step 2) Protect the chest and abdomen of the rat. The specific process is as follows: After the rat is anesthetized, wrap the rat's trunk and limbs in gauze and fix them to the mouse board with tape. Place the rat in a prone position on the bottom wall of the protective box, insert the upper board, and expose the rat's head.

5. The method for constructing a rat traumatic brain injury model induced by explosion shock wave according to claim 1, characterized in that: Step 3) The fixed distance from the explosion center point is 18-30m.

6. The method for constructing a rat traumatic brain injury model induced by explosion shock wave according to claim 1, characterized in that: Step 3) also includes arranging an overpressure sensor on the same arc as the rat, and making the sensitive surface of the overpressure sensor level with the ground, and the electrical signal of the overpressure sensor is connected to the computer.

7. The method for constructing a rat traumatic brain injury model induced by explosion shock wave according to claim 2, characterized in that: The gross anatomical manifestations of brain injury in rats were dilation of blood vessels and macular hemorrhage on the brain surface; the histopathological manifestations were blurred contours of cortical neurons, dark staining of the cytoplasm, and visible nuclear shrinkage and nuclear dissolution.

8. The method for constructing a rat traumatic brain injury model induced by explosion shock wave according to claim 1, characterized in that: The gross anatomy of the rat lungs showed that the lung capsule was smooth, the alveoli of each lobe were well inflated, punctate hemorrhages were visible in some lobes, and no plaque or flaky hemorrhages or exudates were observed; histopathological manifestations showed that the alveolar epithelial cells had regular morphology and were closely arranged, without cell swelling, inflammatory cell infiltration, or pulmonary interstitial congestion.

9. The method for constructing a rat traumatic brain injury model induced by explosion shock wave according to claim 4, characterized in that: The protection box is a rectangular parallelepiped, the mouse plate is placed in the protection box, a through hole is opened on the front side of the protection box to expose the rat's head, and an extension plate is provided on the lower bottom surface of the protection box toward the front side to support the rat's head.

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