Typical heat stroke mouse model and construction method thereof

Abstract

The invention discloses a typical heat stroke mouse model and a construction method thereof, and relates to the technical field of animal model construction.The construction method comprises the steps that male mice are transferred into an artificial climate box in advance to adapt to the environment, and pretreated mice are obtained; on the day of modeling, removing the stress mice according to the initial body temperature to obtain experimental mice; then starting a temperature and humidity control program of the artificial climate box, performing thermal strike on the experimental mouse, measuring the body temperature of the mouse, determining the duration of continuous thermal strike in sections, and recording the behavior state of the mouse; when the body temperature of the mouse is larger than or equal to 43 DEG C, righting reflex disappears or disturbance of consciousness occurs, thermal strike is stopped; detecting the body temperature of the mouse, and taking the body temperature greater than or equal to 43 DEG C as a modeling success standard to obtain a heat stroke mouse model; by optimizing the thermal strike parameters and duration, the modeling effect and death rate of the mouse model are balanced; through the initial anal temperature screening step, the stress mice are removed in time, and the interference of stress reaction on experimental results is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal model construction, and in particular to a classical heat stroke mouse model and a construction method thereof. BACKGROUND

[0002] Heat stroke is a severe acute heat stroke type caused by high temperature and high humidity environment or intense exercise, with rapid core body temperature rise above 40℃, consciousness disorder and multiple organ dysfunction as the main characteristics, and with extremely high morbidity and mortality. With global climate warming and frequent occurrence of extreme high temperature weather, heat stroke has become an important challenge in the field of public health. Due to the complexity of clinical cases and ethical restrictions, it is a key means to study the pathogenesis of heat stroke, develop treatment drugs and develop rescue programs to construct stable and reliable animal models. Mice are the first choice for constructing classical heat stroke models due to their clear genetic background, strong reproductive ability and certain similarity to human physiological characteristics.

[0003] In the prior art, the construction method of the classical heat stroke mouse model has many deficiencies: on the one hand, some methods rely on large climate chambers with high cost, and only a few scientific research institutions have such equipment, which has high purchase cost and strict space requirements, limiting the popularization and application of the technology; on the other hand, the existing methods do not fully consider the influence of mouse stress response, and the sudden change of environment during modeling can easily cause mouse stress, which not only interferes with the experimental results, but also increases the mortality rate during the modeling process; in addition, the temperature setting of heat shock in some schemes is too low, resulting in too long modeling time and low experimental efficiency, and the end point temperature setting in some schemes is unreasonable, which can ensure multiple organ damage, but significantly increases the acute mortality rate of mice, and cannot provide sufficient experimental objects for subsequent research. Therefore, it is urgent to propose a classical heat stroke mouse model and a construction method thereof to solve the problems of poor stability, easy stress, unreasonable end point temperature setting, long modeling time and non-classical pathological characteristics of the classical heat stroke mouse model in the prior art, so as to meet the needs of heat stroke related research. SUMMARY

[0004] The purpose of the present application is to provide a classical heat stroke mouse model and a construction method thereof to solve the problems of poor stability, easy stress, unreasonable end point temperature setting, long modeling time and non-classical pathological characteristics of the heat stroke mouse model in the prior art.

[0005] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme: a construction method of a classical heat stroke mouse model, specifically comprising the following steps:

[0006] S1, select several SPF level male C57BL / 6J mice, and transfer them to an artificial climate chamber before modeling to adapt to the environment, to obtain pretreated mice;

[0007] S2. On the day of modeling, a thermometer probe coated with medical gel coupling agent was inserted 2cm into the rectum of a pretreated mouse using a small back-and-forth rubbing motion to measure the initial body temperature. Mice with an initial body temperature <38.5℃ were selected for modeling, and stressed mice with an initial body temperature ≥38.5℃ were removed. The experimental mice were then deprived of their covers, food, and water, their cages were sealed and fixed, and they were placed back into the artificial climate chamber.

[0008] S3. Heat shock to experimental mice: Start the temperature and humidity control program of the artificial climate chamber and continue for 60-65 minutes until the temperature rises from room temperature to 40°C at a uniform rate, and the relative humidity is maintained at 70% RH.

[0009] S4. Maintain a temperature of 40℃ and a relative humidity of 70%RH for 55-60 minutes. Measure the body temperature of the mice when the heat shock has lasted for 2 hours. Determine the duration of heat shock in segments based on the measured body temperature. Test the righting reflex of the mice after each temperature measurement. At the same time, closely observe and record the behavior of the mice through the viewing window.

[0010] S5. Stop heat therapy if the mouse shows any of the following symptoms: body temperature ≥43℃, loss of righting reflex, or altered consciousness.

[0011] S6. Detect the body temperature of the mice. A body temperature ≥43℃ is used as the standard for successful modeling. Combined with the pathological damage characteristics of multiple organs, the successful model construction is confirmed, and the heatstroke mouse model is obtained.

[0012] Furthermore, the mice in S1 are 12–14 weeks old and weigh 30g ± 3g.

[0013] Furthermore, the conditions for raising mice in the artificial climate chamber in S1 are a temperature of 18–29°C and a humidity of 30%–70%.

[0014] Furthermore, the adaptation time in S1 is ≥24h. During the adaptation period, the original feeding cage, food, water and bedding are retained, and the ventilation window of the cage cover is opened.

[0015] Furthermore, the specific steps for determining the duration of continued thermal impact in segmentation in S4 are as follows:

[0016] When the mouse's body temperature is between 41°C and 42°C, continue the heat shock for 30 to 40 minutes.

[0017] When the mouse's body temperature is between 42℃ and 42.5℃, continue the heat shock for 20 to 30 minutes;

[0018] When the mice's body temperature is between 42.5℃ and 43℃, continue the heat shock for 20 minutes.

[0019] Furthermore, the parameters and functional requirements of the artificial climate chamber are as follows:

[0020] Temperature adjustment range: 10℃~50℃; control accuracy: ±0.2℃.

[0021] Humidity adjustment range: 10%~99% RH; control accuracy: ±10% RH.

[0022] It is equipped with a fan, heat sink and temperature control components, and has a transparent window and cold light source. Indirect ventilation is achieved through the fan, heat sink vents and temperature control component vents. Temperature, humidity and light intensity can be dynamically adjusted by setting a program.

[0023] The parameters required for the thermometer are as follows:

[0024] The temperature measurement range covers 30℃~45℃, and the temperature measurement accuracy is not less than ±0.2℃;

[0025] The probe rod is longer than 2cm and has a diameter of less than 2mm.

[0026] Furthermore, the mouse behavioral status recording items in S4 include activity status, movement posture, respiratory characteristics, appearance, and neurological disorders. Among them, activity status includes curled-up rest, normal activity, hyperactivity, staggering movement, and lying still; movement posture includes arched back and squinting eyes, smearing saliva, digging holes and burying head, standing and scratching the cage, climbing upside down on the cage rack, stereotyped circling, and limp and leaning against the cage; respiratory characteristics include heavy panting, rapid and weak breathing, and weak breathing; appearance includes moist mouth and nose; and neurological disorders include startle reflexes, stiff tail, and coma.

[0027] Furthermore, the mouse consciousness disorder in S5 includes at least one of convulsions, seizures, and coma.

[0028] Furthermore, the pathological damage characteristics of multiple organs in S6 include: hepatocyte steatosis and disordered arrangement, hepatocyte necrosis and inflammatory cell infiltration in some areas; renal tubular epithelial cell peeling, irregular lumen, glomerular atrophy and deformation, and interstitial inflammatory cell infiltration; alveolar wall thickening, inflammatory cell infiltration, and a large number of red blood cells in the alveolar septa and alveoli; unclear villus structure of small intestinal mucosa, disordered arrangement of epithelial cells, and partial villus deformity and damage.

[0029] The present invention also discloses a classic heatstroke mouse model, which is prepared by the above-mentioned method for constructing a classic heatstroke mouse model.

[0030] Compared with existing technologies, the classic heatstroke mouse model and its construction method provided by this invention have the following beneficial effects:

[0031] (1) This invention uses ordinary artificial climate chambers to replace expensive large climate chambers. The equipment purchase cost is low, the market stock is large, the space requirement is low and it is easy to move. It solves the problem of scarce and expensive modeling equipment in the prior art, enabling more scientific research institutions to carry out relevant experiments, and significantly improving the feasibility and popularization of the technology.

[0032] (2) This invention avoids the stimulation of mice by environmental changes during modeling by using the "original feeding cage + 1 day of adaptation". At the same time, the initial anal temperature screening step is added to remove stressed mice in time, which reduces the risk of death of mice due to stress and reduces the interference of stress response on experimental results, making the physiological indicators and pathological characteristics of the model more consistent and improving the stability and reproducibility of the model.

[0033] (3) This invention balances the modeling effect and mortality rate of mouse models by optimizing the heat shock parameters and duration: the modeling time is shortened by optimizing the temperature and humidity program and heat shock parameters, thus solving the problem of low modeling efficiency; at the same time, the heat shock duration is dynamically adjusted according to the real-time anal temperature of the mice, avoiding insufficient or excessive heat shock caused by uniform duration. Under the premise of ensuring that the final body temperature is ≥43℃, the survival rate 1 hour after modeling is improved, thus solving the problem of excessively high mortality rate in the prior art.

[0034] (4) The mouse model provided by this invention has typical pathological features. The mice that have been successfully modeled not only meet the core standard of rectal temperature ≥43℃, but also show pathological features of multi-organ damage similar to those of clinical heatstroke patients, including abnormal blood cell indicators, liver and kidney function damage and typical pathological changes in liver, kidney, lung and small intestine tissues. It provides a highly simulated clinical experimental vehicle for the study of the pathogenesis of heatstroke, drug screening and treatment optimization, and has important scientific research value and application prospects. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0036] Figure 1 The graph showing the change in body temperature (rectal temperature) during mouse modeling provided by this invention;

[0037] Figure 2 This is a graph showing the changes in body weight loss during mouse modeling provided by the present invention;

[0038] Figure 3 The appearance of the mouse model after successful establishment provided by the present invention;

[0039] Figure 4The image shows the results of blood cell analysis in the mouse model provided by this invention.

[0040] Figure 5 The image shows the serum biochemical analysis results of the mouse model provided by this invention.

[0041] Figure 6 Comparison of H&E staining of liver, kidney, lung and small intestine tissues in mice before and after heat shock provided by this invention. Detailed Implementation

[0042] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0043] Please see Figures 1 to 6 :

[0044] This invention provides a method for constructing a classic mouse model of heatstroke, specifically including the following steps:

[0045] S1. Select several SPF-grade male C57BL / 6J mice and transfer them to an artificial climate chamber to adapt to the environment before modeling to obtain pretreated mice;

[0046] S2. On the day of modeling, a thermometer probe coated with a small amount of medical gel coupling agent was inserted into the rectum of the pretreated mouse 2cm with a small back-and-forth rubbing motion to measure the initial body temperature. Mice with an initial body temperature <38.5℃ were selected for modeling, and stressed mice with an initial body temperature ≥38.5℃ were removed. The experimental mice were then deprived of their covers, food, and water, sealed and fixed in their cages, and returned to the artificial climate chamber.

[0047] S3. Heat shock to experimental mice: Start the temperature and humidity control program of the artificial climate chamber and continue for 60-65 minutes until the temperature rises from room temperature to 40°C at a uniform rate, and the relative humidity is maintained at 70% RH.

[0048] S4. Maintain a temperature of 40℃ and a relative humidity of 70%RH for 55-60 minutes. Measure the body temperature of the mice when the heat shock has lasted for 2 hours. Determine the duration of heat shock in segments based on the measured body temperature. Test the righting reflex of the mice after each temperature measurement. At the same time, closely observe and record the behavior of the mice through the viewing window.

[0049] S5. Stop heat therapy if the mouse shows any of the following symptoms: body temperature ≥43℃, loss of righting reflex, or altered consciousness.

[0050] S6. Detect the body temperature of the mice. A body temperature ≥43℃ is used as the standard for successful modeling. Combined with the pathological damage characteristics of multiple organs, the successful model construction is confirmed, and the heatstroke mouse model is obtained.

[0051] Specifically, the mice in S1 were 12–14 weeks old and weighed 30g ± 3g.

[0052] Specifically, the conditions for raising mice in the artificial climate chamber in S1 are a temperature of 18–29°C and a humidity of 30%–70%.

[0053] Specifically, the adaptation time for S1 is ≥24 hours. During the adaptation period, the original feeding cage, food, water and bedding should be retained, and the ventilation window of the cage cover should be opened.

[0054] Specifically, the steps for determining the duration of continued hot strikes in S4 are as follows:

[0055] When the mouse's body temperature is between 41°C and 42°C, continue the heat shock for 30 to 40 minutes.

[0056] When the mouse's body temperature is between 42℃ and 42.5℃, continue the heat shock for 20 to 30 minutes;

[0057] When the mice's body temperature is between 42.5℃ and 43℃, continue the heat shock for 20 minutes.

[0058] Specifically, the parameters and functional requirements of the artificial climate chamber are as follows:

[0059] Temperature adjustment range: 10℃~50℃; control accuracy: ±0.2℃.

[0060] Humidity adjustment range: 10%~99% RH; control accuracy: ±10% RH.

[0061] It is equipped with a fan, heat sink, and temperature control components, and features a transparent window and a cold light source. Indirect ventilation is achieved through the fan, heat sink vents, and temperature control component vents. Temperature, humidity, and light intensity can be dynamically adjusted by setting a program.

[0062] The parameter requirements for a thermometer are as follows:

[0063] The temperature measurement range covers 30℃~45℃, and the temperature measurement accuracy is not less than ±0.2℃;

[0064] The probe rod is longer than 2cm and has a diameter of less than 2mm.

[0065] Specifically, the behavioral status recording items for mice in S4 include activity status, movement posture, respiratory characteristics, appearance, and neurological disorders. Activity status includes curled-up rest, normal activity, hyperactivity, staggering movement, and lying still. Movement posture includes arched back and squinting eyes, smearing saliva, digging holes and burying head, standing and scratching the cage, climbing upside down on the cage rack, stereotyped circling, and limp and leaning against the cage. Respiratory characteristics include heavy panting, rapid and weak breathing, and faint breathing. Appearance includes moist mouth and nose. Neurological disorders include startle reflex, convulsions, stiff tail, and coma.

[0066] Specifically, the consciousness disorders in mice in S5 include at least one of convulsions, seizures, and coma.

[0067] Specifically, the pathological damage characteristics of multiple organs in S6 include: hepatocyte steatosis and disordered arrangement, hepatocyte necrosis and inflammatory cell infiltration in some areas; renal tubular epithelial cell peeling, irregular lumen, glomerular atrophy and deformation, and interstitial inflammatory cell infiltration; alveolar wall thickening, inflammatory cell infiltration, and a large number of red blood cells in the alveolar septa and alveoli; unclear villus structure of small intestinal mucosa, disordered arrangement of epithelial cells, and partial villus malformation and damage.

[0068] The present invention also provides a classic heatstroke mouse model, which is prepared by the above-mentioned method for constructing a classic heatstroke mouse model.

[0069] Example 1:

[0070] Please see Figures 1 to 3 A method for constructing a classic mouse model of heatstroke, specifically including the following steps:

[0071] S1. Select SPF-grade male C57BL / 6J mice under 13 weeks of age and house them at a density of one mouse per cage for at least one week. When the mice reach 12-14 weeks of age, 24 hours before modeling, remove the mice and their original cages from the IVC cage rack, open the ventilation window above the cage lid, retain the original food, water, and bedding, and place the original cages into an artificial climate chamber to continue housing them in advance to adapt to the environment and obtain pre-treated mice.

[0072] Taking a 100L artificial climate chamber as an example, it is advisable to use two mice (cages) for each modeling session. Therefore, when acclimatizing to the environment in advance, three mice (cages) should be placed to allow for replacement if necessary.

[0073] The parameters and functional requirements of the artificial climate chamber are as follows:

[0074] Temperature adjustment range: 10℃~50℃; control accuracy: ±0.2℃.

[0075] Humidity adjustment range: 10%~99% RH; control accuracy: ±10% RH.

[0076] The parameter requirements for small animal thermometers are as follows:

[0077] The temperature measurement range covers 30℃~45℃, and the temperature measurement accuracy is not less than ±0.2℃;

[0078] The probe rod is longer than 2cm and has a diameter of less than 2mm.

[0079] It is equipped with a fan, heat sink, and temperature control components. The fan, heat sink, and temperature control components provide indirect airflow to the outside air, thus avoiding oxygen deficiency in mice during the modeling process. It has a transparent viewing window and six independent cold light sources, which can provide illumination levels 0 to 6. In order to facilitate the observation of the mice's behavior and minimize the stimulation of light on the mice, only level 1 illumination is turned on.

[0080] The specific parameters and procedures for temperature / humidity control are as follows:

[0081] Phase 1: Target temperature 25℃; Target humidity 70% RH; Running time 2 minutes;

[0082] Second stage: target temperature increased by 0.5℃; target humidity 70% RH; running time 2min, this step is repeated 30 times until the temperature reaches 40℃;

[0083] Phase 3: Target temperature 40℃; target humidity 70% RH; the first temperature measurement during the heat shock process was performed on the mice at 60 minutes of running time.

[0084] The above lighting / temperature / humidity control programs only need to be pre-programmed once, and the climate chamber will automatically run according to the program upon subsequent startup.

[0085] S2. On the day of modeling, a thermometer probe coated with a small amount of medical gel coupling agent was inserted into the rectum of a pretreated mouse 2cm with a small back-and-forth rubbing motion to measure the initial body temperature. After the thermometer reading stabilized, the body temperature was read. Mice with an initial body temperature <38.5℃ were selected for modeling, and stressed mice with an initial body temperature ≥38.5℃ were removed to obtain experimental mice.

[0086] Mice with an initial rectal temperature ≥38.5℃ are prone to death during heat shock and are identified as stressed mice, which need to be replaced in time; mice with an initial rectal temperature <38.5℃ are considered normal and can be used for subsequent modeling.

[0087] S3. Remove the cage cover, food, and water bottle, put the mouse back into the cage, fix the cage rack and seal it to prevent escape; then put the cage back into the artificial climate chamber, start the preset temperature and humidity control program, and continue for 60-65 minutes until the temperature rises from room temperature to 40℃ at a uniform rate, and the relative humidity is maintained at 70% RH.

[0088] S4. Maintain a temperature of 40℃ and a relative humidity of 70%RH for 55–60 minutes, until the heat shock has lasted for 2 hours. Measure the mouse's body temperature then, and determine the duration of the heat shock in segments based on the measured body temperature values. The specific steps are as follows:

[0089] When the mouse's body temperature is between 41°C and 42°C, continue the heat shock for 30 to 40 minutes.

[0090] When the mouse's body temperature is between 42℃ and 42.5℃, continue the heat shock for 20 to 30 minutes;

[0091] When the mice's body temperature was between 42.5℃ and 43℃, continue the heat shock for 20 minutes;

[0092] This design can adapt to individual differences in mice, avoiding the problem of insufficient or excessive heat shock for some mice due to uniform heat shock duration. During heat shock, the lid should be removed to improve the heat exchange efficiency inside and outside the cage. At the same time, the cage rack should be sealed and fixed with breathable material to prevent mice from escaping.

[0093] After each temperature measurement, when returning the mouse to the cage, its abdomen should be facing upwards to check whether its righting reflex is normal. At the same time, the mouse's behavior and status should be closely observed and recorded through the viewing window. The disappearance of the righting reflex is considered one of the important signs of termination of the model.

[0094] Behavioral states specifically include: activity status, movement and posture, respiratory characteristics, appearance, and neurological disorders. Activity status includes curling up to rest, normal activity, hyperactivity, unsteady movement, and lying still. Movement and posture include arching back and squinting, smearing saliva, digging holes and burying head, standing and scratching at the cage, climbing backwards on the cage rack, stereotyped circling, and limply leaning against the cage. Respiratory characteristics include heavy panting, rapid and weak breathing, and faint breathing. Appearance includes a moist mouth and nose. Neurological disorders include startle reflexes, convulsions, rigid tail lifting, and coma.

[0095] S5. Stop heat therapy if the mouse shows any of the following symptoms: body temperature ≥43℃, loss of righting reflex, or altered consciousness.

[0096] Disorders of consciousness in mice include at least one of the following: seizures, convulsions, or coma.

[0097] S6. Detect mouse body temperature, using a body temperature ≥43℃ as the standard for successful model establishment. Confirm successful model establishment by combining this with multi-organ pathological damage characteristics, thus obtaining the heatstroke mouse model. The appearance of mice after successful model establishment is as follows: Figure 3 As shown;

[0098] The pathological features of multiple organ damage include: hepatocyte steatosis and disordered arrangement, hepatocyte necrosis and inflammatory cell infiltration in some areas; renal tubular epithelial cell peeling, irregular lumen, glomerular atrophy and deformation, and interstitial inflammatory cell infiltration; alveolar wall thickening, inflammatory cell infiltration, and a large number of red blood cells in the alveolar septa and alveoli; unclear villus structure of small intestinal mucosa, disordered arrangement of epithelial cells, and partial villus malformation and damage.

[0099] The body temperature change curves during mouse modeling (n=10) are shown below. Figure 1As shown: Before heat shock, the rectal temperature of mice was within the normal range of 37.2-38.4℃, and the initial state was consistent with no stress. From 0 to 2 hours, the body temperature slowly increased to 40℃ as the temperature in the climate chamber increased, avoiding acute stress death. From 2 to 3 hours, with the continuation of heat shock, the body temperature of mice entered a plateau period, reflecting that the thermoregulatory mechanism was functioning to resist excessive temperature rise. After breaking through the plateau period, the body temperature rose rapidly, reflecting the failure of the thermoregulatory mechanism. All 10 mice reached the modeling endpoint of above 43℃. The survival rate was high 1 hour after modeling, and the individual change trends were consistent, proving that the modeling method of the present invention is reproducible and stable.

[0100] The changes in behavior, appearance, and weight loss in mice at different body temperatures during the mouse modeling process are shown in Table 1 and... Figure 2 As shown:

[0101] Table 1. Changes in behavior, appearance, and weight loss in mice at different body temperatures.

[0102] Body temperature Behavioral appearance Rate of body weight loss 40℃(n=10) Curling up, normal activity 6.31±1.81% 41℃(n=10) Active, digging, head down, standing, inverted, smearing saliva, wet muzzle and nares, panting, labored breathing 7.16±1.87% 42℃ (n=10) Stumbling, smearing saliva, wet muzzle and nares, head down, standing, inverted, stereotypic circling, hunched, eyes closed, recumbent, labored breathing, weak breathing 7.74±2.29% 43℃ (n=10) Recumbent, weak breathing, jumping, convulsions, stiff tail, coma, loss of righting reflex 11.09±1.51%

[0103] The weight loss rate of the 10 mice showed a significant increasing trend with rising body temperature, and a leap in growth occurred when the body temperature rose from 42°C to 43°C. This change was highly synchronized with the progression of behavioral appearance and pathological damage.

[0104] During the hypothermic phase (40-42℃): the rate of weight loss increases slowly, mainly due to physiological water loss caused by evaporation of body surface water and heat dissipation through respiration in high-temperature environments, as well as energy consumption caused by increased metabolic rate. At this time, the body has not yet suffered serious organic damage, and weight loss can be partially recovered through fluid replacement and other means.

[0105] High body temperature stage (43℃): The weight loss rate increases sharply. The weight loss rate in this stage can be used as one of the indicators to help judge the success of modeling. Together with body temperature, behavioral appearance and subsequent blood cell and biochemical indicators, it constitutes a complete model validation system.

[0106] Example 2:

[0107] Please see Figures 4 to 6 This embodiment provides a technical solution based on embodiment one: a verification experiment for the successful establishment of a heatstroke mouse model, including blood cell analysis experiment, serum biochemical analysis experiment and H&E staining experiment of tissue sections of various organs.

[0108] (a) Blood cell analysis experiments:

[0109] Pre-spray EDTA anticoagulant into the EP tube, then collect 100 μL (approximately 3 drops) of orbital sinus blood from mice and send it to a veterinary hematology analyzer for testing as soon as possible. The experimental results are as follows: Figure 4 As shown:

[0110] Compared with the control group, the heat shock experimental groups (heat shock temperatures of 40℃, 42℃, and 43℃) all showed a trend of decreased white blood cell count, decreased neutrophil count, increased eosinophil count, decreased monocyte count, decreased lymphocyte count, and decreased platelet count; among them, there were statistically significant differences in all indicators between the control group and the 43℃ group.

[0111] (II) Serum biochemical analysis experiments:

[0112] 0.2 ml of orbital sinus blood was collected from mice using sterile, additive-free EP tubes. The blood was allowed to stand at room temperature for at least 1 hour until natural coagulation. Serum was extracted by centrifugation twice at 3000 rpm for 10 minutes. The supernatant was collected and stored in EP tubes at -20°C, avoiding repeated freeze-thaw cycles. After thawing at room temperature, the samples were sent to a biochemical analyzer for testing. The tests included liver function (AST and ALT) and kidney function (BUN and creatinine). Samples could be diluted 2–5 times before analysis. Experimental results are as follows: Figure 5 As shown:

[0113] Compared with the control group, the heat shock experimental groups (heat shock temperatures of 40℃, 41℃, 42℃, and 43℃) all showed an increasing trend of liver and kidney injury markers; among them, there were statistically significant differences in all indicators between the control group and the 43℃ group.

[0114] (III) H&E staining experiment on multiple organ tissue sections:

[0115] Tissue sampling and fixation: After the modeling was terminated, the mice were quickly euthanized and the liver, kidney, lung and small intestine tissues were dissected and separated. The tissues were trimmed into 0.5cm×0.5cm×0.3cm pieces and immediately placed in pre-cooled 4% paraformaldehyde fixative solution and fixed at 4℃ for 24h.

[0116] Tissue dehydration: Take out the tissue and rinse it with distilled water 3 times, 5 minutes each time; then soak it in 70%, 80%, 90%, and 95% ethanol for 1 hour each and 100% ethanol twice for 30 minutes each time to gradually remove water from the tissue.

[0117] Clearing treatment: The dehydrated tissue was immersed in xylene solution twice, 20 minutes each time, for clearing treatment;

[0118] Paraffin embedding: The transparent tissue was immersed twice in molten paraffin in a 60℃ constant temperature paraffin box for 1 hour each time, and then placed in an embedding mold to pour paraffin and cool and solidify to form an embedding block.

[0119] Section preparation: The embedded blocks were fixed on a paraffin microtome and 5μm continuous sections were cut. The sections were flattened on a 40℃ spreader and baked in a 60℃ oven for 2 hours.

[0120] Dewaxing and hydration: The baked slices were dewaxed twice with xylene, 10 min each time; then hydrated with a gradient of ethanol, 100% ethanol twice for 5 min each, 95%, 90%, 80%, and 70% ethanol for 5 min each, and then rinsed with distilled water three times for 5 min each time.

[0121] H&E staining: First, stain with hematoxylin solution at room temperature for 5-10 min, rinse with tap water, differentiate with 1% hydrochloric acid ethanol for 30 seconds, then blue with warm water for 5 min, followed by staining with eosin solution at room temperature for 2-5 min. After staining, dehydrate twice with 95% and 100% ethanol, 3 min each time, then clear with xylene twice, 5 min each time. Mount with neutral resin and air dry. Finally, place the slides under an optical microscope, select typical fields of view for observation, and acquire images for pathological feature analysis. Experimental results are as follows: Figure 6 As shown:

[0122] Liver: In the control group, hepatocytes around the central vein of the liver were arranged radially, with abundant cytoplasm, round nuclei located in the center, and clear lobular structure. In the 43℃ model group, a large number of hepatocytes showed fatty degeneration, disordered arrangement of hepatocytes, unclear intercellular boundaries, blurred lobular structure, and hepatocyte necrosis and inflammatory cell infiltration in some areas.

[0123] Kidneys: In the control group, the glomeruli and tubules were clearly structured, the tubular epithelial cells were neatly arranged, and no obvious inflammatory cell infiltration or fibrosis was observed; in the 43℃ model group, the tubular epithelial cells were peeled off, the lumen was irregular and partially collapsed, the glomeruli were atrophied and deformed, and there was significant inflammatory cell infiltration in the interstitium.

[0124] Lungs: The control group had abundant alveoli around the bronchi, with intact alveolar structure and thin alveolar walls, and the columnar epithelial cells of the bronchi were arranged neatly; the alveolar walls of the 43℃ model group were significantly thickened, accompanied by inflammatory cell infiltration, and the alveolar septa and alveoli contained a large number of red blood cells, indicating lung congestion and edema.

[0125] Small intestine: In the control group, the villi of the small intestinal mucosa were arranged regularly and intact, and the epithelial cells were tightly arranged; in the 43℃ model group, the structure of the small intestinal mucosa villi was unclear, the epithelial cells were arranged in a disordered manner, some villi were deformed and damaged, and a lot of inflammatory cell infiltration was visible, indicating that the intestinal mucosal barrier was damaged.

[0126] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a classic mouse model of heatstroke, characterized in that, Specifically, the following steps are included: S1. Select several SPF-grade male C57BL / 6J mice and transfer them to an artificial climate chamber to adapt to the environment before modeling to obtain pretreated mice; S2. On the day of modeling, a thermometer probe coated with medical gel coupling agent was inserted 2cm into the rectum of a pretreated mouse using a small back-and-forth rubbing motion to measure the initial body temperature. Mice with an initial body temperature <38.5℃ were selected for modeling, and stressed mice with an initial body temperature ≥38.5℃ were removed. The experimental mice were then deprived of their covers, food, and water, their cages were sealed and fixed, and they were placed back into the artificial climate chamber. S3. Heat shock to experimental mice: Start the temperature and humidity control program of the artificial climate chamber and continue for 60-65 minutes until the temperature rises from room temperature to 40°C at a uniform rate, and the relative humidity is maintained at 70% RH. S4. Maintain a temperature of 40℃ and a relative humidity of 70%RH for 55-60 minutes. Measure the body temperature of the mice when the heat shock has lasted for 2 hours. Determine the duration of heat shock in segments based on the measured body temperature. Test the righting reflex of the mice after each temperature measurement. At the same time, closely observe and record the behavior of the mice through the viewing window. S5. Stop heat therapy if the mouse shows any of the following symptoms: body temperature ≥43℃, loss of righting reflex, or altered consciousness. S6. Detect the body temperature of the mice. A body temperature ≥43℃ is used as the standard for successful modeling. Combined with the pathological damage characteristics of multiple organs, the successful model construction is confirmed, and the heatstroke mouse model is obtained.

2. The method for constructing a classic heatstroke mouse model according to claim 1, characterized in that, The mice in S1 were 12–14 weeks old and weighed 30g ± 3g.

3. The method for constructing a classic heatstroke mouse model according to claim 1, characterized in that, The conditions for raising mice in the artificial climate chamber in S1 are a temperature of 18–29°C and a humidity of 30%–70%.

4. The method for constructing a classic heatstroke mouse model according to claim 1, characterized in that, The adaptation time in S1 is ≥24h. During the adaptation period, the original feeding cage, food, water and bedding are retained, and the ventilation window of the cage cover is opened.

5. The method for constructing a classic heatstroke mouse model according to claim 1, characterized in that, The specific steps for determining the duration of continued thermal strike in S4 are as follows: When the mouse's body temperature is between 41°C and 42°C, continue the heat shock for 30 to 40 minutes. When the mouse's body temperature is between 42℃ and 42.5℃, continue the heat shock for 20 to 30 minutes. When the mice's body temperature is between 42.5℃ and 43℃, continue the heat shock for 20 minutes.

6. The method for constructing a classic heatstroke mouse model according to claim 1, characterized in that, The parameters and functional requirements of the artificial climate chamber are as follows: Temperature adjustment range: 10℃~50℃; control accuracy: ±0.2℃. Humidity adjustment range: 10%~99% RH; control accuracy: ±10% RH. It is equipped with a fan, heat sink and temperature control components, and has a transparent window and cold light source. Indirect ventilation is achieved through the fan, heat sink vents and temperature control component vents. Temperature, humidity and light intensity can be dynamically adjusted by setting a program. The parameters required for the thermometer are as follows: The temperature measurement range covers 30℃~45℃, and the temperature measurement accuracy is not less than ±0.2℃; The probe rod is longer than 2cm and has a diameter of less than 2mm.

7. The method for constructing a classic heatstroke mouse model according to claim 1, characterized in that, The mouse behavior status recording items in S4 include activity status, movement posture, respiratory characteristics, appearance and neurological disorders. Activity status includes curling up to rest, normal activity, hyperactivity, staggering, and lying still. Movement posture includes arching back and squinting, smearing saliva, digging holes and burying head, standing and scratching the cage, climbing upside down on the cage rack, stereotyped circling, and limp and leaning against the cage. Respiratory characteristics include heavy, rapid breathing, rapid and weak breathing, and faint breathing; appearance includes a moist mouth and nose; neurological disorders include startle reflexes, convulsions, rigidity, tail curling, and coma.

8. The method for constructing a classic heatstroke mouse model according to claim 1, characterized in that, The consciousness disorder in mice in S5 includes at least one of convulsions, seizures, and coma.

9. The method for constructing a classic heatstroke mouse model according to claim 1, characterized in that, The pathological damage characteristics of multiple organs in S6 include: hepatocyte steatosis and disordered arrangement, hepatocyte necrosis and inflammatory cell infiltration in some areas; renal tubular epithelial cell peeling, irregular lumen, glomerular atrophy and deformation, and interstitial inflammatory cell infiltration; alveolar wall thickening, inflammatory cell infiltration, and a large number of red blood cells in the alveolar septa and alveoli; unclear villus structure of small intestinal mucosa, disordered arrangement of epithelial cells, and partial villus deformity and damage.

10. A classic mouse model of heatstroke, characterized in that, It is prepared by the method for constructing a classic heatstroke mouse model as described in any one of claims 1-9.

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