Establishment method of respirator combined non-exposed type tracheal instillation rat silicosis model

Abstract

The invention discloses an establishment method of a respirator combined non-exposed type tracheal instillation rat silicosis model. The establishment method comprises the following operation steps: step 1, preprocessing; step 2, trachea intubation; step 3, performing tracheal instillation; step 4, combining breathing machines; 5, observing and recording; and step 6, processing and analyzing. The small animal breathing machine is connected to assist ventilation of the rat after each time of tracheal instillation, silica dust can be effectively diffused to pulmonary alveoli of the rat, the success rate of modeling is guaranteed, the rat can breathe smoothly, and therefore the survival rate of the rat is guaranteed.

Description

Technical Field

[0001] The invention relates to the field of silicosis model establishment, and in particular to a method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation. Background Art

[0002] Occupational silicosis (hereinafter referred to as "silicosis") is an ancient and potentially fatal occupational pneumoconiosis, for which there is currently no effective treatment and the pathogenesis is still unclear. The establishment of an animal model is very important for the study of the pathogenesis of silicosis and can lay a theoretical foundation for the subsequent effective treatment of silicosis.

[0003] Existing studies mostly use rats and mice to establish silicosis models. Considering factors such as survival rate, modeling success rate and feeding cost, rats are the most widely used in silicosis modeling. Among the existing modeling methods, the dusting device of the dynamic and static oral and nasal inhalation dusting method is expensive, the dusting time is long, the dusting dose is difficult to control, and the animal infection rate and mortality rate during the dusting process are high; although the extrapulmonary dusting method is simple to operate, it is different from the situation where workers are exposed to dust in the actual workplace; although the non-exposure intranasal instillation method is simple to operate, the instillation dose is not easy to control, and a large instillation dose can easily cause the death of rats. Therefore, the above methods are not suitable for use as models for clinical research on silicosis.

[0004] The intratracheal instillation method is simple to operate, the dust dosage is easy to control, and the success rate is high. It is currently a classic method for silicosis modeling. It can be divided into exposed intratracheal injection method and non-exposed intratracheal injection method. The exposed intratracheal injection method is complicated to operate and requires surgical exposure of the trachea, which can easily cause bleeding, infection, and even death in rats. The non-exposed intratracheal injection method is widely used because it takes a short operation time and is less likely to cause infection and death in animals.

[0005] Domestic and foreign studies have mostly used non-exposed intratracheal infusion to establish a rat silicosis model. Due to the rapid respiratory rate and glottis stenosis of rats, many studies in recent years have improved the non-exposed intratracheal infusion method, such as blind tracheal intubation, but its success rate is low, and frequent and repeated intubation can easily cause laryngeal edema and airway damage or even death in rats.

[0006] In the past, when the traditional intratracheal instillation method was used to establish a rat silicosis model, small animal ventilator-assisted ventilation was not used after endotracheal intubation and instillation, and the ventilation of the rats could not be effectively guaranteed, which to a certain extent increased the risk of death in the rat modeling process. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the defects of the prior art in establishing a rat silicosis model, namely, the complex operation and low success rate, and to provide a method for establishing a rat silicosis model using a ventilator combined with non-exposed tracheal instillation.

[0008] The present invention solves the above technical problems through the following technical solutions:

[0009] The present invention provides a method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation, the method comprising the following steps:

[0010] Step 1: Pretreatment: anesthetize and fix the rats;

[0011] Step 2: Intubation: Use a small laryngoscope to expose the glottis of the rat and gently insert the tracheal cannula into the airway through the glottis.

[0012] Step 3: tracheal instillation: 1.0 mL of 0.9% sodium chloride solution and 50 g / L silica suspension were slowly instilled into the trachea of ​​the two groups of rats through the tracheal intubation needle for 4 times;

[0013] Step 4: Connect the tracheal cannula to the ventilator for assisted ventilation after each tracheal instillation. When the tidal volume reaches 2.0 mL, perform the next instillation. After the last instillation, connect the ventilator. When the tidal volume is about 2.0 mL, remove the tracheal cannula. After the ventilator is removed, place the rat in a cage and wait for it to wake up.

[0014] Step 5: Observe and record the body weights of the two groups of rats before the experiment and at 1, 2, 3, and 4 weeks after modeling;

[0015] Step 6: Processing and analysis: obtain lung tissues of the two groups of rats, and use hematoxylin-eosin staining and Masson staining to analyze the pathological changes of the lung tissues of the two groups of rats.

[0016] In the present technical scheme, a small animal ventilator is connected to assist rat ventilation after each tracheal instillation, which is beneficial to the effective diffusion of silica dust to the rat alveoli, which not only ensures the success rate of modeling, but also enables the rat to breathe smoothly, thereby ensuring its survival rate. The small animal laryngoscope inserts the cannula needle into the trachea through the glottis under direct vision, and then the cannula needle is connected to the infusion tube with 0.1mL of 0.9% sodium chloride solution to determine whether the cannula needle is in the trachea. Through the respiratory rhythmic movement of the rat, a small amount of water column fluctuations in the infusion tube are observed to determine whether the trachea is successfully inserted, and a small amount of 0.9% sodium chloride solution in the infusion tube will not be inhaled into the lungs of the rat. After each tracheal instillation, a small animal ventilator is connected to assist rat ventilation, which is beneficial to the effective diffusion of silica dust to the rat alveoli, which not only ensures the success rate of modeling, but also enables the rat to breathe smoothly, thereby ensuring its survival rate.

[0017] Preferably, in step 1, an even number of rats are selected and randomly divided into an experimental group and a control group, and the number of rats in the experimental group and the control group is the same.

[0018] Preferably, the specific operation steps of the pretreatment in step 1 are:

[0019] S1. Anesthesia: Rats were anesthetized by intraperitoneal injection of 3.0% sodium pentobarbital solution at 45 mg / kg body weight. The depth of anesthesia was determined by the absence of contraction reaction when pinching the skin or toes of the rats.

[0020] S2. Fixation: Use rubber bands to fix the rat's incisor and limbs to the rat fixing table so that its head and neck are in a straight line with the experimenter's line of sight.

[0021] Preferably, in step 2, the needle core is pulled out after the insertion length of the tracheal cannula needle is about 3 / 4 of the cannula needle.

[0022] Preferably, in step 2, an infusion tube containing 0.1 mL of 0.9% sodium chloride solution is connected to the intubation via the tracheal cannula needle. If the liquid in the infusion tube moves back and forth with the rat's breathing, it indicates that the intubation is successful.

[0023] Preferably, the preparation of the silica suspension in step 3 is to take 50 mg of silica particles and grind them thoroughly in an agate mortar so that the silica particles with a particle size of <5.0 μm reach more than 90.0%, dry them at 180° C. for 6 h, and then add 1.0 mL of 0.9% sodium chloride solution and mix well.

[0024] Preferably, in the step S2, after the rat is fixed, the hair of the rat is trimmed from the lower jaw to the neck area above the sternum, and the rat is disinfected with iodine or alcohol.

[0025] Preferably, in step six, hematoxylin-eosin staining, i.e., HE staining, is used. Hematoxylin staining solution is alkaline, which mainly stains the chromatin in the cell nucleus and the ribosomes in the cytoplasm purple-blue; eosin is an acidic dye, which mainly stains the components in the cytoplasm and the extracellular matrix red.

[0026] Preferably, the Masson staining in step six is ​​also called Masson staining, which is related to the size of the anionic dye molecules and the penetration of the tissue. The size of the molecule is reflected by the molecular weight. Small molecular weight can easily penetrate densely structured and low-permeability tissues, while large molecular weight can only enter loosely structured and high-permeability tissues. However, the molecular weight of light green or aniline blue is very large, so after Masson staining, muscle fibers appear red and collagen fibers appear green or blue, which is mainly used to distinguish collagen fibers from muscle fibers.

[0027] Preferably, the selection criteria for the rats are: specific pathogen-free healthy male SD rats, 6 weeks old, with a body weight of (200±20) g, free access to food and water, and after 2 weeks of adaptive feeding, the rats are randomly divided into an experimental group and a control group.

[0028] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0029] The positive and progressive effects of the present invention are:

[0030] The invention connects a small animal ventilator to assist rat ventilation after each tracheal instillation, which is beneficial to the effective diffusion of silica dust to the rat alveoli, ensures the success rate of modeling, and enables the rat to breathe smoothly, thereby ensuring its survival rate. The small animal laryngoscope inserts the cannula needle into the trachea through the glottis under direct vision, and then connects the cannula needle to an infusion tube with 0.1 ml of 0.9% sodium chloride solution to judge whether the cannula needle is in the trachea. Through the respiratory rhythmic movement of the rat, a small amount of water column fluctuation in the infusion tube is observed, thereby judging whether the trachea is successfully inserted. A small amount of 0.9% sodium chloride solution in the infusion tube will not be inhaled into the lungs of the rat. The small animal ventilator is connected to assist rat ventilation after each tracheal instillation, which is beneficial to the effective diffusion of silica dust to the rat alveoli, ensures the success rate of modeling, and enables the rat to breathe smoothly, thereby ensuring its survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure is a schematic diagram of the overall process of the method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation according to an embodiment of the present invention.

[0032] Figure 2 for Figure 1 The schematic diagram of the specific operation flow of the pretreatment of the method for establishing a silicosis model in rats using a ventilator combined with non-exposed tracheal instillation is shown.

[0033] Figure 3 for Figure 1 Schematic diagram of the changes in body weight of two groups of rats in the method for establishing a rat silicosis model using a ventilator combined with non-exposed tracheal instillation.

[0034] Figure 4 for Figure 1 Schematic diagram of the pathological changes in lung tissue HE staining of rats in each group of the method for establishing a rat silicosis model using a ventilator combined with non-exposed tracheal instillation.

[0035] Figure 5 for Figure 1 Schematic diagram of Masson staining pathological changes in lung tissue of each group of rats in the method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation. DETAILED DESCRIPTION

[0036] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0037] Figure 1 to Figure 2Shown is a structural schematic diagram of an embodiment of a method for establishing a rat silicosis model using a ventilator combined with non-exposed tracheal instillation according to the present invention.

[0038] In this embodiment, 20 rats are taken as an example, and the 20 rats are divided into an experimental group and a control group, with 10 rats in each group.

[0039] A method for establishing a silicosis model in rats using a ventilator combined with non-exposed tracheal instillation, the method comprising the following steps:

[0040] Step 1: Pretreatment: anesthetize and fix the rats;

[0041] Step 2: Intubation: Use a small laryngoscope to expose the glottis of the rat and gently insert the tracheal cannula into the airway through the glottis.

[0042] Step 3: tracheal instillation: 1.0 mL of 0.9% sodium chloride solution and 50 g / L silica suspension were slowly instilled into the trachea of ​​the two groups of rats through the tracheal intubation needle for 4 times;

[0043] Step 4: Connect the tracheal cannula to the ventilator for assisted ventilation after each tracheal instillation. When the tidal volume reaches 2.0 mL, perform the next instillation. After the last instillation, connect the ventilator. When the tidal volume is about 2.0 mL, remove the tracheal cannula. After the ventilator is removed, place the rat in a cage and wait for it to wake up.

[0044] Step 5: Observe and record the body weights of the two groups of rats before the experiment and at 1, 2, 3, and 4 weeks after modeling;

[0045] Step 6: Processing and analysis: obtain lung tissues of the two groups of rats, and use hematoxylin-eosin staining and Masson staining to analyze the pathological changes of the lung tissues of the two groups of rats.

[0046] The measurement data in step 6 were tested for normality and were described by xˉ±s. The weight changes of the two groups of rats at different time points were compared by repeated measures analysis of variance. The comparison of the means between multiple groups was performed by one-way analysis of variance, and the comparison of the means within the groups was performed by LSD test. The test level α = 0.05 (bilateral).

[0047] In the present technical scheme, a small animal ventilator is connected to assist rat ventilation after each tracheal instillation, which is beneficial to the effective diffusion of silica dust to the rat alveoli, which not only ensures the success rate of modeling, but also enables the rat to breathe smoothly, thereby ensuring its survival rate. The small animal laryngoscope inserts the cannula needle into the trachea through the glottis under direct vision, and then the cannula needle is connected to the infusion tube with 0.1mL of 0.9% sodium chloride solution to determine whether the cannula needle is in the trachea. Through the respiratory rhythmic movement of the rat, a small amount of water column fluctuation in the infusion tube is observed to determine whether the trachea is successfully inserted, and a small amount of 0.9% sodium chloride solution in the infusion tube will not be inhaled into the lungs of the rat. After each tracheal instillation, a small animal ventilator is connected to assist rat ventilation, which is beneficial to the effective diffusion of silica dust to the rat alveoli, which not only ensures the success rate of modeling, but also enables the rat to breathe smoothly, thereby ensuring its survival rate.

[0048] During the experiment, no rats in the two groups died. The rats in the experimental group showed low spirits after modeling, and their diet and activities were reduced compared with those in the control group. Their body weight increased slowly. Five rats were selected from each group and recorded as follows: Figure 3 shown.

[0049] During the dust exposure process, the rats in the control group had normal lung tissue morphology and structure, normal alveolar cavity size, no inflammatory cell infiltration around, thin alveolar wall, and only a small amount of fine collagen distribution around the pulmonary interstitium and bronchi. The rats in the experimental group had mild thickening of alveolar wall at 2 weeks after modeling, interstitial lymphocytes and macrophage infiltration, mild hyperplasia of fibrous tissue, and a small amount of fibroblasts; at 4 weeks after modeling, the alveolar wall was significantly thickened, fibrous nodules were formed, fibroblasts were contained, and the number of fibroblasts and collagen fibers increased significantly, such as Figure 4 and Figure 5 As shown;

[0050] The results showed that the new non-exposed tracheal instillation method - ventilator combined with non-exposed tracheal instillation method can successfully establish a rat silicosis model and ensure the survival rate of the rats.

[0051] in, Figure 4 Middle, A: Week 2 (×40); the black arrows in the control group indicate the normal alveolar wall structure, and the red arrows in the experimental group indicate the slightly thickened alveolar wall;

[0052] B: Week 4 (×40); the blue arrows in the experimental group indicate pulmonary fibrous nodules;

[0053] C: Week 2 (×200); in the experimental group, the green arrows indicate the proliferating macrophages, and the yellow arrows indicate the proliferating lymphocytes;

[0054] D: Week 4 (×200); the white arrows in the experimental group indicate the proliferating fibroblasts.

[0055] Figure 5 Middle, A: Week 2 (×40);

[0056] B: Week 4 (×40);

[0057] C: Week 2 (×200), the black arrows in the experimental group indicate the deposition of blue collagen fibers;

[0058] D: Week 4 (×200), the white arrows in the experimental group indicate large, dense blue collagen fiber deposition.

[0059] The endotracheal intubation method adopted has made innovations in experimental materials and instruments, which reduces the difficulty of endotracheal intubation and is simple and easy to learn. At the same time, the method of connecting the rat tracheal instillation to a ventilator ensures the survival rate of the rats and the success rate of replicating the silicosis model. This new non-exposed intratracheal instillation method - ventilator combined with non-exposed endotracheal instillation method is simple, safe and effective, and can provide a good foundation for the study of silicosis animal models.

[0060] In the step 1, an even number of rats are selected and randomly divided into an experimental group and a control group, wherein the number of rats in the experimental group and the control group is the same.

[0061] The specific operation steps of the pretreatment in step 1 are:

[0062] S1. Anesthesia: Rats were anesthetized by intraperitoneal injection of 3.0% sodium pentobarbital solution at 45 mg / kg body weight. The depth of anesthesia was determined by the absence of contraction reaction when pinching the skin or toes of the rats.

[0063] S2. Fixation: Use rubber bands to fix the rat's incisor and limbs to the rat fixing table so that its head and neck are in a straight line with the experimenter's line of sight.

[0064] In the step 2, the needle core is pulled out after the insertion length of the tracheal cannula needle is about 3 / 4 of the cannula needle.

[0065] In the step 2, an infusion tube containing 0.1 mL of 0.9% sodium chloride solution is connected to the intubation through the tracheal cannula needle. If the liquid in the infusion tube moves back and forth with the rat's breathing, it indicates that the intubation is successful.

[0066] For endotracheal intubation, an 18-gauge intravenous indwelling needle is converted into an endotracheal cannula needle; a flexible tube of about 3 cm is retained, the distal end is cut into an inclined surface, and the blunted needle core is inserted into the flexible tube, making it slightly shorter than the flexible tube.

[0067] The preparation of the silica suspension in step 3 is to take 50 mg of silica particles and grind them thoroughly in an agate mortar so that the silica particles with a particle size of less than 5.0 μm reach more than 90.0%, dry them at 180° C. for 6 h, and then add 1.0 mL of 0.9% sodium chloride solution and mix well.

[0068] In the step S2, after the rat is fixed, the hair of the rat is trimmed from the lower jaw to the neck area above the sternum, and the rat is disinfected with iodine and alcohol.

[0069] In step six, hematoxylin-eosin staining is HE staining. Hematoxylin staining solution is alkaline, which mainly stains the chromatin in the cell nucleus and the ribosomes in the cytoplasm purple-blue; eosin is an acidic dye, which mainly stains the components in the cytoplasm and the extracellular matrix red.

[0070] The Masson staining in step six is ​​also called Masson staining, which is related to the size of the anionic dye molecules and the penetration of the tissue. The size of the molecule is reflected by the molecular weight. Small molecular weight can easily penetrate densely structured and low-permeability tissues, while large molecular weight can only enter loosely structured and high-permeability tissues. However, the molecular weight of light green or aniline blue is very large, so after Masson staining, muscle fibers appear red and collagen fibers appear green or blue, which is mainly used to distinguish collagen fibers from muscle fibers.

[0071] The observation and analysis of rats were divided into general observation and lung tissue pathology examination;

[0072] General observation: observe the mental state, diet, excrement and other basic conditions of the two groups of rats, and record the body weight of the two groups of rats before the experiment and at the 1st, 2nd, 3rd and 4th week after modeling.

[0073] Pathological examination of lung tissue: At the 2nd and 4th week after modeling, 5 rats in each group were killed by bleeding, and the right lung tissue was obtained and fixed with 4.0% paraformaldehyde solution. After routine rinsing, dehydration, paraffin embedding and sectioning, HE staining and Masson staining were used to observe the pathological changes of lung tissue under an optical microscope.

[0074] The selection criteria for the rats are: specific pathogen-free healthy male SD rats, 6 weeks old, with a body weight of (200±20) g, free access to food and water, and after 2 weeks of adaptive feeding, the rats are randomly divided into an experimental group and a control group.

[0075] Specific pathogen-free healthy male SD rats were from Beijing Spefoc Biotechnology Co., Ltd., with experimental animal use license number: SYXK(E)2016-0057 and experimental animal production license number: SCXK(Beijing)2019-0010.

[0076] In this application, the intravenous indwelling needle cannula is converted into a tracheal cannula needle, and the tracheal intubation is performed under direct vision using a light source animal small laryngoscope to ensure the smooth progress of the subsequent silicosis modeling intratracheal instillation method, and to improve the tracheal intubation method to reduce the difficulty of intubation;

[0077] Connect the cannula needle to the infusion tube with 0.1mL of 0.9% sodium chloride solution, observe the fluctuation of a small amount of water column in the infusion tube through the rat's respiratory rhythmic movement, and judge whether the trachea is successfully inserted, so as to improve the efficiency of tracheal intubation;

[0078] After tracheal instillation, the rats were connected to a small animal ventilator to assist ventilation, which was beneficial for the effective diffusion of silica dust into the rat alveoli and made the rats breathe smoothly, thus ensuring the success rate of modeling and the survival of the rats.

[0079] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that this is only for illustration and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A method for establishing a silicosis model in rats using a ventilator combined with non-exposed tracheal instillation, characterized in that: The establishment method comprises the following steps: Step 1: Pretreatment: anesthetize and fix the rats; Step 2: endotracheal intubation: use a small animal laryngoscope with a light source to expose the rat's glottis, and gently insert the endotracheal cannula needle into the airway through the glottis; Step 3: tracheal instillation: 1.0 mL of 0.9% sodium chloride solution and 50 g / L silica suspension were slowly instilled into the trachea of ​​the two groups of rats through the tracheal intubation needle in four times; Step 4: Connect the tracheal cannula to the ventilator for assisted ventilation after each tracheal instillation. When the tidal volume reaches 2.0 mL, perform the next instillation. After the last instillation, connect the ventilator. When the tidal volume is about 2.0 mL, remove the tracheal cannula. After the ventilator is removed, place the rat in a cage and wait for it to wake up. Step 5: Observe and record the body weights of the two groups of rats before the experiment and at 1, 2, 3, and 4 weeks after modeling; Step 6: Processing and analysis: obtain lung tissues of the two groups of rats, and use hematoxylin-eosin staining and Masson staining to analyze the pathological changes of the lung tissues of the two groups of rats.

2. The method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation as claimed in claim 1, characterized in that: In the step 1, an even number of rats are selected and randomly divided into an experimental group and a control group, wherein the number of rats in the experimental group and the control group is the same.

3. The method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation as claimed in claim 1, characterized in that: The specific operation steps of the pretreatment in step 1 are: S1. Anesthesia: Rats were anesthetized by intraperitoneal injection of 3.0% sodium pentobarbital solution at 45 mg / kg body weight. The depth of anesthesia was determined by the absence of contraction reaction when pinching the skin or toes of the rats. S2. Fixation: Use rubber bands to fix the rat's incisor and limbs to the rat fixing table so that its head and neck are in a straight line with the experimenter's line of sight.

4. The method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation as claimed in claim 1, characterized in that: In the step 2, the needle core is pulled out after the insertion length of the tracheal cannula needle is about 3 / 4 of the cannula needle.

5. The method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation as claimed in claim 1, characterized in that: In the step 2, an infusion tube containing 0.1 mL of 0.9% sodium chloride solution is connected to the intubation through the tracheal cannula needle. If the liquid in the infusion tube moves back and forth with the rat's breathing, it indicates that the intubation is successful.

6. The method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation as claimed in claim 1, characterized in that: The preparation of the silica suspension in step 3 is to take 50 mg of silica particles and grind them thoroughly in an agate mortar so that the silica particles with a particle size of less than 5.0 μm reach more than 90.0%, dry them at 180° C. for 6 h, and then add 1.0 mL of 0.9% sodium chloride solution and mix well.

7. The method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation as claimed in claim 3, characterized in that: In the step S2, after the rat is fixed, the hair of the rat is trimmed from the lower jaw to the neck area above the sternum, and the rat is disinfected with iodine and alcohol.

8. The method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation as claimed in claim 1, characterized in that: In step six, hematoxylin-eosin staining is HE staining. Hematoxylin staining solution is alkaline, which mainly stains the chromatin in the cell nucleus and the ribosomes in the cytoplasm purple-blue; eosin is an acidic dye, which mainly stains the components in the cytoplasm and the extracellular matrix red.

9. The method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation as claimed in claim 1, characterized in that: The Masson staining in step six is ​​also called Masson staining, which is related to the size of the anionic dye molecules and the penetration of the tissue. The size of the molecule is reflected by the molecular weight. Small molecular weight can easily penetrate densely structured and low-permeability tissues, while large molecular weight can only enter loosely structured and high-permeability tissues. However, the molecular weight of light green or aniline blue is very large, so after Masson staining, muscle fibers appear red and collagen fibers appear green or blue, which is mainly used to distinguish collagen fibers from muscle fibers.

10. The method for establishing a silicosis model of rats using a ventilator combined with non-exposed tracheal instillation as claimed in claim 2, characterized in that: The selection criteria for the rats are: specific pathogen-free healthy male SD rats, 6 weeks old, with a body weight of (200±20) g, free access to food and water, and after 2 weeks of adaptive feeding, the rats are randomly divided into an experimental group and a control group.

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