Animal model of brain protein-induced lung edema after brain injury and construction method and application thereof

Abstract

The application belongs to the field of animal model construction, and particularly relates to a brain protein induced lung edema animal model after brain injury and a construction method and application thereof. The construction method comprises the following steps: injecting brain protein into a rat to construct. The brain protein induced lung edema animal model after brain injury can simulate lung edema caused by brain protein leakage after brain injury, and avoids lung edema induced by sympathetic nerve excitation caused by nerve injury. The prepared animal model has the advantages of good stability, good repeatability and long survival time.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction, specifically relating to an animal model of pulmonary edema following brain injury caused by brain protein, its construction method, and its application. Background Technology

[0002] Neurogenic pulmonary edema is a life-threatening complication following central nervous system injury, characterized by acute onset and rapid accumulation of interstitial fluid in the lungs. It is most commonly seen in patients with subarachnoid hemorrhage or other causes of cerebral hemorrhage, traumatic brain injury, ischemic stroke, encephalitis, and epilepsy. Incidence rates reported by different medical teams vary, ranging from 2% to 42.9%, with a mortality rate approaching 10%. The atypical symptoms and lack of specific diagnostic markers of pulmonary edema make rapid identification and accurate diagnosis difficult. Therefore, a deeper understanding of the pathogenesis of neurogenic pulmonary edema, identifying key contributing factors, and developing more effective diagnostic and treatment methods is particularly urgent. The foundation for such research lies in establishing practical, targeted, and reproducible animal models of neurogenic pulmonary edema.

[0003] The link between central nervous system injury and pulmonary edema remains largely unclear. Systemic autoimmune responses following brain injury may play an indispensable role in the development of neurogenic pulmonary edema. Currently, animal models of neurogenic pulmonary edema are mainly induced by central nervous system injury (traumatic brain injury models, subarachnoid hemorrhage models, spinal cord injury models, etc.). However, these animal models have short survival times, poor stability, and low reproducibility. Furthermore, existing methods for constructing neurogenic pulmonary edema animal models suffer from complexity and numerous experimental factors. The pathogenesis of neurogenic pulmonary edema is complex, involving many pathophysiological processes. Complex modeling methods significantly increase the instability of pulmonary edema symptoms and mortality in experimental animals, and may also negatively impact subsequent research on the mechanisms, diagnosis, and treatment based on these animal models. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an animal model of pulmonary edema following brain injury caused by brain protein, its construction method and application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for constructing an animal model of pulmonary edema following brain injury induced by brain protein includes the following steps: injecting brain protein into rats to construct the model.

[0007] The brain protein injection amount is 0.1-5 mg; preferably 2 mg, and the modeling time is 12 h-7 d; preferably 3 d.

[0008] The injection method is a single injection via the tail vein; preferably, the injection solution is a physiological saline solution of brain protein; preferably, the concentration of the injection solution is 0.1-5 mg / mL; the injection volume is 1 mL.

[0009] The brain protein solution was extracted using the following method: (a) after euthanizing the rat, the scalp was dissected along the sagittal line and separated, the skull was removed with a drill, and the brain tissue was exposed and removed; (b) the blood vessels and meningeal tissue on the surface of the brain were removed, and the brain tissue was ground into small pieces after freezing and thawing until the brain cell morphology disappeared; (c) the small pieces of brain tissue were dissolved in physiological saline and filtered to obtain the brain protein solution; (d) the concentration of brain protein was measured.

[0010] The freeze-thaw process in step (b) is performed as follows: freeze at -80°C for 30 min, then thaw in a 37°C water bath for 5 min, and then grind the brain tissue completely in a mortar at 4°C; preferably, the freeze-thaw process is repeated 5 times.

[0011] The method for determining the concentration of brain protein in step (d) is spectrophotometry.

[0012] In step (a), the rats weighed 180-220g and were 6-8 weeks old.

[0013] The filter used in step (c) is a 0.22μm filter.

[0014] The present invention also includes an animal model of pulmonary edema following brain injury caused by brain protein, which is obtained using the construction method described above.

[0015] The present invention also includes the application of an animal model of pulmonary edema following brain injury caused by brain protein, for the study of the mechanism of neurogenic pulmonary edema, and / or the screening of drugs for the treatment and / or prevention of neurogenic pulmonary edema.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] The animal model of pulmonary edema following brain injury caused by brain protein leakage can simulate pulmonary edema caused by brain protein leakage after brain injury, and avoids pulmonary edema induced by sympathetic nerve excitation caused by nerve damage. The animal model prepared has the advantages of good stability, good reproducibility and long survival time.

[0018] Furthermore, the model established in this invention only involves simple brain protein stimulation in experimental animals, reducing experimental factors and improving the controllability of the modeling process and the stability of the model. It is particularly suitable for research on the pathogenesis (including research pathways and pathophysiological mechanisms) and drug screening of immune inflammatory factors in neurogenic pulmonary edema. Attached Figure Description

[0019] Figure 1The results of rat brain protein proteomic analysis provided in Example 1 of this invention;

[0020] Figure 2 The results of peripheral blood alkaline myelin protease-linked immunosorbent assay (ELISA) for each experimental group provided in Example 2 of this invention;

[0021] Figure 3 This is a graph showing the lung tissue water content results for each experimental group provided in Example 3 of the present invention;

[0022] Figure 4 This is a graph showing the protein concentration results of bronchoalveolar lavage fluid in each experimental group provided in Example 3 of the present invention;

[0023] Figure 5 The images show hematoxylin-eosin staining of lung tissue paraffin sections and lung injury scores for each experimental group provided in Example 3 of this invention.

[0024] Figure 6 Immunofluorescence staining images of basic myelin, immunoglobulin IgG, and complement C3 in lung tissues of each experimental group provided in Example 4 of the present invention;

[0025] Figure 7 This is a diagram showing the results of immune co-precipitation of lung tissue from the brain protein animal model provided in Example 4 of the present invention. Detailed Implementation

[0026] 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 and preferred embodiments.

[0027] Example 1

[0028] The method for preparing brain protein includes the following steps: (a) anesthetizing rats (weighing 180-220g, aged 6-8 weeks) and fixing them on a stereotactic device, disinfecting them, and euthanizing them by cervical dislocation; dissecting and separating the scalp along the sagittal line, removing the skull with a drill, exposing and removing the brain tissue; (b) carefully removing the blood vessels and meninges on the surface of the brain, rapidly placing the brain tissue in an environment of -80℃ for 30 min, then thawing it in a water bath at 37℃ for 5 min, and then completely grinding the brain tissue in a mortar at 4℃; repeating the above freeze-thaw steps 5 times, and observing the fragmented brain tissue with an inverted phase contrast microscope to confirm the disappearance of brain cell morphology; (c) dissolving the extracted brain protein in 5 mL of physiological saline and filtering it through a 0.22 μm filter; (d) measuring the protein concentration using a spectrophotometer and adjusting it to the injection concentration with physiological saline. In this application, 1 mL of injection is used as an example for illustration.

[0029] The obtained brain proteins were analyzed by mass spectrometry. The results are as follows: Figure 1As shown, the main components of the brain proteins extracted in this invention were identified. Basic myelin (MBP) was selected as a biomarker for central nervous system injury in subsequent experiments.

[0030] Example 2

[0031] A method for constructing an animal model of pulmonary edema following brain injury induced by brain protein includes the following steps: injecting brain protein into rats. The construction method is as follows: extracting brain protein powder is injected into different rats via the tail vein at doses of 0, 0.1, 1, 2, and 5 mg; peripheral blood samples are collected from each group of experimental animals at 0 h, 12 h, 24 h, 3 d, and 7 d after modeling for enzyme-linked immunosorbent assay (ELISA) to determine the concentration of basic myelin (MBP).

[0032] Example for comparison:

[0033] Six- to eight-week-old SD rats (weighing 180-220g) were subjected to craniocerebral trauma model and brain protein injection model, respectively. The following groups were set up as control groups:

[0034] Control Group 1: The craniocerebral trauma group, modeled using the classic free-fall method. After anesthesia and disinfection, a longitudinal incision was made on the scalp along the midsagittal line of the skull. The rat was then fixed to a stereotactic device, and a circular bone window with a diameter of 2 mm was drilled 1 mm anterior to the coronal suture and 2 mm lateral to the midline. A 400g cylindrical metal hammer was dropped freely from a height of 45 cm along a vertical tube, striking the pin located at the bone window. After the impact, the bone window was sealed with bone wax, and the wound was sutured.

[0035] Control group 2: Sham surgery group. After anesthetizing, fixing and disinfecting the experimental animals, the scalp was cut open to expose the skull. A bone window was drilled out and sealed with bone wax. The scalp was then sutured in layers.

[0036] Control group 2: Carrier group, in which physiological saline of the same volume as the brain protein injected in the example was injected into rats via the tail vein.

[0037] Figure 2 The concentrations of peripheral blood MBP in each group at different time points and at different doses were shown. The results showed that when the injection dose of brain protein was 2 mg, the trend of the change in peripheral blood MBP concentration in rats over time was basically consistent with the trend of MBP change after traumatic brain injury, indicating that the animal model provided by this invention can well simulate the leakage of brain protein after traumatic brain injury.

[0038] Example 3

[0039] The success of establishing an animal model of pulmonary edema following brain injury induced by brain protein was assessed using three indicators: lung tissue water content, lung air-blood barrier permeability, and lung tissue pathological evaluation.

[0040] The lung tissue water content was calculated by measuring the wet and dry weight of rat lung tissue and then using the formula: Lung water content = (wet weight - dry weight) / wet weight × 100%.

[0041] Lung air-blood barrier permeability was assessed by measuring the protein concentration in bronchoalveolar lavage fluid. The specific procedure was as follows: Rats were anesthetized and placed on a foam board. The trachea was exposed and dissected using surgical scissors and curved forceps, then cut open. An indwelling needle was inserted along the tracheal incision and secured with sutures. The lavage fluid was lavaged twice with pre-cooled physiological saline (total volume 5 mL). The recovery rate of the lavage fluid was 80%. The fluid was then centrifuged at 1500g for 10 min, and the protein concentration in the supernatant was determined using a BCA assay kit.

[0042] Lung tissue pathological assessment involved hematoxylin-eosin staining of paraffin-embedded lung tissue sections, followed by microscopic scoring of five variables across 10 random fields of view at 400x magnification: 1. neutrophil infiltration in the alveolar spaces; 2. neutrophil infiltration in the interstitium; 3. alveolar septal thickening; 4. hyaline membrane formation; and 5. presence of protein debris in the alveolar spaces. A lung injury score was then calculated.

[0043] like Figure 3 As shown, rats in the craniocerebral trauma group and the brain protein group had higher lung wet / dry weight ratios at 12h, 24h, 3d, and 7d, suggesting pulmonary edema in this model. Figure 4 In the study, compared with the sham surgery group and the carrier group, the protein concentration in the bronchoalveolar lavage fluid of rats in the craniocerebral trauma group and the brain protein group also reached its peak on day 3. Figure 5 Hematoxylin-eosin staining of lung tissue from these rats showed that, over time, both the craniocerebral trauma group and the brain protein group exhibited varying degrees of lung injury, including alveolar wall thickening, alveolar exudation, interstitial inflammatory cell infiltration, and hyaline membrane formation. Semi-quantitative analysis revealed increased lung injury scores at 12h, 24h, 3d, and 7d in both the craniocerebral trauma group and the brain protein group compared to the sham-operated group and the carrier group, with the severity of injury reaching its peak on day 3. This indicates that intravenous injection of brain protein can induce pulmonary edema in rats. In the figures, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and ns represents no significant difference. The scale bar in the hematoxylin-eosin stained pathological images is 100 μm.

[0044] Example 4

[0045] Immunofluorescence staining and immunoprecipitation techniques were used to further evaluate the deposition of brain proteins and the formation of autoantibodies in animal models of pulmonary edema constructed from the craniocerebral trauma group and the brain proteome. Figure 6As shown, compared with the sham surgery group and the vector group, the lung tissue of the animal models constructed by the craniocerebral trauma group and the brain protein group showed significant MBP deposition, accompanied by the deposition of immunoglobulin IgG and the activation of complement C3. Figure 7 Immunoprecipitation results showed that after precipitation of lung tissue proteins with anti-MBP or anti-IgG antibodies, MBP and IgG were detected by Western blotting, consistent with their interaction in lung tissue. Higher IgG levels were also associated with increased MBP levels. These experimental results indicate the formation and deposition of MBP-IgG immune complexes in both traumatic brain injury and brain protein-induced pulmonary edema. Figure 6 The caliber bar is 50 μm.

[0046] The immunofluorescence staining method described in this embodiment includes: dewaxing paraffin sections of lung tissue with xylene, hydrating them with graded ethanol, immersing the paraffin sections in citrate buffer (pH 6.0), and boiling them in an autoclave at 121°C for 4 minutes to repair antigens. Then, the slides are allowed to cool slowly at room temperature while immersed in citrate buffer, and rinsed with an appropriate amount of phosphate buffer. Non-specific sites are blocked with 5% goat serum. The sections are then incubated overnight at 4°C with rabbit anti-IgG antibody and rabbit anti-C3 antibody. Next, the sections are incubated with fluorescent secondary antibody at room temperature in the dark for 2 hours, and mounted with a DAPI-containing anti-fluorescence quenching mounting medium. The slides are observed and photographed under a confocal microscope. The primary antibody used in this experiment was purchased from Abcam (UK), and the fluorescent secondary antibody was purchased from Thermo Fisher Scientific (USA).

[0047] The immunoprecipitation method described in this embodiment includes: lysing fresh lung tissue from a model animal with pre-chilled RIPA lysis buffer, centrifuging at 14000g for 15 min at 4°C, and transferring the supernatant to a new centrifuge tube. Anti-MBP or anti-IgG antibody is added, and the antigen-antibody mixture is incubated overnight at 4°C with gentle shaking. Then, 100 μL of Protein A / G agarose beads are added to capture the antigen-antibody complex. The antigen-antibody mixture is incubated overnight at 4°C with gentle shaking. After centrifugation at 14000g for 5 seconds, the supernatant is removed, and the agarose bead-antigen-antibody complex is collected. The mixture is washed three times with 800 μL of pre-chilled RIPA lysis buffer, resuspended in 60 μL of 2× loading buffer, and gently mixed. The loaded sample is boiled for 5 min, centrifuged at 14000g, and the remaining agarose beads are obtained. The supernatant is denatured by boiling for 5 min, and then Western blot experiments are performed to assess protein expression.

[0048] This invention creatively proposes a scheme to construct an animal model of pulmonary edema following brain injury using brain proteins. While avoiding neural factors, it simulates the leakage of brain proteins caused by blood-brain barrier disruption following central nervous system injury, thus creating an animal model of neurogenic pulmonary edema induced solely by brain protein leakage. Compared to existing animal models of neurogenic pulmonary edema, this invention reduces experimental factors and creates an environment that better simulates the leakage of brain proteins to the periphery after central nervous system injury. This not only ensures stable pulmonary edema symptoms in rats but also avoids the high mortality rate caused by complex experimental factors.

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of pulmonary edema following brain injury induced by brain protein, characterized in that, The process includes the following steps: injecting brain protein into rats to construct the protein; The injection method is a single injection via tail vein; the injection solution is a physiological saline solution of brain protein; the amount of brain protein injected is 0.1-5 mg; the modeling time is 12 h-7 d; the success of the animal model of pulmonary edema after brain protein-induced brain injury is evaluated by three indicators: lung tissue water content, lung air-blood barrier permeability, and lung tissue pathological assessment. The brain protein solution was extracted using the following method: (a) rats were euthanized and dissected along the sagittal line, the scalp was separated, the skull was removed with a burr, and the brain tissue was exposed and removed; (b) blood vessels and meningeal tissue on the surface of the brain were removed, and the brain tissue was frozen and thawed and then ground into small pieces until the brain cell morphology disappeared; (c) the small pieces of brain tissue were dissolved in physiological saline and filtered to obtain the brain protein solution; (d) the concentration of brain protein was measured. The construction method described herein only stimulates the brain proteins of experimental animals, thus avoiding pulmonary edema induced by sympathetic nerve excitation caused by nerve damage.

2. The method for constructing an animal model of pulmonary edema following brain injury induced by brain protein according to claim 1, characterized in that, The brain protein injection dosage was 2 mg, and the modeling time was 3 days.

3. The method for constructing an animal model of pulmonary edema following brain injury induced by brain protein according to claim 1, characterized in that, The concentration of the injection solution is 0.1-5 mg / mL; the injection volume is 1 mL.

4. The method for constructing an animal model of pulmonary edema following brain injury induced by brain protein according to claim 1, characterized in that, The freeze-thaw process in step (b) is as follows: freeze at -80°C for 30 min, then thaw in a 37°C water bath for 5 min, and then grind the brain tissue completely in a mortar at 4°C; the freeze-thaw process is repeated 5 times.

5. The method for constructing an animal model of pulmonary edema following brain injury induced by brain protein according to claim 1, characterized in that, The method for determining the concentration of brain protein in step (d) is spectrophotometry.

6. The method for constructing an animal model of pulmonary edema following brain injury induced by brain protein according to claim 1, characterized in that, In step (a), the rats weighed 180-220g and were 6-8 weeks old.

7. The method for constructing an animal model of pulmonary edema following brain injury induced by brain protein according to claim 1, characterized in that, The filter used in step (c) is a 0.22μm filter.

8. The application of an animal model of pulmonary edema following brain injury induced by brain protein obtained by the construction method according to any one of claims 1-7, characterized in that, For the study of the mechanism of neurogenic pulmonary edema, and / or for the screening of drugs; the screening of drugs is for the treatment and / or prevention of neurogenic pulmonary edema.

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