A method for constructing an acute pneumonia model in rabbit juveniles

Abstract

This invention aims to develop an animal model that effectively targets the core symptoms of acute pneumonia and provides a good evaluation of the efficacy of medications for acute pneumonia. The method for constructing a chemically induced acute pneumonia model in rabbit juveniles involves intraperitoneal injection of bleomycin and lipopolysaccharide, intravenous injection of cysteine, feeding with a high-protein diet, applying hot and cold stimulation, and spraying with citric acid to obtain the acute pneumonia animal model. This non-single-factor induced acute pneumonia animal model is consistent with the etiological view that acute pneumonia is induced by multiple factors and can better simulate the characteristics of human acute pneumonia. This non-single-factor induced acute pneumonia animal model is an ideal animal model from both a pathophysiological perspective and from the perspectives of simulating typical clinical symptoms and predicting treatment, providing support for understanding the multifactorial pathogenesis of acute pneumonia and offering a theoretical basis for developing new treatment options for acute pneumonia.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction for childhood diseases, and specifically relates to a method for constructing an acute pneumonia rabbit model. Background Technology

[0002] Young animals do not have the same drug metabolism and efficacy as adults. Data obtained using adult animal models are insufficient to support drug evaluation in young animals, which greatly limits basic pathological and pharmacological research on pediatric drugs. Therefore, the establishment of relevant young animal models is particularly important.

[0003] In pneumonia models, mammals are typically chosen as subjects because their lung anatomy and physiological functions are largely similar. When assessing overall physiological cardiovascular monitoring and hemodynamics, large mammals such as rabbits, dogs, pigs, and baboons are preferred pneumonia models for lung biomechanical measurements. Although successful validation has been achieved in baboons, piglets are currently the most frequently used model. In contrast, multimicrobial infections are primarily assessed in rodents, particularly mice and rats. Rodents are small, reproduce rapidly, and offer numerous advantages for laboratory research.

[0004] Based on the etiology, pneumonia can be classified into several types, including viral, bacterial, and chemically induced pneumonia. Wang Xuefeng et al. used ether anesthesia to inoculate young mice intranasally with 0.05 mL of influenza A virus FM1 strain, and verified the influenza virus pneumonia model by dynamically observing the mice's vital signs, lung tissue pathology, and lung index. The advantage of this modeling method is its strong susceptibility, closely resembling the clinical susceptibility characteristics of influenza virus in children, but the model maintenance period is short. Qiu Yu et al. used young rats to inoculate them intranasally with influenza A virus for 3 consecutive days to establish an influenza virus-induced pneumonia model. The model group infected with the intranasal influenza virus showed significantly reduced food and water intake, decreased activity, poor mental state, and increased respiratory rate. The content of fibroblast growth factor-2 in the lung perfusion fluid increased, and the expression of fibroblast growth factor receptor-1 protein in the lung tissue was significantly reduced. Zimmerman et al. established a highly inflammatory bronchopneumonia model in juvenile baboons by infecting them with Bordetella pertussis. The histopathological changes in this model were limited to cell infiltration and mucus changes, while immunohistochemical staining showed that the bacteria were localized on the surface of the ciliated epithelium of the airways. Wang Yanfeng established a pneumonia model in young male C57BL / 6J mice by intratracheal inoculation with a standard strain of Klebsiella pneumoniae. Mice with successful models showed dull fur, lethargy, reduced activity, and decreased appetite; macroscopic examination of the lung tissue revealed congestion and suppuration, and microscopic observation showed varying degrees of inflammatory cell infiltration and alveolar structural damage. The advantages of this modeling method are its simplicity, ease of infection, cost-effectiveness, and wide availability of sources; however, the model maintenance period is short.

[0005] Currently, several animal models that can effectively simulate pneumonia have been successfully constructed, including direct contact, aerosol inoculation, endotracheal injection, oral intubation, and nasal instillation. Each model has its advantages and disadvantages. The advantages of direct contact are: it can accurately replicate the natural spread of infectious infections; the pathogen's virulence can be enhanced through breeding mice, thus reducing the infectious dose for secondary infections; its disadvantages are: the inoculation dose cannot be controlled, and the infection time and number of infected animals cannot be standardized. The advantages of aerosol inoculation are: it simulates the natural spread of airborne infection, allowing simultaneous infection of the entire animal population; the pathogen is evenly distributed in both lungs, and anesthesia is not required; its disadvantages are: aerosol modeling systems are relatively expensive; pathogens may deposit on the mouse's fur and eyes; the survival rate of pathogens in the aerosol is low; and there are limitations on the amount of pathogen that can be inoculated. The advantages of endotracheal injection are: it mimics oropharyngeal aspiration infection, allowing precise drug delivery to the lower respiratory tract; and the experimental procedure is relatively simple; its disadvantages are: it requires anesthesia and tracheal exposure surgery; the pathogen is unevenly distributed in both lungs; and the experimental start time cannot be synchronized. The advantages of oral intubation are: mimicking oropharyngeal aspiration infection, allowing for precise drug delivery to the lower respiratory tract, a relatively simple experimental procedure, and no surgical trauma. Its disadvantages include: requiring general anesthesia, higher technical difficulty, uneven distribution of pathogens in both lungs, inconsistent start times, inability to conduct experiments synchronously, and the risk of oral bacterial contamination. The advantages of nasal instillation are: mimicking oropharyngeal aspiration infection, enabling the construction of upper and lower respiratory tract infection models in mice, and a relatively simple experimental procedure. Its disadvantages include: requiring general anesthesia, higher and harder-to-control pathogen deposition density in the lungs, uneven distribution of pathogens in both lungs, and the risk of nasal bacterial contamination. These modeling methods are commonly used for viral or bacterial induction. Regarding the chemical induction of pneumonia models, current research focuses on intravenous injection or nebulized inhalation of lipopolysaccharide, but this method still suffers from unstable modeling and low success rates. Therefore, there is an urgent need for a simple, controllable, low-cost, reliable, and stable method for constructing animal pneumonia models. Summary of the Invention

[0006] This invention aims to develop an animal model that effectively targets the core symptoms of acute pneumonia and provides a reliable method for evaluating the efficacy of drugs used to treat acute pneumonia. The acute pneumonia rabbit model developed through chemical induction in this invention demonstrates high success rates and stability in inducing classic symptoms of acute pneumonia, as well as accompanying symptoms such as alveolar wall thickening, inflammatory cell infiltration, and interstitial and bronchial hemorrhage and edema. This invention provides a reliable and controllable animal model for evaluating the efficacy of drugs used to treat acute pneumonia.

[0007] This invention is achieved through the following technical solution:

[0008] A method for constructing an acute pneumonia model in juvenile rabbits, characterized by comprising the following steps:

[0009] 1. Drug Injection: Select 35-day-old New Zealand White rabbits weighing approximately 700g and acclimatize them for two weeks. The modeling period is 36 days. Starting from day 1 of modeling, administer bleomycin and lipopolysaccharide intraperitoneally and cysteine ​​intravenously, once every 6 days for a total of 6 injections. The dosage for each injection is: bleomycin 85μg / kg, lipopolysaccharide 7mg / kg, and cysteine ​​2mg / kg;

[0010] 2. High-protein feed feeding: During the modeling period, rabbits were fed a high-protein feed, which consisted of a basic feed supplemented with 9% corn gluten meal, 8% fish meal, and 1% methionine choline by weight of the basic feed.

[0011] 3. Hot and cold stimulation: During the modeling period, rabbits were placed in an 8℃ constant temperature incubator every morning, in a 35℃ constant temperature incubator every afternoon, and in room temperature conditions every evening.

[0012] 4. Citric acid spraying: During the last 6 days of the modeling cycle, i.e., days 31-36, spray the rabbits with a citric acid solution to obtain an acute pneumonia model in young rabbits. Spray the citric acid solution twice a day. The concentration of the citric acid solution is 10%, and the citric acid solution is prepared by dissolving 100g of citric acid in 1000ml of water.

[0013] After 36 days of modeling, the rabbits' physiological state was observed, and the levels of inflammatory factors in the bronchoalveolar lavage fluid (BALF) were measured by HE staining, lung CT, and ELISA. The results showed that the rabbits exhibited typical symptoms of acute pneumonia, which met the criteria for the reliability and effectiveness of the acute pneumonia animal model, namely the cause of the disease, symptoms, and pathophysiology. The acute pneumonia rabbit model was successfully obtained by following the construction method of this invention.

[0014] The technical solution of the present invention has the following advantages:

[0015] 1. In the method for constructing the acute pneumonia rabbit model of the present invention, an acute pneumonia animal model is obtained by intraperitoneal injection of bleomycin and lipopolysaccharide, intravenous injection of cysteine, feeding with a high-protein diet, applying hot and cold stimulation, and spraying with citric acid. This non-single-factor induced acute pneumonia animal model is consistent with the etiological view that acute pneumonia is induced by multiple factors and can better simulate the characteristics of human acute pneumonia. The non-single-factor induced acute pneumonia animal model of the present invention basically meets three effectiveness criteria. Whether from the perspective of pathophysiological theory, simulation of typical clinical symptoms, or treatment prediction, it is a relatively ideal animal model, which will provide support for the multifactorial pathogenesis of acute pneumonia and provide a theoretical basis for developing new treatment options for acute pneumonia.

[0016] 2. The acute pneumonia animal model constructed in this invention exhibits stable performance and minimal individual variability; the synergistic interaction among multiple factors contributes to a more accurate disease model. This model demonstrates high success rates and stability in inducing classic acute pneumonia symptoms (slow and lethargic response, rapid and deep breathing, severe hypoxemia, etc.) as well as accompanying symptoms of acute pneumonia such as alveolar wall thickening, inflammatory cell infiltration, and interstitial and bronchial hemorrhage and edema.

[0017] 3. The acute pneumonia animal model of this invention is a non-single-factor induced acute pneumonia animal model, which will provide support for understanding the multifactorial pathogenesis of acute pneumonia and better simulate the advantages of human acute pneumonia, providing an important theoretical basis for developing a new drug formulation for the effective treatment of acute pneumonia. It can not only be used to explore the scientific implications of multifactorial effects on acute pneumonia, but also provide an animal model that reflects the essence of the disease for new drug screening.

[0018] 4. This invention employs an induction method combining intraperitoneal injection of bleomycin and lipopolysaccharide (LPS) with intravenous injection of cysteine. LPS is a component of the outer wall of Gram-negative bacterial cell walls, composed of lipids and polysaccharides, and is also a type of endotoxin. It can activate inflammatory pathways and induce inflammation through Toll-like receptors present on the cell membrane surface of host cells. The complex of bleomycin and iron intercalates into DNA, causing single-strand and double-strand breaks. Bleomycin can exacerbate the induction of lung inflammation by LPS, greatly increasing its toxic effects on the lungs. Simultaneously, intravenous injection of cysteine ​​further exacerbates lung inflammation in young rabbits and facilitates the synergistic binding of bleomycin and LPS, inducing the activation of inflammatory pathways in the young rabbit lungs by bleomycin and LPS. These three reagents, while exerting their individual effects, can synergistically enhance each other, effectively inducing acute pneumonia. Through numerous experiments and verifications, the inventors optimized the injection time and dosage of the three reagents, ensuring a more accurate acute pneumonia model in young rabbits.

[0019] 5. This invention also employs a combination of hot and cold stimulation and citric acid spraying to better simulate the etiology and symptoms of acute pneumonia in traditional Chinese medicine clinical practice. Combined with the injection of three reagents and feeding a high-protein diet, it effectively induces classic and accompanying symptoms of acute pneumonia. Daily hot and cold stimulation during the modeling period reduces the immunity and resistance of the young rabbits' lungs, making them more sensitive to toxins. The citric acid spraying in the later stages of modeling not only helps the young rabbits absorb high-protein feed but also stimulates their susceptibility to drug injection, thus promoting the induction of lung inflammation by drug injection to some extent. Feeding a high-protein diet during the modeling period can, to some extent, increase the sensitivity of young rabbits, especially their lungs, to drugs. Through numerous creative efforts and scientific experiments, the inventors have selected a high-protein feed formula with 9% corn gluten meal, 8% fish meal, and 1% methionine choline added to the basic feed. This formula is not only highly suitable for feeding young rabbits but also extremely beneficial for the clinical simulation of the etiology and symptoms of acute pneumonia; in particular, methionine choline not only promotes protein absorption in young rabbits but also effectively increases the sensitivity of their lungs. Spraying citric acid solution during the last 6 days of modeling played a crucial role in this study, ensuring a stable acute pneumonia rabbit model.

[0020] This study provides strong theoretical support and a model reference for a deeper understanding of the pulmonary immune mechanisms following acute pneumonia in hosts and for evaluating new anti-infective treatments. The research team has repeatedly validated the model, demonstrating its reliability. The model has also been used to evaluate the efficacy of various drugs, validating its application value. In recent years, research on anti-pneumonia and the development of new drugs have become hot topics. This study provides a reliable animal model for evaluating the efficacy of drugs for acute pneumonia, offering advantages such as low cost, model stability, ease of operation, and non-invasiveness. Detailed Implementation

[0021] A method for constructing an acute pneumonia model in juvenile rabbits, characterized by comprising the following steps:

[0022] 1. Drug Injection: Select 35-day-old New Zealand White rabbits weighing approximately 700g and acclimatize them for two weeks. The modeling period is 36 days. Starting from day 1 of modeling, administer bleomycin and lipopolysaccharide intraperitoneally and cysteine ​​intravenously, once every 6 days for a total of 6 injections. The dosage for each injection is: bleomycin 85μg / kg, lipopolysaccharide 7mg / kg, and cysteine ​​2mg / kg;

[0023] 2. High-protein feed feeding: During the modeling period, rabbits were fed a high-protein feed, which consisted of a basic feed supplemented with 9% corn gluten meal, 8% fish meal, and 1% methionine choline by weight of the basic feed.

[0024] 3. Hot and cold stimulation: During the modeling period, rabbits were placed in an 8℃ constant temperature incubator every morning, in a 35℃ constant temperature incubator every afternoon, and in room temperature conditions every evening.

[0025] 4. Citric acid spraying: During the last 6 days of the modeling cycle, i.e., days 31-36, spray the rabbits with a citric acid solution to obtain an acute pneumonia model in young rabbits. Spray the citric acid solution twice a day. The concentration of the citric acid solution is 10%, and the citric acid solution is prepared by dissolving 100g of citric acid in 1000ml of water.

[0026] The subjects were divided into five groups: experimental group, control group 1, control group 2, control group 3, and normal group. The experimental group rabbits underwent the modeling process described above, while the normal group rabbits received PBS injections, conventional feed and room temperature feeding, and were treated with water spraying. Control group 1 used endotracheal infusion of Klebsiella pneumoniae at a concentration of 2.4 × 10⁹ CFU / mL, administered every 6 days. Control group 2 received intraperitoneal injection of LPS 10 mg / kg every 6 days. Control group 3 received intravenous injection of LPS 10 mg / kg every 6 days.

[0027] After 36 days of modeling, the physiological status of the rabbits was observed, organ HE staining pathological examination was performed, lung CT scans were performed, and the levels of inflammatory factors in bronchoalveolar lavage fluid (BALF) were measured by ELISA. The results are as follows:

[0028] 1. Physiological state of young rabbits

[0029] The healthy group of young rabbits had normal eating and defecating habits, were active, and had glossy fur, with no obvious oral or nasal secretions. The experimental group rabbits, after modeling, had duller fur, huddled together and curled up on one side of their cages, were sluggish and lethargic, had increased nasal and periorbital secretions, faster respiratory rate, and were significantly smaller than the healthy group. Control groups 1, 2, and 3 also showed similar symptoms to the experimental group, but to a lesser degree.

[0030] 2. Histopathological monitoring of young rabbits

[0031] Complete organs (heart, liver, spleen, lung, and kidney) are dissected and harvested as pathological samples. The organs are fixed in 4% paraformaldehyde for at least 24 hours, followed by dehydration, paraffin embedding, sectioning, and HE staining. Pathological sections are prepared and scanned electronically, then submitted to pathology experts for blind review.

[0032] In the normal group of young rabbits, the alveolar structure of the lung tissue was intact, the bronchial structure at all levels was normal, and no obvious pathological changes were observed in the lungs or other tissues. In the experimental group, the lung tissue of the rabbits showed pathological features such as alveolar wall thickening, extensive inflammatory cell infiltration, and interstitial hemorrhage and edema; these lesions worsened over time. Compared with the lung tissue, the lesions in other tissues were milder. The heart showed varying degrees of cardiac chamber dilation, possibly due to compensatory lesions of acute pneumonia; although a small amount of inflammatory infiltration was present, it was mostly located at the organ periphery. A small number of inflammatory cells were occasionally observed in the liver and spleen tissues; no lesions were observed in the kidney tissue. Comparative groups 1, 2, and 3 also showed similar conditions to the experimental group, but the symptoms were milder.

[0033] 3. Changes in the lungs of young rabbits on CT scan

[0034] In the normal group, the young rabbits showed no obvious lung lesions. CT scans showed uniform lung transparency, clear lung markings, and no abnormal linear or patchy shadows. In the experimental group, the rabbits showed blurred and disordered lung markings, with large areas of confluent patchy shadows. Comparative groups 1, 2, and 3 also showed similar conditions to the experimental group, but the symptoms were milder than those in the experimental group.

[0035] 4. Levels of inflammatory factors in bronchoalveolar lavage fluid (BALF) of young rabbits

[0036] The levels of inflammatory factors in bronchoalveolar lavage fluid (BALF) of young rabbits were measured by ELISA. Compared with the normal group, the levels of IL-6, TNF-α, and IL-1β in the experimental group were significantly increased (P < 0.05). The levels of IL-6, TNF-α, and IL-1β in control groups 1, 2, and 3 were also significantly higher than those in the normal group, but slightly lower than those in the experimental group.

[0037] After repeated experiments, the experimental groups all showed the same results. This demonstrates that the rabbits in the experimental groups exhibited typical symptoms of acute pneumonia, meeting the criteria for the reliability and effectiveness of an acute pneumonia animal model, namely, the cause of the disease, symptom presentation, and pathophysiology. Following the construction method of this invention, an acute pneumonia rabbit model can be successfully obtained.

Claims

1. A method for constructing an acute pneumonia model in rabbit juveniles, characterized in that, Includes the following steps: (1) Drug injection: Select New Zealand white rabbits weighing about 700g at 35 days old and acclimatize them for two weeks; the modeling period is 36 days; starting from the first day of modeling, inject bleomycin and lipopolysaccharide into the rabbits intraperitoneally and inject cysteine ​​into the vein, once every 6 days, for a total of 6 injections. The dosage of each injection is: bleomycin 85μg / kg, lipopolysaccharide 7mg / kg, cysteine ​​2mg / kg; (2) High-protein feed feeding: During the modeling period, rabbits were fed a high-protein feed, which consisted of a basic feed with 9% corn gluten meal, 8% fish meal and 1% methionine choline added by weight of the basic feed. (3) Hot and cold stimulation: During the modeling period, the rabbits were placed in an 8℃ constant temperature box every morning, in a 35℃ constant temperature box every afternoon, and in room temperature conditions at night. (4) Spraying citric acid: Spray citric acid solution on the rabbits during the last 6 days of the modeling cycle, i.e., days 31-36, to obtain the acute pneumonia model of young rabbits; spray citric acid solution twice a day, and the concentration of the citric acid solution is 10%.

2. The method for constructing an acute pneumonia rabbit model according to claim 1, characterized in that, The citric acid solution is prepared by dissolving 100g of citric acid in 1000ml of water.