Pulmonary vascular remodeling experimental pig model and construction method thereof

Abstract

The present application relates to the field of animal models, in particular to a pulmonary vascular remodeling experimental pig model and a construction method thereof.The pulmonary vascular remodeling experimental pig model provided by the present application is obtained by injecting anhydrous ethanol into an experimental pig.Anhydrous ethanol is easily metabolized in vivo, which can avoid damage to other organs in the heart-lung circulation while inducing pulmonary hypertension, to some extent, eliminating the interference caused by the original lung injury caused by the injection of hydralazine.Secondly, through long-term induction of anhydrous ethanol, histological changes occur in the pulmonary artery, which not only helps to study the hemodynamics of pulmonary hypertension, but also further explores the mechanism of pulmonary hypertension from the organic changes of pulmonary artery remodeling.Finally, compared with small experimental animals, large experimental animals not only have better tolerance, but also provide convenience for related research of interventional medicine due to their larger vessel diameter.

Description

Technical Field

[0001] This invention relates to the field of animal models, and in particular to a pig model of pulmonary vascular remodeling and its construction method. Background Technology

[0002] Pulmonary hypertension (PH) is a chronic cardiopulmonary disease caused by cell proliferation, vascular remodeling, and fibrosis in the pulmonary arteries. It manifests as elevated pulmonary artery pressure and right heart failure, ultimately leading to death. The incidence of PH is estimated at 15-50 per 1,000,000 people. Its etiology is complex and multifactorial, with the main pathological processes involving pulmonary artery vasoconstriction, vascular remodeling, and thrombosis. Its pathogenesis primarily includes oxidative stress, inflammation, metabolic abnormalities, and endothelial-mesenchymal transition.

[0003] Existing methods for constructing animal models of pulmonary hypertension still have limitations: for example, limonene. Limonene is a dipyrrole alkaloid that, after being converted in the liver by P450 monooxidase, reaches the lungs via the bloodstream, selectively damaging the pulmonary vascular endothelium and inducing chronic vasculitis. However, due to its biotoxicity, limonene can cause pulmonary edema, alveolar septal cell proliferation, and pulmonary vein occlusion, in addition to pulmonary artery damage. In mice, limonene induces interstitial pulmonary fibrosis concurrently with pulmonary hypertension. Furthermore, myocarditis has been observed in limonene-induced pulmonary hypertension mouse models. Moreover, existing animal models of pulmonary hypertension are all constructed using small experimental animals (rats, rabbits), while reports and studies on models using large experimental animals are limited. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a pig model of pulmonary vascular remodeling induced by pulmonary hypertension and a method for constructing the model, in order to solve the problems in the prior art where the original lily alkaloid can induce damage to other organs in the cardiopulmonary circulation while inducing pulmonary hypertension, there are few models of single organic changes in pulmonary artery remodeling, and the small experimental animals have poor tolerance and the small blood vessel diameter is not conducive to related research in interventional medicine.

[0005] To achieve the above and other related objectives, the present invention provides a method for constructing a pig model of pulmonary vascular remodeling, the method comprising repeatedly inducing pulmonary vascular remodeling in experimental pigs with ethanol.

[0006] Preferably, the ethanol induces pulmonary hypertension, thereby triggering pulmonary vascular remodeling.

[0007] Preferably, the ethanol is administered by injection.

[0008] Preferably, the injection site is the jugular vein sheath.

[0009] Preferably, the ethanol is anhydrous ethanol.

[0010] The present invention also provides a pig model of pulmonary vascular remodeling obtained according to the construction method.

[0011] The present invention also provides the use of ethanol in the preparation of products for constructing experimental pig models of pulmonary vascular remodeling.

[0012] Preferably, the histological features of the experimental pig model of pulmonary vascular remodeling include one or more of the following: thickening of the pulmonary artery intima, disordered arrangement of pulmonary artery smooth muscle cells, or infiltration of inflammatory cells in the pulmonary artery compared with the control group.

[0013] Preferably, the histological characteristics of the experimental pig model of pulmonary vascular remodeling further include one or more of the following: compared with the control group, there are no pathological changes in muscle tissue, liver tissue, lung tissue, or kidney tissue.

[0014] This invention also provides the use of pulmonary vascular remodeling experimental pig models for screening drugs to prevent pulmonary hypertension.

[0015] This invention also provides the use of screening drugs for preventing pulmonary vascular remodeling in experimental pig models of pulmonary vascular remodeling.

[0016] As described above, the experimental pig model of pulmonary vascular remodeling induced by pulmonary hypertension and its construction method of the present invention have the following beneficial effects:

[0017] 1. The mechanism by which pulmonary hypertension is induced during endovascular ethanol therapy remains unclear. The establishment of this animal model can help investigate the mechanism of pulmonary hemodynamic changes in pulmonary hypertension. Simultaneously, it can help explore the pathophysiological mechanism of pulmonary artery remodeling caused by long-term pulmonary hypertension.

[0018] 2. The existing animal models of pulmonary hypertension induced by lily alkaloids have certain defects. In the model of pulmonary hypertension induced by anhydrous ethanol, ethanol can be metabolized by animals in a short period of time without causing damage to the cardiopulmonary organs. Therefore, the animal model in this invention can eliminate interfering factors in related studies. Attached Figure Description

[0019] Figure 1This invention presents the results of acute increases in pulmonary artery pressure and femoral artery pressure induced by anhydrous ethanol. AC represents a comparison of pulmonary artery systolic pressure (sPAP), pulmonary artery diastolic pressure (dPAP), and mean pulmonary artery pressure (mPAP) after injection in the blank group and the ethanol group; DF represents a comparison of femoral artery systolic pressure (sFAP), femoral artery diastolic pressure (dFAP), and mean femoral artery pressure (mFAP) after injection in the blank group and the ethanol group; GI represents a comparison of pulmonary artery systolic pressure (sPAP), pulmonary artery diastolic pressure (dPAP), and mean pulmonary artery pressure (mPAP) before and after injection in the ethanol group; JL represents a comparison of femoral artery systolic pressure (sFAP), femoral artery diastolic pressure (dFAP), and mean femoral artery pressure (mFAP) before and after injection in the ethanol group.

[0020] Figure 2 This represents the result of long-term injection of anhydrous ethanol to induce stable pulmonary hypertension in this invention. A represents the systolic pressure gradient of the pulmonary artery (ΔsPAP); B represents the diastolic pressure gradient of the pulmonary artery (ΔdPAP); and C represents the mean pulmonary artery pressure gradient (ΔmPAP).

[0021] Figure 3 These are the results of pulmonary artery remodeling induced by long-term injection of anhydrous ethanol in this invention. A is the H&E staining result of the blank group; B is the immunohistochemical detection result of CD31 in the blank group; C is the immunohistochemical detection result of α-smooth muscle actin in the blank group; D is the immunohistochemical detection result of tumor necrosis factor-α (TNF-α) in the blank group; E is the H&E staining result of the ethanol group; F is the immunohistochemical detection result of CD31 in the ethanol group; G is the immunohistochemical detection result of α-smooth muscle actin in the ethanol group; H is the immunohistochemical detection result of tumor necrosis factor-α (TNF-α) in the ethanol group.

[0022] Figure 4 These are staining images of the heart, liver, lung, and kidney tissues from experimental pigs used in this invention. A shows the H&E staining result for the heart; B shows the Sirius red staining result; C shows the H&E staining result for the liver; D shows the periodate-Schiff (PAS) staining result for the liver; E shows the H&E staining result for the lungs; F shows the Masson staining result for the lungs; G shows the H&E staining result for the kidneys; and H shows the Masson staining result for the kidneys.

[0023] Figure 5 This invention demonstrates the drug's ability to inhibit pulmonary artery remodeling induced by long-term anhydrous ethanol injection. AD indicates that losartan does not affect the tissue morphology of the experimental pig pulmonary arteries; EH indicates that losartan can partially reverse pulmonary intimal hyperplasia, smooth muscle disorder, and inflammatory cell infiltration induced by long-term anhydrous ethanol injection. Detailed Implementation

[0024] This invention provides a method for constructing a pig model of pulmonary vascular remodeling, the method comprising repeatedly inducing pulmonary vascular remodeling in experimental pigs with ethanol.

[0025] Furthermore, the ethanol induces pulmonary hypertension, which in turn induces pulmonary vascular remodeling.

[0026] Furthermore, the ethanol is administered by injection.

[0027] Furthermore, the injection site is the jugular vein sheath.

[0028] Preferably, the ethanol is anhydrous ethanol.

[0029] The ethanol induction includes the following steps:

[0030] 1) Preparations before the experiment;

[0031] 2) Injection of anhydrous ethanol;

[0032] 3) Post-experiment processing.

[0033] Furthermore, step 1) of the pre-experiment preparation includes the following steps:

[0034] 1a) Laboratory animals should be fasted from food and water before experiments;

[0035] 1b) Induction of anesthesia by intramuscular injection of anesthetic drugs;

[0036] 1c) Maintain anesthesia with gas anesthetic drugs;

[0037] 1d) Monitoring of physiological indicators of experimental animals.

[0038] In some specific implementations, the fasting period in step 1a) is 8-24 hours before surgery.

[0039] In some specific embodiments, the anesthetic drug injected intramuscularly in step 1b) is selected from ketamine and / or thiamethoxam hydrochloride.

[0040] In some specific embodiments, the gas anesthetic drug in step 1c) is selected from isoflurane or sevoflurane.

[0041] In some specific embodiments, the physiological indicators of the experimental animals in step 1d) are selected from rectal temperature, heart rhythm, heart rate and / or blood oxygen.

[0042] Further, in step 2), anhydrous ethanol is injected via the jugular vein sheath. The anhydrous ethanol is an ethanol solution with a volume content of 88%-90%, 90%-92%, 92%-94%, 94%-96%, or 96%-99%. Preferably, hemodynamic parameters are recorded before and after the anhydrous ethanol injection.

[0043] In some specific embodiments, the dose of anhydrous ethanol injected in step 2) is 0.08-0.12 ml / kg.

[0044] In some specific embodiments, the duration of a single injection of anhydrous ethanol in step 2) is 48-72 seconds.

[0045] In some specific embodiments, the time interval between the two anhydrous ethanol injections in step 2) is 8-12 minutes.

[0046] In some specific embodiments, the number of times anhydrous ethanol is injected in step 2) is 4-6.

[0047] In some specific embodiments, the hemodynamic parameters in step 2) are selected from pulmonary artery systolic pressure (sPAP), pulmonary artery diastolic pressure (dPAP), mean pulmonary artery pressure (mPAP), femoral artery systolic pressure (sFAP), femoral artery diastolic pressure (dFAP), or mean femoral artery pressure (mFAP).

[0048] Furthermore, the postoperative management in step 3) includes analgesia and / or anti-inflammatory treatment.

[0049] In some specific embodiments, analgesia and / or anti-inflammation are achieved in step 3) using a drug. Preferably, the drug is ibuprofen and / or cefoxitin sodium.

[0050] Furthermore, the ethanol-induced induction is repeated more than four times.

[0051] The experimental pigs were selected from either large or small pigs.

[0052] Furthermore, the large pig is selected from one or more of the following: Large Yorkshire pig, Duroc pig, Landrace pig, Shanghai White pig, Hubei White pig, Zhejiang Medium White pig, or Nanchang White pig; and / or, the small pig is selected from one or more of the following: Göttingen Miniature pig, Minnesota Homer Miniature pig, Pittman Moore Miniature pig, Yucatan Miniature pig, Sinclair Miniature pig, Guangxi Bama Miniature pig, Xishuangbanna Miniature pig, or Guizhou Miniature pig.

[0053] In some specific embodiments, the experimental pigs are 4-5, 5-6, 6-7, or 7-8 months old, and weigh 28-30, 30-32, 32-35, 35-37, or 37-40 kg; preferably, the experimental pigs are male. More preferably, the experimental pigs are 6-7 months old and weigh 30-35 kg.

[0054] The present invention also provides the use of the ethanol in the preparation of experimental pig models for pulmonary vascular remodeling.

[0055] The histological features of the above-mentioned pig model of pulmonary vascular remodeling include one or more of the following: compared with the control group, pulmonary artery intima thickening, disordered arrangement of pulmonary artery smooth muscle cells, or inflammatory cell infiltration of pulmonary artery.

[0056] The histological characteristics of the above-mentioned pig model of pulmonary vascular remodeling also include one or more of the following: compared with the control group, there are no pathological changes in myocardial tissue, liver tissue, lung tissue, or kidney tissue.

[0057] This invention also provides the use of pulmonary vascular remodeling experimental pig models for screening drugs to prevent pulmonary hypertension.

[0058] This invention also provides the use of screening drugs for preventing pulmonary vascular remodeling in experimental pig models of pulmonary vascular remodeling.

[0059] The aforementioned pulmonary hypertension or pulmonary vascular remodeling is induced by ethanol.

[0060] The above-mentioned drugs are selected from one or more of losartan, valsartan, irbesartan, enalapril, benazepril, fosinopril, amlodipine, levamlodipine besylate, nivadipine, nifedipine, urapidil, sodium nitroprusside, tolol, bisoprolol, or propranolol.

[0061] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0062] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0063] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0064] Example 1: Induction Procedure via Anhydrous Ethanol Injection into the Jugular Vein

[0065] Induction of anhydrous ethanol injection via jugular vein includes the following steps:

[0066] a. Selection of experimental materials and pre-experimental preparation:

[0067] Male white pigs were selected, approximately 6 months old and weighing 30-35 kg. Animals were fasted from food and water the night before surgery. Anesthesia was induced by intramuscular injection of ketamine (20 mg / kg) and thiamethoxam hydrochloride (2.0 mg / kg), and intravenous access was established in the ear vein using a 20G cannula. A 7.5 mm endotracheal tube was inserted, and mechanical ventilation was maintained using an anesthesia machine. Anesthesia was maintained using 1.5%-2.5% isoflurane containing 0.45-0.55% oxygen, and intravenous infusion of normal saline (15 ml / kg / h) was maintained throughout the experiment. A warming blanket was used to maintain the rectal temperature of the experimental animals at 37°C. Heart rhythm and rate were continuously monitored using a lead II electrocardiogram. A pulse oximeter was placed under the lower lip to monitor changes in blood oxygen levels throughout the procedure.

[0068] b. Anhydrous ethanol injection and hemodynamic parameter recording:

[0069] Anhydrous ethanol (≥99.5%) was injected via a transjugular vein puncture sheath at a dose of 0.1 ml / kg body weight over 60 seconds, with 10-minute intervals between injections, for a total of five injections. Hemodynamic parameters were recorded before each injection, including: pulmonary artery systolic pressure (sPAP), pulmonary artery diastolic pressure (dPAP), mean pulmonary artery pressure (mPAP), femoral artery systolic pressure (sFAP), femoral artery diastolic pressure (dFAP), and mean femoral artery pressure (mFAP). Hemodynamic changes were recorded after each injection.

[0070] c. Postoperative management:

[0071] The experimental pigs were fed according to the standards required by the laboratory, and ibuprofen and cefoxitin sodium were used for postoperative analgesia and anti-inflammation, respectively.

[0072] Example 2: Acute increase in pulmonary artery pressure and femoral artery pressure induced by anhydrous ethanol

[0073] Sixteen male white pigs (average weight: 32.9±1.6 kg) were selected and administered anhydrous ethanol via intravenous bolus injection as described above, for a total of 5 times. The average dose of anhydrous ethanol per administration was 16.5±0.8 mL. The control group received physiological saline instead of anhydrous ethanol via bolus injection. Results are as follows... Figure 1As shown in AC, the pulmonary artery systolic pressure (sPAP), pulmonary artery diastolic pressure (dPAP), and mean pulmonary artery pressure (mPAP) in the ethanol group were significantly higher than those in the control group (P<0.05). These changes were evident after the first injection and tended to stabilize after subsequent injections. Figure 1 GI). In addition, femoral artery pressure also changes, directly reflecting systemic arterial pressure. Although changes in femoral artery systolic pressure (sFAP), femoral artery diastolic pressure (dFAP), and mean femoral artery pressure (mFAP) are similar to those in pulmonary artery pressure (…). Figure 1 DF (pulmonary artery pressure), but they cannot reflect ethanol-induced blood pressure changes as accurately and promptly as pulmonary artery pressure. Figure 1 Therefore, ethanol can effectively induce acute changes in pulmonary artery hemodynamics.

[0074] Example 3: Long-term injection of anhydrous ethanol to induce stable pulmonary hypertension

[0075] After the first week of administration, experimental pigs were given the same intravenous injection of anhydrous ethanol weekly for four consecutive weeks. The pulmonary artery pressure at the end of each subsequent administration was compared to the baseline pulmonary artery pressure of the white pigs to obtain the pulmonary artery systolic pressure gradient (ΔsPAP), pulmonary artery diastolic pressure gradient (ΔdPAP), and mean pulmonary artery pressure gradient (ΔmPAP). Results are as follows: Figure 2 As shown, compared with the control group, the pulmonary artery pressure remained at a higher level after continuous induction with anhydrous ethanol. After the fourth induction, the pulmonary artery hypertension of the experimental pigs remained at a stable high level.

[0076] Example 4: Long-term injection of anhydrous ethanol to induce pulmonary artery remodeling

[0077] One month after the first experiment, pulmonary artery tissue was collected from the experimental pigs. The pulmonary artery tissue was first stained with H&E to observe its overall morphology. Figure 3 As shown, compared with the control group (A), the pulmonary artery intima of the ethanol-treated pigs was significantly thickened (E). This result was further validated by immunohistochemical detection of CD31 (B, F). Next, immunohistochemical detection of α-smooth muscle actin (α-SMA) in the pulmonary artery tissue was performed, as shown in Figure 1. Figure 3 As shown in C and G, after repeated ethanol induction, the arrangement of pulmonary artery smooth muscle cells became significantly disordered, and the tissue morphology became irregular. Finally, the inflammatory response in the pulmonary artery tissue was observed using immunohistochemical detection of tumor necrosis factor-α (TNF-α). Compared with the blank group (D), the pulmonary artery of the pigs in the ethanol-induced pulmonary hypertension experiment showed significant inflammatory cell infiltration (H). All of the above histological results are consistent with the pathological phenotype of pulmonary artery remodeling induced by pulmonary hypertension.

[0078] Example 5: Long-term injection of anhydrous ethanol does not cause pathological changes in cardiopulmonary tissue.

[0079] Heart, liver, lung, and kidney tissues were collected from experimental pigs. H&E and Sirius red staining revealed no significant abnormalities in the myocardial tissue structure of the experimental pigs. Figure 4 A, B); No obvious abnormalities were observed in the liver tissue of the experimental pigs by H&E and periodic acid-Schiff (PAS) staining. Figure 4 (C, D); H&E and Masson staining showed no significant pathological changes in the lung and kidney tissues of the experimental pigs. These results confirm that, compared to lily alkaloids, anhydrous ethanol is safer for inducing pulmonary hypertension and does not cause significant side effects on the cardiopulmonary system.

[0080] Example 6: Losartan partially inhibits pulmonary artery remodeling induced by long-term anhydrous ethanol injection.

[0081] Three days prior to ethanol administration, experimental pigs were given losartan orally at a dose of 10 mg / kg body weight once daily for three consecutive days. Subsequently, as in the previous experiment, ethanol was administered intravenously via the jugular vein. After the first week of the experiment, the pigs received the same prophylactic administration of losartan and intravenous injection of anhydrous ethanol weekly for four consecutive weeks. Pulmonary artery tissue was collected one month later. Figure 5 As shown in AD, losartan alone does not affect the tissue morphology of the pulmonary artery in experimental pigs. However, losartan can partially reverse pulmonary intimal hyperplasia induced by long-term injection of anhydrous ethanol. Figure 5 E, F), disordered smooth muscle arrangement ( Figure 5 G) and infiltration of inflammatory cells ( Figure 5 H).

[0082] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method for constructing a pig model of pulmonary vascular remodeling, characterized in that, The construction method involves repeatedly inducing pulmonary vascular remodeling in experimental pigs with ethanol; inducing pulmonary hypertension through ethanol induction, thereby inducing pulmonary vascular remodeling; the ethanol is administered by injection. It also includes the following features: a) The dosage of ethanol for a single injection is 0.08-0.12 ml / kg; b) The duration of a single ethanol injection is 48-72 seconds; c) One induction involves multiple ethanol injections, with an interval of 8-12 minutes between two ethanol injections; d) The number of ethanol injections during a single induction is 4-6.

2. The construction method according to claim 1, characterized in that, The injection site is the jugular vein sheath; and / or, the ethanol is anhydrous ethanol.

3. The construction method according to claim 1, characterized in that, The ethanol-induced number of times is more than 4.

4. The construction method according to claim 1, characterized in that, The experimental pigs included one or more of the following characteristics: age 4-8 months, weight 28-37 kg, and male.

5. The construction method according to claim 4, characterized in that, The experimental pigs were selected from one or more of the following: Large White, Duroc, Landrace, Shanghai White, Hubei White, Zhejiang Medium White, or Nanchang White; and / or one or more of the following: Göttingen Miniature Pig, Minnesota Homer Miniature Pig, Pittman Moore Miniature Pig, Yucatan Miniature Pig, Sinclair Miniature Pig, Guangxi Bama Miniature Pig, Xishuangbanna Miniature Pig, or Guizhou Miniature Pig.

6. The use of the pulmonary vascular remodeling experimental pig model obtained by the construction method according to any one of claims 1-5 in screening drugs for the prevention of pulmonary hypertension; or, the use of the pulmonary vascular remodeling experimental pig model obtained by the construction method according to any one of claims 1-5 in screening drugs for the prevention of pulmonary vascular remodeling.

7. The use of ethanol in the preparation of products for constructing experimental pig models of pulmonary vascular remodeling, characterized in that, The histological features of the pulmonary vascular remodeling experimental pig model include one or more of the following: compared with the control group, pulmonary artery intima thickening, disordered arrangement of pulmonary artery smooth muscle cells or inflammatory cell infiltration of pulmonary artery; and / or, compared with the control group, no pathological changes in myocardial tissue, liver tissue, alveolar tissue or kidney tissue. Ethanol is administered by injection; It also includes the following features: a) The dosage of ethanol for a single injection is 0.08-0.12 ml / kg; b) The duration of a single ethanol injection is 48-72 seconds; c) One induction involves multiple ethanol injections, with an interval of 8-12 minutes between two ethanol injections; d) The number of ethanol injections during a single induction is 4-6.

Images