A method for constructing an animal model of cardiomyopathy induced by circulatory system and application thereof

Abstract

This invention relates to the field of medical animal model construction technology, and in particular to a method for constructing an animal model of cardiomyopathy induced by the circulatory system and its application. This invention, through adventitia-35 injection, establishes for the first time an animal model of "Aβ25-35-induced cardiomyopathy via the adventitia-35 circulatory system" in SD rats. In this model, Aβ25-35 perfusion via the adventitia-35 myocardium of the lower limbs induces diffuse myocardial fibrosis and a decrease in ejection fraction in SD rats, consistent with epidemiological data. This model can be used to further study the pathogenic mechanism of Aβ25-35-induced cardiomyopathy and novel treatment methods using adventitia-35 transport as a pathway, while also providing a new perspective for studying the pathogenic mechanisms and diagnostic and treatment methods of AD or cerebrovascular diseases.

Description

Technical Field

[0001] This invention relates to the field of medical animal model construction technology, and in particular to a method for constructing an animal model of cardiomyopathy induced by the circulatory system and its application. Background Technology

[0002] Beta-amyloid protein not only plays a crucial role in the pathogenesis of cognitive impairment diseases (such as Alzheimer's disease, AD), but since 2016, extensive epidemiological data have confirmed a strong correlation between plasma beta-amyloid levels and cardiac dysfunction. In human specimens from AD patients, Aβ40 and Aβ42 have been found deposited in cardiomyocytes and interstitial spaces of the heart tissue. Most AD patients also exhibit varying degrees of cardiac dysfunction. Furthermore, studies have shown that the accumulation of Aβ40 in the blood, blood vessel walls, and myocardial tissue is closely associated with cardiac dysfunction, coronary artery disease, and the incidence of heart failure. However, current epidemiological data only demonstrate a correlation between circulatory Aβ40 levels and cardiac dysfunction; they do not prove that Aβ40 can directly impair cardiac function through circulatory cardiac perfusion. There are also no reported animal models of cardiac lesions induced by circulatory injection of Aβ40.

[0003] In the mammalian circulatory system, besides blood and lymphatic circulation, Starling proposed in 1896 that tissue fluid, derived from blood plasma, enters the extracellular matrix after filtration through capillaries and can flow continuously within the matrix. Studies have shown that tissue fluid, in addition to free diffusion, can also be transported long distances within organs or tissues, such as perivascular tissues of the brain or thymus, and interstitial spaces within tumor tissues. In 2018, Benias et al. discovered that spaces filled with tissue fluid are supported by complex bundles of collagen fibers. This structure is mainly found in the submucosa of the sinuses, gastrointestinal tract, and bladder, the dermis, and various fibrous connective tissues (such as peribronchial, periarterial, and fascia).

[0004] Constructing animal models of cardiomyopathy is an excellent way to study the pathogenesis of cardiomyopathy and can also be used to screen drugs for its treatment. Since there are currently no animal models of Aβ-induced cardiac lesions induced by intravascular injection, this invention aims to construct an Aβ-induced rat model of cardiomyopathy by injecting the Aβ25-35 peptide into the adventitia. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing an animal model of cardiomyopathy induced by the circulatory system and its application, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for constructing an animal model of cardiomyopathy induced by the circulatory system, comprising the following steps:

[0008] SD rats were injected subcutaneously with Aβ25-35 solution every 2 days for a total of 4 injections to obtain the aforementioned animal model of cardiomyopathy.

[0009] Preferably, the subcutaneous injection site is the adventitia of the blood vessels in the lower extremities.

[0010] Preferably, the injection volume of the Aβ25-35 solution is 400 μL per injection.

[0011] Preferably, the concentration of Aβ25-35 in the Aβ25-35 solution is 1 mg / mL.

[0012] Aβ25-35 is a key peptide in Aβ40 or Aβ42 that can promote the aggregation of Aβ40 or Aβ42 and has similar physicochemical properties to Aβ40 or Aβ42.

[0013] This invention provides the application of the cardiomyopathy animal model obtained by the above construction method in studying the pathogenic mechanism of Aβ-induced cardiomyopathy.

[0014] This invention provides the application of the cardiomyopathy animal model obtained using the above-described construction method in screening drugs for the prevention and / or treatment of cardiomyopathy.

[0015] This invention provides the application of the cardiomyopathy animal model obtained using the above-described construction method in evaluating the safety of drugs for the prevention and / or treatment of cardiomyopathy.

[0016] This invention provides the application of the animal model of cardiomyopathy obtained by the above construction method in evaluating the efficacy of drugs for the prevention and / or treatment of cardiomyopathy.

[0017] The present invention discloses the following technical effects:

[0018] This invention establishes, for the first time, an animal model of "Aβ25-35-induced cardiomyopathy via the adventitia-35 transport system" in SD rats through adventitia-35 injection. In this model, Aβ25-35 perfusion via the lower limb adventitia-35 induces diffuse myocardial fibrosis and a decrease in ejection fraction in SD rats, consistent with epidemiological data. The construction method is simple, and Aβ25-35 fragments are readily available as commercially available powders that can be formulated into working solutions. The adventitia-35 injection method is simple, mature, and easy to perform. The model establishment period provided by this invention is relatively short, with a decrease in ejection fraction appearing approximately 30 days after the first injection and diffuse fibrosis appearing after 60 days. This model allows for further research into the pathogenic mechanism of Aβ25-35-induced cardiomyopathy and novel treatment methods using adventitia-35 transport as a pathway. It also provides a new perspective for studying the pathogenic mechanisms and diagnostic methods of Alzheimer's disease (AD) or cerebrovascular diseases. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The results are shown in the following figures: A is an echocardiogram of the heart (left ventricular long axis) of rats injected with Aβ25-35SD via the adventitia; B is an echocardiogram of the heart (left ventricular long axis) of rats injected with saline via the adventitia; C is an echocardiogram of the heart (left ventricular long axis) of rats injected with Aβ25-35SD via intravenous injection; D is an echocardiogram of the heart (left ventricular long axis) of rats injected with saline via intravenous injection; E is a curve showing the change in left ventricular ejection fraction (EF%) in rats injected with Aβ25-35SD or saline via the adventitia; F is a curve showing the change in left ventricular ejection fraction (EF%) in rats injected with Aβ25-35SD or saline via intravenous injection.

[0021] Figure 2 The images show Masson staining results and myocardial fibrosis scores. A1 and A2 are Masson staining images of SD rats injected adventitiously with Aβ25-35; B1 and B2 are Masson staining images of SD rats injected adventitiously with saline; C1 and C2 are Masson staining images of SD rats injected intravascularly with Aβ25-35; D1 and D2 are Masson staining images of rats injected intravascularly with saline; Interstitia represents the myocardial interstitium; Perivascular represents the perivascular area; E represents the myocardial fibrosis score of SD rats under different treatments; Adventitial infusion represents adventitious injection; Intravenous infusion represents intravascular injection; Saline represents either the adventitious injection control group or the intravascular injection control group; scale bar = 250 μm. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Unless otherwise specified, all materials used in this invention are commonly purchased by those skilled in the art, and all methods used in this invention are well known to those skilled in the art.

[0028] Example 1: Construction of an animal model of Aβ protein-induced cardiac disease (cardiomyopathy) via the circulatory system

[0029] 1. Preparation of Aβ25-35 working solution:

[0030] Aβ25-35 powder was added to physiological saline to prepare a working solution with a concentration of 1 mg / mL, and sonication was used to aid dissolution.

[0031] 2. Treat with Aβ25-35 working solution at 37℃ for 5-7 days.

[0032] 3. Adventitia Injection: SD rats (250-300g) were injected adventitia of their hind limbs with Aβ25-35 (1mg / mL), 400μL per injection, once every 3 days (i.e., once every 2 days), for a total of 4 injections. This group was designated as the Aβ25-35 adventitia induction group. The adventitia control group received an equal volume of physiological saline in the adventitia. The adventitia injection site was in the subcutaneous tissue space of the rat's paw and ankle. After injection of the Aβ25-35 solution at this site, the Aβ25-35 fragments could flow centripetally along the adventitia of the hind limb blood vessels, thereby entering the heart.

[0033] 4. Intravascular Injection: The procedure is the same as step "3. Adventitia Injection", except that the injection site is intravascular, i.e., intravenous injection, and is designated as the Aβ25-35 Intravascular Induction Group. An intravascular injection control group was also set up, which received an equal volume of physiological saline via intravascular injection.

[0034] 5. Echocardiography was performed 30 and 60 days after the first injection to measure parameters such as left ventricular ejection fraction. The echocardiogram results 30 days after injection were as follows: Figure 1 As shown in AD, the ejection fraction change curve is as follows: Figure 1 As shown in Figures E and F. The results showed that the left ventricular ejection fraction (LVEF) of SD rats in the Aβ25-35 adventitia-induced group decreased significantly compared to the adventitia-injected control group. While the LVEF of SD rats in the Aβ25-35 intravascular induction group also showed a decreasing trend, it was not significantly different from the intravascular injection control group. Therefore, adventitia-injection of Aβ25-35 into the hind limbs of SD rats can induce cardiac dysfunction (decreased ejection fraction).

[0035] 5. Sixty days after the first injection, the heart of the SD rat was harvested, embedded in paraffin, and longitudinally cut into 5 sections along the largest surface of the heart. Masson staining was used to analyze myocardial fibrosis.

[0036] The Masson staining procedure is as follows:

[0037] 1) Dewax sections to water using standard procedures; 2) Stain with prepared Weigert iron hematoxylin staining solution for 5-10 minutes; 3) Rinse thoroughly with water; if overstained, differentiate with hydrochloric acid alcohol; 4) Re-blue with Masson's blue solution for 3-5 minutes, then rinse with water; 5) Rinse with distilled water for 1 minute; 6) Stain with Ponceau S and fuchsin for 5-10 minutes; 7) Prepare a weak acid working solution with a volume ratio of distilled water to weak acid solution of 2:1 during the above operations, and wash with the weak acid working solution for 1 minute; 8) Rinse with 1% phosphomolybdic acid solution for 1-2 minutes; 9) Rinse with the prepared weak acid working solution for 1 minute; 10) Immerse directly in aniline blue staining solution for 1-2 minutes without rinsing with water; 11) Rinse with the prepared weak acid working solution for 1 minute; 12) Rapidly dehydrate with 95% (v / v) ethanol; 13) Dehydrate three times with anhydrous ethanol, 5-10 seconds each time; 14) Clear with xylene three times, 1-2 minutes each time; 15) Mount with neutral resin.

[0038] Next, the degree of myocardial fibrosis was scored, and the scoring criteria for the degree of fibrosis are as follows:

[0039] 0: Normal fibroblasts, without excessive proliferation;

[0040] 1: Scattered myocardial interstitial fibroblasts proliferate in small amounts;

[0041] 2: Moderate proliferation of myocardial interstitial fibroblasts and perivascular fibroblasts;

[0042] 3: Extensive proliferation of myocardial interstitial fibroblasts and perivascular fibroblasts.

[0043] Myocardial fibrosis analysis results as follows Figure 2 As shown in the figure. The results indicate that Aβ25-35-induced myocardial fibrosis is mainly concentrated in the perivascular and myocardial interstitial areas, characterized by excessive proliferation of fibroblasts, belonging to diffuse fibrosis. Figure 2 A1 in the study showed the proliferation of myocardial interstitial fibroblasts in Aβ25-35SD rats injected with the adventitia; Figure 2 A2 in the study showed perivascular fibroblast proliferation in Aβ25-35SD rats injected with the adventitia; Figure 2 B1 in the study showed that the myocardial interstitial fibroblasts in rats injected with saline were normal. Figure 2 B2 in the image shows that the perivascular fibroblasts in rats injected with saline are normal. Figure 2 C1 in the figure shows that intravascular injection of Aβ25-35SD rat myocardial interstitial fibroblasts resulted in only minimal proliferation. Figure 2 C2 in the study showed that perivascular fibroblasts in Aβ25-35SD rats injected intravascularly exhibited only minimal proliferation. Figure 2D1 in the study showed that the myocardial interstitial fibroblasts in rats injected with saline were normal; Figure 2 D2 in the study showed normal perivascular fibroblasts in rats injected with saline. The SD rats in the Aβ25-35 adventitia-induced group had high myocardial fibrosis scores, significantly different from the adventitia control group. Similarly, while the myocardial fibrosis scores in the Aβ25-35 intravascular induction group were also elevated, they were not significantly different from the intravascular injection control group. This demonstrates that intravascular injection cannot successfully establish an animal model of Aβ protein-induced cardiac disease (cardiomyopathy) via the circulatory system in the short term. The method provided in this invention (adventitia injection) can successfully establish an animal model of Aβ protein-induced cardiac disease (cardiomyopathy) via the circulatory system, and the model establishment time is shorter, with a decrease in ejection fraction appearing approximately 30 days after the first injection and diffuse fibrosis appearing at 60 days.

[0044] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing an animal model of cardiomyopathy induced by the circulatory system, characterized in that, Includes the following steps: SD rats were used, and Aβ 25-35 solution was injected into the adventitia of the hind limbs every 2 days for a total of 4 injections to obtain the cardiomyopathy animal model. The injection volume of Aβ 25-35 solution was 400 μL each time. The concentration of Aβ 25-35 in the Aβ 25-35 solution was 1 mg / mL. The adventitia injection site was in the subcutaneous tissue space of the rat's foot and ankle.

2. The application of the animal model of cardiomyopathy obtained by the construction method described in claim 1 in the study of the pathogenic mechanism of Aβ-induced cardiomyopathy.

3. The application of the animal model of cardiomyopathy obtained by the construction method of claim 1 in screening drugs for the prevention and / or treatment of cardiomyopathy.

4. The application of the animal model of cardiomyopathy obtained by the construction method of claim 1 in evaluating the safety of drugs for the prevention and / or treatment of cardiomyopathy.

5. The application of the animal model of cardiomyopathy obtained by the construction method of claim 1 in evaluating the efficacy of drugs for the prevention and / or treatment of cardiomyopathy.

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