A viable non-compaction left ventricular animal model and its construction method and application

Abstract

This invention discloses a method for constructing a viable fetal left ventricular noncompaction animal model, comprising the following steps: on day 12.5 of embryonic development, all-trans retinoic acid injection is injected into the peritoneal cavity of pregnant mice, and the mice are fed normally until delivery to obtain a viable fetal left ventricular noncompaction animal model. The animal model constructed using this method is viable after birth. The ratio of the thickness of the noncompact myocardium to the thickness of the compact myocardium in the region from the level of the left ventricular papillary muscles to the apex of the heart in the model mice is N / C > 2, meeting the requirements for modeling and effectively solving the problem that fetal mice cannot survive after modeling using existing methods.

Description

Technical Field

[0001] This invention belongs to the field of animal model preparation technology, specifically relating to a viable fetal left ventricular compaction insufficiency animal model, its construction method, and its application. Background Technology

[0002] Left ventricular non-compaction (LVNC) is a congenital cardiomyopathy. Its pathogenesis involves disruption of the embryonic myocardial compaction process, resulting in the formation of a thick non-compact trabecular layer and a thin compact layer in the ventricular wall. Clinically, a ratio of non-compact to compact layer thickness (N / C) greater than 2 is usually used as the diagnostic criterion for LVNC.

[0003] The clinical manifestations of ventricular dystrophic heart disease (LVNC) are heterogeneous, presenting as heart failure, arrhythmias, thromboembolism, etc.; however, it can also be asymptomatic in the early stages or in some individuals. Studies have shown that LVNC has significant genetic heterogeneity, with various gene mutations (such as the taffazin gene (TAZ) encoding cardiac structural proteins and the α-dystrophin gene (DTNA)) contributing to its development. Furthermore, imaging examinations such as echocardiography can reveal excessive intracardiac trabeculae and decreased cardiac function in patients with LVNC. This disease is not extremely rare in pediatric patients, with imaging showing a significant increase in ventricular wall trabeculae and impaired cardiac function. Due to limited clinical sample availability and disease heterogeneity, the pathogenesis of LVNC, perinatal structural-functional evolution, and intervention strategies still require systematic research using reproducible and standardized animal models.

[0004] However, existing animal models of left ventricular noncompaction (LVNC) have certain limitations. For example, some researchers induced LVNC in pregnant rat embryos by administering a high dose of RA (70 mg / kg) at 8.5 days of gestation, but this method carries a high risk of teratogenicity, leading to heart malformations in the fetuses, and a large number of fetuses die during pregnancy, rarely surviving to birth. Another study used a hyperglycemic environment during pregnancy (streptozotocin induction) to construct a fetal rat LVNC model and compared it with the RA-induced method. The results showed that embryos in both models exhibited significant left ventricular noncompaction characteristics, with a significantly higher N / C ratio than normal. The streptozotocin-induced NVM model showed a significantly reduced embryo survival rate and a higher perinatal mortality rate.

[0005] Early embryonic lethality leads to a severe "transformation gap," preventing researchers from longitudinally observing the natural course of LVNC after birth, studying compensatory mechanisms of adult-onset cardiac dysfunction, and providing a sufficient time window for screening candidate drugs and evaluating their efficacy. Furthermore, placental defects (such as Daam1) or vascular abnormalities (such as Ogt) associated with some systemic knockout models also increase the complexity of phenotypic analysis.

[0006] Therefore, it is particularly urgent to develop an LVNC animal model that is easy to operate, has a typical phenotype, and can survive for a long time after birth. Summary of the Invention

[0007] To address the aforementioned shortcomings in the prior art, this invention provides a method for constructing a viable fetal left ventricular incomplete compaction animal model. The animal model constructed using this method is viable after birth, and the ratio of the thickness of the non-compact myocardium to the thickness of the compact myocardium in the region from the level of the left ventricular papillary muscle to the apex of the heart in the model mouse is N / C > 2, which meets the modeling requirements and effectively solves the problem that fetal mice cannot survive after modeling using the prior art methods.

[0008] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: A method for constructing a viable fetal left ventricular compaction insufficiency animal model includes the following steps: on day 12.5 of embryonic development, all-trans retinoic acid injection is injected into the abdominal cavity of pregnant mice, and the mice are fed normally until delivery to obtain a viable fetal left ventricular compaction insufficiency animal model.

[0009] Furthermore, the solvent for all-trans retinoic acid injection includes corn oil at a volume percentage of 95% and DMSO at a volume percentage of 5%.

[0010] Furthermore, the dosage is 40-50 mg / kg.

[0011] Furthermore, pregnant mice were injected with all-trans retinoic acid injection at a volume of 8 ml / kg.

[0012] A viable animal model of left ventricular compaction insufficiency was obtained using the method described above.

[0013] The above-mentioned viable animal models of left ventricular non-compaction can be used to screen or evaluate candidate drugs for the prevention and / or treatment of left ventricular non-compaction.

[0014] The above-mentioned viable animal model of left ventricular incompression was used to study the pathogenesis of left ventricular incompression.

[0015] The beneficial effects of this invention are as follows: The method of this invention only requires a single intraperitoneal administration to pregnant mice at E12.5 days. It is simple to operate, has a short cycle, and good reproducibility. The resulting animal model shows that the thickness of the non-compact layer in the region from the level of the papillary muscle to the apex of the left ventricle exceeds twice the thickness of the compact layer (N / C > 2), proving the successful model establishment. The newborn mice obtained from the model can survive for a long time, which is convenient for postnatal functional assessment and follow-up. The lesion area can involve the region below the level of the papillary muscle and the apex of the heart, which is consistent with the characteristics of common affected segments in LVNC. It is suitable for mechanism research and candidate drug screening and evaluation, and has good industrial applicability and scientific research promotion value. Attached Figure Description

[0016] Figure 1 Anatomical views of P1 mice from different groups on day 1 after birth; Figure 2 Anatomical views of the overall appearance of P0 mice in different groups after birth; Figure 3 High-frequency ultrasound long-axis section of the left ventricle of P1 mice in the ATRA model group on day 1 after birth; Figure 4 High-frequency ultrasound long-axis section of the left ventricle of P1 mice on day 1 after birth (blank control group). Figure 5 High-frequency ultrasound long-axis section of the left ventricle of P1 mice on day 1 postnatal, representing the vector control group. Figure 6 High-frequency ultrasound short-axis section of the heart at the left ventricular papillary muscle level in P1 mice of the ATRA model group on day 1 after birth; Figure 7 This is a short-axis section of the heart at the left ventricular papillary muscle level, taken by high-frequency ultrasound of the heart of P1 mice on day 1 after birth, which is the blank control group. Figure 8 High-frequency ultrasound short-axis section of the heart at the left ventricular papillary muscle level of P1 mice on day 1 after birth, serving as the vector control group. Figure 9 High-frequency ultrasound spot tracking analysis of the heart of P1 mice in the ATRA model group on day 1 after birth; Figure 10 High-frequency ultrasound spot tracking analysis of the heart of P1 mice on day 1 after birth, which is the blank control group. Figure 11 High-frequency ultrasound spot tracking analysis of the heart of P1 mice in the vector control group on day 1 after birth; Figure 12 Gross anatomical view of the heart of P1 mice in the ATRA model group on day 1 after birth; Figure 13 Gross anatomical view of the heart of P1 mice on day 1 after birth, which is the blank control group. Figure 14Gross anatomical view of the heart of P1 mice in the vector control group on day 1 after birth; Figure 15 Short-axis pathological tissue sections at the left ventricular papillary muscle level of P1 mice from different groups on day 1 after birth; Figure 16 Short-axis pathological tissue sections below the level of the papillary muscles of the left ventricle of P1 mice from different groups on day 1 after birth; Figure 17 This image shows pathological tissue sections of the left ventricular apex level short axis of the heart of P1 mice from different groups on day 1 after birth. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0018] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0019] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0020] The features and performance of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0021] Example 1 A viable fetal left ventricular compaction insufficiency animal model is constructed by the following steps: on day 12.5 (E12.5) of embryonic development, all-trans retinoic acid injection is injected intraperitoneally into pregnant mice, and the mice are fed normally until delivery to obtain a viable fetal left ventricular compaction insufficiency animal model. The solvent of the all-trans retinoic acid injection includes corn oil with a volume percentage of 95% and DMSO with a volume percentage of 5%. The dosage is 40 mg / kg, and the pregnant mice are injected with all-trans retinoic acid injection at a volume of 8 ml / kg.

[0022] Example 2 A viable fetal left ventricular compaction insufficiency animal model is constructed by the following steps: on day 12.5 (E12.5) of embryonic development, all-trans retinoic acid injection is injected intraperitoneally into pregnant mice, and the mice are fed normally until delivery to obtain a viable fetal left ventricular compaction insufficiency animal model. The solvent of the all-trans retinoic acid injection includes corn oil with a volume percentage of 95% and DMSO with a volume percentage of 5%. The dosage is 45 mg / kg, and the pregnant mice are injected with all-trans retinoic acid injection at a volume of 8 ml / kg.

[0023] Example 3 A viable fetal left ventricular compaction insufficiency animal model is constructed by the following steps: on day 12.5 (E12.5) of embryonic development, all-trans retinoic acid injection is injected intraperitoneally into pregnant mice, and the mice are fed normally until delivery to obtain a viable fetal left ventricular compaction insufficiency animal model. The solvent of the all-trans retinoic acid injection includes corn oil with a volume percentage of 95% and DMSO with a volume percentage of 5%. The dosage is 50 mg / kg, and the pregnant mice are injected with all-trans retinoic acid injection at a volume of 8 ml / kg.

[0024] Comparative Example 1 An animal model of fetal left ventricular compaction insufficiency is established by the following steps: on day 8.5 (E8.5) of embryonic development, all-trans retinoic acid injection is injected intraperitoneally into pregnant mice, and the mice are fed normally until delivery; wherein, the solvent of all-trans retinoic acid injection includes corn oil with a volume percentage of 95% and DMSO with a volume percentage of 5%, the dosage is 50 mg / kg, and the pregnant mice are injected with all-trans retinoic acid injection at a volume of 8 ml / kg.

[0025] Comparative Example 2 An animal model of fetal left ventricular compaction insufficiency is established by the following steps: on day 12.5 (E12.5) of embryonic development, all-trans retinoic acid injection is injected intraperitoneally into pregnant mice, and the mice are fed normally until delivery; wherein, the solvent of all-trans retinoic acid injection includes corn oil with a volume percentage of 95% and DMSO with a volume percentage of 5%, the dosage is 70 mg / kg, and the pregnant mice are injected with all-trans retinoic acid injection at a volume of 8 ml / kg.

[0026] Test case (1) Animal mating and gestational age determination: C57BL / 6 female mice were selected and mated with male mice in the same cage; the vaginal plugs were checked the next morning, and those with vaginal plugs were recorded as pregnant and recorded as E0.5. Pregnant female mice were housed in individual cages and managed with routine feeding and care.

[0027] (2) Drug preparation First, all-trans retinoic acid (ATRA) is dissolved in DMSO, then corn oil is added and mixed thoroughly to prepare ATRA injection solution.

[0028] (3) Grouping and administration of experimental animals: The ATRA model group was operated according to the method in Example 3; The carrier control group was prepared by omitting all-trans retinoic acid from the method described in Example 3. The blank control group was not given any drugs or solvents; Comparative Example 1 was performed using the method described in Comparative Example 1. Comparative Example 2 was performed using the same method as in Comparative Example 2.

[0029] In the above groups, fetal mice that died within 24 hours after giving birth were marked as P0, and those that survived after 24 hours were marked as P1.

[0030] (4) Observation of parturition and survival: Pregnant mice were allowed to gestate naturally to full term and give birth naturally. The birth status of newborn mice was recorded. Newborn mice that were spontaneously breathing and had good vitality in P1 were determined to be P1 surviving individuals. Figures 1-2 ).

[0031] Figure 1 Anatomical images of P1 mice on day 1 after birth in different groups; A is the ATRA model group, showing that the structure of each part of the mouse body is fully developed and there are no surface deformities, but the overall body size is significantly smaller than that of the control group; B is the blank control group, showing the appearance of P1 mice with normal development and full body size; C is the vector control group, showing the same normal appearance as the blank control group, with no significant difference in body size.

[0032] Figure 2Anatomical images of P0 mice after birth in different groups; A is comparative group 1, showing that P0 mice cannot survive after birth and have poor limb development. B is comparative group 2, showing that P0 mice cannot survive after birth and have relatively good limb development. C is the blank control group, showing the appearance of normally developed P1 mice, with a full body shape and spontaneous breathing.

[0033] (5) High-frequency echocardiography of P1 mice: High-frequency echocardiography (VEVO 3100, 30-50MHz) was performed on P1 newborn mice to measure the thickness of the compact and non-compact layers of the left ventricle and calculate the N / C ratio. At the same time, parameters related to left ventricular systolic function and myocardial strain parameters were evaluated to obtain structural-functional evidence (Table 1). Figure 3-11 ).

[0034] Table 1: Echocardiographic parameter analysis of all-trans-retinoic acid (ATRA) induced LVNC mouse model Note: Data are expressed as Mean ± SD; Left Ventricular Diameter; N: Non-compacted layer; C: Compacted layer; N / C: Non-compacted / Compacted Ratio; EF: Ejection Fraction; FS: Fractional Shortening; GLS: Global Longitudinal Strain; EDV: End-Diastolic Volume; ESV: Systolic Volume; SV: Stroke Volume; CO: Cardiac Output; Compared with the blank control group: *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0035] Figure 3 High-frequency ultrasound long-axis section of the left ventricle of P1 mice in the ATRA model group on day 1 after birth shows significant enlargement of the left ventricular cavity; Figure 4 The image shows a high-frequency ultrasound long-axis section of the left ventricle of P1 mice on day 1 after birth, representing the blank control group, showing a normal left ventricular morphology. Figure 5 The image shows a high-frequency ultrasound long-axis section of the left ventricle of P1 mice on day 1 after birth, which is similar to the blank control group and shows no obvious enlargement.

[0036] Figure 6 A high-frequency ultrasound short-axis section of the heart at the level of the left ventricular papillary muscle of P1 mice in the ATRA model group on day 1 after birth, showing a significant thickening of the non-compact layer and a thinning of the compact layer. Figure 7 The image shows a short-axis section of the left ventricular papillary muscle level in the heart of P1 mice on day 1 after birth, representing the blank control group, showing normal ventricular wall structure. Figure 8 This is a short-axis section of the heart at the left ventricular papillary muscle level of a P1 mouse on day 1 after birth, used as the vector control group. It shows a normal structure consistent with the blank control group.

[0037] Figure 6-8 Explanation of markings: 1 indicates a non-compacted layer (N); 2 indicates a compacted layer (C). Figure 9 The high-frequency ultrasound speckle tracking analysis of the heart of P1 mice in the ATRA model group on day 1 after birth shows that the amplitude of the long axis strain curve of each segment of the left ventricle is reduced and the absolute value of GLS is significantly reduced. Figure 10 High-frequency ultrasound speckle tracking analysis of the heart of P1 mice on day 1 after birth, which is the blank control group, shows the normal left ventricular long axis strain curve and strain value. Figure 11 The image shows a high-frequency ultrasound spot tracking analysis of the heart of P1 mice on day 1 after birth in the vector control group, which shows normal strain characteristics consistent with the blank control group.

[0038] (6) HE staining and morphological verification: P1 neonatal rat heart tissue was routinely fixed, dehydrated, paraffin-embedded, sectioned, and stained with HE. Sections below the level of the mammary muscle and at the apex of the heart were selected to measure the thickness of the non-compact and compact layers and calculate the N / C ratio. A significant thickening of the non-compact layer and an N / C ratio greater than 2 were observed (Table 2). Figure 12-17 ).

[0039] Table 2: Myocardial pathological model Note: Data are expressed as Mean ± SD; compared with the blank control group: *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0040] Figure 12 This is a gross anatomical view of the heart of P1 mice in the ATRA model group on day 1 after birth. It shows that the overall outline of the heart is full, with pathological spherical changes, a blunt apex, and an increased volume compared to the normal control group. Figure 13This is a gross anatomical view of the heart of P1 mice on day 1 after birth, showing the normal conical heart appearance of the blank control group. Figure 14 This is a gross anatomical view of the heart of P1 mice on day 1 after birth in the vector control group, showing a normal heart appearance consistent with the blank control group. Figure 12-13 The red scale represents 1 mm.

[0041] Figure 15 The images show short-axis pathological sections of the left ventricular papillary muscle level of P1 mice on day 1 after birth in different groups. A is the ATRA model group, showing a large number of coarse trabeculae in the ventricular wall and a significant thickening of the non-compact layer. B is the blank control group, showing a compact ventricular wall structure, mainly composed of the compact layer. C is the carrier control group, showing a normal ventricular wall structure that is consistent with the blank control group. Figure 15 Measurement markings: Blue line indicates the thickness of the non-compacted layer (N); black line indicates the thickness of the compacted layer (C).

[0042] Figure 16 The images show short-axis pathological sections below the papillary muscle level of the left ventricular papillary muscles of P1 mice from different groups on day 1 after birth. A is the ATRA model group, showing a large number of coarse trabeculae in the ventricular wall and a significant thickening of the non-compact layer. B is the blank control group, showing a compact ventricular wall structure, mainly composed of the compact layer. C is the carrier control group, showing a normal ventricular wall structure that is consistent with the blank control group. Figure 16 Measurement markings: Blue line indicates the thickness of the non-compacted layer (N); black line indicates the thickness of the compacted layer (C).

[0043] Figure 17 The following are pathological tissue sections of the left ventricular apex level short axis of P1 mice on day 1 after birth in different groups: A is the ATRA model group, showing a large number of coarse trabeculae in the ventricular wall and a significant thickening of the non-compact layer; B is the blank control group, showing a compact ventricular wall structure, mainly composed of the compact layer; C is the carrier control group, showing a normal ventricular wall structure that is consistent with the blank control group. Figure 17 Measurement markings: Blue line indicates the thickness of the non-compacted layer (N); black line indicates the thickness of the compacted layer (C).

Claims

1. A method for constructing a viable fetal left ventricular tamponade animal model, characterized in that, Includes the following steps: On day 12.5 of embryonic development in pregnant mice, all-trans retinoic acid injection was injected into the peritoneal cavity of the pregnant mice, and they were fed normally until delivery to obtain a viable fetal left ventricular incomplete compaction animal model. The dosage of all-trans retinoic acid was 40-50 mg / kg. Pregnant mice were injected with all-trans retinoic acid injection at a volume of 8 ml / kg.

2. The method for constructing a viable animal model of left ventricular compaction insufficiency as described in claim 1, characterized in that, The solvent in all-trans retinoic acid injection includes corn oil (95% by volume) and DMSO (5% by volume).

3. The method for constructing a viable animal model of left ventricular compaction insufficiency as described in claim 1, characterized in that, The pregnant mice were C57BL / 6 pregnant mice.

4. The application of the method for constructing a viable animal model of left ventricular compaction insufficiency as described in claim 1 in screening or evaluating candidate drugs for the prevention and / or treatment of left ventricular compaction insufficiency.

5. The application of the method for constructing a viable animal model of left ventricular non-compaction as described in claim 1 in the study of the pathogenesis of left ventricular non-compaction.

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