A mouse model of intrauterine growth restriction during pregnancy and a preparation method thereof

Abstract

This invention belongs to the technical field of animal intrauterine models, specifically relating to a mouse intrauterine growth restriction model and its preparation method. The preparation method includes: Step 1: Preparing a low molecular weight racemic polylactic acid (PDLLA, MW~1000) solution using corn oil as a solvent; Step 2: Obtaining pregnant mice by adapting them to the feed and then co-culturing them; Step 3: Randomizing the pregnant mice into groups and administering the prepared low molecular weight racemic polylactic acid (PDLLA) solution by gavage daily from day 0 to day 18 of gestation, obtaining the intrauterine growth restriction model on day 18 of gestation. The method proposed in this invention is simple to operate, has a high success rate, a low stillbirth rate, and causes minimal damage to the mother. The model obtained by this method closely approximates the intrauterine growth restriction condition under human exposure conditions, which is of great significance for studying the pathogenesis, physiological and pathological changes, and treatment methods of fetal intrauterine growth restriction.

Description

Technical Field

[0001] This invention belongs to the field of animal intrauterine model technology, specifically relating to a mouse intrauterine growth restriction model during pregnancy and its preparation method. Background Technology

[0002] Intrauterine growth restriction (IUGR), also known as small for gestational age (SGA), refers to newborns whose birth weight is below the 10th percentile or two standard deviations below the mean birth weight for their gestational age. The average incidence of IUGR in the Chinese population is 6.39%. IUGR is a common complication of pregnancy and a significant cause of perinatal mortality and morbidity. Furthermore, intrauterine growth restriction can have multiple adverse effects on offspring's metabolic function, cardiovascular function, and neurodevelopment, both in the short and long term. Therefore, finding a simple, rapid, and effective method to establish an intrauterine growth restriction model that closely resembles human clinical pathological characteristics is of great significance for studying the pathogenesis, physiological and pathological changes, and treatment methods of fetal intrauterine growth restriction.

[0003] Currently, there are several models for preparing intrauterine growth restriction, but each has its own shortcomings.

[0004] 1. Uterine artery ligation: This method reduces blood supply to fetal mice through manipulation, thereby affecting their normal development. While simple, it is technically challenging to perform. Overly tight ligation can cause momentary interruption of blood supply, often leading to intrauterine death and increased miscarriage and mortality rates. Overly loose ligation reduces the incidence of intrauterine growth retardation. Previous studies have reported a low surviving rate and high mortality rate with uterine artery ligation. Therefore, the overall success rate of this method for replicating the model is not yet ideal.

[0005] 2. Dietary nutrition structure change during pregnancy: For example, a low-protein diet induces intrauterine growth restriction (IUGR). Low-protein diets used to create IUGR models resulted in increased stillbirths and birth defects in newborn mice, with an IUGR rate of less than 50%. However, compared to other modeling methods, this method is more difficult to control regarding the maternal dietary nutrient levels and the duration of restriction. Furthermore, maternal nutritional restriction may alter placental function and fetal growth and development.

[0006] 3. Genetic engineering method: This includes traditional ES targeting, TALEN, CRISPR / Cas9, and the new TetraOne™ gene knockout technology. These methods are very expensive and labor-intensive, requiring the combination of high-throughput sequencing and analysis technologies. They are rarely used in the preparation of IUGR model animals in China. These technologies are suitable for establishing specific intrauterine growth restriction models.

[0007] Each of the above methods has its advantages and disadvantages. Some have high fetal mortality rates, some are cumbersome to operate, some are highly toxic, and some have low rates of IUGR. Given the shortcomings of current methods for preparing intrauterine growth restriction (IUGR) models, such as high fetal mortality, high toxicity, and cumbersome operation, there is an urgent need to construct an animal model of IUGR that is simple to operate, has a high success rate, a low stillbirth rate, causes minimal damage to the mother, and closely approximates human exposure conditions. Polylactic acid (PLA) is a novel bio-based and renewable biodegradable material derived from the fermentation of starch and starch-based polymers. PLA can degrade in vivo into low molecular weight racemic polylactic acid (PDLLA). According to relevant studies, PDLLA can be completely and harmlessly decomposed in the human body, with the main decomposition products being carbon dioxide and water. This decomposition is completed during human metabolism, thus exhibiting good biocompatibility. Therefore, we chose low molecular weight racemic polylactic acid (PDLLA) for modeling. Summary of the Invention

[0008] To comprehensively address the aforementioned problems, this invention proposes a mouse intrauterine growth restriction model and its preparation method. This method is simple to operate, has a high success rate, a low stillbirth rate, and causes minimal damage to the mother. Furthermore, this model closely approximates the intrauterine growth restriction condition under human exposure conditions.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A mouse model of intrauterine growth restriction during pregnancy was developed by preparing a low molecular weight racemic polylactic acid (PDLLA) solution using corn oil as a solvent. After adaptive feeding, pregnant mice were obtained by co-culturing with the mice. The pregnant mice were randomly divided into groups and administered the low molecular weight racemic polylactic acid (PDLLA) solution by gavage daily from day 0 to day 18 of pregnancy. The intrauterine growth restriction model was obtained on day 18 of pregnancy.

[0011] A method for preparing a mouse model of intrauterine growth restriction during pregnancy includes:

[0012] Step 1: Prepare a low molecular weight racemic polylactic acid (PDLLA) solution using corn oil as a solvent;

[0013] Step 2: After acclimatizing the mice, mate them in the same cage to obtain pregnant mice;

[0014] Step 3: Pregnant mice were randomly divided into groups. From day 0 to day 18 of gestation, they were administered the PDLLA solution prepared in step 1 by gavage at a ratio of 100g body weight / 0.5 mL. An intrauterine growth restriction model was obtained on day 18 of gestation.

[0015] Preferably, in step 1, 1 mL of corn oil and 0.02 mg of PDLLA powder are weighed.

[0016] Preferably, the concentration of the PDLLA solution in step 1 is 0.02 mg / mL.

[0017] Preferably, the mouse strain used in step 2 is ICR, with a microbial grade of SPF, and an age of 8-9 weeks.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. This invention is the first to establish a mouse model of intrauterine growth restriction during pregnancy that simulates human dietary intake of PDLLA;

[0020] 2. This invention uses SPF-grade ICR strain pregnant mice. The ICR strain was bred by Hauschka based on the Swiss mouse population, with a focus on high productivity. This strain of mice is highly adaptable, robust, has strong reproductive capacity, fast growth rate, and good experimental reproducibility. The average litter size of a pregnant mouse is about 10 pups. They have strong reproductive and survival abilities, excellent maternal instincts, and are generally able to raise their offspring to survival. They are an internationally recognized closed-group mouse strain. This model was established using an oral gavage method, which is simple to operate, causes minimal harm to the pregnant mice, allows for precise control of the intake of pregnant mice, and can simulate the process of human dietary intake of low molecular weight racemic polylactic acid (PDLLA) to a high degree. The model success rate and model establishment rate were 29.8%, and the reproducibility reached 100%. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0022] In the attached diagram:

[0023] Figure 1 This is a flowchart of the preparation method of this application;

[0024] Figure 2 This is a general description of the pregnant mice used in this invention.

[0025] Figure 3 The phenotypes of fetal mice in the control and treatment groups of the model of this invention;

[0026] Figure 4 The control and treatment groups of the model of this invention are: (A) fetal mouse weight, (B) fetal mouse body length, (C) placental weight, (D) placental diameter (5) IUGR incidence. Detailed Implementation

[0027] The following is in conjunction with the appendix Figure 1-3 The preferred embodiments of the present invention will be described herein. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0028] A mouse model of intrauterine growth restriction during pregnancy was developed by preparing a low molecular weight racemic polylactic acid (PDLLA) solution using corn oil as a solvent. After adaptive feeding, pregnant mice were obtained by co-culturing with the mice. The pregnant mice were randomly divided into groups and administered the low molecular weight racemic polylactic acid (PDLLA) solution by gavage daily from day 0 to day 18 of pregnancy. The intrauterine growth restriction model was obtained on day 18 of pregnancy.

[0029] Using corn oil as a solvent can avoid negatively impacting the growth and health of laboratory animals. Corn oil possesses certain dissolving properties, capable of dissolving fat-soluble drugs, making them easier for laboratory animals to absorb and utilize. Choosing corn oil as a solvent ensures the safety and health of laboratory animals, while also improving drug efficacy and experimental reliability.

[0030] A method for preparing a mouse model of intrauterine growth restriction during pregnancy includes:

[0031] Step 1: Weigh 0.02 mg of low molecular weight racemic polylactic acid (PDLLA) fluid into a container, dissolve it in 1 mL of corn oil, gently vortex to mix, and obtain a 0.02 mg / mL low molecular weight racemic polylactic acid (PDLLA) solution. Store the solution in a sealed container at 4°C away from light.

[0032] Step 2: After acclimatizing the mice, mate them in the same cage to obtain pregnant mice. The specific procedure is as follows:

[0033] Purchase 8-9 week old SPF-grade ICR male and female mice from a qualified and reputable laboratory animal company. Before the formal experiment, all mice were placed in an observation room for one week for acclimatization feeding, with free access to food and water. The environmental conditions were a temperature of 23±2℃, humidity of 50±5%, and no noise interference. After the acclimatization feeding period, mice with abnormal weight gain, abnormal phenotypes, or diseases were culled. The remaining healthy mice were marked using a back hair staining method, with picric acid dye for the units digit and fuchsin solution for the tens digit. Male and female mice that reached the required weight were mated together at 8 PM in a 1:2 ratio. The following morning at 8 AM, the female mice were checked for vaginal plugs. Female mice with detected vaginal plugs were placed in new cages, and the date was recorded as Gestational day (GD0). Female mice without detected vaginal plugs continued to be mated together in the same cage every evening. The frequency of mating was 4 consecutive days of mating and 3 days of rest per week.

[0034] Step 3: Pregnant mice were randomly divided into groups and administered the PDLLA solution prepared in Step 1 via gavage daily from day 0 to day 18 of gestation. An intrauterine growth restriction model was obtained on day 18 of gestation. Specifically:

[0035] Pregnant rats at gestational age (GD0) were randomly divided into a control group and a high-dose group according to their body weight using a serpentine grouping method. During gestational age (GD0-GD18), the high-dose group was administered PDLLA solution at a dose of 0.1 mg / kg / day via gavage at regular intervals.

[0036] The control group was administered corn oil solution by gavage at a dose of 100 g / 0.5 mL daily.

[0037] Furthermore, all the above-mentioned procedures on mice should be performed gently to avoid stress, and non-exposure factors should be avoided from affecting the fetal mice.

[0038] Example:

[0039] A method for preparing a mouse model of intrauterine growth restriction during pregnancy.

[0040] Step 1: Weigh 0.02 mg of low molecular weight racemic polylactic acid (PDLLA) fluid into a plastic container, dissolve it in 1 mL of corn oil, gently vortex to mix, and ensure it is fully dissolved and homogeneous to obtain a 0.02 mg / mL low molecular weight racemic polylactic acid (PDLLA) solution. Store the solution in a sealed container at 4°C away from light.

[0041] Step 2: After acclimatizing the mice, they are housed together to obtain pregnant mice. The specific procedure is as follows:

[0042] (1) Selection of experimental animals:

[0043] SPF-grade ICR mice (Beijing Vital River Laboratory Technology Co., Ltd.), female, 8 weeks old, weighing 28-30g.

[0044] SPF-grade ICR mice (Beijing Vital River Laboratory Technology Co., Ltd.), male, 8 weeks old, weighing 38-40g.

[0045] (2) Arrangement of the breeding environment:

[0046] Soak the rat cage in disinfectant, rinse it thoroughly with clean water, let it air dry, and then place clean, dry SPF-grade bedding inside. Place the rat cage in an SPF-grade animal room environment with a temperature of 23±2℃ and humidity of 50±5%, free from noise pollution.

[0047] (3) Reception and adaptive feeding of experimental mice:

[0048] Mice were received, their vital signs were observed, and their weight was recorded. Before the formal experiment, all mice were placed in an observation room for one week of acclimatization feeding, during which time they could eat and drink freely. After the acclimatization feeding ended, the mice's vital signs were observed and their weight was recorded again. Mice with abnormal weight gain, abnormal characteristics, or diseases were removed. The remaining healthy mice were marked using the back hair staining method, with picric acid dye marking the units place and fuchsin solution marking the tens place.

[0049] (4) Mating together in cages:

[0050] Two days after the numbering period ended, female mice weighing 28g were mated with male mice weighing 38g at a ratio of 1:2 at 8 PM. The following morning at 8 AM, the female mice's vaginal opening was checked for vaginal plugs. Female mice with vaginal plugs were placed in new cages, and the date was recorded as Gestational day (GD0). Female mice without vaginal plugs continued to be mated in the same cage at night. The mating frequency was 4 consecutive days with a 3-day rest period per week.

[0051] Step 3: Model Preparation Process

[0052] (1) Pregnant mice were grouped

[0053] The pregnant mice were separated into groups of 2-3 and 8-12 per group, and weighed daily.

[0054] (2) Post-grouping processing

[0055] Pregnant mice at gestational age (GD0) were randomly divided into a control group and a high-dose group based on their body weight. The high-dose group received a corn oil solution at 0.1 mg / kg / day via gavage at 8:00 AM daily from GD0 to GD18. The control group received a corn oil solution at a dose of 100 g / 0.5 mL via gavage daily at 8:00 AM.

[0056] Experiment and results:

[0057] Figure 2 Exposure to low molecular weight racemic polylactic acid (PDLLA) solution throughout pregnancy had no effect on the weight or diet of pregnant mice.

[0058] Figure 3 The results showed that the phenotypic results of the fetal mice in the control group and the treatment group were significantly different. The mice in the low molecular weight racemic polylactic acid (PDLLA) solution treatment group were significantly smaller than the control group, indicating that the model was successfully established.

[0059] Figure 4 Compared with the control group, treatment with low molecular weight racemic polylactic acid (PDLLA) solution significantly reduced fetal weight and body length, as well as placental weight and diameter. Furthermore, the incidence of intrauterine growth restriction (IUGR) was significantly increased in PDLLA-treated mice compared to the control group.

[0060] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a mouse model of intrauterine growth restriction during pregnancy, characterized by: include: Step 1: Prepare using corn oil as solvent; weigh 1 mL of corn oil as solvent and 0.02 mg of low molecular weight racemic polylactic acid (PDLLA) fluid; Prepare a low molecular weight racemic polylactic acid (PDLLA) solution with a concentration of 0.02 mg / mL. Step 2: After acclimatizing the mice, mate them in the same cage to obtain pregnant mice; the mice should be of ICR strain, SPF grade, and 8-9 weeks old. Step 3: Pregnant mice were randomly divided into groups. From day 0 to day 18 of gestation, they were administered the PDLLA solution prepared in step 1 by gavage at a ratio of 100g body weight / 0.5 mL daily. An intrauterine growth restriction model was obtained on day 18 of gestation.

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