Construction method of animal model for ovarian lymphatic drainage disorder

Abstract

The invention discloses a construction method of an ovarian lymphatic drainage disorder animal model. The method comprises the following steps: performing anesthesia and disinfection treatment on female Balb / c mice of 11-13 weeks old; performing an operation opening at a position 0.5 + / -0.1 cm away from the spinal side of the mouse, and exposing a fat pad around the ovary, the ovary and a fallopian tube structure; injecting Evans blue staining fluid into the ovary of the mouse to develop the ovary drainage lymphatic vessel; identifying and microscopically separating the ovarian drainage lymphatic vessel in the fat pad near the ovarian door, then ligating the ovarian drainage lymphatic vessel by using a fine line, suturing a surgical wound and performing postoperative recovery; injecting a fluorescent macromolecular dye to evaluate the blocking condition of the ovarian lymphatic drainage function; and monitoring general conditions and ovarian functions of the mouse to evaluate the effectiveness of the model so as to obtain the animal model with ovarian lymphatic drainage disorder. The model can simulate the pathological state of the ovarian lymphatic drainage disorder, and provides a reliable experimental tool for researching the correlation between the ovarian function and the lymphatic system.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and particularly relates to a method for constructing an animal model of ovarian lymphatic drainage obstruction. Background Art

[0002] The lymphatic system plays an important role in physiological processes such as maintaining fluid balance and immune regulation, but its dysfunction is associated with various diseases (such as edema and tumor metastasis). The functional study of ovarian lymphatics is limited due to the lack of effective models. In the prior art, lymphatic ligation methods for organs such as the kidney and brain have been reported, but the imaging and ligation techniques for ovarian drainage lymphatics have not been publicly reported. The lymphatic system has multiple functions such as maintaining fluid balance, immune regulation, and lipid absorption. Excess fluid, macromolecules, and immune cells in tissues are recycled through lymphatics and returned to the blood circulation after filtration by lymph nodes. Blockage of lymphatic drainage can cause fluid accumulation in tissue spaces, leading to hereditary lower limb edema, filariasis, secondary edema after tumor resection, etc., and aggravating the pathological processes of neurodegenerative diseases, myocardial infarction, silicosis, etc. Existing studies have shown that ovarian lymphatics may be involved in regulating physiological processes such as follicle maturation, embryonic development, and corpus luteum degradation. It has been reported that ovarian lymphatic drainage is the pathway for inhibin to reach the peripheral circulation. Hironori et al. detected 3β-HSD positive luteal cells in lymphatic drainage fluid by injecting dye into the subcutaneous interstitial tissue of bovine ovaries, and speculated that lymphatics may be involved in the regression process of ovarian corpus luteum.

[0003] In other tissues and organs, by ligating lymphatics to block the lymphatic drainage path, the role of the lymphatic system in the pathophysiological process of the organ can be evaluated. Pathological modeling by lymphatic ligation can help to deeply understand the pathogenesis of diseases related to lymphatic reflux obstruction. For example, ligation of renal drainage lymphatics causes apoptosis of renal tubular epithelial cells and damage to the integrity of renal tubules, resulting in increased urinary albumin and serum creatinine. Ligation of the deep cervical brain drainage lymphatics leads to a decrease in the drainage of brain tumor cells by the lymphatic system, an increase in T cells in the tissue, and a reduction in the therapeutic effect of brain tumors. Mouse lower limb or tail lymphatic ligation models are also commonly used to study secondary lymphedema caused by surgery, cancer treatment, infection, trauma, or obesity. On the other hand, ligation is also a treatment method for some diseases. For example, chylothorax caused by injury to the thoracic duct secondary to cardiac surgery can cause severe lymphopenia and hypogammaglobulinemia, and ligation of the thoracic duct is one of the clinical surgical methods for treating chylothorax. Performing lymphatic microsurgical prophylactic healing to create a preferential lymphatic-venous anastomosis during axillary lymph node dissection for breast cancer can reduce the possibility of subsequent lymphedema in the ipsilateral upper arm. Ligation of mouse corneal lymphatics can inhibit the occurrence of corneal transplant rejection. However, at present, there is no literature report on the ligation study of ovarian drainage lymphatics, and there is still a lack of an effective method for constructing a model of ovarian drainage lymphatic ligation.

[0004] Therefore, to solve the above technical problems, it is necessary to provide a method for constructing an animal model of ovarian lymphatic drainage disorder. Summary of the Invention

[0005] The object of the present invention is to provide a method for constructing an animal model of ovarian lymphatic drainage disorder, which can simulate the pathological state of ovarian lymphatic drainage disorder and provide a reliable experimental tool for studying the association between ovarian function and the lymphatic system.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for constructing an animal model of ovarian lymphatic drainage disorder, the method comprising:

[0008] Anesthetize and disinfect female Balb / c mice at 11 - 13 weeks of age;

[0009] Under a stereomicroscope, make a surgical incision at 0.5 ± 0.1 cm lateral to the mouse spine to expose the adipose tissue around the ovary and the ovarian and fallopian tube structures;

[0010] Inject a staining solution into the mouse ovary to visualize the ovarian draining lymphatics;

[0011] Identify and microdissect the ovarian draining lymphatics in the adipose tissue near the ovarian hilum, then ligate the bilateral ovarian draining lymphatics with a fine thread, suture the surgical wound and perform postoperative recovery;

[0012] Inject a fluorescent macromolecular dye to evaluate the blockade of ovarian lymphatic drainage function; and monitor the general condition and ovarian function of the mice to evaluate the effectiveness of the model, thereby obtaining an animal model of ovarian lymphatic drainage disorder.

[0013] Further, the anesthetic is 1% sodium pentobarbital, and the dose is 0.05 g / kg by intraperitoneal injection.

[0014] Further, the staining solution includes one of methylene blue, patent blue, and Evans blue; the concentration of the Evans blue staining solution is 1%, which is prepared by mixing 100 mg of Evans blue with 10 mL of normal saline, and is stored at -80 °C after aliquoting.

[0015] Further, the macromolecular dye includes at least one of OVA-647, FITC-dextran, and indocyanine green; the use concentration of OVA-647 is 0.2 μg / μL, and the fluorescence intensity of the tracer in the ovarian draining lymph nodes is quantitatively analyzed by a fluorescence imaging device.

[0016] Further, the fine thread is a 10-0 type surgical suture.

[0017] Furthermore, the method further includes: comprehensively evaluating the change of ovarian function by detecting serum biochemical indexes, ovarian index, estrous cycle, progesterone and AMH levels, and oocyte quality.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0019] A method for constructing an animal model of ovarian lymphatic drainage disorder provided by the present invention injects Evans blue dye into the mouse ovary to develop the draining lymphatic vessels and draining lymph nodes of the ovary, microdissects and ligates the draining lymphatic vessels of the ovary, injects the fluorescent macromolecular dye OVA-647 to evaluate the blockage of ovarian lymphatic drainage function, and detects the general condition of the mouse and ovarian function to evaluate the impact of ovarian lymphatic vessel ligation on the body. This innovative experimental method has good reference significance for subsequent scientific research on the function of the ovarian lymphatic system, especially lymphatic drainage-related research. Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 Schematic diagram of ovarian draining lymphatic vessel ligation surgery;

[0022] Figure 2 For lymphatic vessel imaging and ligation effect; A and B. After injecting Evans blue dye into the ovary, the draining lymphatic vessels near the ovarian hilum are developed. C. The deep abdominal direction of the ovarian draining lymphatic vessels. D. The abdominal location of the ovarian draining lymph nodes. E. Schematic diagram of ovarian draining lymphatic vessel ligation. F. Injecting Evans blue dye again after ligation of the ovarian draining lymphatic vessels. G. OVA-647 fluorescence imaging of ovarian draining lymph nodes in the control group (CON group) and the ovarian draining lymphatic vessel ligation group (LA group). H. Quantitative fluorescence statistics of OVA-647 in ovarian draining lymph nodes of the CON group and the LA group. *P<0.05, **P<0.01, ***P<0.001, ns indicates no statistical difference. (○ indicates the ovary, □ indicates the ovarian draining lymph node, blue △ indicates the lymphatic vessel, red △ indicates the blood vessel, ☆ indicates the kidney).

[0023] Figure 3Physiological indices of mice at 30 days after surgery; body weights of mice in the CON group and LA group 30 days after ligation. B. Serum alanine aminotransferase (ALT) level. C. Serum aspartate aminotransferase (AST) level. D. Serum blood urea nitrogen (BUN) level of mice. E. Serum creatinine (CREA) level. F. Serum creatine kinase (CK) level. G. Serum lactate dehydrogenase (LDH) level. ns indicates no statistical difference.

[0024] Figure 4 Data on the changes in ovarian function after ligation of ovarian drainage lymphatics. A is the ovarian index of the CON group and LA (i.e., the ratio of ovarian weight to body weight). B. Proportion of regular and irregular estrous cycles. C. Serum AMH level. D. Serum progesterone level. E. Statistical count of the number of ovarian follicles at all levels in mice, PMF, primordial follicle, primary follicle; PF, primary follicle, secondary follicle; SF, secondary follicle, antral follicle; ANF, antral follicle, atretic follicle; CL, corpus luteum, corpus luteum. F. Statistical count of the cohabitation and litter production experiment and the average litter size. G. Representative images of in vitro culture of oocytes and statistical count of the first polar body extrusion rate (PB1). H. Fluorescent staining of oocyte spindle tubulin and statistical count of the spindle aberration rate. *P<0.05, **P<0.01, ***P<0.001, ns indicates no statistical difference. Detailed implementation manners

[0025] The following will specifically elaborate on the embodiments of the present invention in combination with the detailed implementation manners and examples, and the advantages and various effects of the embodiments of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the embodiments of the present invention, rather than limiting the embodiments of the present invention.

[0026] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the embodiments of the present invention belong. In case of contradiction, this specification shall prevail.

[0027] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the embodiments of the present invention can be obtained through market purchase or can be obtained by existing methods.

[0028] The following will specifically describe in detail a method for constructing an animal model of ovarian lymphatic drainage obstruction of the present application in combination with examples and experimental data.

[0029] Example 1: Method for constructing a model of ligation of ovarian drainage lymphatic vessels

[0030] I. Experimental method

[0031] Twelve-week-old female Balb / c mice were used as the research objects. In a sterile environment of the SPF-class experimental animal center, imaging and ligation surgeries were performed on the ovarian drainage lymphatic vessels, and the fluorescent macromolecular dye OVA-647 was injected to evaluate the blockade of ovarian lymphatic drainage function. All animal experiments were approved by the Animal Experiment Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Approval No.: TJH-202207047).

[0032] The operation steps are as follows:

[0033] (1) Anesthesia: Twelve-week-old Balb / c mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.05 g / kg), and the success of anesthesia was confirmed by pinching the toes of the four limbs.

[0034] (2) Disinfection: Place the mice in the prone position, shave the hair on the dorsal side of the lumbar spine to expose the surgical field, and disinfect with 75% alcohol.

[0035] (3) Surgical incision: Under a stereomicroscope, cut the skin, peritoneal wall and muscle layer at about 0.5±0.1 cm on the side of the spine, avoiding blood vessels, and find structures such as the periovarian fat pad, ovary, and fallopian tube (as Figure 1 shown). The ovary is located below the fimbrial end of the fallopian tube and is surrounded by adipose tissue. The ovarian hilum blood vessels (red arrows) run parallel to the lymphatic vessels (blue arrows). Since lymph is transparent and the lumen of ovarian lymphatic drainage is narrow, the lymphatic vessels wandering in the surrounding fat and connective tissues are not easily visible to the naked eye.

[0036] (4) Dye injection: A 1% (mass / volume) solution was prepared by mixing Evans blue powder (HY-B1102, MCE, New Jersey, USA) with physiological saline, that is, 100 mg of Evans blue was fully mixed with 10 mL of physiological saline, and after aliquoting, it was stored in a -80°C refrigerator. The ovary was fixed with microforceps, and under a stereomicroscope (SZ27, Olympus, Tokyo, Japan), about 1-2 μL of Evans blue solution was slowly and gently injected into the ovarian parenchyma at a rate of 5 μl / min using a BD insulin syringe (BD, New Jersey, USA), and a fine blue network could be seen immediately filling the ovarian surface.

[0037] (5) Ligation of ovarian lymphatic drainage vessels: In the fat pad near the ovarian hilum, a relatively thick blue vessel can be seen accompanying the red blood vessels. This is the ovarian lymphatic drainage vessel. Along the course of the blue vessel, it can be observed that the blue dye is injected into the lymph nodes on the dorsal side of the upper pole of the kidney at the bifurcation of the renal artery of the abdominal aorta. Carefully separate the lymphatic drainage vessels near the ovarian hilum with micro forceps and ligate them with 10-0 fine thread (perform the operation gently to avoid damaging the blood vessels near the ovarian hilum, as Figure 1 shown). The control group of mice was given a similar surgical procedure, injected with an equal amount of Evans blue dye, and the fat around the lymphatic drainage vessels was separated, but not ligated. The same operation was performed on both ovaries.

[0038] (6) Surgical suture: Rinse the surgical field with normal saline, blot the excess liquid with a wet gauze, suture the muscle layer with 8-0 needle-containing sutures, and suture the skin with 4-0 needle-containing sutures. Then place the mice on a heating pad to promote recovery from anesthesia, and return them to the cage after their activity resumes.

[0039] (7) Evaluation of ovarian lymphatic drainage of OVA-647: 2 mg of OVA-647 (OVA-647, O34784, Thermo Fisher Scientific, Massachusetts, USA) was resuspended in 500 μl of PBS to form a stock solution of 4 mg / mL, aliquoted, and stored at -80 °C in the refrigerator. When used, it was diluted 1:20 (concentration 0.2 μg / μL). Fix the ovary with micro forceps during the surgical operation as in steps (1-3), and slowly and gently inject 5 μl of the OVA-647 dilution into the ovary with a micro Gauge 33 Hamilton syringe. After injection, stop the needle for two minutes to reduce leakage. Evaluate the ovarian lymphatic drainage function through the fluorescence intensity of OVA-647. After a certain period of time, isolate the ovary or ovarian lymphatic drainage lymph nodes under a stereomicroscope, take pictures and observe under a small animal fluorescence imaging device Xenogen IVIS Spectrum (Xenogen, Alameda, California, USA), and analyze the fluorescence intensity with Living Image software.

[0040] (8) Functional monitoring: Continuously monitor the general condition, diet, and activity of the mice after ligation, evaluate the serum biochemical indicators of the heart, liver, and kidneys, and detect the ovarian endocrine and reproductive functions, etc.

[0041] II. Experimental results of ligation of ovarian lymphatic drainage vessels

[0042] 1. Evaluation of ligation and drainage blockage of ovarian lymphatic drainage vessels

[0043] Lymphatic vessels often run parallel to blood vessels and are located in connective tissues such as fat. Since lymph is colorless, the tissue structure of mice is small, and the draining lymphatic vessels are delicate and not easily observable by the naked eye. There is currently a lack of literature reports on the draining lymphatic vessels of the mouse ovary. Referring to the studies on the draining lymphatic vessels of the mouse kidney, the deep cervical cerebral draining lymphatic vessels, and the ligation of the lower limb lymphatic vessels, the draining lymphatic vessels of the mouse ovary were visualized and ligated. By microinjecting Evans blue dye into the ovary, a fine blue network was immediately seen to fill the ovarian surface ( Figure 2 A, as shown by the black circle), and then a thinner blue vessel was visible beside the red blood vessels ( Figure 2 A, as shown by the red arrow) surrounded by adipose tissue near the ovarian hilum. This was the draining lymphatic vessel of the mouse ovary ( Figure 2 A, as shown by the blue arrow). The blue lymphatic vessel near the ovarian hilum ran parallel to the red blood vessel and flowed towards the proximal end ( Figure 2 B), and then it was observed that the blue dye was injected into the ovarian draining lymph node (draining lymph node, dLN) behind the posterior side of the upper pole of the kidney at the bifurcation of the renal artery of the abdominal aorta ( Figure 2 C, D). The draining lymphatic vessel of the ovary near the ovarian hilum was isolated and ligated with a fine thread ( Figure 2 E). In addition, after ligating the draining lymphatic vessel of the ovary and injecting Evans blue dye again, the blood vessels near the ovarian hilum were normal as usual, but no blue lymphatic vessel was filled ( Figure 2 F), indicating that the ligation of the draining lymphatic vessel of the ovary was successful. To evaluate the degree of blockade of ovarian lymphatic drainage after ligation of the draining lymphatic vessel of the ovary, we injected the macromolecular fluorescent tracer OVA-647 into the ovary and then evaluated the fluorescence intensity of OVA-647 in the draining lymph node. Compared with the control group, the fluorescence tracer of OVA-647 homing to the draining lymph node in the ligation group (LA group) was significantly reduced at 1 hour, 2 hours, and 3 hours after injection ( Figure 2 G, H). These results indicate that local lymphatic return in the ovary is significantly impaired after ligation of the draining lymphatic vessel of the ovary.

[0044] 2. Physiological indices 30 days after ligation of the draining lymphatic vessel of the ovary

[0045] As Figure 3 shown in A, there was no significant difference in the body weight of mice between the ligation group (LA group) and the control group (CON group) (P = 0.28).

[0046] Liver function: ALT ( Figure 3 B) and AST ( Figure 3 C) levels showed no statistical difference between the two groups (P > 0.05).

[0047] Renal function: BUN ( Figure 3 D) and CREA ( Figure 3E) There was no significant change horizontally (P > 0.05).

[0048] Cardiac function: CK( Figure 3 F) and LDH( Figure 3 G) levels showed no difference between the two groups (P > 0.05).

[0049] The above data indicate that the surgery has no significant impact on the functions of important organs such as the heart, liver, and kidneys, and the model constructed by the present invention has good safety.

[0050] 3. Effects of ovarian lymphatic drainage ligation on ovarian function

[0051] (1) Ovarian index:

[0052] As Figure 4 shown in A, 30 days after ovarian lymphatic drainage ligation, compared with the CON group, the ovarian index (ovary weight / body weight) in the LA group was 0.085 ± 0.012, significantly higher than 0.062 ± 0.008 in the CON group (*P < 0.05). The ovarian index in the LA group increased significantly, indicating that ovarian lymphatic drainage ligation blocked the drainage function of lymphatic vessels for fluids and macromolecules, and had a certain impact on the local tissue homeostasis of the ovary.

[0053] (2) Estrus cycle:

[0054] As Figure 4 shown in B, the irregular estrus cycle rate in the LA group (62.5%) was significantly higher than that in the CON group (18.3%, *P < 0.01).

[0055] (3) Hormone levels:

[0056] As Figure 4 shown in C - 4D, the levels of serum AMH( Figure 4 C) and progesterone( Figure 4 D) decreased by 48.7% and 51.2% respectively in the LA group (both *P < 0.001).

[0057] (4) Follicular development:

[0058] As Figure 4 shown in E, the number of growing follicles (PMF / PF / SF) decreased by 65.3% (*P < 0.001), and the number of atretic follicles (ATF) increased by 2.1 - fold (*P < 0.01). That is, the number of growing follicles (including primary follicles, secondary follicles, and antral follicles) decreased significantly, while the number of atretic follicles increased significantly.

[0059] (5) Fertility:

[0060] As Figure 4 shown in F, the co - housing experiment showed that the average litter size in the LA group decreased from 5.6 to 3.3, indicating a decrease in fertility.

[0061] (6) Oocyte quality:

[0062] As Figure 4 shown in G, the oocytes isolated from the ovaries and cultured in vitro showed that the meiotic arrest of oocytes in the LA group was accompanied by a decrease in the first polar body extrusion rate (PB1) to 38.9% (82.4% in the CON group, *P<0.001).

[0063] As Figure 4 shown in F, the abnormal spindles increased significantly, and the abnormal spindle rate increased to 57.6% (12.3% in the CON group, *P<0.001).

[0064] In summary, it can be seen that ovarian lymphatic drainage obstruction leads to inhibition of ovarian endocrine function, abnormal follicular development, and decline in oocyte quality, resulting in reduced fertility.

[0065] Finally, it should also be noted that the term "comprises", "comprising", or any other variation thereof is intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a series of elements includes not only those elements but also other elements not expressly listed, or elements that are inherent to such process, method, article, or apparatus.

[0066] Although the preferred embodiments of the embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the embodiments of the present invention.

[0067] Obviously, those skilled in the art can make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Thus, if these modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalent technologies, the embodiments of the present invention are also intended to include these changes and modifications.

Claims

1. A method for constructing an animal model of ovarian lymphatic drainage disorder, characterized in that, The method includes: Anesthetizing and disinfecting female Balb / c mice at 11-13 weeks of age; Under a stereomicroscope, making a surgical incision at 0.5±0.1 cm on the side of the mouse spine to expose the periovarian fat pad and the ovarian and fallopian tube structures; Injecting a staining solution into the mouse ovary to visualize the ovarian draining lymphatics; Identifying and microdissecting the ovarian draining lymphatics in the fat pad near the ovarian hilum, then ligating the bilateral ovarian draining lymphatics with a fine thread, suturing the surgical wound and performing postoperative recovery; Injecting a fluorescent macromolecular dye to evaluate the blockade of ovarian lymphatic drainage function; and monitoring the general condition of the mice and ovarian function to evaluate the effectiveness of the model, thereby obtaining an animal model of ovarian lymphatic drainage disorder.

2. The method for constructing an animal model of ovarian lymphatic drainage disorder according to claim 1, characterized in that, The anesthetic is 1% sodium pentobarbital, and the dosage is 0.05 g / kg by intraperitoneal injection.

3. The construction method of an animal model of ovarian lymphatic drainage disorder according to claim 1, characterized in that, The staining solution includes one of methylene blue, patent blue, and Evans blue; the concentration of the Evans blue staining solution is 1%, which is prepared by mixing 100 mg of Evans blue with 10 mL of normal saline, and after aliquoting, it is stored at -80 °C.

4. The method for constructing an animal model of ovarian lymphatic drainage disorder according to claim 1, wherein The macromolecular dye includes one of OVA-647, FITC-dextran, and indocyanine green ICG; the usage concentration of OVA-647 is 0.2 μg / μL.

5. The construction method of an animal model of ovarian lymphatic drainage disorder according to claim 1, characterized in that, The method further includes: comprehensively evaluating the changes in ovarian function by detecting serum biochemical indexes, ovarian index, estrous cycle, progesterone and AMH levels, and oocyte quality.

6. The construction method of an animal model of ovarian lymphatic drainage disorder according to claim 1, characterized in that, The fine thread is 10-0 fine thread.

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