The experiment is divided into two parts. As shown in Fig. 1, in the first place, we established a novel PGAD mouse model through vaginal dilation and ovariectomized. Then, screened out objective indicators for quantifying mouse sexual behavior and other parameters through a variety of detection methods. Finally, we used this novel model to verify the causal relationship between microvascular injury and PGAD.
Animal recruitment
Experiments were conducted in 118 gravid female C57BL6J mice (Laboratory Animal Center of Ningxia Medical University, Yinchuan, Ningxia, China) weighing 8 weeks old. All mice were randomized the day after giving birth to sham (sham operation), VD2H (vaginal dilation for 2 hours), VD4H (vaginal dilation for 4 hours), OVX (ovariectomy), OVX& VD2H (Vaginal dilation administered at 2 hours following ovariectomy), OVX&&VD4H (Vaginal dilation administered at 4 hours following ovariectomy). The above groupings have 1-month and 2-month time gradients.
This study was reviewed by the Ethical Review Committee of Ningxia Medical University (IACUC-NYLAC-2021-052). All experiments were performed in accordance with relevant Laboratory Animal Welfare and Ethics guidelines and regulations. And we confirmed that complied with the ARRIVE guidelines.
Methods and materials used.
Ovariectomized: Made a 2cm incision above the pubic symphysis to expose the uterine horns, find the ovaries along both sides of the uterus, ligated the ovarian arteries and removed the ovaries. Vaginal dilation: Used 8Fr urinary catheter (Zhanjiang Shida Industrial Co., Ltd., Guangdong Province, China), applied lubricant and inserted it into the vagina, injected 0.6ml warm saline into the water bladder, the urinary catheter is sutured to the vaginal opening with 4 − 0 sutures, and a 30 g weight is suspended from the end of the catheter to hold the catheter under tension (in order to allow the bladder to fully expand the vaginal tissue and not get stuck on the pubic symphysis). Changes before and after vaginal dilation are shown in Supplementary Fig. 1A-C. Vascular occlusion: As indicated by Supplementary Fig. 2, we isolated the blood vessels on both sides of the vagina, and then used high frequency electro tome to divide these vessels into unilateral and bilateral severing.
Sexual behavior and other related sexual responses measured
In this part of the experiment, we refer to the method of Snoeren E and Hernández-Munive A.22,23. The process of sexual behavioral analysis can be stated with two conditions to made mice sexually aroused: One with intraperitoneal hormone therapy (24 hours before the test, 3 mg per mouse of estradiol benzoate subcutaneously; followed by progesterone, 1 mg per mouse subcutaneously, 4 hours before the test), and second with nerve stimulation. After then testing sniff in the first 5 minutes, receptive behavior (called lordosis, which refers to the dorsiflexion of the spinal cord), preceptive behaviors (hopping, darting, and ear wiggling) and aggressive behaviors (boxing, bites and lateral postures) in 30 minutes. In addition, we also recorded the return latencies, defined as the time the female needs to return to the male’s chamber after an exit, and the time that the female spends in the male’s compartment. All tests were conducted between 1:00 PM and 5:30 PM during the dark cycle in a glass box (80×60×60cm) that had a door and black covering cloth. Attach a remote-controlled camera to the side wall of the cage, and after 5 minutes, place an 8-week-old male C57BL6J mouse with normal sexual function.
Vaginal lubrication test
In this part, we innovatively used our own absorbent paper (0.3*1.0cm) to directly observe the changes in the degree of vaginal lubrication (Supplementary Fig. 1F). Cut the scaled absorbent paper into 0.3cm*1cm lengths, inserted into the same depth at the proximal end of the vagina, and the mice were artificially sexually aroused by stimulating the vaginal branch of the pelvic nerve (Supplementary Fig. 2), each stimulation was 10 seconds, and the interval was 1 minute, observe for 10 minutes.
Vaginal temperature test
The room temperature was always kept at 36.0°C, an electronic thermometer (unit: 0.1°C) was inserted into the vagina, the mice were sexually aroused by stimulating the vaginal branch of the pelvic nerve, and the reading was waited for 1 minute (Supplementary Fig. 1E). After three consecutive measurements, the average value was taken as the temperature of the vagina when the mice were sexually aroused.
Vaginal surface tension
Use the MD3000 biological information acquisition system (Beijing Zhimo Duobao Biotechnology Co., Ltd. China) to detect the surface tension of the vagina, give electrical stimulation with the corresponding pulse width, frequency, and energy (The stimulator voltage was 6V, the pulse interval was 0.8ms, the stimulation duration was 30s, the stimulation frequency was 10 Hz, and the stimulation interval was 5 minutes), and slowly place the vaginal recording electrode in the center of the mouse channel, adjust the electrode position to make the metal probe close to the vaginal wall, to stimulated the vaginal branch of the pelvic nerve and observe changes in vaginal surface tension.
Vaginal morphology
We use different staining, including H&E staining, Masson staining, Glycogen dyeing, Glycine silver staining and transmission electron microscopy to observe the effect of vaginal dilation and ovariectomy on vaginal morphology.
Immunological staining
Immunological chemical analysis: The dehydrated tissue was first subjected to antigen retrieval (boiled with 0.01mol/L sodium citrate buffer for 15min in a microwave oven), and incubated with 0.3% Triton-x 100 for 15min. The serum working solution was blocked at room temperature for 1 hour, and the antibody CD31(1:200, Abcam, USA), VEGF-A (1:200, Abcam, USA), MMP-9(1:500, Cell Signaling, Beverly, MA, USA) was added dropwise, overnight at 4°C; on the second day, rewarmed for 30-60min, added Rabbit anti-mouse IgG/HRP (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., China), incubated at 37°C for 30min, and then incubated with DAB developer solution. Immunofluorescence: Except that the secondary antibody uses Goat Anti-Rabbit IgG H&L, other experimental methods and reagents are similar to immunological chemical analysis.
Enzyme-linked immunosorbent assay (ELISA)
Blood was drawn from the heart, centrifuged at 12000 r/min for 10 min, serum was separated, and ELISA detection kit (Wuhan Elite Biotechnology Co., Ltd. China) was used. The serum to be tested in each group was added to the reaction well, 100µl per well, and 4 duplicate wells were set for each group, and incubated at 37°C. 90 min, discard the liquid in the well, add 100 µL of biotinylated antibody working solution, incubate at 37°C for 60 min, discard the liquid in the well, wash 3 times, then add 100 µL of enzyme conjugate working solution, incubate at 37°C for 30 min, then discard remove the liquid in the wells, wash 5 times, add 90 µL of substrate solution to each well, incubate at 37°C for 15 min, add 50 µL of stop solution, detect at 450 nm wavelength, and calculate the content of T, E2 and DA in the serum of mice in each group according to the standard curve.
Western blot
Anterior vagina tissue protein samples were prepared by homogenizing in RIPA lysis buffer. Equal protein (20µg/lane) was electrophoresed on 10% SDS-PAGE and then transferred to polyvinylidene fluoride membrane (Millipore Corp, Bedford, MA, USA). Western blot was performed with antibodies against MMP-9 (1: 1000) (Abcam, Cambridge, MA, USA), VEGF (1:1000) (Abcam, Cambridge, MA, USA), CD31(1: 1000) (Abcam, Cambridge, MA, USA) and β-actin(1:1000) (Santa Cruz Biotechnology, CA, USA).
Estrous Cycle Determination
This part of the experiment is only in the experiment of the relationship between blood flow and PGAD. Starting from the first day, testing every 3 days until the day before the end of the experiment, a daily vaginal smear was collected (13:00–14:00 PM) and examined under a light microscope to determine the stage of the estrous cycle. The estrous cycle phase was characterized by vaginal cytologic study based on Cora M 24.
Statistics
The statistical analysis is made using SPSS23.0 software. The results were expressed as the mean ± SD. The normal distribution of the data was checked with the Kolmogorov-Smirnov test. This study was a mixed within/between design, One-Way ANOVA analysis of variance was used for comparison between groups and Tukey Post Hoc test is used to perform multiple comparisons, and use the two-way ANOVA with repeated measures with one within (test-time) and six between (sham, VD2H, VD4H, OVX, OVX&VD2H, OVX&VD4H) factors. Means were considered significantly different at P < 0.05.