Animal models and interventions
Forty female immature Wistar rats aged 22 days-old (D22) were provided by the Institute of Genome Engineered Animal Models for Human Disease of Dalian Medical University (Dalian, China). They were kept under a controlled 12h light/12h dark life cycle and were fed with a standard laboratory chow diet, with free access to food and water.
The animals were randomly divided into eight groups (n=5), and there was no difference in the initial body weight of each group (P = 0.382). The interventions of each group were conducted as below: (1) CON, control rats received intraperitoneal injection of saline (0.5mL/100g) for 5 consecutive days (D22-D26); (2) OHSS, OHSS model rats received 10 IU equine chorionic gonadotropin (eCG, Solarbio, China, 0.5mL/100g) for 4 consecutive days (D22-D25) and 30 IU of hCG (Ningbo Renjian Pharmaceutical Group Co.Ltd., China, 0.5mL/100g) on the 5th day (D26); (3) EGb761+OHSS, prophylactic treatment group received (intraperitoneal injection) three doses of EGb761 (50mg/kg/d, 100mg/kg/d, 200mg/kg/d) one hour before injection of eCG or hCG for 7 consecutive days; (4) OHSS+EGb761, therapeutic treatment group received (intraperitoneal injection) three doses of EGb761 (50mg/kg/d, 100mg/kg/d, 200mg/kg/d) 48 hours after injection of eCG or hCG for 7 consecutive days. Medications were stored and used according to the manufacturers’ instructions. All doses are recommended to be efficacy in humans and extrapolated from animal’s weight.
Measurement of peritoneal and ovarian capillary permeability
Alterations in vascular permeability were measured by the Evans Blue (EB) dye method according to a protocol slightly modified from that described by Ujioka et al. [10]. Rats were inhalation anesthetized with diethyl ether and kept in thermal blanket to avoid hypothermia. EB dye powder (E8010, Solarbio, China) was diluted in distilled water at a final concentration of 5mM. A fixed volume of 0.2mL was administered via the caudal vein in each rat. Thirty min after the EB injection, the peritoneal cavity was irrigated with 5mL of 0.9% normal saline (21°C). The peritoneal irrigated fluid was collected into tubes and was centrifuged at 900g for 12 minutes at room temperature. Immediately after the peritoneal irrigation, blood was obtained by puncture of the inferior vena cava for hormone assays as described below. Then, the right atrium was incised to allow the outflow of perfusate, and the left apex was slowly injected with 50mL saline for systemic circulation perfusion. After that, the ovaries were removed, weighed and incubated in 65°C N,N-Dimethylformamide (D112007, Aladdin, China) for 24h. EB concentrations were determined by measuring the dye absorption at 600nm with a microplate reader (Spectra MR, DynexTechnologies, USA).
Hormone assay
The serum was collected from blood samples after centrifugation with 3000rpm for 15min. All samples were quick-frozen in liquid nitrogen and stored at -80°C for further use. Estradiol (E2) and progesterone (PRG) levels were determined by the electrochemiluminescence assay (Roche, German) on an automatic electrochemical luminescence immunoassay system (Cobas e 601, Roche, Switzerland). The inter- and intra-assay coefficients of variation as indicated by manufactures were 13.8% and 8.5% for E2 respectively, 10.7% and 7.2% for PRG respectively.
Histologyanalysis
Hematoxylin and eosin (H&E) staining: Ovaries and kidneys isolated from animals were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin as previously described [11]. Paraffin-embedded tissues were serially sectioned at 5μm thickness and stained with H&E routinely for morphological observation. Three rats in each group have been randomly selected for detection of follicular development, and the images were obtained blind with the microscopy (Olympus BX63, Japan) and Image Pro Plus software (Media Cybernetics, USA).
Immunohistochemical (IHC) analysis: Paraffin-embedded ovarian and renal tissues were performed using the DAB Detection Kit (ZLI-9018, ZSGB-BIO, China) according to the manufacturer’s instructions. Sections were deparaffinized, hydrated and rinsed before heat-mediated antigen retrieval in 0.01M pH 6.0 sodium citrate buffer (C1010, Solarbio, China). After quenching of endogenous peroxidases with 3% H2O2, sections were incubated in nonspecific staining blockers for 15min and with primary antibody against VEGF (WL00009b, Wanleibio, China) at a dilution of 1:100 at 4°C overnight. Sections were then incubated in biotinylated secondary antibody for 15min followed by avidin and biotinylated HRP (1:1) mixed solution incubations for 15min. Finally, the sections were visualized with DAB and counterstained with hematoxylin, and antigen distribution was examined under light microscope.
Western blotting (WB)
Ovary and kidney samples were homogenized and lysed in lysis buffer (KGP2100, KeyGEN BioTECH, China). Protein extract was collected and subjected to BCA protein assays (Thermo, USA). Thirty microgram of protein was separated by SDS-PAGE and subsequently transferred to PVDF membrane. Afterwards, the membranes were blotted with 10% skim milk for 1h at 37℃ and incubated with primary antibodies against VEGF (WL00009b, Wanleibio, China) and VEGFR1 (AF6204, Affinity Biosciences, USA) overnight at 4°C. After incubation with the horseradish peroxidase-conjugated goat anti-rabbit IgG (ZB-2301, ZSGB-BIO, China) for 2h at room temperature, the membranes were visualized with an enhanced chemiluminescence plus kit (P10100, NCM Biotech, China) and Bio-Rad ChemiDoc MP imaging system. Band density was quantified using ImageJ software.
Statistical analysis
The data were expressed as the means ± standard error of mean (SEM) and analyzed using the SPSS 20.0 statistical software (SPSS Inc. USA). The comparisons between groups were conducted using ANOVA followed by least-significant difference (LSD) post hoc analysis, and P ≤ 0.05 was considered statistically significant.