Research and Publication Ethics: This study was conducted at the Experimental Animals Research and Application Center, Balikesir University, following ethical approval from the Balikesir University Experimental Animal Ethics Committee (date no. 26/12/2024, approval no. 2024/12-5).
Materials: Boric acid was obtained from Merck (Boric acid, Merck 1.00165 Cas No: 1 0043-35-3). Xylazine was procured from Alba Farma and ketamine was purchased from Doğa-Ilaç.
Animals: Twenty-four Wistar albino adult (8-12 weeks of age) female rats weighing 220-250 g were used as material. Animals were kept at a room temperature of 22±2ºC and 45-55% humidity, 12 hours light/dark period under standard laboratory conditions. No feed and water restriction was applied before the experiment.
Experimental Protocol: The sample size was determined based on the study design assumptions (α = 0.05, 1 - beta = 0.90), resulting in 8 animals per group. Although the Kruskal-Wallis test was ultimately used due to the non-normal distribution of the data, the sample size is expected to provide sufficient statistical power to detect meaningful differences among the groups. Before the experimental procedures, estrous cycles of all animals were monitored by vaginal smear method and animals showing regular estrus at least 3 times were identified and the experiment was performed during estrus phase22,23.
Group 1 (Sham, n=8): Saline was given intraperitoneally, and laparotomy was performed 10 minutes later.
Group 2 (IR, n=8): Saline was administered intraperitoneally, followed by 45 minutes of uterine ischemia and 1 hour of reperfusion.
Group 3 (BA+IR, n=8): Boric acid (200 mg/kg) dissolved in saline, was administered intraperitoneally 10 min before 45 minutes of uterine ischemia. Afterwards, reperfusion was performed for 1 hour.
The boric acid dose was based on previous studies17,18,24,25. In accordance with Kar et al.26, it was freshly prepared by dissolving in saline, and the administration route and dosage reported by Güler et al.25 were applied.
Surgery and Pharmacological Applications: All procedures were performed under general anesthesia and sterile conditions. Xylazine-ketamine protocol was used for anesthesia. Before the operation, 80 mg/kg ketamine hydrochloride (Keta-Control; Doğa İlaç) and 10 mg/kg xylazine hydrochloride (Beltazyn 2%; Alba Farma) were given intramuscularly. After anesthesia was achieved, the abdominal area was shaved and povidone-iodine was used for disinfection and laparotomy was performed with a 2-3 cm vertical incision in the midline of the lower abdomen. In the groups in which ischemia was to be induced, the distal abdominal aorta was clamped with an atraumatic microvascular clamp (Bulldog clamp; Aesculap (r), B. Braun Melsungen) approximately 0.5 cm from the bifurcation and left for 45 minutes. Bilateral ovaries were also clamped to prevent collateral circulation. Then, the incision was closed with a single suture and covered with sterile saline-soaked gauze to minimize intra-abdominal heat and fluid loss. At the end of 45 minutes, clamps were opened for reperfusion. The uteruses were reperfused for one hour. Following reperfusion, the uterine tissue was excised27.
After reperfusion, rats were sacrificed by exsanguination method under anesthesia. The uterine tissues were then removed and placed in buffered formalin for histopathological examination. The evaluations were performed blindly by the same pathologist including edema, inflammatory cells, congestion, necrosis and endometrial cell loss.
Histopathological Analysis: The excised uterine tissue samples were first washed under running tap water overnight to remove residual formalin. Subsequently, the samples were subjected to routine tissue processing, including dehydration through a graded ethanol series, clearing in xylene, and embedding in paraffin blocks. Sections of 5 μm thickness were obtained using a microtome (Leica RM 2125 RT). For systematic evaluation, the first three sections and every tenth section thereafter were selected and mounted on glass slides. The slides were deparaffinized and rehydrated through xylene and graded ethanol series, then stained with hematoxylin and eosin (HE). All histopathological evaluations were conducted under a light microscope at 40-400 fold magnification (Olympus BX53-DIC with DP-73 camera, Tokyo, Japan). In each sample, 10 fields were randomly selected for scoring. Tissue sections were evaluated semi-quantitatively for edema, inflammation, congestion, necrosis, and endometrial injury. Each parameter was scored based on severity using a standardized scale: 0 = none, 1 = mild, 2 = moderate, 3 = severe. This scoring system was adapted from previously published protocols28.
Statistical Analysis: GraphPad Prism 8 program (GraphPad Software, USA) was used for the statistical analyses. Since the histopathological parameters obtained from the groups in the study were semi-quantitative data, nonparametric tests were employed. Differences between the groups were first evaluated by Kruskal-Wallis test. Since significant results were obtained, post-hoc Dunn's multiple comparisons test was applied. p<0.05 was accepted as significant.
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