Investigation of the Effects of Boric Acid Against Post Operative Testicular Adhesion Caused by Experimental Laporotomy in Rats

Investigation of the Effects of Boric Acid Against Post Operative Testicular Adhesion Caused by Experimental Laporotomy in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigation of the Effects of Boric Acid Against Post Operative Testicular Adhesion Caused by Experimental Laporotomy in Rats İsmail BOLAT, Merve BOLAT, Metin KİLİÇLİOĞLU, Sıtkıcan OKUR, Ayşe GÖLGELİ, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4741801/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Sep, 2024 Read the published version in Biological Trace Element Research → Version 1 posted 14 You are reading this latest preprint version Abstract Post-operative intra-abdominal adhesions, significantly affecting testicular tissue, are a prevalent and serious complication following laparoscopic surgery. This study investigated the efficacy of boric acid, known for its antioxidant, anti-inflammatory, and anti-apoptotic properties, in preventing post-operative testicular adhesions. Forty rats, weighing between 230 and 290 grams, were divided into four groups: control, laparoscopy (LA), boric acid (BA), and LA + BA. Following laparoscopic surgery, BA treatment was administered for seven days. Adhesion scores were markedly higher in the LA group, whereas the LA + BA group exhibited a significant reduction in adhesion scores. Testicular tissues were analyzed using immunohistochemistry, immunofluorescence, Masson's trichrome staining, biochemical assays, and histopathological evaluation. In the LA group, malondialdehyde (MDA) levels increased while superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels decreased; these parameters normalized with BA treatment. Additionally, the LA group exhibited reduced levels of IL-10, Bcl-2, Kisspeptin-1, and GnRH, alongside elevated levels of inflammatory markers IL-1β, IL-6, TNF-α, JNK, BAX, and Caspase 3. Boric acid treatment significantly restored these levels to normal. In conclusion, oxidative stress, inflammation, and apoptosis in testicular tissues were associated with post-operative testicular adhesions. Boric acid demonstrated potential as an anti-adhesive agent, reducing testicular adhesions and normalizing biochemical and histological parameters following laparoscopic surgery. Boric acid Laparotomy Post-operative intra-abdominal adhesions Testis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Highlight Points Impact of Post-operative Adhesions on Testicular Tissue: Post-operative intra-abdominal adhesions significantly affect testicular tissue, leading to suppressed spermatogenesis due to oxidative stress, inflammation, fibrosis, and apoptosis. Protective Role of Boric Acid: Boric acid (BA) demonstrated significant protective effects against post-operative testicular adhesions, normalizing oxidative stress markers, inflammatory cytokines, and apoptotic proteins. Experimental Findings: In a rat model, BA treatment significantly reduced adhesion scores and attenuated tissue damage, inflammation, and fibrosis, as confirmed by histopathological, immunohistochemical, and biochemical analyses. Clinical Implications: These findings suggest that BA could be a promising therapeutic agent for preventing postoperative adhesions and preserving testicular function. Further research is needed to optimize its clinical application. Introduction Laparoscopic surgical intervention is an accepted reference method for gallstone surgery that has been widely used in intra-abdominal operations in later times. Recently it becomes very popular in genito-urinary surgery. Although this method has surgical advantages, it is also known to cause various complications, especially intraperitoneal adhesions [ 1 , 2 ]. While many adhesions remain clinically silent, they can lead to significant complications, including chronic abdominal pain, recurrent intestinal obstruction, and infertility [ 3 , 4 ]. Moreover, the intra-abdominal pelvic adhesions are leading causes of infertility, chronic pelvic pain, and bowel obstruction. Although the prevalence of postoperative adhesions is not precisely known, it has been confirmed that the incidence after abdominal or pelvic surgery ranges between 55% and 95%. [ 5 ]. Laparoscopic procedures in the scrotal area, such as La Barrosa scopic operations, pose significant challenges due to the formation of scrotal adhesions. These adhesions can lead to complications such as chronic pain, infertility, hematocele, and potential injury to the spermatic cord and testicle during hernia sac removal [ 6 ]. Postoperative abdominal adhesions, fibrous bands of scar tissue that form between abdominal tissues and organs, are a common consequence of abdominal surgery. These adhesions are characterized by abnormal fibrous tissue connections between peritoneal surfaces, often resulting from surgical intervention, trauma, inflammation, infection, or the introduction of foreign bodies into the peritoneal cavity [ 7 ]. Intra-abdominal surgeries initiate complex processes involving cytokine release, inflammatory cell recruitment, and adhesion molecule expression, leading to regional collagen fiber accumulation and adhesion formation [ 8 ]. Additionally, elevated levels of reactive oxygen species (ROS) contribute significantly to oxidative stress, exacerbating the development of intra-abdominal adhesions [ 7 ]. Therefore, effective strategies targeting inflammation suppression and oxidative stress mitigation are crucial in preventing intra-abdominal adhesions [ 9 ]. To further address the challenge of postoperative abdominal adhesions, researchers are exploring various anti-adhesive agents. Ideal anti-adhesive agents must exhibit efficacy, safety, and applicability to both open and laparoscopic surgical procedures. Currently, researchers are exploring a range of materials for surgical anti-adhesion purposes, including gelatin-based polymers, synthetic absorbable films, hydrogels, fibrous membranes, and chitosan-based materials [ 10 ]. Despite their potential advantages, these materials face ongoing challenges in achieving optimal performance and safety. Consequently, efforts are focused on advancing alternative solutions capable of effectively reducing adhesion formation and enhancing patient recovery [ 3 , 4 ]. In light of these challenges and the ongoing search for optimal anti-adhesive materials, boric acid emerges as a promising candidate due to its significant importance in medicine, particularly within surgical contexts. Boric acid is known for its broad spectrum of antiseptic properties and diverse therapeutic applications. Its absorption is relatively quick, and it is primarily excreted through urine, with an average half-life of approximately one day [ 11 ]. Traditionally used in wound care, vaginal health, eye care, swimming pool maintenance, ear infections, and foot odor control [ 12 ], boric acid has also gained attention as a component in the development of surgical anti-adhesive polymers [ 13 ]. This versatility underscores its potential in healthcare by potentially preventing post-operative adhesion formation, thereby contributing to improved surgical outcomes and patient rehabilitation. Furthermore, borates, including boric acid, are increasingly used in nutritional supplements as a source of boron, an essential trace element widely distributed in nature. These compounds, particularly boric acid salts (H3BO3), are commonly found in laundry detergents, cleaning agents, and fertilizers [ 14 ]. They play a crucial role in regulating hormone levels, including steroids, thyroid hormones, estrogen, and testosterone, as well as maintaining various minerals such as calcium and vitamin D, and protecting bone tissue [ 15 ]. Additionally, these compounds are effective in numerous metabolic processes, including DNA damage repair mechanisms and the regulation of oxidative stress. Studies have demonstrated their anti-inflammatory, antioxidant, antibacterial, anticarcinogenic, and antiviral activities [ 16 , 17 ]. These multifaceted properties further highlight the potential of boric acid and its derivatives in enhancing surgical outcomes and patient rehabilitation by preventing post-operative adhesion formation. Mechanistically, boric acid interferes with protein synthesis, activates MAP kinase cascades, inhibits cell cycle progression, and induces morphological changes, contributing to significant antiproliferative effects in specific cancer cell lines [ 18 ]. Upon entering cells at physiological pH, boric acid hydrolyzes into borate ions, which subsequently lower intracellular pH [ 19 ]. This acidification process can lead to cellular inhibition and apoptosis. Borate ions, rather than boric acid itself, are recognized for their roles as cell-signaling molecules, co-factors for enzymes, contributors to redox reactions, and structural components of the cytoskeleton [ 20 ]. Boric acid exhibits inhibitory effects on several enzymes with those involved in progression of prostate cancer [ 21 , 22 ]. On a molecular level, boric acid functions as a Lewis acid, capable of forming complexes with hydroxyl-group-bearing amino acids within tissue protein polypeptides [ 12 , 23 ]. This interaction highlights its potential to cross-link these polypeptides, facilitated by boric acid's ability to establish strong bonds with hydroxyl groups due to its vacant d-orbital in boron. These reactions with nucleophiles enable the formation of cross-linked complexes, creating new covalent bonds between boric acid and protein molecules. Such interactions may hinder further protein-protein interactions between adjacent tissue surfaces, thereby potentially preventing the formation of de novo adhesions. Despite these promising mechanisms, to the best of our knowledge, no previous studies have investigated the protective effects of boric acid against post-operative testicular adhesions. Therefore, this study aims to evaluate the protective effects of boric acid treatment against testicular adhesions induced by laparoscopic surgery in rats. Materials and Methods Animals and Experimental Groups The experimental animals used in the study were obtained from the Atatürk University Medical Experimental Application and Research Center. In the study, 40 Sprague Dawley rats weighing 230–290 grams were used. The rats were acclimatized to the environmental conditions for 1 week before the experiment. The rats were fed freely with standard feed and drinking water at room temperature in a 12-hour light and 12-hour dark cycle. The rats were randomly divided into 4 groups with 10 animals in each group. Ethics committee permission for the study was obtained from Atatürk University Animal Experiments Local Ethics Committee (Ethics Number: 2024/156). Control group No surgical intervention was performed on the rats and 2 ml physiological saline was given intra-abdominally for 7 days (n = 10). Laparotomy (LA) group Only the abdominal cavity of the rats was opened and closed again (n = 10). Boric Acid (BA) group No surgical intervention was performed on the rats, and BA at a dose of 8 mg/kg dissolved in 2 ml physiological saline was administered intra-abdominally for 7 days (n = 10) [ 24 ]. LA + BA group After the abdominal cavity of the rats was opened and closed, BA at a dose of 8 mg/kg dissolved in 2 ml physiological saline was administered intra-abdominally for 7 days (n = 10). Laparotomy Procedures Butorphanol was administered subcutaneously at a dose of 1 mg/kg before laparotomy to all rats except the control and BA groups [ 25 ]. Peritoneal Button Creation (PBC) model was applied to create an adhesion model in rats [ 26 ]. Before the intervention, rats were anesthetized with 10 mg/kg xylazine followed by 100 mg/kg ketamine. For laparotomy, a 3 cm long incision was made from the middle part of the abdominal region to cover the peritoneum, then strangulation was created with the help of hemostatic forceps to cover the parietal peritoneum of approximately 1 cm2 and the bottoms of these strangulations were ligated with 6.0 proline threads. This procedure was repeated 4 times at 0.2 cm intervals. Subcutaneous tissue was then closed with the continuous suture method (PDS 4/0) and skin was closed with a separate suture method (Nylon 4/0) [ 27 ]. At the end of the 7th day, all rats were sacrificed under general anesthesia. The abdominal cavity of the rats was opened and the presence of adhesion in the testicular tissue was checked and the scoring was done according to Table 1 [ 28 ]. After macroscopic evaluation, testicular tissues of all rats were collected; It was examined by biochemical, histopathological, Massons's trichrome, immunohistochemical and immunofluorescence methods. Table 1 Intra-abdominal adhesion scoring model calculation in rats Score Adhesion Assessment 0 No adhesion 1 Presence of a single fibrous bridge between internal organs or between the internal organ and the abdominal wall 2 Presence of two fibrous bridges between internal organs or between the internal organ and the abdominal wall 3 More than two fibrous bridges between the viscera or between the viscera and the abdominal wall, or the formation of a single mass of intestines without adhesion to the abdominal wall 4 Internal organs are directly attached to the abdominal wall, regardless of their number and extension. Enzyme-Linked ImmunoSorbent Assay (ELISA) By following the manufacturer's instructions, the ELISA kits (SUNREDBIO) were used to measure the concentrations of MDA, SOD, CAT, GPx, IL-1β, IL-6, IL-10, and TNF-α in the supernatants of brain tissue homogenates. Histopathological Examinations Testis tissue samples obtained from rats were fixed in a 10% buffered formalin solution and subjected to routine tissue tracing procedures. The prepared sections were blocked and 4 µm thick sections were taken from each of the blocks. The sections were stained with Hematoxylin & Eosin (H&E) and Masson's Trichrome stains and evaluated under a light microscope (OLYMPUS BX51). In Masson's Trichrome staining, fibrosis formation was evaluated as absent (-), mild (+), moderate (+++), and severe (++++). Testicular tissues were subjected to histopathologic evaluation using Johnsen's mean testicular biopsy score method (a semi-quantitative method). Each tubule was given a score between 0 and 10 according to epithelial maturation (Table 2 ) [ 29 ]. Additionally, each sample was examined in 10 randomly selected fields of approximately x20 objective. Scores were as follows: Grade 0 = − (negative); Grade 1 = + 1 (mild); Grade 2 = + 2 (moderate); Grade 3 = + 3 (severe); and Grade 4 = + 4 (most severe) [ 30 ]. Table 2 Testicular biopsy score criteria Skor Description 1 No cells 2 Sertoli cells without germ cells 3 Only spermatogonia 4 Only a few spermatocytes 5 Many spermatocytes 6 Only a few early spermatids 7 Many early spermatids 8 Few late spermatids 9 Many late spermatids 10 Full spermatogenesis Immunohistochemical Examinations Tissue sections taken on adhesive (poly-L-Lysin) slides for immunoperoxidase examination were treated with primary antibodies (GnRH I Cat No: sc-32292, Dilution Ratio: 1/100, US; Kisspeptin 1 (KiSS 1) Cat No: sc-101246, Dilution Ratio: 1/100, US; Bcl-2 Cat. No: sc-7382, Dilution Ratio: 1/100, US) were added and incubated according to the instructions for use. 3–3' Diaminobenzidine (DAB) chromogen was used as chromogen in the tissues. Stained sections were examined by light microscopy (Zeıss AXIO GERMANY) [ 31 ]. Double Immunofluorescence Examinations For immunofluorescence examination, tissue sections were placed on adhesive (poly-L-Lysin) slides. Following standard protocols, primary antibodies (8-OHdG Cat. No: sc-66036, Dilution Ratio: 1/100, US; BAX Cat. No: sc-780, Dilution Ratio: 1/100, US) were added, and the samples were incubated by the usage instructions. Immunofluorescence secondary antibody was used as secondary marker (FITC Cat No: ab6785 Dilution Ratio: 1/1000, UK) and kept in the dark for 45 min. The tissues were then treated with the second primary antibody (JNK Cat No: sc-7345, Dilution Ratio: 1/100, US; Caspase 3 Cat No: sc-56053 Dilution Ratio: 1/100, US) and incubated according to the instructions for use. Immunofluorescence secondary antibody was used as secondary marker (Texas Red Cat No: ab6719 Dilution Ratio: 1/1000 UK) and kept in the dark for 45 minutes. Then, DAPI with mounting medium (Cat no: D1306 Dilution Ratio: 1/200 UK) was added to the sections and kept in the dark for 5 min and the sections were covered with coverslips. The stained sections were examined under a fluorescence attachment microscope (Zeıss AXIO GERMANY) [ 32 ]. Statistical Analysis The data were assessed using GraphPad Prism 8.0.2 for statistical analysis, with a significance level of p < 0.05. The non-parametric Mann-Whitney U test was used to assess group differences in non-parametric data, while the non-parametric Kruskal-Wallis test was used to determine differences among groups. One-way ANOVA and the Tukey test were used to assess parametric data. Results Macroscopic findings Upon concluding the experiment and inspecting the abdominal region of the animals, no adhesions were detected in either the control group (Fig. 1 a) or the boric acid (BA) treated group (Fig. 1 c). In contrast, the laparotomy (LA) group (Fig. 1 b) exhibited multiple adhesions (bands) in the testicular tissues. However, in the LA + BA group (Fig. 1 d), these adhesion bands were significantly reduced (< 0.001) compared to the LA group. Histopathological Findings Hematoxylin-eosin staining of testicular tissue samples revealed normal histological structures in both the control group (Fig. 2 a) and the boric acid (BA) treated group (Fig. 2 c). In contrast, the laparotomy (LA) group (Fig. 2 b) exhibited severe spermatocyte degeneration, necrosis, tissue edema, and inflammation. However, these histopathological findings were markedly reduced in the LA + BA group (Fig. 2 d). For fibrosis assessment, Masson's trichrome staining showed severe fibrosis in the area of adhesion formation in the LA group (Fig. 3 b). Notably, fibrosis development was significantly attenuated in the LA + BA group (Fig. 3 d). For quantitative analysis, the detailed adhesion scoring, histopathological findings, and fibrosis development scoring, along with corresponding statistical analyses, are summarized in Fig. 4 . Immunohistochemical and Immunofluorescence Findings In immunohistochemical and immunofluorescence analyses of testicular tissues, high levels of Bcl-2 (Fig. 5 ), GnRH (Fig. 6 ), and KiSS1 (Fig. 7 ) expressions were observed in the control and BA groups, as well as mild expressions of 8-OHdG, JNK (Fig. 8 ), BAX, and Caspase 3 (Fig. 9 ). The LA group had high expressions of 8-OHdG, JNK, BAX, and Caspase 3, but mild expressions of Bcl-2, GnRH, and KiSS1. In the LA + BA group, it was determined that expressions of 8-OHdG, JNK, BAX, and Caspase 3 were significantly decreased (< 0.0001), and expressions of Bcl-2, GnRH, and KiSS1 were significantly increased (< 0.0001). Results of immunohistochemical and immunofluorescence staining findings, along with statistical analysis data, are presented in Fig. 10 . Effect of Laparotomy-induced Adhesion on Lipid Peroxidation/antioxidant Status The activities of SOD (Fig. 11 B), CAT (Fig. 11 C), and GPx (Fig. 11 D) were significantly reduced in the LA group compared to the control, LA + BA groups. Treatment with BA resulted in an increase in these enzyme activities, with a particularly notable effect observed in the high-dose BA group. Evaluation of MDA levels revealed a significant increase in the LA group compared to other groups; however, BA treatment led to a dose-dependent reduction in MDA (Fig. 11 A) levels. Effect of Laparotomy-induced Adhesion on the Inflammatory Response Based on the assessments depicted in Fig. 12 , the LA-exposed group exhibited substantial increases in proinflammatory mediators TNF-α (Fig. 12 A), IL-1β (Fig. 12 B), and IL-6 (Fig. 12 C) compared to both control and LA + BA treated groups. Treatment with BA demonstrated a dose-dependent reduction in these mediators, with the highest efficacy observed in the LA + BA group. Evaluation of IL-10 (Fig. 12 D) levels revealed a significant decrease in the LA group, which showed a dose-dependent reversal with BA treatment. Discussion Adhesions are abnormal fibrous connections that form between adjacent tissues or organs, often as a result of surgery, trauma, or inflammation. These fibrous bands can lead to serious complications such as bowel obstruction and infertility [ 5 ]. In contrast, normal fibrosis is a natural healing response characterized by collagen deposition to facilitate tissue repair. However, excessive fibrosis, seen in conditions like liver cirrhosis or pulmonary fibrosis, can impair organ function. Intra-abdominal adhesions specifically refer to abnormal fibrous connections within the abdominal cavity, commonly occurring following surgical procedures. Despite their prevalence, effective preventive measures remain challenging to implement. These adhesions can affect various abdominal tissues and organs, underscoring their clinical significance [ 33 , 34 ]. The genital organs, particularly the testicular tissues, are vulnerable to post-operative adhesions, potentially leading to infertility due to oxidative stress, inflammation, and apoptosis [ 35 ]. Histopathological studies have revealed severe inflammation, degenerative changes in spermatocytes, and extensive fibrosis in testicular tissues affected by adhesions [ 36 , 37 ]. During this scenario, the oxidative stress and inflammation play pivotal roles in the pathogenesis of post-operative adhesions, triggering increases in reactive oxygen species (ROS), inflammatory cytokines (IL-1β, TNF-α, IL-6), and markers of oxidative stress (8-OHdG, SOD, CAT, GPx) in affected tissues [ 38 , 39 ]. This imbalance between ROS production and antioxidant defenses contributes to tissue damage and adhesion formation. Morevover, following surgery, a pronounced inflammatory response develops in testicular tissues, characterized by elevated levels of pro-inflammatory markers such as JNK, IL-1β, TNF-α, and IL-6, alongside decreased IL-10 levels [ 40 ]. This inflammatory cascade is exacerbated by increased oxidative stress post-surgery, further contributing to adhesion formation within testicular tissue. Consistently, our study observed a pronounced inflammatory response in testicular tissues by the 7th day post-surgery, marked by elevated levels of JNK, IL-1β, TNF-α, and IL-6, alongside decreased IL-10 levels. This inflammatory cascade in the testis was exacerbated by increased oxidative stress post-surgery, which not only triggered inflammation but potentially initiates testicular adhesion. During this post-surgery inflammatory process, macrophages, as key initiators of the inflammatory response, release pro-inflammatory cytokines like IL-1, IL-6, and TNF-α, which recruit inflammatory cells and exacerbate tissue damage [ 41 , 42 ]. Experimental studies have highlighted the role of cytokines such as TNF-α and IL-6 in the pathogenesis of postoperative peritoneal adhesions [ 43 – 45 ]. Apoptosis, on the other hand, plays a significant role in tissue damage during adhesion formation, involving the TNF-α-induced JNK pathway, caspase 3 activation, and altered regulation of Bcl-2 family proteins [ 46 , 47 ]. Caspase 3 is central to the final stages of apoptosis, while Bcl-2 family proteins regulate mitochondrial integrity and cell fate decisions [ 48 , 49 ]. Therefore, it is logically to recognize severe caspase 3 expressions in the testicular tissue, especially in spermatocytes, during post-operative testicular adhesion with marked apoptosis. In addition to the inflammatory and apoptotic pathways involved in adhesion formation, reproductive hormones such as GnRH and its stimulators, Kisspeptins, play crucial roles in regulating testicular function. GnRH is essential for regulating the reproductive axis and stimulating gonadotropin synthesis [ 50 ]. Kisspeptins, potent stimulators of GnRH neurons, also play a significant role in the synthesis and secretion of GnRH, crucially influencing spermatogenesis and overall reproductive function [ 51 – 53 ]. Our findings align with previous research indicating that decreases in GnRH and Kisspeptin levels are associated with impaired testicular function following adhesion formation. This provides a plausible explanation for the observed decrease in testicular spermatogenesis induced by laparotomy in our study. In contrary, boric acid exhibits protective effects against oxidative stress, inflammation, fibrosis, and apoptosis in various cellular contexts [ 54 ]. These properties are potentially relevant in the context of postoperative adhesions, where BA's antioxidant capabilities mitigate oxidative stress and its anti-inflammatory properties reduce the inflammatory response [ 12 , 23 ]. By forming cross-linked barriers between peritoneal surfaces, BA might has the ability to prevent adhesion formation, thereby protecting tissues from the detrimental effects of surgery-induced inflammatory cascades and apoptotic pathways observed in our study [ 41 , 42 , 46 , 47 ]. As demonstrated in our results, BA effectively mitigated laparotomy-induced testicular adhesions and associated pathologies. Macroscopically, BA-treated groups showed reduced adhesion bands compared to untreated controls, while histopathological analysis revealed attenuated tissue damage, inflammation, and fibrosis. Immunohistochemical and immunofluorescence analyses confirmed BA's ability to modulate oxidative stress, inflammation, and apoptosis pathways, restoring the balance of pro- and anti-apoptotic proteins. Additionally, BA treatment enhanced antioxidant enzyme activities and reduced lipid peroxidation in a dose-dependent manner, thereby preserving testicular function and preventing infertility. These findings collectively suggest that BA presents a promising therapeutic strategy for preventing postoperative complications in sensitive tissues like the testes. Conclusion In this study, we identified that postoperative testicular adhesions suppress spermatogenesis through mechanisms involving oxidative stress, inflammation, fibrosis, and apoptosis in testicular tissue. Moreover, boric acid demonstrated protective effects in testicular tissue by attenuating adhesion formation. These findings suggest that boric acid holds promise as a protective agent against postoperative intra-abdominal adhesions, which currently lack effective preventive strategies. BA's diverse protective mechanisms underscore its potential as a therapeutic intervention to mitigate postoperative adhesions and maintain testicular function. Future research efforts should prioritize elucidating BA's molecular pathways and optimizing its clinical application to enhance therapeutic outcomes. 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Gynécologie Obs Fertil 35:290–296. https://doi.org/10.1016/j.gyobfe.2007.02.011 Chung JH, Chung Y, Cha YJ, et al (2022) Anti-adhesion agent to prevent of post-operative adhesion and fibrosis after vasectomy: a study using a rat model. Transl Androl Urol 11:1234–1244. https://doi.org/10.21037/tau-21-1170 Pattaras JG, Moore RG, Landman J, et al (2002) Incidence of postoperative adhesion formation after transperitoneal genitourinary laparoscopic surgery. Urology 59:37–41. https://doi.org/10.1016/S0090-4295(01)01474-1 Zhang T, Huang Y, Gong Y, et al (2024) A ROS-responsive and scavenging hydrogel for postoperative abdominal adhesion prevention. Acta Biomater. https://doi.org/10.1016/j.actbio.2024.06.027 Wu Y, Duan X, Gao Z, et al (2022) AICAR attenuates postoperative abdominal adhesion formation by inhibiting oxidative stress and promoting mesothelial cell repair. PLoS One 17:e0272928. https://doi.org/10.1371/journal.pone.0272928 Rafael A. Fissore, Manabu Kurokawa JK, Mao Zhang and JS (2022) Mechanisms underlying oocyte activation and postovulatory ageing. Reproduction 124:745–754 Haney A. (2000) Identification of macrophages at the site of peritoneal injury: evidence supporting a direct role for peritoneal macrophages in healing injured peritoneum. Fertil Steril 73:988–995. https://doi.org/10.1016/S0015-0282(00)00490-8 Tsai JM, Shoham M, Fernhoff NB, et al (2019) Neutrophil and monocyte kinetics play critical roles in mouse peritoneal adhesion formation. Blood Adv 3:2713–2721. https://doi.org/10.1182/bloodadvances.2018024026 Corona R, Verguts J, Schonman R, et al (2011) Postoperative inflammation in the abdominal cavity increases adhesion formation in a laparoscopic mouse model. Fertil Steril 95:1224–1228. https://doi.org/10.1016/j.fertnstert.2011.01.004 Turza KC, Butler CE (2012) Adhesions and Meshes. Plast Reconstr Surg 130:206S-213S. https://doi.org/10.1097/PRS.0b013e3182638d48 Bian Y-Y, Yang L-L, Yan Y, et al (2020) Identification of candidate biomarkers correlated with pathogenesis of postoperative peritoneal adhesion by using microarray analysis. World J Gastrointest Oncol 12:54–65. https://doi.org/10.4251/wjgo.v12.i1.54 Wu Q, Wu W, Jacevic V, et al (2020) Selective inhibitors for JNK signalling: a potential targeted therapy in cancer. J Enzyme Inhib Med Chem 35:574–583. https://doi.org/10.1080/14756366.2020.1720013 Obeng E (2021) Apoptosis (programmed cell death) and its signals - A review. Brazilian J Biol 81:1133–1143. https://doi.org/10.1590/1519-6984.228437 Cory S, Adams JM (2002) The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2:647–656. https://doi.org/10.1038/nrc883 Qian S, Wei Z, Yang W, et al (2022) The role of BCL-2 family proteins in regulating apoptosis and cancer therapy. Front Oncol 12:. https://doi.org/10.3389/fonc.2022.985363 Gruenewald DA, Naai MA, Hess DL, Matsumoto AM (1994) The Brown Norway Rat as a Model of Male Reproductive Aging: Evidence for Both Primary and Secondary Testicular Failure. J Gerontol 49:B42–B50. https://doi.org/10.1093/geronj/49.2.B42 Han S-K, Gottsch ML, Lee KJ, et al (2005) Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for the Onset of Puberty. J Neurosci 25:11349–11356. https://doi.org/10.1523/JNEUROSCI.3328-05.2005 HERBISON A (2006) Physiology of the Gonadotropin-Releasing Hormone Neuronal Network. In: Knobil and Neill’s Physiology of Reproduction. Elsevier, pp 1415–1482 Anjum S, Krishna A, Sridaran R, Tsutsui K (2012) Localization of Gonadotropin‐Releasing Hormone (GnRH), Gonadotropin‐Inhibitory Hormone (GnIH), Kisspeptin and GnRH Receptor and Their Possible Roles in Testicular Activities From Birth to Senescence in Mice. J Exp Zool Part A Ecol Genet Physiol 317:630–644. https://doi.org/10.1002/jez.1765 Tekin A, Güner A, Akkan T (2024) Protective Effect of Boric Acid Against Ochratoxin A-Induced Toxic Effects in Human Embryonal Kidney Cells (HEK293): A Study on Cytotoxic, Genotoxic, Oxidative, and Apoptotic Effects. Biol Trace Elem Res. https://doi.org/10.1007/s12011-024-04194-5 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 Sep, 2024 Read the published version in Biological Trace Element Research → Version 1 posted Editorial decision: Revision requested 08 Sep, 2024 Reviews received at journal 06 Sep, 2024 Reviews received at journal 02 Sep, 2024 Reviewers agreed at journal 28 Aug, 2024 Reviews received at journal 26 Aug, 2024 Reviewers agreed at journal 25 Aug, 2024 Reviewers agreed at journal 25 Aug, 2024 Reviewers agreed at journal 24 Aug, 2024 Reviews received at journal 11 Aug, 2024 Reviewers agreed at journal 28 Jul, 2024 Reviewers invited by journal 23 Jul, 2024 Editor assigned by journal 16 Jul, 2024 Submission checks completed at journal 15 Jul, 2024 First submitted to journal 15 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4741801","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":335837113,"identity":"e77592d3-d6ef-4d2f-a432-fd3926b25c13","order_by":0,"name":"İsmail BOLAT","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYBACNhCRUMCQwMAOZHwAizE2QMXxaTEAamFmYGycwWCA0MKD1y6olmYesBaoUbi08PGfTvzwwIAhj5+Z+fljm5o/8ga3mxsYPpQdZrCXPoDdYRK5myWADiuWbGYzbM45ZmC44c7BBsYZ5w4z8PAl4NDCuwGkJXHDYQagFjYDxg03EhuYeduAWnC4jI3/7OYfEC3sH5st/hnYg7X8xaeFIXcb1BYew2bGNoNEsBZGfFokcrdZJBhIAP3CUzizt884eSZQy8Gec+k8PGewa5HvP7v55o8Kmzx+9vYNH358k7Ptu5H+8MGPMms59h7sWqBAApV7gIFQTI6CUTAKRsEowAsAXgRZLXlaLW0AAAAASUVORK5CYII=","orcid":"","institution":"Atatürk University","correspondingAuthor":true,"prefix":"","firstName":"İsmail","middleName":"","lastName":"BOLAT","suffix":""},{"id":335837115,"identity":"2e4b6daf-27ee-4c73-9529-1e679da2cc14","order_by":1,"name":"Merve BOLAT","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Merve","middleName":"","lastName":"BOLAT","suffix":""},{"id":335837117,"identity":"59f0c847-00bd-49f1-8271-37ef2f87ad7d","order_by":2,"name":"Metin KİLİÇLİOĞLU","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Metin","middleName":"","lastName":"KİLİÇLİOĞLU","suffix":""},{"id":335837119,"identity":"10ee201d-1471-413a-bdee-502e01ad9b5b","order_by":3,"name":"Sıtkıcan OKUR","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Sıtkıcan","middleName":"","lastName":"OKUR","suffix":""},{"id":335837121,"identity":"28c1dd9b-6cc4-4e7b-b6b8-fae6d47911d8","order_by":4,"name":"Ayşe GÖLGELİ","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Ayşe","middleName":"","lastName":"GÖLGELİ","suffix":""},{"id":335837124,"identity":"e6a6efe1-e8a0-42ed-8396-495e03ccc4ba","order_by":5,"name":"Berrah GÖZEGİR","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Berrah","middleName":"","lastName":"GÖZEGİR","suffix":""},{"id":335837125,"identity":"2b73874b-9118-4932-89c2-9f63e810e928","order_by":6,"name":"Selim ÇOMAKLI","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Selim","middleName":"","lastName":"ÇOMAKLI","suffix":""},{"id":335837127,"identity":"1d0462fa-8508-496a-982b-f0db7a064c06","order_by":7,"name":"Serkan YILDIRIM","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Serkan","middleName":"","lastName":"YILDIRIM","suffix":""},{"id":335837128,"identity":"e88c8511-e6a6-4353-9ab2-b86c13ccf284","order_by":8,"name":"Yavuz Selim SAĞLAM","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Yavuz","middleName":"Selim","lastName":"SAĞLAM","suffix":""},{"id":335837131,"identity":"a51ab051-fb25-4d47-90ae-2d267076636a","order_by":9,"name":"Mohamad WARDA","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Mohamad","middleName":"","lastName":"WARDA","suffix":""}],"badges":[],"createdAt":"2024-07-15 09:10:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4741801/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4741801/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12011-024-04400-4","type":"published","date":"2024-09-28T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62632671,"identity":"5aa13cf0-f7d6-4da9-a0e7-3e9e2584b08c","added_by":"auto","created_at":"2024-08-16 16:14:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":422273,"visible":true,"origin":"","legend":"\u003cp\u003ePostoperative macroscopic findings. Control (a), LA (b), BA (c), and LA+BA (d). Testicular adhesion (arrows).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/27b1d28d6394f5a9d782d622.png"},{"id":62632675,"identity":"53ffefbc-aded-4bce-9ebf-705832c5910f","added_by":"auto","created_at":"2024-08-16 16:14:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":407807,"visible":true,"origin":"","legend":"\u003cp\u003eTesticular tissue histopathological findings. Control (a), LA (b), BA (c), and LA+BA (d). Adhesion zone (asterisk), edema (asterix), degeneration (arrowhead), necrosis (arrow) and inflammation (zigzag arrow), H\u0026amp;E, Bar:200µm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/103c5d3961754f935d9c7828.png"},{"id":62632672,"identity":"0270d0c8-0a05-44aa-ac88-8f23cb0c48c4","added_by":"auto","created_at":"2024-08-16 16:14:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":401440,"visible":true,"origin":"","legend":"\u003cp\u003eMassons's trichrome staining findings in testicular tissue. Control (a), LA (b), BA (c), and LA+BA (d). Fibrosis (asterix), MT, Bar:200µm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/914760d04f44d0b3b0057dc6.png"},{"id":62632670,"identity":"9de026d9-05f6-47e7-a3d9-2361453d1704","added_by":"auto","created_at":"2024-08-16 16:14:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":119643,"visible":true,"origin":"","legend":"\u003cp\u003eAdhesion scoring, Johnsen scoring, histopathological scoring and fibrosis scoring. Statistical analysis data of scoring (n=10). Results are expressed as mean ± SEM.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/64fcebd1eb01d60c9d7033aa.png"},{"id":62632680,"identity":"d4dd4db3-2903-427d-ad50-277af704d8b3","added_by":"auto","created_at":"2024-08-16 16:14:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":346776,"visible":true,"origin":"","legend":"\u003cp\u003eTesticular tissue, Control (a), LA (b), BA (c), and LA+BA (d). Bcl-2 expressions (arrowhead), IHC-P, Bar:50µm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/8e6aef4502bc1cc1b3145bc7.png"},{"id":62632676,"identity":"53f73013-b348-4ed0-8ff2-8048b619a27a","added_by":"auto","created_at":"2024-08-16 16:14:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":382295,"visible":true,"origin":"","legend":"\u003cp\u003eTesticular tissue, Control (a), LA (b), BA (c), and LA+BA (d). GnRH expressions (arrowhead), IHC-P, Bar:50µm.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/d06137012b66dd9c08331a48.png"},{"id":62632674,"identity":"26bda3af-bcc5-49ee-8ac6-dd03a0dbff9f","added_by":"auto","created_at":"2024-08-16 16:14:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":406317,"visible":true,"origin":"","legend":"\u003cp\u003eTesticular tissue, Control (a), LA (b), BA (c), and LA+BA (d). KiSS1 expressions (arrowhead), IHC-P, Bar:50µm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/236a9943986eca961163ed6c.png"},{"id":62632677,"identity":"de5f2034-ab56-48d3-b5ea-98ffd917f4a4","added_by":"auto","created_at":"2024-08-16 16:14:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":494411,"visible":true,"origin":"","legend":"\u003cp\u003eTesticular tissue, Control (a), LA (b), BA (c), and LA+BA (d). 8-OHdG expressions (FITC) and JNK expressions (Texas Red), IF, Bar:50µm.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/7ab966c1fd1cca888713e495.png"},{"id":62632681,"identity":"d3fbb7db-9d27-4d0a-b11b-cc6274435c01","added_by":"auto","created_at":"2024-08-16 16:14:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":491216,"visible":true,"origin":"","legend":"\u003cp\u003eTesticular tissue, Control (a), LA (b), BA (c), and LA+BA (d). BAX expressions (FITC) and Caspase 3 expressions (Texas Red), IF, Bar:50µm\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/e8fb13dcad96f230c62fb356.png"},{"id":62632678,"identity":"c122e776-392f-4297-a939-7abef171a633","added_by":"auto","created_at":"2024-08-16 16:14:26","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":120054,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical and immunofluorescence staining data and statistical analysis findings in testicular tissue. (n=10). Results are expressed as mean ± SEM.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/5840a488517872ce5a80010b.png"},{"id":62632673,"identity":"6bf9e8c3-b499-42a4-86b8-f4f71b4602c1","added_by":"auto","created_at":"2024-08-16 16:14:25","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":86918,"visible":true,"origin":"","legend":"\u003cp\u003eIllustrates MDA (A), SOD (B), CAT (C), and GPx (D) levels in testis tissues (n = 10). Results are expressed as mean ± SEM.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/c33a437f6bec4d1c84dcd773.png"},{"id":62632679,"identity":"90efb7b5-6f34-47a5-9ca4-018100917042","added_by":"auto","created_at":"2024-08-16 16:14:26","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":86694,"visible":true,"origin":"","legend":"\u003cp\u003eIllustrates TNFα (A), IL-1ß (B), IL-6 (C), and IL-10 (D) levels in testis tissues (n = 10). Results are expressed as mean ± SEM.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/920163d68075157e99214c77.png"},{"id":65627241,"identity":"325b5819-6b4c-469f-801c-c3f063aa00a5","added_by":"auto","created_at":"2024-09-30 16:13:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4241846,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4741801/v1/46f57472-8df7-487c-af2c-b9e94c7febda.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of the Effects of Boric Acid Against Post Operative Testicular Adhesion Caused by Experimental Laporotomy in Rats","fulltext":[{"header":"Highlight Points","content":"\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003e\u003cstrong\u003eImpact of Post-operative Adhesions on Testicular Tissue:\u003c/strong\u003e Post-operative intra-abdominal adhesions significantly affect testicular tissue, leading to suppressed spermatogenesis due to oxidative stress, inflammation, fibrosis, and apoptosis.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eProtective Role of Boric Acid:\u003c/strong\u003e Boric acid (BA) demonstrated significant protective effects against post-operative testicular adhesions, normalizing oxidative stress markers, inflammatory cytokines, and apoptotic proteins.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eExperimental Findings:\u003c/strong\u003e In a rat model, BA treatment significantly reduced adhesion scores and attenuated tissue damage, inflammation, and fibrosis, as confirmed by histopathological, immunohistochemical, and biochemical analyses.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eClinical Implications:\u003c/strong\u003e These findings suggest that BA could be a promising therapeutic agent for preventing postoperative adhesions and preserving testicular function. Further research is needed to optimize its clinical application.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eLaparoscopic surgical intervention is an accepted reference method for gallstone surgery that has been widely used in intra-abdominal operations in later times. Recently it becomes very popular in genito-urinary surgery. Although this method has surgical advantages, it is also known to cause various complications, especially intraperitoneal adhesions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While many adhesions remain clinically silent, they can lead to significant complications, including chronic abdominal pain, recurrent intestinal obstruction, and infertility [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, the intra-abdominal pelvic adhesions are leading causes of infertility, chronic pelvic pain, and bowel obstruction. Although the prevalence of postoperative adhesions is not precisely known, it has been confirmed that the incidence after abdominal or pelvic surgery ranges between 55% and 95%. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Laparoscopic procedures in the scrotal area, such as La Barrosa scopic operations, pose significant challenges due to the formation of scrotal adhesions. These adhesions can lead to complications such as chronic pain, infertility, hematocele, and potential injury to the spermatic cord and testicle during hernia sac removal [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePostoperative abdominal adhesions, fibrous bands of scar tissue that form between abdominal tissues and organs, are a common consequence of abdominal surgery. These adhesions are characterized by abnormal fibrous tissue connections between peritoneal surfaces, often resulting from surgical intervention, trauma, inflammation, infection, or the introduction of foreign bodies into the peritoneal cavity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Intra-abdominal surgeries initiate complex processes involving cytokine release, inflammatory cell recruitment, and adhesion molecule expression, leading to regional collagen fiber accumulation and adhesion formation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, elevated levels of reactive oxygen species (ROS) contribute significantly to oxidative stress, exacerbating the development of intra-abdominal adhesions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, effective strategies targeting inflammation suppression and oxidative stress mitigation are crucial in preventing intra-abdominal adhesions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo further address the challenge of postoperative abdominal adhesions, researchers are exploring various anti-adhesive agents. Ideal anti-adhesive agents must exhibit efficacy, safety, and applicability to both open and laparoscopic surgical procedures. Currently, researchers are exploring a range of materials for surgical anti-adhesion purposes, including gelatin-based polymers, synthetic absorbable films, hydrogels, fibrous membranes, and chitosan-based materials [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite their potential advantages, these materials face ongoing challenges in achieving optimal performance and safety. Consequently, efforts are focused on advancing alternative solutions capable of effectively reducing adhesion formation and enhancing patient recovery [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn light of these challenges and the ongoing search for optimal anti-adhesive materials, boric acid emerges as a promising candidate due to its significant importance in medicine, particularly within surgical contexts. Boric acid is known for its broad spectrum of antiseptic properties and diverse therapeutic applications. Its absorption is relatively quick, and it is primarily excreted through urine, with an average half-life of approximately one day [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Traditionally used in wound care, vaginal health, eye care, swimming pool maintenance, ear infections, and foot odor control [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], boric acid has also gained attention as a component in the development of surgical anti-adhesive polymers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This versatility underscores its potential in healthcare by potentially preventing post-operative adhesion formation, thereby contributing to improved surgical outcomes and patient rehabilitation.\u003c/p\u003e \u003cp\u003eFurthermore, borates, including boric acid, are increasingly used in nutritional supplements as a source of boron, an essential trace element widely distributed in nature. These compounds, particularly boric acid salts (H3BO3), are commonly found in laundry detergents, cleaning agents, and fertilizers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. They play a crucial role in regulating hormone levels, including steroids, thyroid hormones, estrogen, and testosterone, as well as maintaining various minerals such as calcium and vitamin D, and protecting bone tissue [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, these compounds are effective in numerous metabolic processes, including DNA damage repair mechanisms and the regulation of oxidative stress. Studies have demonstrated their anti-inflammatory, antioxidant, antibacterial, anticarcinogenic, and antiviral activities [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These multifaceted properties further highlight the potential of boric acid and its derivatives in enhancing surgical outcomes and patient rehabilitation by preventing post-operative adhesion formation.\u003c/p\u003e \u003cp\u003eMechanistically, boric acid interferes with protein synthesis, activates MAP kinase cascades, inhibits cell cycle progression, and induces morphological changes, contributing to significant antiproliferative effects in specific cancer cell lines [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Upon entering cells at physiological pH, boric acid hydrolyzes into borate ions, which subsequently lower intracellular pH [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This acidification process can lead to cellular inhibition and apoptosis. Borate ions, rather than boric acid itself, are recognized for their roles as cell-signaling molecules, co-factors for enzymes, contributors to redox reactions, and structural components of the cytoskeleton [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Boric acid exhibits inhibitory effects on several enzymes with those involved in progression of prostate cancer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn a molecular level, boric acid functions as a Lewis acid, capable of forming complexes with hydroxyl-group-bearing amino acids within tissue protein polypeptides [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This interaction highlights its potential to cross-link these polypeptides, facilitated by boric acid's ability to establish strong bonds with hydroxyl groups due to its vacant d-orbital in boron. These reactions with nucleophiles enable the formation of cross-linked complexes, creating new covalent bonds between boric acid and protein molecules. Such interactions may hinder further protein-protein interactions between adjacent tissue surfaces, thereby potentially preventing the formation of de novo adhesions.\u003c/p\u003e \u003cp\u003eDespite these promising mechanisms, to the best of our knowledge, no previous studies have investigated the protective effects of boric acid against post-operative testicular adhesions. Therefore, this study aims to evaluate the protective effects of boric acid treatment against testicular adhesions induced by laparoscopic surgery in rats.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Experimental Groups\u003c/h2\u003e \u003cp\u003eThe experimental animals used in the study were obtained from the Atat\u0026uuml;rk University Medical Experimental Application and Research Center. In the study, 40 Sprague Dawley rats weighing 230\u0026ndash;290 grams were used. The rats were acclimatized to the environmental conditions for 1 week before the experiment. The rats were fed freely with standard feed and drinking water at room temperature in a 12-hour light and 12-hour dark cycle. The rats were randomly divided into 4 groups with 10 animals in each group. Ethics committee permission for the study was obtained from Atat\u0026uuml;rk University Animal Experiments Local Ethics Committee (Ethics Number: 2024/156).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eControl group\u003c/strong\u003e \u003cp\u003eNo surgical intervention was performed on the rats and 2 ml physiological saline was given intra-abdominally for 7 days (n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLaparotomy (LA) group\u003c/strong\u003e \u003cp\u003eOnly the abdominal cavity of the rats was opened and closed again (n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBoric Acid (BA) group\u003c/strong\u003e \u003cp\u003eNo surgical intervention was performed on the rats, and BA at a dose of 8 mg/kg dissolved in 2 ml physiological saline was administered intra-abdominally for 7 days (n\u0026thinsp;=\u0026thinsp;10) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLA\u0026thinsp;+\u0026thinsp;BA group\u003c/strong\u003e \u003cp\u003eAfter the abdominal cavity of the rats was opened and closed, BA at a dose of 8 mg/kg dissolved in 2 ml physiological saline was administered intra-abdominally for 7 days (n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLaparotomy Procedures\u003c/h2\u003e \u003cp\u003eButorphanol was administered subcutaneously at a dose of 1 mg/kg before laparotomy to all rats except the control and BA groups [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Peritoneal Button Creation (PBC) model was applied to create an adhesion model in rats [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Before the intervention, rats were anesthetized with 10 mg/kg xylazine followed by 100 mg/kg ketamine. For laparotomy, a 3 cm long incision was made from the middle part of the abdominal region to cover the peritoneum, then strangulation was created with the help of hemostatic forceps to cover the parietal peritoneum of approximately 1 cm2 and the bottoms of these strangulations were ligated with 6.0 proline threads. This procedure was repeated 4 times at 0.2 cm intervals. Subcutaneous tissue was then closed with the continuous suture method (PDS 4/0) and skin was closed with a separate suture method (Nylon 4/0) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. At the end of the 7th day, all rats were sacrificed under general anesthesia. The abdominal cavity of the rats was opened and the presence of adhesion in the testicular tissue was checked and the scoring was done according to Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. After macroscopic evaluation, testicular tissues of all rats were collected; It was examined by biochemical, histopathological, Massons's trichrome, immunohistochemical and immunofluorescence methods.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIntra-abdominal adhesion scoring model calculation in rats\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdhesion Assessment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo adhesion\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePresence of a single fibrous bridge between internal organs or between the internal organ and the abdominal wall\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePresence of two fibrous bridges between internal organs or between the internal organ and the abdominal wall\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMore than two fibrous bridges between the viscera or between the viscera and the abdominal wall, or the formation of a single mass of intestines without adhesion to the abdominal wall\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInternal organs are directly attached to the abdominal wall, regardless of their number and extension.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-Linked ImmunoSorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003eBy following the manufacturer's instructions, the ELISA kits (SUNREDBIO) were used to measure the concentrations of MDA, SOD, CAT, GPx, IL-1β, IL-6, IL-10, and TNF-α in the supernatants of brain tissue homogenates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Examinations\u003c/h2\u003e \u003cp\u003eTestis tissue samples obtained from rats were fixed in a 10% buffered formalin solution and subjected to routine tissue tracing procedures. The prepared sections were blocked and 4 \u0026micro;m thick sections were taken from each of the blocks. The sections were stained with Hematoxylin \u0026amp; Eosin (H\u0026amp;E) and Masson's Trichrome stains and evaluated under a light microscope (OLYMPUS BX51). In Masson's Trichrome staining, fibrosis formation was evaluated as absent (-), mild (+), moderate (+++), and severe (++++). Testicular tissues were subjected to histopathologic evaluation using Johnsen's mean testicular biopsy score method (a semi-quantitative method). Each tubule was given a score between 0 and 10 according to epithelial maturation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Additionally, each sample was examined in 10 randomly selected fields of approximately x20 objective. Scores were as follows: Grade 0 = \u0026minus; (negative); Grade 1\u0026thinsp;=\u0026thinsp;+\u0026thinsp;1 (mild); Grade 2\u0026thinsp;=\u0026thinsp;+\u0026thinsp;2 (moderate); Grade 3\u0026thinsp;=\u0026thinsp;+\u0026thinsp;3 (severe); and Grade 4\u0026thinsp;=\u0026thinsp;+\u0026thinsp;4 (most severe) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTesticular biopsy score criteria\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSkor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo cells\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSertoli cells without germ cells\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOnly spermatogonia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOnly a few spermatocytes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMany spermatocytes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOnly a few early spermatids\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMany early spermatids\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFew late spermatids\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMany late spermatids\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFull spermatogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical Examinations\u003c/h2\u003e \u003cp\u003eTissue sections taken on adhesive (poly-L-Lysin) slides for immunoperoxidase examination were treated with primary antibodies (GnRH I Cat No: sc-32292, Dilution Ratio: 1/100, US; Kisspeptin 1 (KiSS 1) Cat No: sc-101246, Dilution Ratio: 1/100, US; Bcl-2 Cat. No: sc-7382, Dilution Ratio: 1/100, US) were added and incubated according to the instructions for use. 3\u0026ndash;3' Diaminobenzidine (DAB) chromogen was used as chromogen in the tissues. Stained sections were examined by light microscopy (Zeıss AXIO GERMANY) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDouble Immunofluorescence Examinations\u003c/h2\u003e \u003cp\u003eFor immunofluorescence examination, tissue sections were placed on adhesive (poly-L-Lysin) slides. Following standard protocols, primary antibodies (8-OHdG Cat. No: sc-66036, Dilution Ratio: 1/100, US; BAX Cat. No: sc-780, Dilution Ratio: 1/100, US) were added, and the samples were incubated by the usage instructions. Immunofluorescence secondary antibody was used as secondary marker (FITC Cat No: ab6785 Dilution Ratio: 1/1000, UK) and kept in the dark for 45 min. The tissues were then treated with the second primary antibody (JNK Cat No: sc-7345, Dilution Ratio: 1/100, US; Caspase 3 Cat No: sc-56053 Dilution Ratio: 1/100, US) and incubated according to the instructions for use. Immunofluorescence secondary antibody was used as secondary marker (Texas Red Cat No: ab6719 Dilution Ratio: 1/1000 UK) and kept in the dark for 45 minutes. Then, DAPI with mounting medium (Cat no: D1306 Dilution Ratio: 1/200 UK) was added to the sections and kept in the dark for 5 min and the sections were covered with coverslips. The stained sections were examined under a fluorescence attachment microscope (Zeıss AXIO GERMANY) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data were assessed using GraphPad Prism 8.0.2 for statistical analysis, with a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The non-parametric Mann-Whitney U test was used to assess group differences in non-parametric data, while the non-parametric Kruskal-Wallis test was used to determine differences among groups. One-way ANOVA and the Tukey test were used to assess parametric data.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eMacroscopic findings\u003c/h2\u003e\n \u003cp\u003eUpon concluding the experiment and inspecting the abdominal region of the animals, no adhesions were detected in either the control group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea) or the boric acid (BA) treated group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). In contrast, the laparotomy (LA) group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb) exhibited multiple adhesions (bands) in the testicular tissues. However, in the LA\u0026thinsp;+\u0026thinsp;BA group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed), these adhesion bands were significantly reduced (\u0026lt;\u0026thinsp;0.001) compared to the LA group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eHistopathological Findings\u003c/h2\u003e\n \u003cp\u003eHematoxylin-eosin staining of testicular tissue samples revealed normal histological structures in both the control group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea) and the boric acid (BA) treated group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). In contrast, the laparotomy (LA) group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb) exhibited severe spermatocyte degeneration, necrosis, tissue edema, and inflammation. However, these histopathological findings were markedly reduced in the LA\u0026thinsp;+\u0026thinsp;BA group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). For fibrosis assessment, Masson\u0026apos;s trichrome staining showed severe fibrosis in the area of adhesion formation in the LA group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). Notably, fibrosis development was significantly attenuated in the LA\u0026thinsp;+\u0026thinsp;BA group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed). For quantitative analysis, the detailed adhesion scoring, histopathological findings, and fibrosis development scoring, along with corresponding statistical analyses, are summarized in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunohistochemical and Immunofluorescence Findings\u003c/h2\u003e\n \u003cp\u003eIn immunohistochemical and immunofluorescence analyses of testicular tissues, high levels of Bcl-2 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), GnRH (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), and KiSS1 (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e) expressions were observed in the control and BA groups, as well as mild expressions of 8-OHdG, JNK (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e), BAX, and Caspase 3 (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). The LA group had high expressions of 8-OHdG, JNK, BAX, and Caspase 3, but mild expressions of Bcl-2, GnRH, and KiSS1. In the LA\u0026thinsp;+\u0026thinsp;BA group, it was determined that expressions of 8-OHdG, JNK, BAX, and Caspase 3 were significantly decreased (\u0026lt;\u0026thinsp;0.0001), and expressions of Bcl-2, GnRH, and KiSS1 were significantly increased (\u0026lt;\u0026thinsp;0.0001). Results of immunohistochemical and immunofluorescence staining findings, along with statistical analysis data, are presented in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of Laparotomy-induced Adhesion on Lipid Peroxidation/antioxidant Status\u003c/h2\u003e\n \u003cp\u003eThe activities of SOD (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eB), CAT (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eC), and GPx (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eD) were significantly reduced in the LA group compared to the control, LA\u0026thinsp;+\u0026thinsp;BA groups. Treatment with BA resulted in an increase in these enzyme activities, with a particularly notable effect observed in the high-dose BA group. Evaluation of MDA levels revealed a significant increase in the LA group compared to other groups; however, BA treatment led to a dose-dependent reduction in MDA (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eA) levels.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of Laparotomy-induced Adhesion on the Inflammatory Response\u003c/h2\u003e\n \u003cp\u003eBased on the assessments depicted in Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e, the LA-exposed group exhibited substantial increases in proinflammatory mediators TNF-\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eA), IL-1\u0026beta; (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eB), and IL-6 (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eC) compared to both control and LA\u0026thinsp;+\u0026thinsp;BA treated groups. Treatment with BA demonstrated a dose-dependent reduction in these mediators, with the highest efficacy observed in the LA\u0026thinsp;+\u0026thinsp;BA group. Evaluation of IL-10 (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eD) levels revealed a significant decrease in the LA group, which showed a dose-dependent reversal with BA treatment.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAdhesions are abnormal fibrous connections that form between adjacent tissues or organs, often as a result of surgery, trauma, or inflammation. These fibrous bands can lead to serious complications such as bowel obstruction and infertility [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In contrast, normal fibrosis is a natural healing response characterized by collagen deposition to facilitate tissue repair. However, excessive fibrosis, seen in conditions like liver cirrhosis or pulmonary fibrosis, can impair organ function. Intra-abdominal adhesions specifically refer to abnormal fibrous connections within the abdominal cavity, commonly occurring following surgical procedures. Despite their prevalence, effective preventive measures remain challenging to implement. These adhesions can affect various abdominal tissues and organs, underscoring their clinical significance [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genital organs, particularly the testicular tissues, are vulnerable to post-operative adhesions, potentially leading to infertility due to oxidative stress, inflammation, and apoptosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Histopathological studies have revealed severe inflammation, degenerative changes in spermatocytes, and extensive fibrosis in testicular tissues affected by adhesions [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. During this scenario, the oxidative stress and inflammation play pivotal roles in the pathogenesis of post-operative adhesions, triggering increases in reactive oxygen species (ROS), inflammatory cytokines (IL-1β, TNF-α, IL-6), and markers of oxidative stress (8-OHdG, SOD, CAT, GPx) in affected tissues [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This imbalance between ROS production and antioxidant defenses contributes to tissue damage and adhesion formation.\u003c/p\u003e \u003cp\u003eMorevover, following surgery, a pronounced inflammatory response develops in testicular tissues, characterized by elevated levels of pro-inflammatory markers such as JNK, IL-1β, TNF-α, and IL-6, alongside decreased IL-10 levels [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This inflammatory cascade is exacerbated by increased oxidative stress post-surgery, further contributing to adhesion formation within testicular tissue. Consistently, our study observed a pronounced inflammatory response in testicular tissues by the 7th day post-surgery, marked by elevated levels of JNK, IL-1β, TNF-α, and IL-6, alongside decreased IL-10 levels. This inflammatory cascade in the testis was exacerbated by increased oxidative stress post-surgery, which not only triggered inflammation but potentially initiates testicular adhesion. During this post-surgery inflammatory process, macrophages, as key initiators of the inflammatory response, release pro-inflammatory cytokines like IL-1, IL-6, and TNF-α, which recruit inflammatory cells and exacerbate tissue damage [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Experimental studies have highlighted the role of cytokines such as TNF-α and IL-6 in the pathogenesis of postoperative peritoneal adhesions [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Apoptosis, on the other hand, plays a significant role in tissue damage during adhesion formation, involving the TNF-α-induced JNK pathway, caspase 3 activation, and altered regulation of Bcl-2 family proteins [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Caspase 3 is central to the final stages of apoptosis, while Bcl-2 family proteins regulate mitochondrial integrity and cell fate decisions [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Therefore, it is logically to recognize severe caspase 3 expressions in the testicular tissue, especially in spermatocytes, during post-operative testicular adhesion with marked apoptosis.\u003c/p\u003e \u003cp\u003eIn addition to the inflammatory and apoptotic pathways involved in adhesion formation, reproductive hormones such as GnRH and its stimulators, Kisspeptins, play crucial roles in regulating testicular function. GnRH is essential for regulating the reproductive axis and stimulating gonadotropin synthesis [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Kisspeptins, potent stimulators of GnRH neurons, also play a significant role in the synthesis and secretion of GnRH, crucially influencing spermatogenesis and overall reproductive function [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Our findings align with previous research indicating that decreases in GnRH and Kisspeptin levels are associated with impaired testicular function following adhesion formation. This provides a plausible explanation for the observed decrease in testicular spermatogenesis induced by laparotomy in our study.\u003c/p\u003e \u003cp\u003eIn contrary, boric acid exhibits protective effects against oxidative stress, inflammation, fibrosis, and apoptosis in various cellular contexts [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These properties are potentially relevant in the context of postoperative adhesions, where BA's antioxidant capabilities mitigate oxidative stress and its anti-inflammatory properties reduce the inflammatory response [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. By forming cross-linked barriers between peritoneal surfaces, BA might has the ability to prevent adhesion formation, thereby protecting tissues from the detrimental effects of surgery-induced inflammatory cascades and apoptotic pathways observed in our study [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs demonstrated in our results, BA effectively mitigated laparotomy-induced testicular adhesions and associated pathologies. Macroscopically, BA-treated groups showed reduced adhesion bands compared to untreated controls, while histopathological analysis revealed attenuated tissue damage, inflammation, and fibrosis. Immunohistochemical and immunofluorescence analyses confirmed BA's ability to modulate oxidative stress, inflammation, and apoptosis pathways, restoring the balance of pro- and anti-apoptotic proteins. Additionally, BA treatment enhanced antioxidant enzyme activities and reduced lipid peroxidation in a dose-dependent manner, thereby preserving testicular function and preventing infertility. These findings collectively suggest that BA presents a promising therapeutic strategy for preventing postoperative complications in sensitive tissues like the testes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we identified that postoperative testicular adhesions suppress spermatogenesis through mechanisms involving oxidative stress, inflammation, fibrosis, and apoptosis in testicular tissue. Moreover, boric acid demonstrated protective effects in testicular tissue by attenuating adhesion formation. These findings suggest that boric acid holds promise as a protective agent against postoperative intra-abdominal adhesions, which currently lack effective preventive strategies. BA's diverse protective mechanisms underscore its potential as a therapeutic intervention to mitigate postoperative adhesions and maintain testicular function. Future research efforts should prioritize elucidating BA's molecular pathways and optimizing its clinical application to enhance therapeutic outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eİ.B., S.Y., Y.S.S., and M.W. They wrote the main text and edited the main textİ.B., M.K., B.G., and S.\u0026Ccedil;. They performed and evaluated histopathological analyzesİ.B., M.W., and M.B. They performed and evaluated biochemical analyzesS.O., and A.G. They performed surgical interventions\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBoyd‐Carson H, Gana T, Lockwood S, et al (2020) A review of surgical and peri‐operative factors to consider in emergency laparotomy care. Anaesthesia 75:. https://doi.org/10.1111/anae.14821\u003c/li\u003e\n\u003cli\u003eHaddad S, Ghadimi K, Abrishamkar R, Asl NSM (2021) Comparing laparoscopy and laparotomy procedures in the radical hysterectomy surgery for endometrial cancer: a basic review. Am J Transl Res 13:2456\u0026ndash;2461\u003c/li\u003e\n\u003cli\u003eK\u0026Ouml;M M (2013) Effect of Hyaluronic Acid/Carboxymethylcellulose and Flunixin Meglumine Combination on the Prevention of Postoperative Intraabdominal Adhesions: An Experimental Study in Rabbits. Kafkas Univ Vet Fak Derg. https://doi.org/10.9775/kvfd.2013.8406\u003c/li\u003e\n\u003cli\u003eTabibian N, Swehli E, Boyd A, et al (2017) Abdominal adhesions: A practical review of an often overlooked entity. 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Plast Reconstr Surg 130:206S-213S. https://doi.org/10.1097/PRS.0b013e3182638d48\u003c/li\u003e\n\u003cli\u003eBian Y-Y, Yang L-L, Yan Y, et al (2020) Identification of candidate biomarkers correlated with pathogenesis of postoperative peritoneal adhesion by using microarray analysis. World J Gastrointest Oncol 12:54\u0026ndash;65. https://doi.org/10.4251/wjgo.v12.i1.54\u003c/li\u003e\n\u003cli\u003eWu Q, Wu W, Jacevic V, et al (2020) Selective inhibitors for JNK signalling: a potential targeted therapy in cancer. J Enzyme Inhib Med Chem 35:574\u0026ndash;583. https://doi.org/10.1080/14756366.2020.1720013\u003c/li\u003e\n\u003cli\u003eObeng E (2021) Apoptosis (programmed cell death) and its signals - A review. Brazilian J Biol 81:1133\u0026ndash;1143. https://doi.org/10.1590/1519-6984.228437\u003c/li\u003e\n\u003cli\u003eCory S, Adams JM (2002) The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2:647\u0026ndash;656. https://doi.org/10.1038/nrc883\u003c/li\u003e\n\u003cli\u003eQian S, Wei Z, Yang W, et al (2022) The role of BCL-2 family proteins in regulating apoptosis and cancer therapy. Front Oncol 12:. https://doi.org/10.3389/fonc.2022.985363\u003c/li\u003e\n\u003cli\u003eGruenewald DA, Naai MA, Hess DL, Matsumoto AM (1994) The Brown Norway Rat as a Model of Male Reproductive Aging: Evidence for Both Primary and Secondary Testicular Failure. J Gerontol 49:B42\u0026ndash;B50. https://doi.org/10.1093/geronj/49.2.B42\u003c/li\u003e\n\u003cli\u003eHan S-K, Gottsch ML, Lee KJ, et al (2005) Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for the Onset of Puberty. J Neurosci 25:11349\u0026ndash;11356. https://doi.org/10.1523/JNEUROSCI.3328-05.2005\u003c/li\u003e\n\u003cli\u003eHERBISON A (2006) Physiology of the Gonadotropin-Releasing Hormone Neuronal Network. In: Knobil and Neill\u0026rsquo;s Physiology of Reproduction. Elsevier, pp 1415\u0026ndash;1482\u003c/li\u003e\n\u003cli\u003eAnjum S, Krishna A, Sridaran R, Tsutsui K (2012) Localization of Gonadotropin‐Releasing Hormone (GnRH), Gonadotropin‐Inhibitory Hormone (GnIH), Kisspeptin and GnRH Receptor and Their Possible Roles in Testicular Activities From Birth to Senescence in Mice. J Exp Zool Part A Ecol Genet Physiol 317:630\u0026ndash;644. https://doi.org/10.1002/jez.1765\u003c/li\u003e\n\u003cli\u003eTekin A, G\u0026uuml;ner A, Akkan T (2024) Protective Effect of Boric Acid Against Ochratoxin A-Induced Toxic Effects in Human Embryonal Kidney Cells (HEK293): A Study on Cytotoxic, Genotoxic, Oxidative, and Apoptotic Effects. Biol Trace Elem Res. https://doi.org/10.1007/s12011-024-04194-5\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Boric acid, Laparotomy, Post-operative intra-abdominal adhesions, Testis","lastPublishedDoi":"10.21203/rs.3.rs-4741801/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4741801/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePost-operative intra-abdominal adhesions, significantly affecting testicular tissue, are a prevalent and serious complication following laparoscopic surgery. This study investigated the efficacy of boric acid, known for its antioxidant, anti-inflammatory, and anti-apoptotic properties, in preventing post-operative testicular adhesions. Forty rats, weighing between 230 and 290 grams, were divided into four groups: control, laparoscopy (LA), boric acid (BA), and LA\u0026thinsp;+\u0026thinsp;BA. Following laparoscopic surgery, BA treatment was administered for seven days. Adhesion scores were markedly higher in the LA group, whereas the LA\u0026thinsp;+\u0026thinsp;BA group exhibited a significant reduction in adhesion scores. Testicular tissues were analyzed using immunohistochemistry, immunofluorescence, Masson's trichrome staining, biochemical assays, and histopathological evaluation. In the LA group, malondialdehyde (MDA) levels increased while superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels decreased; these parameters normalized with BA treatment. Additionally, the LA group exhibited reduced levels of IL-10, Bcl-2, Kisspeptin-1, and GnRH, alongside elevated levels of inflammatory markers IL-1β, IL-6, TNF-α, JNK, BAX, and Caspase 3. Boric acid treatment significantly restored these levels to normal. In conclusion, oxidative stress, inflammation, and apoptosis in testicular tissues were associated with post-operative testicular adhesions. Boric acid demonstrated potential as an anti-adhesive agent, reducing testicular adhesions and normalizing biochemical and histological parameters following laparoscopic surgery.\u003c/p\u003e","manuscriptTitle":"Investigation of the Effects of Boric Acid Against Post Operative Testicular Adhesion Caused by Experimental Laporotomy in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-16 16:14:20","doi":"10.21203/rs.3.rs-4741801/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-09T01:26:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-07T01:43:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-02T09:50:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30938159269201175410042895179112715010","date":"2024-08-29T00:58:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-26T19:01:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243663702440035336353606284302758857869","date":"2024-08-25T14:06:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194447569510113427591118214772297018745","date":"2024-08-25T07:57:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257646178884163026345720245414700675353","date":"2024-08-24T21:43:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-11T21:05:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147547270717861588056193273552061945530","date":"2024-07-28T21:44:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-23T20:42:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-16T13:00:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-16T01:39:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Trace Element Research","date":"2024-07-15T09:08:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4d87f4ff-5ddc-4d24-8365-004eb9ca2a3b","owner":[],"postedDate":"August 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:03:15+00:00","versionOfRecord":{"articleIdentity":"rs-4741801","link":"https://doi.org/10.1007/s12011-024-04400-4","journal":{"identity":"biological-trace-element-research","isVorOnly":false,"title":"Biological Trace Element Research"},"publishedOn":"2024-09-28 15:57:35","publishedOnDateReadable":"September 28th, 2024"},"versionCreatedAt":"2024-08-16 16:14:20","video":"","vorDoi":"10.1007/s12011-024-04400-4","vorDoiUrl":"https://doi.org/10.1007/s12011-024-04400-4","workflowStages":[]},"version":"v1","identity":"rs-4741801","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4741801","identity":"rs-4741801","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}