Transcriptomics Changes in the Peritoneum of Mice with Lipopolysaccharide-Induced Peritonitis

Peritonitis caused by LPS is a severe clinical challenge, which causes organ damage and death. However, the mechanism of LPS-induced peritonitis has not been fully revealed yet. Here, we investigated the transcriptome profile of the peritoneal tissue of LPS-induced peritonitis in mice. A model of LPS-induced peritonitis in mice was established (LPS 10 mg/kg, i.p.), and the influence of TAK 242 (TLR4 inhibitor) on the level of inflammatory cytokines in mouse peritoneal lavage fluid was investigated by using an ELISA test. Next, the peritoneal tissues of the three groups of mice (Control, LPS, and LPS+TAK 242) (n = 6) were isolated and subjected to RNA-seq, followed by a series of bioinformatics analyses, including differentially expressed genes (DEGs), enrichment pathway, protein-protein interaction, and transcription factor pathway. Then, qPCR verified-hub genes that may interact with TAK 242 were obtained. Subsequently, the three-dimensional structure of hub proteins was obtained by using homology modeling and molecular dynamics optimization (300 ns). Finally, the virtual docking between TAK 242 and hub proteins was analyzed. Our results showed that TAK 242 significantly inhibited the production of inflammatory cytokines in the peritoneal lavage fluid of mice with peritonitis, including IL-6, IFN-γ, IL-1β, NO, and TNF-α. Compared with the Control group, LPS treatment induced 4201 DEGs (2442 down-regulated DEGs and 1759 up-regulated DEGs). Compared with the LPS group, 30 DEGs were affected by TAK 242 (8 down-regulated DEGs and 22 up-regulated DEGs). A total of 10 TAK 242-triggered hub genes were obtained, and the possible docking modes between TAK 242 and hub proteins were acquired. Overall, our data demonstrated that a large number of DEGs were affected in LPS-triggered peritonitis mice. Moreover, the TLR4 inhibitor TAK 242 is capable of suppressing the inflammatory response of LPS-induced peritonitis. Our work provides clues for understanding the pathogenesis of LPS-induced peritonitis in mice.

A Novel Ex Vivo Peritoneal Model to Investigate Mechanisms of Peritoneal Metastasis in Gastric Adenocarcinoma

Peritoneal metastases (PM) portend limited survival in patients with Gastric Adenocarcinoma (GCa), and strategies to prevent and/or more effectively treat PM are needed. Existing models are limited in recapitulating key elements of the peritoneal metastatic cascade. To explore the underlying cellular and molecular mechanisms of PM, we have developed an ex vivo human peritoneal explant model. Fresh peritoneal tissue samples were obtained from patients undergoing abdominal surgery and suspended, mesothelial layer down but without direct contact, above a monolayer of red-fluorescent stained AGS human GCa cells for 24hrs, then washed and cultured for a further 3 days. Implantation and invasion of GCa cells within the explant were examined using real-time confocal fluorescence microscopy. Superficial implantation of AGS GCa cells within the mesothelial surface was readily detected, and colonies expanded over 3 days. To investigate the sensitivity of the model to altered GCa cellular implantation, we stably transfected AGS cells with E-Cadherin, restoring the E-Cadherin that they otherwise lack. This markedly suppressed implantation and invasion of AGS cells into the submesothelial mesenchymal layer. Here we show that this ex vivo human peritoneal explant model is responsive to manipulation of genetic factors that regulate peritoneal implantation and invasion by GCa cells, with reproducible results.

Adipose Mesenchymal Stem Cell-Derived Exosomes Relieve Postoperative Abdominal Adhesion By Activating the MAPK-ERK 1/2 and PI3K-Akt Pathways

Background: Mesenchymal stem cells (MSCs) have shown potential for peritoneal repair and regeneration. However, the roles of exosomes (Exos) derived from MSCs are not well understood. Therefore, our aim was to elucidate the therapeutic effect of MSC-derived Exos on peritoneal injury. Methods: Adipose-derived mesenchymal stem cells-derived Exos (ADSC-Exos) were isolated using ultracentrifugation and then injected through the tail vein. Rat peritoneal mesothelial cells (RPMCs) were extracted using enzymatic digestion of the peritoneal cavity. Postsurgical adhesions were brought on by scratching rat cecums. The anti-adhesion effects of ADSC-Exos in rats were evaluated using the Nair’s scoring system at 8 and 15 days after abdominal surgery. In addition, the levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in rat peritoneal fluid were determined via enzyme-linked immunosorbent (ELISA) assays at 8 days after operation. Expression differences in inflammatory and apoptotic markers were detected using immunofluorescence at 4 days after surgery. H&E staining and immunohistochemistry were employed to observe histological changes and expression changes of extracellular matrix (ECM)-related indices after 15 days. In vitro, ADSC-Exo-driven RPMC proliferation was assessed via 5-ethynyl-2-deoxyuridine assay. Cell migration was determined using scratch-wound and transwell assays. Related signaling networks were estimated based on sequencing and bioinformatics analyses. The roles of the MAPK-ERK1/2 and PI3K-Akt signaling networks were analyzed using immunoblotting.Results: ADSC-Exos were incorporated into RPMCs, where they induced cell proliferation and migration in proportion to dosage. This was likely mediated via stimulation of the MAPK-ERK 1/2 and PI3K-Akt axes. In vivo, the ADSC-Exos group had a lower adhesion score than the control group at 15 days. At postoperative day 4, inflammation and apoptosis were markedly inhibited by ADSC-Exos. At 8 days, ADSC-Exos treatment decreased PAI-1 expression and increased tPA expression in peritoneal fluid. At 15 days, ADSC-Exos potentially helped regulate the ECM balance.Conclusions: ADSC-Exos may promote the healing of injured peritoneal tissue, suggesting a promising therapeutic approach for peritoneal adhesions.

Sulodexide Prevents Peritoneal Fibrosis by Downregulating the Expression of TGF-β1 and Its Signaling Pathway Molecules

Peritoneal dialysis is one of the main renal replacement treatments. However, long-term peritoneal dialysis keeps the peritoneum in contact with the sugar-containing nonphysiological peritoneal fluid, which leads to recurrent peritonitis, peritoneal fibrosis, and failure of ultrafiltration. Transforming growth factor-β1 (TGF-β1), related cytokines, and inflammatory factors are closely related to peritoneal fibrosis. Sulodexide (SLX) is a new type of glycosaminoglycan preparation, which is involved in the formation of an anionic charge barrier and can maintain the selective permeability of vascular endothelial cells. In this study, the innovative analysis of SLX specifically prevents the process of peritoneal dialysis peritoneal fibrosis by downregulating the expression of TGF-β1 and its signaling pathway molecules. We randomly divided 30 rats into three groups. The blank control group received no treatment. The peritoneal dialysis model group was injected with 4.25% peritoneal dialysate (PDF) 20 ml daily, and the SLX group was injected with 4.25% PDF 20 ml + sulodexide (SLX) 20 mg/kg daily. After 8 weeks of dialysis, the rats were sacrificed, and the peritoneal function test was performed to determine the amount of glucose transport and ultrafiltration. The thickness of peritoneal per unit area was observed under high magnification. The level of inflammation in peritoneal tissue and the expression of TGF-β1/Smad were detected. The results showed that SLX can significantly improve peritoneal tissue thickening and inflammation, can downregulate the expression of TGF-β1, Smad2, Smad3, and Smad7 in peritoneal tissue, and improve the progression of peritoneal fibrosis.

Water removal during automated peritoneal dialysis assessed by remote patient monitoring and modelling of peritoneal tissue hydration

Water removal which is a key treatment goal of automated peritoneal dialysis (APD) can be assessed cycle-by-cycle using remote patient monitoring (RPM). We analysed ultrafiltration patterns during night APD following a dry day (APDDD; no daytime fluid exchange) or wet day (APDWD; daytime exchange). Ultrafiltration for each APD exchange were recorded for 16 days using RPM in 14 patients. The distributed model of fluid and solute transport was applied to simulate APD and to explore the impact of changes in peritoneal tissue hydration on ultrafiltration. We found lower ultrafiltration (mL, median [first quartile, third quartile]) during first and second vs. consecutive exchanges in APDDD (−61 [−148, 27], 170 [78, 228] vs. 213 [126, 275] mL; p < 0.001), but not in APDWD (81 [−8, 176], 81 [−4, 192] vs. 115 [4, 219] mL; NS). Simulations in a virtual patient showed that lower ultrafiltration (by 114 mL) was related to increased peritoneal tissue hydration caused by inflow of 187 mL of water during the first APDDD exchange. The observed phenomenon of lower ultrafiltration during initial exchanges of dialysis fluid in patients undergoing APDDD appears to be due to water inflow into the peritoneal tissue, re-establishing a state of increased hydration typical for peritoneal dialysis.

Influence of pre-analytical sample preparation on drug concentration measurements in peritoneal tissue: an ex-vivo study

Objectives Biopsy morphology (surface/depth ratio) and sample processing might affect pharmacological measurements in peritoneal tissue. Methods This is an ex-vivo study on inverted bovine urinary bladders (IBUB). We compared cisplatin (CIS) and doxorubicin (DOX) concentration in 81 standardized transmural punch biopsies of different diameters (6 and 12 mm). Then, we assessed the effect of dabbing the peritoneal surface before analysis. After automatized tissue homogenization with ceramic beads followed by lyophilisation, DOX concentration was quantified by high-performance liquid chromatography (HPLC), CIS concentration by atomic absorption spectroscopy. Experiments were performed in triplicate; the analysis was blinded to the sample origin. Comparisons were performed using non-parametric tests. Results Concentrations are given in mean (CI 5–95%). Results were reproducible between experiments (for CIS p=0.783, for DOX p=0.235) and between different localizations within the IBUB (for CIS p=0.032, for DOX p=0.663). Biopsy diameter had an influence on CIS tissue concentration (6 mm biopsies: 23.2 (20.3–26.1), vs. 12 mm biopsies: 8.1 (7.2–9.2) ng/mg, p<0.001) but not on DOX: (0.46, 0.29–0.62) vs. 0.43 (0.33–0.54) ng/mg respectively, p=0.248). Dabbing the peritoneal surface reduced DOX tissue concentration (dry biopsies: 0.28 (0.12–0.43) vs. wet biopsies: 0.64 (0.35–0.93) ng/mg, p=0.025) but not CIS (23.5 (19.0–28.0) vs. 22.9 (18.9–26.9) ng/mg, respectively, p=0.735). Conclusions Measurements of drug concentration in peritoneal tissue can be influenced by the biopsy’s surface/depth ratio and after drying the biopsy’s surface. This influence can reach a factor three, depending on the drug tested. The biopsy technique and the pre-analytical sample preparation should be standardized to ensure reliable pharmacological measurements in peritoneal tissue.

Confocal Laser Microscopy for in vivo Intraoperative Application: Diagnostic Accuracy of Investigator and Machine Learning Strategies

<b><i>Background:</i></b> Confocal laser microscopy (CLM) is one of the optical techniques that are promising methods of intraoperative in vivo real-time tissue examination based on tissue fluorescence. However, surgeons might struggle interpreting CLM images intraoperatively due to different tissue characteristics of different tissue pathologies in clinical reality. Deep learning techniques enable fast and consistent image analysis and might support intraoperative image interpretation. The objective of this study was to analyze the diagnostic accuracy of newly trained observers in the evaluation of normal colon and peritoneal tissue and colon cancer and metastasis, respectively, and to compare it with that of convolutional neural networks (CNNs). <b><i>Methods:</i></b> Two hundred representative CLM images of the normal and malignant colon and peritoneal tissue were evaluated by newly trained observers (surgeons and pathologists) and CNNs (VGG-16 and Densenet121), respectively, based on tissue dignity. The primary endpoint was the correct detection of the normal and cancer/metastasis tissue measured by sensitivity and specificity of both groups. Additionally, positive predictive values (PPVs) and negative predictive values (NPVs) were calculated for the newly trained observer group. The interobserver variability of dignity evaluation was calculated using kappa statistic. The F1-score and area under the curve (AUC) were used to evaluate the performance of image recognition of the CNNs’ training scenarios. <b><i>Results:</i></b> Sensitivity and specificity ranged between 0.55 and 1.0 (pathologists: 0.66–0.97; surgeons: 0.55–1.0) and between 0.65 and 0.96 (pathologists: 0.68–0.93; surgeons: 0.65–0.96), respectively. PPVs were 0.75 and 0.90 in the pathologists’ group and 0.73–0.96 in the surgeons’ group, respectively. NPVs were 0.73 and 0.96 for pathologists’ and between 0.66 and 1.00 for surgeons’ tissue analysis. The overall interobserver variability was 0.54. Depending on the training scenario, cancer/metastasis tissue was classified with an AUC of 0.77–0.88 by VGG-16 and 0.85–0.89 by Densenet121. Transfer learning improved performance over training from scratch. <b><i>Conclusions:</i></b> Newly trained investigators are able to learn CLM images features and interpretation rapidly, regardless of their clinical experience. Heterogeneity in tissue diagnosis and a moderate interobserver variability reflect the clinical reality more realistic. CNNs provide comparable diagnostic results as clinical observers and could improve surgeons’ intraoperative tissue assessment.

P–300 Metformin: new therapeutic approach for endometriosis-associated infertility

Study question Could endometriosis-associated infertility be mitigated by metformin? Summary answer Metformin treatment restored fertility to control rates in endometriosis-induced mice that present lower fertility. No effect was observed on sham-operated mice. What is known already Endometriosis is a gynaecological disorder characterized by ectopic vascularized endometrial tissue growth, mainly in pelvic cavity, which provokes pain and infertility. Corroborating observations in women, endometriosis decreases oocyte quality and pregnancy success in rodents, without affecting the number of ovulations, resorption rate and fetal weight. Thus, animal models of endometriosis constitute a valuable tool to elucidate the pathophysiology of the disease and putative pharmacological therapies. Metformin is widely used for diabetes type–2 treatment, reducing glucose, oxidative stress and inflammation. Due to its antioxidant properties, metformin has shown to induce regression of endometrial implants in a rat model of endometriosis. Study design, size, duration B6CBA/F1 female mice were randomly divided in groups and subjected to treatment: 1-Endometriosis (n = 20); 2-Sham-operated (n = 12); 3-Endometriosis with metformin (n = 20); 4-Sham-operated with metformin (n = 20). Endometriosis was surgically induced by heterologous transplantation of endometrium from one donor in receptors from the same strain mice. Implants were confirmed and monitored by ultrasound. 50mg/kg/day of metformin was orally administrated during 3 months to Groups 3 and 4. Half of mice in each group were mated to fertility study. Participants/materials, setting, methods Endometriomas were monitored at 3 timepoints during the experiments. Biometric parameters of mice and number of implantation sites, fetuses and fetal weight were recorded. Fertility rates were assessed by the average number of fetuses in each group. Histological characterization of ovary, uterus, endometriomas, and peritoneal tissue at the implant site was performed by Hemathoxylin & Eosin (H&E) staining. Statistical study among groups was carried out and significant differences were considered for student t-test &lt; 0.05. Main results and the role of chance A decrease of 30% of fertility rate was verified in mice with endometriosis (p = 0,01); treatment with metformin was able to revert this decrease (p = 0,04). Interestingly, no differences in fertility were found in sham-operated mice under metformin treatment relatively with those of group 2 (p = 0,16). Although the number of absorptions observed in mice of the endometriosis group was higher, no statistical difference was reached comparatively with other groups. No biometrical differences were found between mice with endometriosis receiving metformin and those that do not receive the drug. Regarding H&E staining we verified that endometriomas showed histologically resemblances to uterus. Moreover, endometriomas from mice without treatment with metformin were dark brown, recalling for human endometriomas called “chocolate” cysts, while the endometriomas from mice who were treated with metformin were visually clearly. We postulate that these findings owes to a metformin-mediated decrease of oxidative imbalance and inflammatory response, induction of regression of endometriomas and regulation of oestrogen secretion. Limitations, reasons for caution Extrapolation of data from animal models to human needs caution, considering that endometriosis pattern differs between species. Also, further investigation, focused in identification of molecular targets of metformin and molecular pathways activated in endometriosis, is needed and in course. Wider implications of the findings: With these results, we indicate metformin as a novel and safe strategy to mitigate endometriosis-related oxidative stress and indeed could be used as a valid pharmacological approach to ameliorate endometriosis-associated infertility. Trial registration number Not applicable

Matrix Metalloproteinase-2, COL1A1, and COL3A1 mRNA Expression in Aponeurosis Musculus obliquus Externus Abdominis of Adult Inguinal Hernias

BACKGROUND: The ratio change of type I and type III collagen in the peritoneal tissue can be associated with defects in collagen synthesis caused by the extracellular matrix’s degradation. Matrix metalloproteinase-2 (MMP-2) is an enzyme that contributes primarily to the degradation of this extra cell. AIM: This study aimed to analyze the differences in expression of COL1A1, COL3A1, and MMP-2 mRNA and the relationship between these expressions in adult inguinal hernias and the expression ratio between the COL1A1/COL3A1 genes. METHODS: This study was an observational study with a cross-sectional comparative study design, where the sample was adult inguinal hernia patients who were taken from the aponeurosis tissue m. external obliquus performed at the time of surgery, while control was a non-herniated patient. The sample RNA was isolated, followed by cDNA synthesis, and examined by real-time polymerase chain reaction. RESULTS: The mean values of expression for COL1A1, COL3A1, and MMP-2 in the case group were 40.02 ± 181.38 copy number, 33.70 ± 143.62 copy number, and 31.78 ± 84.47 copy number. Meanwhile, the expression values for COL1A1, COL3A1, and MMP-2 in the control group were 40.247 ± 162.837 copy number, 13.35 ± 37.43 copy number, and 20.58 ± 48.95 copy number. CONCLUSIONS: Our study showed a difference in COL3A1 expression between the hernia and non-hernia groups, and no difference was found in the expression of COL1A1 and MMP2 between the hernia and non-hernia groups.

Importance of Laparoscopy to Solve the Diagnostic Dilemma of Abdominal Tuberculosis

Background: Diagnosis of abdominal tuberculosis as well as histopathological confirmation is difficult because of suboptimal access to the intraperitoneal pathology. Laparoscopy provides minimally invasive access to the peritoneal cavity and materials can be collected for confirmation of diagnosis. Objectives: To study the importance of laparoscopy as a tool for the diagnosis of abdominal tuberculosis and initiation of appropriate treatment without delay. Materials & Methods: In this study 25 patients with suspected abdominal tuberculosis were selected within the period of May, 2014 to October, 2014. Diagnostic laparoscopy performed on all patients with biopsy of tissue from accessible sites. Results: Diagnostic laparoscopy with biopsy confirmed the diagnosis in 24 (96%) patients, 23 of these patients (96%) had nodules at different site of abdominal cavity and 19 of these patients (76%) had ascites. In two cases there were nodules over liver surface; biopsy was taken also from both liver nodules. One nodule revealed fibrosis and another nodule revealed tuberculosis. Conclusion: Imaging and culture of ascitic fluid may fail to confirm or exclude abdominal tuberculosis in clinically suspected cases. Laparoscopy with peritoneal tissue biopsy provided rapid and correct diagnosis of abdominal tuberculosis and should be performed early in suspected cases. KYAMC Journal.2021;12(01): 14-17