A new bicornuate model of rat uterus transplantation.

INTRODUCTION: Uterus transplantation has revolutionized reproductive medicine for women with absolute uterine factor infertility, resulting in more than 40 reported successful live births worldwide to date. Small animal models are pivotal to refine this surgical and immunological challenging procedure aiming to enhance safety for both the mother and the child. MATERIAL AND METHODS: We established a syngeneic bicornuate uterus transplantation model in young female Lewis rats. All surgical procedures were conducted by an experienced and skilled microsurgeon who organized the learning process into multiple structured steps. Animals underwent meticulous preoperative preparation and postoperative care. Transplant success was monitored by sequential biopsies, monitoring graft viability and documenting histological changes long-term. RESULTS: Bicornuate uterus transplantation were successfully established achieving an over 70% graft survival rate with the passage of time. The bicornuate model demonstrated safety and feasibility, yielding outcomes comparable to the unicornuate model in terms of ischemia times and complications. Longitudinal biopsies were well-tolerated, enabling comprehensive monitoring throughout the study. CONCLUSIONS: Our novel bicornuate rat uterus transplantation model provides a distinctive opportunity for sequential biopsies at various intervals after transplantation and, therefore, comprehensive monitoring of graft health, viability, and identification of potential signs of rejection. Furthermore, this model allows for different interventions in each horn for comparative studies without interobserver differences contrary to the established unicornuate model. By closely replicating the clinical setting, this model stands as a valuable tool for ongoing research in the field of uterus transplantation, promoting further innovation and deeper insights into the intricacies of the uterus transplant procedure.

Notch Signaling Pathway in Pancreatobiliary Tumors.

Background and objectives: The Notch signaling pathway plays an important role both in the development of the ductal systems of the pancreas and the bile ducts as well as in cancer development and progression. The aim of this study was to examine the expression of central proteins of the Notch signaling pathway in pancreatobiliary tumors and its influence on patient survival. Materials and Methods: We compared the receptors (Notch1, Notch4), activating splicing factors (ADAM17), and target genes (HES1) of the Notch pathway and progenitor cell markers with relevance for the Notch signaling pathway (CD44, MSI1) between pancreatic adenocarcinomas (PDAC, n = 14), intrahepatic cholangiocarcinoma (iCC, n = 24), and extrahepatic cholangiocarcinoma (eCC, n = 22) cholangiocarcinomas via immunohistochemistry and ImageJ software-assisted analysis. An Immunohistochemistry (IHC)-score was determined by the percentage and intensity of stained (positive) cells (scale 0-7) and normal and malignant tissue was compared. In the IHC results, patients' (gender, age) and tumor (TNM Classification of Malignant Tumors, Union Internationale contre le Cancer (UICC) stages, grading, and lymphangitic carcinomatosa) characteristics were correlated to patient survival. Results: For eCC, the expression of CD44 (p = 0.043, IHC-score 3.94 vs. 3.54) and for iCC, the expression of CD44 (p = 0.026, IHC-score 4.04 vs. 3.48) and Notch1 (p < 0.001, IHC-score 2.87 vs. 1.78) was significantly higher in the tumor compared to non-malignant tissue. For PDAC, the expression of ADAM17 (p = 0.008, IHC-score 3.43 vs. 1.73), CD44 (p = 0.012, IHC-score 3.64 vs. 2.27), Notch1 (p = 0.012, IHC-score 2.21 vs. 0.64), and Notch4 (p = 0.008, IHC-score 2.86 vs. 0.91) was significantly higher in the tumor tissue. However, none of the analyzed Notch-signaling related components showed an association to patient survival. Conclusion: A significant overexpression of almost all studied components of the Notch signaling pathway can be found in the tumor tissue, however, without a significant influence on patient survival. Therefore, further studies are warranted to draw conclusions on Notch pathway's relevance for patient survival.

DNA Cytometry for Differentiation Between Low- and Medium-grade Dysplasia in Intraductal Papillary Mucinous Neoplasms.

BACKGROUND/AIM: The indication for resection of cystic pancreatic lesions is usually performed by sectional imaging criteria, such as the Sendai criteria. The aim of this study was to analyze a possible correlation between DNA cytometry and Sendai criteria for the differentiation between low-grade intraductal papillary mucinous neoplasms (IPMN-A) and medium-grade dysplasia (IPMN-B). MATERIALS AND METHODS: Histopathological analysis, DNA index and preoperative Sendai criteria were determined in 16 patients who underwent pancreatic resection for IPMN. RESULTS: All patients with IPMN-B showed aneuploid histograms with DNA indices ≥1.3, whereas three out of four patients with IPMN-A had diploid DNA indices ≤1.3. All 11 patients with one or more high-risk stigmata and aneuploid histograms had IPMN-Bs, whereas both patients who were Sendai-negative and diploid in the DNA analysis had an IPMN-A. CONCLUSION: DNA index may be an important diagnostic tool for the differentiation of different IPMN types beyond the traditional Sendai criteria.

Prognostic significance of DNA cytometry for adjuvant therapy response in pancreatic cancer.

BACKGROUND AND OBJECTIVES: The continuous progress in treatment options for pancreatic adenocarcinoma has lead to a re-evaluation of prognostic markers. In this study the prognostic relevance of DNA Index and classical histopathological parameters with regard to disease-free (DFS) and overall survival (OS) was analyzed within the CONKO-001 patient population. METHODS: One hundred forty three fresh-frozen paraffin-embedded tissue samples of the resected tumor specimen of the CONKO-001 patient population were available for DNA index analysis to evaluate its impact on patient outcome. RESULTS: Median DFS (7.3 vs. 14.3 months; P = 0.004) and median OS (16.6 vs. 29.2 months; P = 0.011) were significantly decreased in patients with a high DNA index (>1.4). Multivariate analysis revealed both DNA index (DFS: P = 0.002; OS: P = 0.019) and tumor grading (DFS: P = 0.004; OS: P = 0.004) as individual prognostic markers for DFS and OS. The following prognostic subgroups were identified: good (low DNA Index + G1/2 tumor grading), intermediate (low DNA Index + G3 tumor grading or high DNA Index + G1/2 tumor grading), poor (high DNA Index + G3 tumor grading). CONCLUSION: The DNA index/tumor grading constellation may serve as a helpful guide for personalized treatment recommendations for adjuvant therapy of patients with pancreatic adenocarcinoma.

Postoperative bile leakage inhibits liver regeneration after 70% hepatectomy in rats.

BACKGROUND: Postoperative bile leakage is a typical complication in liver surgery. The influence of small bile leakage and concomitant bile peritonitis on the regenerative capacity of the liver remnant has not yet been investigated thoroughly. MATERIAL AND METHODS: Fifty-four rats were randomized in the following groups: Sham operation (Sh), 70% liver resection (LR), and 70% LR with simultaneous induction of a small bile leakage. Animals were euthanized 6, 24, 48, and 96 hr after surgery. Liver regeneration was measured by relative liver weight, mitotic index, Ki-67 immunohistochemistry, and BrdU labeling index. Liver function was evaluated by thromboplastin time, serum bilirubin, and albumin levels as well as indocyanine green plasma disappearance rate (ICG-PDR). The inflammatory response was characterized by assessment of the hepatic transcription of TNF-α, IL-6, and TGF-ß and the serum concentration of IL-1ß. In addition, myeloperoxidase (MPO) activity in liver tissue was measured. Transaminases and histological sections of the liver were used as markers for hepatocellular damage, and the bacterial concentration in different organs was quantified. RESULTS: With a small bile leakage, mitotic index was reduced by 89% ( p < .05) and the number of Ki-67 positive hepatocytes was reduced by 92% ( p < .05) 24 hr after LR. Likewise, the ICG-PDR dropped by 57% ( p < .05). No differences in liver histology were observed between the groups. With bile leakage, the postoperative transcription of cytokines was markedly higher. A bacterial superinfection could be excluded. CONCLUSION: Small intraabdominal bile leakage can suppress liver function and impair the regenerative capacity of the liver.

Safety and efficacy of new integrated bipolar and ultrasonic scissors compared to conventional laparoscopic 5-mm sealing and cutting instruments.

BACKGROUND: Hemostasis is a central issue in laparoscopic surgery. Ultrasonic scissors and bipolar clamps are commonly used, with known advantages with each technique. METHODS: The prototype of new surgical scissors, delivering ultrasonically generated frictional heat energy and bipolar heat energy simultaneously (THUNDERBEAT(®) [TB]), was compared to ultrasonic scissors (Harmonic ACE(®) [HA]) and an advanced bipolar device (LigaSure(®) [LS]) using a pig model. As safety parameters, temperature profiles after single activation and after a defined cut were determined. As efficacy parameters, seal failures and the maximum burst pressure (BP) were measured after in vivo sealing of vessels of various types and diameters (categories 2-4 and 5-7 mm). Moreover, the vertical width of the tissue seal was measured on serial histological slices of selected arteries. The cutting speed was measured during division of isolated arteries and during dissection of a defined length of compound tissue (10 cm of mesentery). Burst-pressure measurement and histological analysis were performed by investigators blinded to the used sealing device. RESULTS: Using the TB, the burst pressure in larger arteries was significantly higher (734 ± 64 mmHg) than that of the HA (453 ± 50 mmHg). No differences in the rate of seal failures were observed. The cutting speed of the TB was significantly higher than that of all other devices. Safety evaluation revealed temperatures below 100 °C in the bipolar device. The maximum temperature of the HA and the TB was significantly higher. No relevant differences were observed between the HA and the TB. CONCLUSIONS: The ultrasonic and bipolar technique of the TB has the potential to surpass the dissection speed of ultrasonic devices with the sealing efficacy of bipolar clamps. However, heat production that is comparable to conventional ultrasonic scissors should be minded for clinical use.

Curcumin attenuates oxidative stress and inflammatory response in the early phase after partial hepatectomy with simultaneous intraabdominal infection in rats.

BACKGROUND: Curcumin is a nontoxic, hepatoprotective antioxidant. It has been shown to efficiently scavenge oxygen free radicals, increase intracellular glutathione concentrations, and prevent lipid peroxidation in rat hepatocytes. Moreover, it has strong anti-inflammatory effects. In the present study we assessed its effect in a model of liver regeneration impaired by bacterial infections. MATERIAL AND METHODS: Male Sprague-Dawley rats underwent sham operation, cecal ligation and puncture (CLP), synchronous partial hepatectomy (PH), and CLP or synchronous PH+CLP with perioperative application of curcumin (100 mg per kg bodyweight per d) 48 h before surgery. Rats were sacrificed 24 h after surgery. Liver function was analyzed by measuring the serum albumin, serum bilirubin, and bile production. The local inflammatory response in the liver tissue was evaluated by quantification of TNF-alpha, IL-6 mRNA, and quantification of IL-1beta by ELISA. In addition, hepatic concentrations of reduced glutathione (GSH) and the oxidized disulfide dimer of glutathione (GSSG) were measured for determination of the redox state. RESULTS: After simultaneous PH+CLP curcumin significantly reduced the expression of TNF-alpha and IL-6 mRNA in the liver tissue. The IL-1beta concentration in the liver was also slightly, but not significantly, lower in the curcumin group. A severe depletion of hepatic glutathione was found in the PH+CLP group. This was reversed by curcumin application, after which the GSH to GSSG ratio increased markedly. The hepatocellular damage, measured by ALT liberation, was significantly lower in the curcumin treated group. The relative liver weight in the curcumin group was significantly higher 24 h after PH+CLP. However, hepatocellular proliferation parameters were not significantly improved by antioxidative treatment with curcumin. Only the Ki-67 index was slightly higher in the curcumin treated PH+CLP group (14+/-3%) than in the untreated PH+CLP group (7%+/-3%). The hepatocyte density was significantly lower in the curcumin group than in the corresponding untreated group. CONCLUSION: In the present model, curcumin revealed significant hepatoprotective effects with stabilization of redox state, reduced liberation of liver enzymes, and attenuated expression of pro-inflammatory cytokines. However, the hepatocellular proliferation was not significantly influenced.

Inhibitory effect of curcumin on early liver regeneration following partial hepatectomy in rats.

BACKGROUND: Curcumin (Cur) is a nontoxic, hepatoprotective antioxidant. Recent investigations have demonstrated a protective effect of curcumin pretreatment during cold ischemia of hepatocytes, but its impact on liver regeneration per se has not been investigated so far. MATERIAL AND METHODS: Male Sprague-Dawley rats (n = 6 per group) underwent sham operation, 70% partial hepatectomy (PH), or PH with curcumin application (100 mg per kg bodyweight per day) starting 48 h before surgery. Rats were sacrificed 24 h after surgery. Liver regeneration was analyzed by measurement of relative liver weight, mitotic-index, bromo-deoxy-uridine (BrdU)-incorporation and Ki-67 expression. RESULTS: The relative liver weight 24 h after surgery was similar in the PH groups with and without curcumin treatment. Also, a comparably high number of Ki-67 positive proliferating hepatocytes was detected in both groups. In contrast, the mitotic index in the untreated PH group (83 +/- 20 mitosis/2000 hepatocytes) was significantly higher than in the curcumin treated group (21 +/- 6). The BrdU labeling index was slightly higher in the curcumin treated group with PH (24% +/- 5%) than in the untreated group (16% +/- 2%). The hepatocyte density as marker of cellular hypertrophy was significantly lower in the curcumin group (474 +/- 23) than in the untreated group (609 +/- 22). CONCLUSIONS: Curcumin inhibits cell cycle progression during normal liver regeneration in rats, predominantly at the level of the G2/M transition point. However, the total liver mass and function was not significantly altered. Nevertheless, application of curcumin in conditions of high physiological cell proliferation should be performed with caution.

Sex-specific differences in the expression levels of estrogen receptor subtypes in colorectal cancer.

BACKGROUND: Altered expression of estrogen receptors alpha and beta (ERalpha and ERbeta) has been hypothesized to play a role in carcinogenesis. However, little is known about the sex-specific differences of ER expression in colorectal cancer (CRC). OBJECTIVE: This study examined ERalpha and ERbeta protein levels in male and female patients with CRC. METHODS: Using Western blot analysis, the intensity of ERalpha and ERbeta protein levels was determined in tumor tissue and in corresponding normal colon mucosa from patients with CRC. RESULTS: All 64 white patients (33 men, mean [SEM] age 64.1 [13.1] years, age range 26-90 years; 31 women, mean age 68.5 [14.5] years, age range 39-91 years [4 were premenopausal at time of surgery]) expressed ERalpha and ERbeta protein in normal colon mucosa, and there were no significant differences between men and women. In tumor tissue, a significantly increased ERalpha protein level was observed in men (P = 0.02 vs normal tissue), whereas in women, the ERalpha level did not differ significantly between tumor and normal tissue. The level of ERbeta protein in CRC was significantly reduced in both men and women, but more so in men (P = 0.04 vs women). Furthermore, in men, the ERbeta level was significantly lower in poorly differentiated tumors than in moderately differentiated tumors (P < 0.03), whereas in women, poor differentiation of the tumor was not associated with a significant decrease of ERbeta level. CONCLUSIONS: Altered levels of ER subtypes resulting in an increased ERalpha:ERbeta ratio were found in patients with CRC. The observation of significantly greater alterations in men than in women supports the hypothesis of sex-specific differences in the pathogenesis of CRC.

Synergistic effect of erythropoietin but not G-CSF in combination with curcumin on impaired liver regeneration in rats.

INTRODUCTION: The effect of erythropoietin (Epo) and granulocyte colony-stimulating factor (G-CSF) alone or in combination with the hepatoprotective antioxidant curcumin (Cur) was evaluated in a model of delayed liver regeneration. MATERIALS AND METHODS: Sprague Dawley rats underwent 70% liver resection with simultaneous cecal ligation and puncture and were randomised to five groups: no treatment, G-CSF (100 microg/kg), Epo (1,000 IU/kg), each alone or in combination with Cur (100mg/kg). Twenty-four hours after surgery, blood and tissue samples were collected. Markers of liver regeneration (liver weight, mitotic index, Ki-67 index), function (bilirubin, bile flow) and hepatocellular damage (liver enzymes, histomorphology) were determined. In addition, cytokine expression and hepatic glutathione concentrations were measured. RESULTS: Liver regeneration was not improved by G-CSF or Epo monotherapy. Epo more effectively increased liver weight and regeneration markers, but the difference was not significant. Whereas liver regeneration was slightly inhibited in the G-CSF plus Cur group, Epo plus Cur significantly improved liver regeneration. This was accompanied by reduced oxidative stress. Liver function and the expression of pro-inflammatory cytokines were comparable in all treatment groups. CONCLUSION: In the present model, Epo, at a relatively low dosage, did not improve liver regeneration. However, the combination of Epo and Cur showed a synergistic effect with highly significant stimulation of liver regeneration.

Quercetin protects human hepatocytes from ethanol-derived oxidative stress by inducing heme oxygenase-1 via the MAPK/Nrf2 pathways.

BACKGROUND/AIMS: Flavonoids, including quercetin, have been reported to have potent hepatoprotective effects, which may be associated with HO-1 induction. However, since the effect and signaling pathway of quercetin involved in HO-1 induction against alcoholic liver damage are still not fully understood, this is the target of the present study. METHODS: Human hepatocytes were incubated with ethanol (100 mM) and quercetin (10-200 microM), and cellular damage and HO-1 activity were measured. Nrf2 expression in cytosolic and nuclear fractions was studied following the incubation with MAPK inhibitor(s). RESULTS: Ethanol exposure resulted in a sustained glutathione depletion, malondialdehyde elevation, and evident release of cellular LDH and AST. Quercetin exerted a dose-dependent protective effect against alcoholic oxidative stress, and increased the EC50 of ethanol by approx. 40%, which is parallel to HO-1 induction with quercetin. Zinc protoporphyrin-9 abrogated the protective effect and dramatically enhanced ethanol cytotoxicity. SB203580 (p38 inhibitor) and especially PD98059 (ERK inhibitor) blocked quercetin-derived HO-1 induction and Nrf2 translocation, and subsequently inhibited the quercetin-related protection. CONCLUSIONS: HO-1 up-regulation by quercetin protected human hepatocytes from ethanol-induced oxidative stress. Among MAPK signaling pathways, p38 and ERK mediated quercetin-derived Nrf2 translocation into nuclei and subsequent induction of HO-1 activity, and the latter showed a stronger mediating effect.

Intraabdominal bacterial infections significantly alter regeneration and function of the liver in a rat model of major hepatectomy.

BACKGROUND: No systematic investigations of interactions of postoperative infections and liver regeneration after resection are available. MATERIALS AND METHODS: Male Sprague-Dawley rats underwent sham operation, 70% partial hepatectomy (PH), cecal ligation and puncture (CLP), or synchronous PH + CLP and were killed at regular intervals. Liver regeneration and function were measured by the mitotic index, Bromo-deoxy-uridine labeling, and Ki-67 as well as bilirubin, albumin, and indocyanine green plasma disappearance rate. The inflammatory response was evaluated by determination of IL-1beta and myeloperoxidase (MPO) activity. Bacterial concentrations in different organs were quantified. RESULTS: Simultaneous CLP + PH resulted in a significantly delayed regeneration kinetic, which was most pronounced at 24 h. This was preceded by hyperinflammation with increased liberation of pro-inflammatory cytokines in the PH + CLP group at 6 h. After 48 h, the pro-inflammatory response declined, and regeneration proceeded also in the PH + CLP group. Liver function was found impaired in both groups; however, it was significantly worse in the PH + CLP group. Especially after 48 h, when regeneration peaked in this group, liver function significantly declined. At 96 h, only minor differences were seen, but the persistently elevated proliferative activity indicated the delay of regeneration after PH + CLP. CONCLUSION: The present analysis shows that infectious conditions delay liver regeneration. Our data suggest a cross-linkage of both conditions via the functional liver capacity. A direct role of microorganisms seems unlikely; however, the inhibitory effect of the pro-inflammatory cytokines may be involved.

Fluorometric measurement of nitrite/nitrate by 2,3-diaminonaphthalene.

We describe a step-by-step protocol for measuring the stable products of the nitric oxide (NO) pathway: nitrite, nitrite plus nitrate and nitrate. This described protocol is easy to apply and is about 50 times more sensitive than the commonly used Griess reaction or commercially available assay kits based on the Griess reaction. It also allows the study of minimal changes in the NO pathway. With this method, it takes about 3 h to analyze the above-mentioned stable products in culture supernatants or in various body fluids, and the method has a sensitive linear range of 0.02-10.0 microM. This restricted linear range suggests that the technique is useful for studying small changes of nitrite and nitrate, rather than for routine diagnostic measurements.

Steroid administration before partial hepatectomy with temporary inflow occlusion does not influence cyclin D1 and Ki-67 related liver regeneration.

BACKGROUND AND AIMS: If temporary inflow occlusion is required during liver resection, the postoperative course might be complicated by ischaemia-reperfusion injury. Steroids protect against ischaemia-reperfusion injury; however, due to its anti-proliferative character concerns exist on its use on liver regeneration after resection. We investigated the effects of methylprednisolone on hepatocyte proliferation after partial hepatectomy with temporary inflow occlusion. PATIENTS AND METHODS: Prior to surgery, one group of Wistar rats received methylprednisolone, while a second group served as non-treated controls. Ischaemia-reperfusion injury was indicated by AST, ALT, and GLDH at 6 h after surgery. Immunohistochemistry tools were used to determine the mitotic index and Ki-67 expression, while cyclin D1 expression characterized the proliferative activity on days 1, 4, 7, and 10. RESULTS: The post-ischaemic liver enzyme release had significantly decreased in the methylprednisolone group, while expression of cyclin D1, percentage of Ki-67-positive cells, and mitotic cell index were comparable in both groups. Similar results were found for bilirubin and albumin and for weight of proliferating liver. CONCLUSION: Although steroid administration significantly reduced ischaemia-reperfusion-associated tissue injury, it has no apparent effects on hepatic regeneration. Thus, steroids could be recommended if a temporary liver ischaemia is required during surgery, in order to reduce complications caused by severe ischaemia-related organ dysfunction.

Probiotics partly reverse increased bacterial translocation after simultaneous liver resection and colonic anastomosis in rats.

BACKGROUND: Bacterial translocation is one important cause of nosocomial infections following major abdominal surgery. Oral administration of probiotics has been proposed to diminish bacterial translocation. MATERIAL AND METHODS: In total 68 rats were divided into seven groups: five of the groups received standard rat chow and were subjected to either sham-operation, 70% liver resection, colonic anastomosis, or a combination of 30 or 70% liver resection with synchronous colonic anastomosis, respectively. In two additional groups with synchronous operation, a combination of four different lactic acid bacteria and four fibers was administered two times daily pre- and postoperatively. Bacterial concentrations in cecum, mesenteric lymph nodes, liver, and spleen were analyzed and blood cultures were taken 48 h after operation. Furthermore, the following parameters were assessed: histological changes in the intestine, intestinal paracellular permeability (Ussing chamber), bursting pressure of the colonic anastomosis, and mitosis rate of the remnant liver. RESULTS: Bacterial translocation was observed in all rats, except in the sham group. Following liver resection, the highest bacterial concentrations were seen in liver and spleen, following colon anastomosis in the mesenteric lymph nodes. Bacterial translocation was increased in the animals with combined operation, in parallel to the extent of liver resection. In rats with colon anastomosis, bacterial concentration in the cecum was also higher than in the sham group. Application of probiotics significantly decreased bacterial concentration in the lymph nodes. In addition, animals with a high cecal concentration of lactobacilli had less translocation than the others. No histological changes were observed in the intestine. Paracellular permeability for ions, but not for the larger molecule lactulose, was increased in the colon in all groups with colon anastomosis. The bursting pressure of the colon anastomosis was not significantly different between the groups. Seventy percent liver resection led to a high rate of hepatocyte mitosis, whereas combination with colon anastomosis impaired the regeneration process. CONCLUSION: Synchronous liver resection and colon anastomosis led to increased bacterial translocation compared to the single operations in the rat model. It is possible to diminish this process by oral administration of probiotics. Bacterial overgrowth in the cecum and impaired hepatic regeneration, but not histological changes or alterations of paracellular permeability, are potential pathogenic mechanisms for translocation in this setting.