Effects of aurantiamide on a rat model of renovascular arterial hypertension.

Asperglaucide (ASP) is an aurantiamide, an effective constituent of purslane (Portulaca oleracea L.), a safe to eat greenery. Effects of ASP on endothelial function, endothelial nitric oxide synthase (eNOS) expression, vascular fluidity, renal and vascular reactive oxygen, and nitrogen species (ROS/RNS) production was examined in the two-kidney one-clip (2 K-1C) rat model of renovascular arterial hypertension. ASP toxicity, dose dependent eNOS gene expression and protein levels were also analyzed in human umbilical vein endothelial cells (HUVEC). The 2 K-1C model of hypertension was created via surgery and mean blood pressure (MBP) was measured by tail-cuff method during four weeks of ASP treatment. Erythrocyte deformability was monitored by rotational ektacytometry, while vascular constrictor and dilator responses were determined in organ baths. eNOS gene expression and protein levels were assessed in thoracic aorta and HUVEC. MBP was significantly decreased in hypertensive rats treated with ASP. Endothelium dependent vascular dilator and constrictor responses were also considerably improved following ASP treatment. There was a notable increase in red blood cell deformability in hypertensive rats treated with ASP as compared to hypertensive rats alone. A significant increase was observed in eNOS gene expression and protein levels in both normotensive and hypertensive rats treated with ASP. Treatment of HUVEC with 3 µM ASP notably increased eNOS mRNA and protein levels. In conclusion, ASP lowered blood pressure, improved endothelium-mediated relaxation, decreased renovascular ROS/RNS production in hypertensive rats. ASP also increased eNOS protein expression in aorta and HUVEC at nontoxic doses. ASP may have future potential as an anti-hypertensive agent.

Organ function, sphingolipid levels and inflammation in tunicamycin induced endoplasmic reticulum stress in male rats.

Disorders of the endoplasmic reticulum (ER) lead to cellular damage but can cause cell death if ER dysfunction is prolonged. We aimed to examine liver/kidney functions, neutral sphingomyelinase (N-SMase) activity, sphingolipid levels, cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2) protein expression in rats under ER stress. ER stress was induced by tunicamycin (TM) and the ER stress inhibitor taurodeoxycholic acid (TUDCA) was injected before induction of ER stress. ER stress was confirmed by increased tissue levels of GRP78. Hematological and biochemical profiles were measured by autoanalyzers while hepatic and renal injury was evaluated via microscopy and histopathological scoring. Tissue levels of C16-C24 sphingomyelins (SM), C16-C24 ceramides (CERs) and sphingosine-1-phosphate (S1P) were determined by LC-MS/MS. Tissue cPLA2 and COX-2 were measured by western blot and activity assays. Tunicamycin treatment caused kidney and liver function test abnormalities, increased hematocrit and hemoglobin levels but decreased white blood cell counts. Histopathological findings showed hepatic necroinflammation and renal tubular damage in rats treated with TM. TUDCA administration attenuated WBC abnormalities and TM- induced hepatic/renal functional impairment in ER stress, as evident by significantly restored serum ALT, AST, creatinine, and total bilirubin levels. A significant increase was observed in N-SMase activity, tissue levels of C16-C24 CERs, cPLA2 and COX-2 expression in liver and kidney tissue under ER stress. TUDCA administration decreased tissue CER levels, cPLA2 and COX-2 expression as well as prostaglandin E2 (PGE2) formation. These results signify that ER stress causes hepatic and renal toxicity as well as CER-induced PGE2 formation in liver and kidney.

Diclofenac induced gastrointestinal and renal toxicity is alleviated by thymoquinone treatment.

The aim of this study was to investigate whether thymoquinone (TQ) could alleviate diclofenac (DCLF)-induced gastrointestinal and renal toxicity in rats. Diclofenac was administered via intramuscular injection twice daily for 5 days and TQ was given by gavage for the same period. Hematological and biochemical profiles were measured with autoanalyzers while reactive oxygen/nitrogen species (ROS/RNS) generation and total antioxidant capacity (TAC) were assayed by standard kits. Tissue injuries were evaluated by microscopy and histopathological scoring. Diclofenac treatment caused kidney and liver function test abnormalities, reduced hematocrit and hemoglobin levels but increased WBC and platelet counts. Histopathological findings showed renal tubular damage, gastrointestinal lesions and increased fibrosis in DCLF treated rats. Thymoquinone administration, along with DCLF treatment, attenuated hematological test abnormalities and DCLF induced renal functional impairment as evident by significantly restored serum creatinine and blood urea nitrogen levels. Similarly, TQ treatment significantly alleviated liver function test abnormalities and decreased tissue injury in the stomach and duodenum. Diclofenac treatment caused increased ROS/RNS formation and decreased TAC in the kidney, stomach and duodenal tissue. Thymoquinone administration increased gastrointestinal and renal TAC in DCLF treated rats. These results indicate that TQ could ameliorate gastrointestinal and renal toxicity induced by high dose DCLF treatment.

ADAM proteases involved in inflammation are differentially altered in patients with gastritis or ulcer.

ADAM metallopeptidase domain (ADAM)9, 10 and 17 have α-secretase activity that regulates ectodomain shedding of factors involved in inflammation, cell proliferation, angiogenesis, and wound healing. The secretase activity of ADAM proteins is known to induce an inflammatory response. However, under certain conditions, a lack of secretase activity may induce inflammation suggesting differential roles of ADAM proteins with secretase activity. To the best of our knowledge, the present study evaluated the changes in α-secretase activity and expression of associated ADAM proteases (ADAM9, 10 and 17) in the gastric mucosa of patients with gastritis and ulcers, for the first time. Gastroduedonal mucosal samples from 42 patients were snap-frozen to determine changes in α-secretase activity. Twenty-four of these patients had gastritis, 9 patients had duedonal ulcers and 9 patients did not have any pathological changes. Paraffin-embedded gastric specimens (n=32) were used for immunohistochemical detection of ADAM9, ADAM10 and ADAM17. α-secretase activity of the gastric mucosa of healthy subjects was significantly higher compared with the uninvolved mucosa of patients with gastritis or ulcer. These results were associated with the immunohistochemical staining results, which demonstrated that ADAM10 expression markedly decreased in glandular epithelial cells and ADAM9 expression was lost in foveolar epithelial cells of gastric mucosa adjacent to ulcer. However, ADAM17 expression was increased in the normal gastric mucosa of patients with bleeding peptic ulcers and in the gastric mucosa adjacent to the ulcer suggesting a counteracting role of ADAM17. Decreased ADAM9 and 10 expression, and an associated decrease in α-secretase activity may predispose to chronic gastritis and ulcer. Further studies are required to determine the possible etiological role of increased ADAM17 expression.

Investigating viability of intestine using spectroscopy: a pilot study.

BACKGROUND: The differentiation of "viable" from "nonviable" bowel remains a challenge in the treatment of acute mesenteric ischemia. In this study, diffuse reflectance spectroscopy (DRS) was used to investigate the viability of bowel tissue after ischemia and reperfusion in an animal model in vivo and in real time. METHODS: A total of 25 females Sprague-Dawley rats were divided into five groups based on different bowel ischemia times. In each study group for four of them, the superior mesenteric artery was occluded using a vascular clamp for a different period (i.e., 30, 45, 60, and 90 min; n = 5 for each group). Intestinal reperfusion was accomplished by releasing the clamps after the given occlusion period for each group. Spectra were acquired by gently touching the optical fiber probe to the bowel tissue before the induce ischemia, at the end of the induced ischemia, and after the reperfusion. The data acquired before the ischemia were used as a control group. Without occluding the superior mesenteric artery, the spectra were acquired on the bowel with the same time intervals of the experiments were used as a sham group (n = 5). Subsequently, the same bowel segments were sent for histopathologic examination. RESULTS: Based on the correlation between the spectra acquired from the bowel segments and the results from the histopathologic investigation, DRS is able to differentiate the histopathologic grading that appears when the Chiu/Park score ≥5 (i.e., high-level ischemic injury) than Chiu/Park score <5. Eight out of nine low-level ischemic injury tissue samples were correctly defined using the spectroscopic classification system. All eleven high-level ischemic injury tissues that were histopathologically assigned grade 5 and above were correctly defined using the spectroscopic classification system in the ischemia-reperfusion groups. CONCLUSIONS: DRS could potentially be used intraoperatively for the assessment of bowel viability in real time. These preliminary findings suggest that DRS has the potential to reduce unnecessary resection of viable tissue or insufficient resection of nonviable tissues may reduce the mortality and morbidity rates of intestinal ischemia-reperfusion as acute mesenteric ischemia.

Pentoxifylline therapy attenuates intestinal injury in rat pups with hypoxic ischemic encephalopathy.

AIM: The aim of this study was to evaluate the effects of post-ischemic pentoxifylline (PTX) therapy on the gut injury in neonatal rat model of hypoxic ischemic encephalopathy (HIE). METHODS: Seven-day-old Wistar rat pups (n = 24) of either sex, delivered spontaneously, were used in this experimental study. Seven-day-old rat pups were randomly divided into three groups. Control group (n = 8): after median neck incision was made, neither ligation nor hypoxia was performed. Hypoxia group (n = 8): 0.5 ml of saline was injected intraperitoneally immediately after hypoxia. Pentoxifylline + Hypoxia group (n = 8): the rat pups were administered intraperitoneally 60 mg/kg of PTX immediately after hypoxia. Eight rats from all groups were sacrificed 24 h after drug administration. The ischemic injury was scored at least six sections at three different levels using histopathologic injury scores (HIS). RESULTS: Induction of hypoxia/reoxygenation (H/R) increased mean HIS levels significantly at 24 h in the intestinal tissue samples in the hypoxia group as compared with the control group. Induction of H/R decreased means HIS levels significantly at 24 h in the intestinal tissue samples in the PTX + hypoxia group as compared with the hypoxia group. CONCLUSION: In this experimental study, PTX significantly attenuated H/R-induced intestinal injury in neonatal rat model of HIE. These findings indicate that PTX can reduce the intestinal H/R injury.

Differential changes in Substance P, VIP as well as neprilysin levels in patients with gastritis or ulcer.

The protective effect of capsaicin-sensitive sensory nerve (CSSN) activation was recently demonstrated in human gastric mucosa. We here examined changes in neuropeptides, specifically Substance P (SP), calcitonin-gene related peptide (CGRP) and vasoactive intestinal peptide (VIP) in patients with chronic gastritis or ulcer. Furthermore changes in neprilysin levels, which hydrolyse these neuropeptides, were determined. Gastric biopsies were obtained from both lesion- and normal-appearing mucosa of 57 patients. The presence of H. pylori infection was verified with rapid urease assay. Neuronal and non-neuronal levels of SP, VIP, CGRP and neprilysin activity were determined in freshly frozen biopsies. Immunohistochemical localization of neprilysin was performed in 30 paraffin embedded specimens. We here found that neuronal SP levels decreased significantly in normally appearing mucosa of patients with gastritis while levels of non-neuronal SP increased in diseased areas of gastritis and ulcer. The presence of H. pylori led to further decreases of SP levels. The content of VIP in both disease-involved and uninvolved mucosa, and expression of neprilysin, markedly decreased in patients with gastritis or ulcer. Since VIP, as well as SP fragments, formed following hydrolysis with neprilysin is recognized to have gastroprotective effects, decreased levels of VIP, SP and neprilysin may predispose to cellular damage.

The repair of a large duodenal defect by a pedicled gastric seromuscular flap.

PURPOSE: The aim was to evaluate the efficacy of a pedicled gastric seromuscular flap for the closure of a large duodenal defect. METHODS: A large defect of the second duodenal part was repaired by a gastric seromuscular flap. Of 35 rats, 9 rats were euthanized at 2 weeks, 12 rats at 2 months, and 14 rats at 4 months for the histopathological evaluation of the patch and normal duodenum (control) adjacent to the patch. RESULTS: All rats survived. The patch was completely covered by neomucosa in all of the 4-month rats, and in 8 of the 12 2-month rats. The villous height of the neomucosa was significantly higher in the 4-month rats in comparison to the other rats (P < 0.001). However, a normal duodenum had higher villi than in that of the patches (P < 0.001). The crypt density of the neomucosa was significantly increased in the 4-month rats in comparison to the 2-week and the 2-month rats (P < 0.001 and P < 0.05 group, respectively). The crypt density was higher in the controls than in the neomucosa covered patch of the 2-week and the 2-month rats (P < 0.001 and P < 0.05, respectively). The crypt depth of the neomucosa increased significantly in the 4-month rats and in the controls versus the 2-week rats (P < 0.05). CONCLUSION: The new mucosal barrier overlaying the patch appeared to be satisfactory. This technique, which has not been described previously, is likely to be useful for the repair of the large duodenal defect.

High TRAIL death receptor 4 and decoy receptor 2 expression correlates with significant cell death in pancreatic ductal adenocarcinoma patients.

OBJECTIVES: The importance of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL receptor expression in pancreatic carcinoma development is not known. To reveal the putative connection of TRAIL and TRAIL receptor expression profile to this process, we analyzed and compared the expression profile of TRAIL and its receptors in pancreatic tissues of both noncancer patients and patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: Thirty-one noncancer patients and 34 PDAC patients were included in the study. TRAIL and TRAIL receptor expression profiles were determined by immunohistochemistry. Annexin V binding revealed the apoptotic index in pancreas. Lastly, the tumor grade, tumor stage, tumor diameter, perineural invasion, and number of lymph node metastasis were used for comparison purposes. RESULTS: TRAIL decoy receptor 2 (DcR2) and death receptor 4 expression were up-regulated in PDAC patients compared with noncancer patients, and the ductal cells of PDAC patients displayed significant levels of apoptosis. In addition, acinar cells from PDAC patients had higher DcR2 expression but lower death receptor 4 expression. Increased DcR2 expression was also observed in Langerhans islets of PDAC patients. CONCLUSIONS: Differential alteration of TRAIL and TRAIL receptor expression profiles in PDAC patients suggest that the TRAIL/TRAIL receptor system may play a pivotal role during pancreatic carcinoma development.

High levels of endogenous tumor necrosis factor-related apoptosis-inducing ligand expression correlate with increased cell death in human pancreas.

OBJECTIVES: Type 1 diabetes (T1D) has been characterized by the T cell-mediated destruction of pancreatic beta cells. Although various members of the tumor necrosis factor (TNF) family, such as Fas ligand or TNF, have recently been implicated in the development of T1D, the lack of TNF-related apoptosis-inducing ligand (TRAIL) expression or function facilitates the onset of T1D. Thus, the goal of the present study was to investigate the expression profiles of TRAIL and its receptors in human pancreas. METHODS: Pancreata of 31 patients were analyzed by immunohistochemistry using antibodies developed against TRAIL and its receptors. Apoptosis was confirmed by Annexin V-fluorescein isothiocyanate binding and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assays. RESULTS: Acinar cells displayed high levels of TRAIL and death receptor 4, but only low levels of death receptor 5. In contrast, only TRAIL and TRAIL decoy receptors (DcR1, DcR2) were detected in ductal cells. Similarly, Langerhans islets expressed only TRAIL and TRAIL decoy receptor. High levels of TRAIL expression in pancreas correlated with increased number of apoptotic cells. CONCLUSIONS: Although the expression of TRAIL decoy receptors might be necessary for defense from TRAIL-induced apoptosis, high levels of TRAIL may provide protection for Langerhans islets from the immunological attack of cytotoxic T cells.

Laparoscopic appendectomy without clip or ligature. An experimental study.

BACKGROUND: We aimed to test the efficacy and safety of closure of the appendeceal stump with only laparoscopic bipolar electrocautery in rats. METHODS: In this study, 40 female Wistar-Albino rats were used. In group I (n = 10), appendix vermiformis, approximately 1 cm in width, was completely ligated with 3/0 silk suture close to cecum, and removed. In group II (n = 20) and group III (n = 10), the appendeceal stump was coagulated by bipolar cautery. The coagulation of 70 mA took 10 s, and was repeated one more time. The stump was divided, and checked to ensure complete occlusion. Groups I and II underwent relaparotomy at 15 days, cecum was taken out, and the burst pressure of the stump was measured. Group III did not undergo relaparotomy; the burst pressure was measured during the first laparotomy. RESULTS: All rats survived. At relaparotomy, no intra-abdominal complications were detected, including intestinal obstruction, abscess, and leakage. Omentum and fatty tissue of uterus was adhered to the appendix stump in group I, but only fatty tissue of uterus was adhered on the stump in group II. Although the intracecal pressure reached 30 cmH(2)O, at which pressure the cecum was highly stretched, ligated (group I) or coagulated (group II) stumps did not burst or opened. In group III, the burst or opening pressure of the stump (11.2 +/- 2.7 cmH(2)O) was significantly lower than in groups I and II (p < 0.001). Of group II rats, 80% had complete epithelial regeneration at the coagulated stump sites in contrast to ligated rats (p < 0.001) with severe inflammatory changes, abscess, and necrosis. CONCLUSIONS: At late course, coagulated stumps did not allow the leakage or burst, unlike ligated stumps. However, coagulation of the stump seemed to contribute more to epithelial healing. This experimental model suggests that the closure of the stump with only bipolar coagulation was a safe and feasible method.

Does noncomplicated acute appendicitis cause bacterial translocation?

It is unknown whether noncomplicated acute appendicitis cause bacterial translocation. In this study, we aimed to test development of the bacterial translocation in the patients who were operated for acute appendicitis. In this prospective study, 10 control patients who underwent elective operations because of other reasons, and 18 patients with noncomplicated acute appendicitis were evaluated. No patients took prophylactic antibiotic. After laparotomy, samples were obtained from peritoneal leaf just close to wound edge, and peritoneal swab culture from right paracolic region. Before appendectomy, a mesenteric lymph node (MLN) adjacent to the terminal ileum was taken out. Tissue samples were placed in a sterile container for microbiological analysis, and 10% formalin for histopathological analysis. Control samples had no bacterial translocation. Only 3 of 18 (16.6%) patients with appendicitis included bacterial translocation to MLN. There was no significant difference between both groups. No bacterial colonization was detected in the peritoneal tissue and peritoneal swab culture. Peritoneal tissue injury score was 2 +/- 1.4 in controls and 2.8 +/- 1.7 in the patients with appendicitis (P>0.05). MLN injury score was 2.5 +/- 1.3 in controls and 3.2 +/- 1.5 in the patients with appendicitis (P>0.05). No patient developed wound and systemic infection. No significant bacterial translocation frequency and tissue injury score was identified in the children with noncomplicated acute appendicitis. This result suggests that antibiotic prophylaxis may be unnecessary in such patients.

Omentum pediculed musculo-peritoneal flap (OPMP) for growing intestinal neomucosa in a rat model.

BACKGROUND/PURPOSE: To construct a musculo-peritoneal flap adequately vascularised through attached omentum and to evaluate whether this flap is functional for growing new intestinal mucosa. METHODS: 24 rats underwent two surgical procedures. 1) A musculo-peritoneal flap was prepared, omentum was fixed onto the muscular side and a silicon graft was placed on the peritoneal side. 2) After one month the so constructed OPMP flap was mobilised and attached to the jejunum. Animals were harvested at 2, 4 and 8 months for macroscopic and microscopic evaluation. RESULTS: One rat died at 7 months due to intestinal volvulus. In the remainder the circumference of the patched intestine significantly increased compared to that of normal intestine (p<0.001). The flaps were markedly contracted in the 4 month rats (p<0.01). All flaps were completely covered by neomucosa except in the 2 month rats. Crypt depth and crypt density of the neomucosa was significantly lower in 2 month rats compared to other groups (p<0.05). Similarly neomucosal villous height was markedly low in the 2 month rats (p<0.01). CONCLUSIONS: To date, we have found no reports of small bowel patching using this technique. In this context, use of OPMP flap is likely to be useful for increasing intestinal surface area.

Androgen receptor levels in the sebaceous glands of papulopustular lesions from patients with Behçet's disease and acne vulgaris: a controlled study.

AIMS: Male sex and a younger age of onset in Behçet's disease (BD) are associated with more severe disease. Eye diseases, folliculitis and thrombophlebitis are also more common among males. The disease rarely developes before puberty or after the age of 50. Clinical manifestations of BD, with the exception of eye symptoms, tend to get better with time. Clinical and histological pattern of the papulopustular lesions (PPL), the most common type of skin lesions in BD and the difference from ordinary acne is contradictory. Therefore, BD may be androgen driven to some degree. In the present study, using acne vulgaris (AV) as a prototype of an androgen driven disease, we investigated such a possibility between PPL and androgen receptor (AR) levels in the sebaceous glands (SG) of PPL from patients with BD and AV. METHODS: Eighteen patients with BD (10 women, 8 men; mean +/- SD age, 34 +/- 11 years), 16 patients with AV (10 women, 6 men; mean +/- SD age, 25 +/- 6.2 years), and 10 healthy volunteers (5 women, 5 men; mean +/- SD age, 36 +/- 12 years), were included in this controlled study with blind histological study. Biopsies of the PPL and adjacent (approximately 2 cm distant) normal-appearing skin in patients with BD and AV, and of the normal-appearing skin from healthy volunteers were performed from the interscapular region, thus minimizing differences in androgenic activity. Follicle-based acneiform lesions were excluded for biopsies from patients with BD. Nuclear AR levels of SG were studied by an immunohistochemical technique, using monoclonal antibodies. The percentage of positively staining cells were recorded as the androgen receptor index (ARI). MAIN RESULTS: The lesional skin of the patients with BD showed a higher ARI than non-lesional skin of those patients (34.3% and 23.82%), or the healthy controls (21.37%). AR levels in the lesional skin of active patients was also higher than inactive patients (39.82 and 27.12%), but the differences were not statistically significant. ARI values in the lesional skin (78.58%) of acne patients were found to be significantly higher as compared with non-lesional skin (38.88%) and other groups (p<0.01). CONCLUSION: Our findings indicate that androgens may play a role at least in the formation of PPL and disease activity in patients with BD. Androgen metabolism takes place peripherally in the SG, being indipendent of the central endocrine regulation. Target organ sensitivity and excess number of nuclear AR seem to be responsible for the development of AV.

Hepatic epithelioid hemangioendothelioma treated with orthotopic liver transplantation: a case report.

Hepatic epithelioid hemangiendothelioma is a rare vascular tumor. The clinical course is unpredictable and different treatment modalities are offered depending on the patients condition. Orthotopic liver tranplantation is the choice of treatment in diffuse cases without metastases. A 32 year old woman was admitted to hospital with multiple mass lesions diagnosed by ultrasonography of the liver. Physical examination was normal except for a painless hepatomegaly, and her biochemical tests were within the normal range. Computed tomographic scanning showed the presence of multiple lesions in both lobes, some of which were accompanied by a small degree of calcification. Although these findings were suggestive of hepatic epithelioid hemangioendothelioma, ultrasonographic guided fine needle aspiration biopsy failed to diagnose the exact nature of the lesions. The diagnosis of hepatic epithelioid hemangioendothelioma was confirmed by diagnostic laparotomy and immunohistochemical examination of the specimen by FVIII-RAg, CD34 and CD 31 markers. The patient was treated by orthotopic liver transplantation and had no evidence of tumor 18 months after transplantation. The problems in differential diagnosis and treatment options are discussed in this report of the first case of this rare tumor, treated by orthotopic liver transplantation in Turkey.