Mechanisms of ischemic neuronal and vascular injury remain obscure. Here we test the hypothesis that thrombin, a blood-borne coagulation factor, contributes to neurovascular injury during acute focal ischemia. Stroke was induced in adult Sprague Dawley rats by occluding the middle cerebral artery. Intra-arterial thrombin infusion during ischemia significantly increased vascular disruption and cellular injury. Intravenous infusion of argatroban, a direct thrombin inhibitor, alleviated neurovascular injury. Immunostaining showed thrombin on neurons in the ischemic core. Using an activatable cell penetrating peptide engineered to detect thrombin activity, we discovered that thrombin proteolytic activity was specifically associated with neuronal damage during ischemia. Protease activated receptor-1, the presumptive thrombin receptor, appeared to mediate ischemic neurovascular injury. Furthermore, rats receiving thrombin during ischemia showed cognitive deficit whereas rats receiving argatroban retained intact learning and memory. These results suggest a potential role for thrombin contributing to neurovascular injury and several potential avenues for neuroprotection.

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Although in vitro and in vivo experiments have suggested that dietary fiber might have beneficial effects on health, results on the association between fiber intake and all-cause mortality in epidemiologic studies have been inconsistent. Therefore, we conducted a meta-analysis of prospective cohort studies to quantitatively assess this association. Pertinent studies were identified by searching articles in PubMed and Web of Knowledge through May 2014 and reviewing the reference lists of the retrieved articles. Study-specific risk estimates were combined using random-effects models. Seventeen prospective studies (1997-2014) that had a total of 67,260 deaths and 982,411 cohort members were included. When comparing persons with dietary fiber intakes in the top tertile with persons whose intakes were in the bottom tertile, we found a statistically significant inverse association between fiber intake and all-cause mortality, with an overall relative risk of 0.84 (95% confidence interval: 0.80, 0.87; I(2) = 41.2%). There was a 10% reduction in risk for per each 10-g/day increase in fiber intake (relative risk = 0.90; 95% confidence interval: 0.86, 0.94; I(2) = 77.2%). The combined estimate was robust across subgroup and sensitivity analyses. No publication bias was detected. A higher dietary fiber intake was associated with a reduced risk of death. These findings suggest that fiber intake may offer a potential public health benefit in reducing all-cause mortality.

Purpose The aim of the study was to characterize enterocolitis in the Ednrb-null (Ednrb−/−) mouse with aganglionosis of the colon and to develop and validate a semiquantitative histopathologic grading system to assess enterocolitis. Methods We isolated colon and ileal specimens of Ednrb−/− and control mice (Ednrb+/+) and performed histochemical staining (H&E) on tissue sections. After establishing inflammation grading criteria, 2 blinded pathologists independently assessed the severity and depth of inflammation of proximal colon segments on 2 separate occasions. Interclass correlations (ICCs) and coefficient of variation (CV) were calculated to determine interrater and intrarater agreement. We then prospectively applied the enterocolitis grading system to Ednrb−/− mice that became clinically ill. A cohort of Ednrb−/− mice were observed until they developed clinical illness, at which time they were euthanized and had multiple organ homogenates cultured for bacteria, and colon and small bowel were histopathologically graded for enterocolitis. Spearman’s rank correlations comparing enterocolitis scores with level of bacteremia were performed. Results Intra- and interrater ICCs of the histologic scoring system were satisfactory (0.61 and 0.94, respectively), as were intra- and interrater CVs (18% and 9%, respectively). Of the Ednrb−/− mice, 65% developed bacteremia. Those with bacteremia had significantly higher enterocolitis scores than those without bacteremia (P < .01). Ednrb−/− mice that developed bacteremia showed a strong positive correlation between total enterocolitis scores and number of bacterial colony forming units in peritoneal lavage, liver, kidney, and aerobic spleen. Conclusions The Ednrb−/− mouse with aganglionosis develops enterocolitis and has features similar to Hirschsprung-associated enterocolitis in humans. Our grading system is a reliable way to assess enterocolitis. By performing microsurgical pull-through, we can now perform controlled, hypothesis-driven, mechanistic studies to evaluate etiologic factors affecting enterocolitis in the Ednrb−/− mouse.

Protease activated receptors (PARs) populate neurons and astrocytes in the brain. The serine protease thrombin, which activates PAR-1 during the first hours after stroke, appears to be associated with the cytotoxicity. Thrombin antagonists and PAR-1 inhibitors have been correlated with reduced cell death and behavioral protection after stroke, but no data yet supports a mechanistic link between PAR-1 action and benefit. We sought to establish the essential role of PAR-1 in mediating ischemic damage. Using a short hairpin mRNA packaged with green fluorescent protein in a lentivirus vector, we knocked downPAR-1 in the medial caudate nucleus prior to rat middle cerebral artery occlusion (MCAo) and in rat neurons prior to oxygen-glucose deprivation. We also compared aged PAR-1 knockout mice with aged PAR-3, PAR-4 mice and young wild-type mice in a standard MCAo model. Silencing PAR-1 significantly reduced neurological deficits, reduced endothelial barrier leakage, and decreased neuronal degeneration in vivo during MCAo. PAR-1 knock-down in the ischemic medial caudate allowed cells to survive the ischemic injury; infected cells were negative for TUNEL and c-Fos injury markers. Primary cultured neurons infected with PAR-1 shRNA showed increased neuroprotection during hypoxic/aglycemic conditions with or without added thrombin. The aged PAR-1 knockout mice showed decreased infarction and vascular disruption compared to aged controls or young wild types. We demonstrated an essential role for PAR-1 during ischemia. Silencing or removing PAR-1 significantly protected neurons and astrocytes. Further development of agents that act at PAR-1or its downstream pathways could yield powerful stroke therapy.

BackgroundEpidemiologic studies have evaluated the association between cruciferous vegetables(CV) intake and the risk of renal cell carcinoma(RCC); however, the existing results are controversial. The aim of this meta-analysis was to investigate the association between CV intake and RCC risk.MethodsA literature search was carried out using PUBMED and EMBASE database between January 1966 and March 2013. Fixed-effect and random-effect models were used to estimate summary relative risks (RR) and the corresponding 95% confidence intervals (CIs). Potential sources of heterogeneity were detected by meta-regression. Subgroup analyses, sensitivity analysis and cumulative meta-analysis were also performed.ResultsA total of 12 studies (six cohorts, six case–control) contributed to the analysis, involving 1,228,518 participants and 5,773 RCC cases. When all studies were pooled, we observed a significantly inverse association between CV intake and RCC risk (RR = 0.81, 95% CI [0.72, 0.91]). This association was also significant when analyses were restricted to six high-quality studies (RR = 0.89, 95% CI [0.82, 0.98]). In subgroup analyses, CV intake was significantly associated with reduced RCC risk among studies conducted in America (RR = 0.77, 95%CI [0.70, 0.86]); however, CV intake had no significant association with RCC risk among studies conducted in Europe (RR = 0.87, 95%CI [0.71, 1.07]). Furthermore, sensitivity analysis confirmed the stability of results.ConclusionsThe findings of this meta-analysis suggested that high intake of CV was inversely associated with RCC risk among Americans. More studies, especially high quality cohort studies with larger sample size, well controlled confounding factors are warranted to confirm this association.

Background Hirschsprung disease (HD) results from aganglionosis of the colon, is linked to acute and chronic enterocolitis (known as Hirschsprung-associated enterocolitis) despite successful corrective surgery, and can lead to bacteremia and even death. The genetic and molecular mechanisms underlying these disorders are largely unknown. Methods We developed a microsurgical corrective pull-through procedure in mice, and applied that to Ednrb−/− mice, which manifest aganglionic megacolon that is very similar to HD. Wild-type littermates (Ednrb+/+) also underwent identical surgery. At prespecified time points postoperatively, mice were sacrificed, and histopathologic analyses of intestinal inflammation were performed. Mice of both genotypes were sacrificed after the postoperative recovery period to determine if corrective surgery itself caused inflammation. Stooling patterns were assessed as well to determine if intestinal function normalized after surgery. Results: There was no difference in histopathological enterocolitis scores after recovery from surgery. Stooling patterns in Ednrb−/− and Ednrb+/+ mice were similar postoperatively, suggesting normalization of intestinal function. However, with time, approximately 40% of Ednrb−/− mice developed clinical illness consistent with enterocolitis. No control (Ednrb+/+) mice developed clinical enterocolitis. Histopathological enterocolitis scores in the 40% of Ednrb−/− mice that developed clinical enterocolitis postoperatively were significantly worse than those of healthy postoperative Ednrb−/− mice. In contrast, none of the Ednrb+/+ control mice exhibited postoperative long-term inflammation. Conclusions Microsurgical pull-through operation in Ednrb−/− mice produces a mouse model that closely resembles key features of Hirschsprung-associated enterocolitis, enabling controlled study of genetic and molecular mechanisms in Ednrb−/− mice and other genotypes that produce similar phenotypes.

Background and Purpose We showed previously robust neuroprotection with the thrombin inhibitor argatroban, and now sought additional support for its neuroprotective potential. Methods We used behavioral and histological endpoints; rigorously blinded the study groups; extended the treatment window to 3 hours following ischemia onset; and used 2 separate models. First, 2-h filament MCAo in 64 male Sprague-Dawley rats was followed by learning and memory testing and quantitative histomporphometry. Randomly assigned treatment was 0.45mg argatroban, saline, or 0.4U thrombin. Second, we used the quantal bioassay (n=272) after 2-hour MCAo to detect the longest time delay after which therapy failed. Results Argatroban powerfully and significantly reversed learning and memory deficits due to focal ischemia compared to saline or thrombin (p<0.03, ANOVA). Argatroban was significantly (p<0.05, t-test with Bonferroni) protective when given immediately or after 1, 2, 3 but not 4 hours delay. Conclusions We obtained supportive evidence for argatroban protection of the neurovascular unit using behavioral and histological measurements at realistic therapeutic time windows.