Nox2 Oxidase Activity Accounts for the Oxidative Stress and Vasomotor Dysfunction in Mouse Cerebral Arteries following Ischemic Stroke

Background and Purpose: Post-ischemic oxidative stress and vasomotor dysfunction in cerebral arteries may increase the likelihood of cognitive impairment and secondary stroke. However, the underlying mechanisms of post-stroke vascular abnormalities, as distinct from those causing primary brain injury, are poorly understood. We tested whether augmented superoxide-dependent dysfunction occurs in the mouse cerebral circulation following ischemia-reperfusion, and evaluated the role of Nox2 oxidase. Methods: Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and Nox2-deficient (Nox2 -/-) mice by middle cerebral artery occlusion (MCAO; 0.5 h), followed by reperfusion (23.5 h). Superoxide production by MCA was measured by L-012-enhanced chemiluminescence. Nitric oxide (NO) function was assessed in cannulated and pressurized MCA via the vasoconstrictor response to N ω-nitro-L-arginine methyl ester (L-NAME; 100 μmol/L). Expression of Nox2, the nitration marker 3-nitrotyrosine, and leukocyte marker CD45 was assessed in cerebral arteries by Western blotting. Results: Following ischemia-reperfusion, superoxide production was markedly increased in the MCA of WT, but not Nox2 -/- mice. In WT mice, L-NAME-induced constriction was reduced by ~50% in ischemic MCA, whereas ischemia-reperfusion had no effect on responses to L-NAME in vessels from Nox2 -/- mice. In ischemic MCA from WT mice, expression of Nox2 and 3-nitrotyrosine were ~1.4-fold higher than in the contralateral MCA, or in ischemic or contralateral vessels from Nox2 -/- mice. Vascular CD45 levels were unchanged by ischemia-reperfusion. Conclusions: Excessive superoxide production, impaired NO function and nitrosative stress occur in mouse cerebral arteries after ischemia-reperfusion. These abnormalities appear to be exclusively due to increased activity of vascular Nox2 oxidase

Pathophysiology, treatment, and animal and cellular models of human ischemic stroke

Stroke is the world's second leading cause of mortality, with a high incidence of severe morbidity in surviving victims. There are currently relatively few treatment options available to minimize tissue death following a stroke. As such, there is a pressing need to explore, at a molecular, cellular, tissue, and whole body level, the mechanisms leading to damage and death of CNS tissue following an ischemic brain event. This review explores the etiology and pathogenesis of ischemic stroke, and provides a general model of such. The pathophysiology of cerebral ischemic injury is explained, and experimental animal models of global and focal ischemic stroke, and in vitro cellular stroke models, are described in detail along with experimental strategies to analyze the injuries. In particular, the technical aspects of these stroke models are assessed and critically evaluated, along with detailed descriptions of the current best-practice murine models of ischemic stroke. Finally, we review preclinical studies using different strategies in experimental models, followed by an evaluation of results of recent, and failed attempts of neuroprotection in human clinical trials. We also explore new and emerging approaches for the prevention and treatment of stroke. In this regard, we note that single-target drug therapies for stroke therapy, have thus far universally failed in clinical trials. The need to investigate new targets for stroke treatments, which have pleiotropic therapeutic effects in the brain, is explored as an alternate strategy, and some such possible targets are elaborated. Developing therapeutic treatments for ischemic stroke is an intrinsically difficult endeavour. The heterogeneity of the causes, the anatomical complexity of the brain, and the practicalities of the victim receiving both timely and effective treatment, conspire against developing effective drug therapies. This should in no way be a disincentive to research, but instead, a clarion call to intensify efforts to ameliorate suffering and death from this common health catastrophe. This review aims to summarize both the present experimental and clinical state-of-the art, and to guide future research directions

Fasting triglycerides are positively associated with cardiovascular mortality risk in people with diabetes

Aims: We investigated the association of fasting triglycerides with cardiovascular disease (CVD) mortality. Methods and results: This cohort study included US adults from the National Health and Nutrition Examination Surveys from 1988 to 2014. CVD mortality outcomes were ascertained by linkage to the National Death Index records. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of triglycerides for CVD mortality. The cohort included 26 570 adult participants, among which 3978 had diabetes. People with higher triglycerides had a higher prevalence of diabetes at baseline. The cohort was followed up for a mean of 12.0 years with 1492 CVD deaths recorded. A 1-natural-log-unit higher triglyceride was associated with a 30% higher multivariate-adjusted risk of CVD mortality in participants with diabetes (HR, 1.30; 95% CI, 1.08–1.56) but not in those without diabetes (HR, 0.95; 95% CI, 0.83–1.07). In participants with diabetes, people with high triglycerides (200–499 mg/dL) had a 44% (HR, 1.44; 95% CI, 1.12–1.85) higher multivariate-adjusted risk of CVD mortality compared with those with normal triglycerides (<150 mg/dL). The findings remained significant when diabetes was defined by fasting glucose levels alone, or after further adjustment for the use of lipid-lowering medications, or after the exclusion of those who took lipid-lowering medications. Conclusion: This study demonstrates that fasting triglycerides of ≥200 mg/dL are associated with an increased risk of CVD mortality in patients with diabetes but not in those without diabetes. Future clinical trials of new treatments to lower triglycerides should focus on patients with diabetes

Dietary fatty acids and mortality risk from heart disease in US adults: an analysis based on NHANES

We investigated the association of dietary intake of major types of fatty acids with heart disease mortality in a general adult cohort with or without a prior diagnosis of myocardial infarction (MI). This cohort study included US adults who attended the National Health and Nutrition Examination Surveys from 1988 to 2014. Heart disease mortality was ascertained by linkage to the National Death Index records through 31 December 2015. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of fatty acid intake for heart disease mortality. This cohort included 45,820 adults among which 1,541 had a prior diagnosis of MI. Participants were followed up for 532,722 person-years (mean follow-up, 11.6 years), with 2,313 deaths recorded from heart disease being recorded. Intake of saturated (SFAs) and monounsaturated fatty acids (MUFAs) was associated with heart disease mortality after adjustment for all the tested confounders. In contrast, a 5% higher calorie intake from polyunsaturated fatty acids (PUFAs) was associated with a 9% (HR, 0.91; 95% CI 0.83–1.00; P = 0.048) lower multivariate-adjusted risk of heart disease mortality. Sub-analyses showed that this inverse association was present in those without a prior diagnosis of MI (HR,0.89; 95% CI 0.80–0.99) but not in those with the condition (HR, 0.94; 95% CI 0.75–1.16). The lack of association in the MI group could be due to a small sample size or severity and procedural complications (e.g., stenting and medication adherence) of the disease. Higher PUFA intake was associated with a favourable lipid profile. However, further adjustment for plasma lipids did not materially change the inverse association between PUFAs and heart disease mortality. Higher intake of PUFAs, but not SFAs and MUFAs, was associated with a lower adjusted risk of heart disease mortality in a large population of US adults supporting the need to increase dietary PUFA intake in the general public

Proteasome inhibition reduces plasma cell and antibody secretion, but not angiotensin II-induced hypertension

IntroductionDepletion of mature B cells affords protection against experimental hypertension. However, whether B cell-mediated hypertension is dependent on differentiation into antibody-secreting cells (ASCs) remains unclear. Using the proteasome inhibitor, bortezomib, the present study tested the effect of ASC reduction on angiotensin II-induced hypertension.MethodsMale C57BL6/J mice were infused with angiotensin II (0.7 mg/kg/day; s.c.) for 28 days via osmotic minipump to induce hypertension. Normotensive control mice received saline infusion. Bortezomib (750 μg/kg) or vehicle (0.1% DMSO) was administered (i.v.) 3 days prior to minipump implantation, and twice weekly thereafter. Systolic blood pressure was measured weekly using tail-cuff plethysmography. Spleen and bone marrow B1 (CD19+B220−), B2 (B220+CD19+) and ASCs (CD138hiSca-1+Blimp-1+) were enumerated by flow cytometry. Serum immunoglobulins were quantified using a bead-based immunoassay.ResultsBortezomib treatment reduced splenic ASCs by ∼68% and ∼64% compared to vehicle treatment in normotensive (2.00 ± 0.30 vs. 0.64 ± 0.15 × 105 cells; n = 10–11) and hypertensive mice (0.52 ± 0.11 vs. 0.14 ± 0.02 × 105 cells; n = 9–11), respectively. Bone marrow ASCs were also reduced by bortezomib in both normotensive (4.75 ± 1.53 vs. 1.71 ± 0.41 × 103 cells; n = 9–11) and hypertensive mice (4.12 ± 0.82 vs. 0.89 ± 0.18 × 103 cells; n = 9–11). Consistent with ASC reductions, bortezomib reduced serum IgM and IgG2a in all mice. Despite these reductions in ASCs and antibody levels, bortezomib did not affect angiotensin II-induced hypertension over 28 days (vehicle: 182 ± 4 mmHg vs. bortezomib: 177 ± 7 mmHg; n = 9–11).ConclusionReductions in ASCs and circulating IgG2a and IgM did not ameliorate experimental hypertension, suggesting other immunoglobulin isotypes or B cell effector functions may promote angiotensin II-induced hypertension

Anakinra reduces blood pressure and renal fibrosis in one kidney/DOCA/salt-induced hypertension

OBJECTIVE: To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal inflammation, structural damage and blood pressure (BP) in mice with established hypertension. METHODS: Hypertension was induced in male mice by uninephrectomy, deoxycorticosterone acetate (2.4mg/d,s.c.) and replacement of drinking water with saline (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water. 10days post-surgery, mice commenced treatment with anakinra (75mg/kg/d, i.p.) or vehicle (0.9% saline, i.p.) for 11 days. Systolic BP was measured by tail cuff while qPCR, immunohistochemistry and flow cytometry were used to measure inflammatory markers, collagen and immune cell infiltration in the kidneys. RESULTS: By 10 days post-surgery, 1K/DOCA/salt-treated mice displayed elevated systolic BP (148.3+/-2.4mmHg) compared to control mice (121.7+/-2.7mmHg; n=18, P\u3c0.0001). The intervention with anakinra reduced BP in 1K/DOCA/salt-treated mice by approximately 20mmHg (n=16, P\u3c0.05), but had no effect in controls. In 1K/DOCA/salt-treated mice, anakinra modestly reduced ( approximately 30%) renal expression of some (CCL5, CCL2; n=7-8; P\u3c0.05) but not all (ICAM-1, IL-6) inflammatory markers, and had no effect on immune cell infiltration (n=7-8, P \u3e 0.05). Anakinra reduced renal collagen content (n=6, P\u3c0.01) but paradoxically appeared to exacerbate the renal and glomerular hypertrophy (n=8-9, P\u3c0.001) that accompanied 1K/DOCA/salt-induced hypertension. CONCLUSION: Despite its anti-hypertensive and renal anti-fibrotic actions, anakinra had minimal effects on inflammation and leukocyte infiltration in mice with 1K/DOCA/salt-induced hypertension. Future studies will assess whether the anti-hypertensive actions of anakinra are mediated by protective actions in other BP-regulating or salt-handling organs such as the arteries, skin and brain

Experimental design and analysis and their reporting II: updated and simplified guidance for authors and peer reviewers

This article updates the guidance published in 2015 for authors submitting papers to British Journal of Pharmacology (Curtis et al., 2015) and is intended to provide the rubric for peer review. Thus, it is directed towards authors, reviewers and editors. Explanations for many of the requirements were outlined previously and are not restated here. The new guidelines are intended to replace those published previously. The guidelines have been simplified for ease of understanding by authors, to make it more straightforward for peer reviewers to check compliance and to facilitate the curation of the journal's efforts to improve standards