Reduction of lipoxidative load by secretory phospholipase A2 inhibition protects against neurovascular injury following experimental stroke in rat

<p>Abstract</p> <p>Background</p> <p>In animal models, ischemia reperfusion (IR) injury triggers membrane lipid degradation and accumulation of lipoxidative exacerbations in neurovascular unit, leading to blood brain barrier (BBB) damage and neurologic deficits. In this study, we investigated whether impeding membrane lipid breakdown by inhibiting secretory phospholipase A2 (sPLA2) activity reduces BBB leakage, leading to neuroprotection and functional recovery.</p> <p>Methods</p> <p>Focal cerebral IR injury was induced by middle cerebral artery occlusion (MCAO) in adult male rats. A sPLA2 inhibitor, 7,7-dimethyleicosadienoic acid (DEDA), was administered following IR injury. DEDA-treated animals were compared with vehicle-treated in terms of BBB leakage, edema, infarct volume, and neurological deficit. Membrane lipid degradation and the expression/activity of sPLA2 were also assessed. The role of one of the sPLA2 products, arachidonic acid (AA), on the morphology of the differentiated neuronal cell PC12 was examined by light microscopy.</p> <p>Results</p> <p>Treatment with DEDA after IR injury not only reduced BBB leakage but also decreased infarct volume and improved neurologic function. The treatment attenuated both the activity of sPLA2 and the levels of sPLA2-derived oxidized products. The metabolites of lipid oxidation/peroxidation, including the protein carbonyl, were reduced as well. The treatment also restored the levels of glutathione, indicating attenuation of oxidative stress. I<it>n vitro </it>treatment of PC12 cells with DEDA did not restore the AA-mediated inhibition of neurite formation and the levels of glutathione, indicating that effect of DEDA is up stream to AA release.</p> <p>Conclusion</p> <p>sPLA2-derived oxidative products contribute to significant neurovascular damage, and treatment with sPLA2 inhibitor DEDA ameliorates secondary injury by reducing exacerbations from lipoxidative stress.</p

Protective Effect of Solanum nigrum

The prophylactic or curative antioxidant efficacy of crude extract and the active constituent of S. nigrum leaves were evaluated in modulating inherent antioxidant system altered due to immobilization stress in rat brain tissues, in terms of measurement of glutathione (GSH), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), and free radical scavenging enzymes activities. Rats were treated with single dose of crude extract of S. nigrum prior to and after 6 h of immobilization stress exposure. Exposure to immobilization stress resulted in a decrease in the brain levels of glutathione, SOD, GST, and catalase, with an increase in thiobarbituric acid reactive substances (TBARS) levels. Treatment of S. nigrum extract and its active constituents to both pre- and poststressed rats resulted in significant modulation in the above mentioned parameters towards their control values with a relative dominance by the latter. Brain is vulnerable to stress induced prooxidant insult due to high levels of fat content. Thus, as a safe herbal medication the S. nigrum leaves extract or its isolated constituents can be used as nutritional supplement for scavenging free radicals generated in the brain due to physical or psychological stress or any neuronal diseases per se

Increased Innate Lymphoid Cells in Periodontal Tissue of the Murine Model of Periodontitis: The Role of AMP-Activated Protein Kinase and Relevance for the Human Condition

Innate lymphoid cells (ILCs) are master regulators of immune and inflammatory responses, but their own regulatory mechanisms and functional roles of their subtypes (i.e., ILC1s–ILC3s) remain largely unresolved. Interestingly, AMP-activated protein kinase (AMPK), influences inflammatory responses, but its role in modulation of ILCs is not known. Periodontitis is a prevalent disorder with impairment of immune and inflammatory responses contributing importantly to its pathogenesis; however, neither the role of ILCs nor AMPK has been explored in this condition. We tested the hypotheses that (a) periodontitis increases ILCs and expression of relevant cytokines thereby contributing to inflammation and (b) knockdown of AMPK worsens indices of periodontitis in association with further increases in subtypes of ILCs and cytokine expression. The studies utilized wild-type (WT) and AMPK knockout (KO) mice, subjected to ligature-induced periodontitis or sham operation, in association with the use of micro-CT for assessment of bone loss, immunogold electron microscopy to show presence of ILCs in periodontal tissues, flow cytometry for quantitative assessment of subtypes of ILCs and RT-polymerase chain reaction analyses to measure mRNA expression of several relevant cytokines. The results for the first time show (a) presence of each subtype of ILCs in periodontal tissues of sham control and periodontitis animals, (b) that periodontitis is associated with increased frequencies of ILC1s–ILC3s with the effect more marked for ILC2s and differential phenotypic marker expression for ILC3s, (c) that AMPK KO mice display exacerbation of indices of periodontitis in association with further increases in the frequency of subtypes of ILCs with persistence of ILC2s effect, and (d) that periodontitis increased mRNA for interleukin (IL)-33, but not IL-5 or IL-13, in WT mice but expression of these cytokines was markedly increased in AMPK KO mice with periodontitis. Subsequently, we showed that human periodontitis is associated with increases in each ILCs subtype with the effect more marked for ILC2s and that mRNA expressions for IL-33 and IL-5 are markedly greater for sites affected by periodontitis than healthy sites. Collectively, these novel observations indicate a pivotal role for ILCs in pathogenesis of periodontitis and that AMPK is a regulator of their phenotype expression in this condition

Optimized and validated spectrophotometric methods for the determination of lisinopril in pharmaceutical formulations using ninhydrin and ascorbic acid

Two simple and sensitive spectrophotometric methods are described for the determination of lisinopril in pure form and pharmaceutical preparations. The first method is based on the reaction of the drug with ninhydrin in N,N'-dimethylformamide (DMF) medium at room temperature which is followed spectrophotometrically by measuring the increase in absorbance at 595 nm as a function of time. The initial-rate, rate-constant and fixed-time (at 10 minutes) procedures were used for constructing the calibration graphs to determine the concentration of the drug. The initial-rate and fixed-time procedures showed a linear response over the concentration range 10-50 µg mL-1 whereas rate-constant procedure was applicable in the range 10-40 µg mL-1. In the second method, the drug reacts with ascorbic acid in DMF medium resulting in the formation of a coloured product, which absorbed maximally at 530 nm. Beer's law is obeyed in the concentration 5-50 µg mL-1 of lisinopril with molar absorptivity of 4.548<FONT FACE=Symbol>&acute;</FONT>10³ L mol-1 cm-1. The variables affecting the development of the colour are optimized and the developed methods are validated statistically and through recovery studies. The proposed methods have been successfully applied to the determination of lisinopril in commercial tablets

Pharmacological Inhibition of Class III Alcohol Dehydrogenase 5: Turning Remote Ischemic Conditioning Effective in a Diabetic Stroke Model

The restoration of cerebral blood flow (CBF) to achieve brain tissue oxygenation (PbtO2) is the primary treatment for ischemic stroke, a significant cause of adult mortality and disability worldwide. Nitric oxide (NO) and its bioactive s-nitrosylated (SNO) reservoirs, such as s-nitrosoglutathione (GSNO), induce hypoxic vasodilation to enhance CBF during ischemia. The endogenous pool of SNOs/GSNO is enhanced via the activation of endothelial NO synthase (eNOS/NOS3) and by the suppression of class III alcohol dehydrogenase 5 (ADH5), also known as GSNO reductase (GSNOR). Remote ischemic conditioning (RIC), which augments NOS3 activity and SNO, is an emerging therapy in acute stroke. However, RIC has so far shown neutral effects in stroke clinical trials. As the majority of stroke patients are presented with endothelial dysfunctions and comorbidities, we tested the hypothesis that NOS3 dysfunction and diabetes will abolish the protective effects of RIC therapy in stroke, and the prior inhibition of GSNOR will turn RIC protective. Our data demonstrate that RIC during thrombotic stroke failed to enhance the CBF and the benefits of thrombolysis in NOS3 mutant (NOS3+/&minus;) mice, a genetic model of NOS3 dysfunction. Interestingly, thrombotic stroke in diabetic mice enhanced the activity of GSNOR as early as 3 h post-stroke without decreasing the plasma nitrite (NO2&minus;). In thrombotic stroke, neither a pharmacological inhibitor of GSNOR (GRI) nor RIC therapy alone was protective in diabetic mice. However, prior treatment with GRI followed by RIC enhanced the CBF and improved recovery. In a reperfused stroke model, the GRI&ndash;RIC combination therapy in diabetic mice augmented PbtO2, a translatory signature of successful microvascular reflow. In addition, RIC therapy unexpectedly increased the inflammatory markers at 6 h post-stroke in diabetic stroke that were downregulated in combination with GRI while improving the outcomes. Thus, we conclude that preexisting NOS3 dysfunctions due to comorbidities may neutralize the benefits of RIC in stroke, which can be turned protective in combination with GRI. Our findings may support the future clinical trial of RIC in comorbid stroke. Further studies are warranted to test and develop SNO reservoirs as the blood-associated biomarker to monitor the response and efficacy of RIC therapy in stroke

Remote ischemic conditioning: A treatment for vascular cognitive impairment

There is a strong link between hypoperfusion and white matter (WM) damage in patients with leukoaraiosis and vascular cognitive impairment (VCI). Other than management of vascular risk factors, there is no treatment for WM damage and VCI that delays progression of the disease process to dementia. Observational studies suggest that exercise may prevent or slow down the progression of Alzheimer′s disease (AD) and VCI. However, getting patients to exercise is challenging, especially with advancing age and disability. Remote ischemic conditioning, an "exercise equivalent," allows exercise to be given with a "device" at home for long periods of time. Since remote ischemic conditioning (RIC) increases cerebral blood flow (CBF) in preclinical studies and in humans, RIC may be an ideal therapy to treat VCI and WM disease and perhaps even sporadic AD. By using magnetic resonance imaging (MRI) imaging of WM progression, a sample size in the range of about 100 subjects per group could determine if RIC has activity in WM disease and VCI

Nebulization of Low-Dose S-Nitrosoglutathione in Diabetic Stroke Enhances Benefits of Reperfusion and Prevents Post-Thrombolysis Hemorrhage

The COVID-19 pandemic has escalated the occurrence of hypoxia including thrombotic stroke worldwide, for which nitric oxide (NO) therapy seems very promising and translatable. Therefore, various modes/routes of NO-delivery are now being tested in different clinical trials for safer, faster, and more effective interventions against ischemic insults. Intravenous (IV) infusion of S-Nitrosoglutathione (GSNO), the major endogenous molecular pool of NO, has been reported to protect against mechanical cerebral ischemia-reperfusion (IR); however, it has been never tested in any kind of “clinically” relevant thromboembolic stroke models with or without comorbidities and in combination with the thrombolytic reperfusion therapy. Moreover, “IV-effects” of higher dose of GSNO following IR-injury have been contradicted to augment stroke injury. Herein, we tested the hypothesis that nebulization of low-dose GSNO will not alter blood pressure (BP) and will mitigate stroke injury in diabetic mice via enhanced cerebral blood flow (CBF) and brain tissue oxygenation (PbtO2). GSNO-nebulization (200 μg/kgbwt) did not alter BP, but augmented the restoration of CBF, improved behavioral outcomes and reduced stroke injury. Moreover, GSNO-nebulization increased early reoxygenation of brain tissue/PbtO2 as measured at 6.5 h post-stroke following thrombolytic reperfusion, and enervated unwanted effects of late thrombolysis in diabetic stroke. We conclude that the GSNO-nebulization is safe and effective for enhancing collateral microvascular perfusion in the early hours following stroke. Hence, nebulized-GSNO therapy has the potential to be developed and translated into an affordable field therapy against ischemic events including strokes, particularly in developing countries with limited healthcare infrastructure