Mrinalini Singh

Hypoxia preconditioning by cobalt chloride enhances endurance performance and protects skeletal muscles from exercise-induced oxidative damage in rats

Aim:  Training under hypoxia has several advantages over normoxic training in terms of enhancing the physical performance. Therefore, we tested the protective effect of hypoxia preconditioning by hypoxia mimetic cobalt chloride against exercise‐induced oxidative damage in the skeletal muscles and improvement of physical performance.

Hypoxic preconditioning with cobalt ameliorates hypobaric hypoxia induced pulmonary edema in rat

Exposure to high altitude results in hypobaric hypoxia which is considered as an acute physiological stress and often leads to high altitude maladies such as high altitude pulmonary edema (HAPE) and high altitude cerebral edema (HACE). The best way to prevent high altitude injuries is hypoxic preconditioning which has potential clinical usefulness and can be mimicked by cobalt chloride. Preconditioning with cobalt has been reported to provide protection in various tissues against ischemic injury. However, the effect of preconditioning with cobalt against high altitude induced pulmonary edema has not been investigated in vivo. Therefore, in the present study, rats pretreated with saline or cobalt (12.5mg/kg body weight) for 7days were exposed to hypobaric hypoxia of 9142m for 5h at 24°C. Formation of pulmonary edema was assessed by measuring transvascular leakage of sodium fluorescein dye and lung water content. Total protein content, albumin content, vascular endothelial growth factor (VEGF) and cytokine levels were measured in bronchoalveolar lavage fluid. Expression of HO-1, MT, NF-κB DNA binding activity and lung tissue pathology were evaluated to determine the effect of preconditioning on HAPE. Hypobaric hypoxia induced increase in transvascular leakage of sodium fluorescein dye, lung water content, lavage total protein, albumin, VEGF levels, pro-inflammatory cytokine levels, tissue expression of cell adhesion molecules and NF-κB DNA binding activity were reduced significantly after hypoxic preconditioning with cobalt. Expression of anti-inflammatory protein HO-1, MT, TGF-β and IL-6 were increased after hypoxic preconditioning. These data suggest that hypoxic preconditioning with cobalt has protective effect against HAPE.

Hypoxic preconditioning with cobalt attenuates hypobaric hypoxia-induced oxidative damage in rat lungs.

Shukla, Dhananjay, Saurabh Saxena, Purushotman Jayamurthy, Mustoori Sairam, Mrinalini, Singh, Swatantra Kumar Jain, Anju Bansal, and Govindaswamy Ilavazaghan. High Alt. Med. Biol. 10:57-69, 2009.-Hypoxic preco759nditioning (HPC) provides robust protection against injury from subsequent prolonged hypobaric hypoxia, which is a characteristic of high altitude and is known to induce oxidative injury in lung by increasing the generation of reactive oxygen species (ROS) and decreasing the effectiveness of the antioxidant defense system. We hypothesize that HPC with cobalt might protect the lung from subsequent hypobaric hypoxia-induced lung injury. HPC with cobalt can be achieved by oral feeding of CoCl(2) (12.5 mg kg(-1)) in rats for 7 days. Nonpreconditioned rats responded to hypobaric hypoxia (7619 m) by increased reactive oxygen species (ROS) generation and a decreased GSH/GSSG ratio. They also showed a marked increase in lipid peroxidation, heat-shock proteins (HSP32, HSP70), metallothionins (MT), levels of inflammatory cytokines (TNF-alpha, IFN-gamma, MCP-1), and SOD, GPx, and GST enzyme activity. In contrast, rats preconditioned with cobalt were far less impaired by severe hypobaric hypoxia, as observed by decreased ROS generation, lipid peroxidation, and inflammatory cytokine release and an inceased GSH/GSSG ratio. Increased expression of antioxidative proeins Nrf-1, HSP-32, and MT was also observed in cobalt- preconditioned animals. A marked increase in the protein expression and DNA binding activity of hypoxia-inducible transcriptional factor (HIF-1alpha) and its regulated genes, such as erythropoietin (EPO) and glucose transporter-1 (glut-1), was observed after HPC with cobalt. We conclude that HPC with cobalt enhances antioxidant status in the lung and protects from subsequent hypobaric hypoxia-induced oxidative stress.

Hypoxic preconditioning facilitates acclimatization to hypobaric hypoxia in rat heart.

Objectives  Acute systemic hypoxia induces delayed cardioprotection against ischaemia‐reperfusion injury in the heart. As cobalt chloride (CoCl2) is known to elicit hypoxia‐like –responses, it was hypothesized that this chemical would mimic the preconditioning effect and facilitate acclimatization to hypobaric hypoxia in rat heart.

Cordyceps sinensis increases hypoxia tolerance by inducing heme oxygenase-1 and metallothionein via Nrf2 activation in human lung epithelial cells.

Cordyceps sinensis, an edible mushroom growing in Himalayan regions, is widely recognized in traditional system of medicine. In the present study, we report the efficacy of Cordyceps sinensis in facilitating tolerance to hypoxia using A549 cell line as a model system. Treatment with aqueous extract of Cordyceps sinensis appreciably attenuated hypoxia induced ROS generation, oxidation of lipids and proteins and maintained antioxidant status similar to that of controls via induction of antioxidant gene HO1 (heme oxygenase-1), MT (metallothionein) and Nrf2 (nuclear factor erythroid-derived 2-like 2). In contrast, lower level of NFκB (nuclear factor kappaB) and tumor necrosis factor-α observed which might be due to higher levels of HO1, MT and transforming growth factor-β. Further, increase in HIF1 (hypoxia inducible factor-1) and its regulated genes; erythropoietin, vascular endothelial growth factor, and glucose transporter-1 was observed. Interestingly, Cordyceps sinensis treatment under normoxia did not regulate the expression HIF1, NFκB and their regulated genes evidencing that Cordyceps sinensis per se did not have an effect on these transcription factors. Overall, Cordyceps sinensis treatment inhibited hypoxia induced oxidative stress by maintaining higher cellular Nrf2, HIF1 and lowering NFκB levels. These findings provide a basis for possible use of Cordyceps sinensis in tolerating hypoxia.

Exogenous Sphingosine-1-Phosphate Boosts Acclimatization in Rats Exposed to Acute Hypobaric Hypoxia: Assessment of Haematological and Metabolic Effects

Background The physiological challenges posed by hypobaric hypoxia warrant exploration of pharmacological entities to improve acclimatization to hypoxia. The present study investigates the preclinical efficacy of sphingosine-1-phosphate (S1P) to improve acclimatization to simulated hypobaric hypoxia. Experimental Approach Efficacy of intravenously administered S1P in improving haematological and metabolic acclimatization was evaluated in rats exposed to simulated acute hypobaric hypoxia (7620m for 6 hours) following S1P pre-treatment for three days. Major Findings Altitude exposure of the control rats caused systemic hypoxia, hypocapnia (plausible sign of hyperventilation) and respiratory alkalosis due to suboptimal renal compensation indicated by an overt alkaline pH of the mixed venous blood. This was associated with pronounced energy deficit in the hepatic tissue along with systemic oxidative stress and inflammation. S1P pre-treatment improved blood oxygen-carrying-capacity by increasing haemoglobin, haematocrit, and RBC count, probably as an outcome of hypoxia inducible factor-1α mediated erythropoiesis and renal S1P receptor 1 mediated haemoconcentation. The improved partial pressure of oxygen in the blood could further restore aerobic respiration and increase ATP content in the hepatic tissue of S1P treated animals. S1P could also protect the animals from hypoxia mediated oxidative stress and inflammation. Conclusion The study findings highlight S1P’s merits as a preconditioning agent for improving acclimatization to acute hypobaric hypoxia exposure. The results may have long term clinical application for improving physiological acclimatization of subjects venturing into high altitude for occupational or recreational purposes.

Co-expression of S. Typhi GroEL and IL-22 gene augments immune responses against Salmonella infection

Recombinant DNA vaccines represent a novel method for generating in situ expression of vaccine antigens. Intramuscular injections of naked DNA are able to elicit potent humoral and cellular immune responses but still numerous factors limit the immunogenicity of DNA vaccines. Co‐expression of cytokines with antigen encoding genes in DNA vectors can improve the immune responses and modify Th1/Th2 balance. In this study, the immunomodulatory effect of Interleukin 22 (IL‐22) as an adjuvant was studied by DNA vaccination with S. Typhi Heat shock protein 60 (HSP60/GroEL) in mice. Further, DNA construct of IL‐22 gene fused with GroEL was developed and immunization studies were carried out in mice. DNA vaccination with GroEL alone stimulated humoral and cell‐mediated immune responses. Co‐immunization (IL‐22+GroEL) further resulted in increase in T‐cell proliferative responses, antibody titres (IgG, IgG1, IgG2a) and secretion of IFNγ (Th1), IL‐1β and Th2 (IL‐4, IL‐6) cytokines. Co‐expression (IL‐22‐GroEL DNA) also promoted antibody titres and cytokine levels were significantly higher as compared to co‐immunized group. A reduction in bacterial load in spleen, liver and intestine was seen in all the immunized groups as compared to control, with least organ burden in fusion DNA construct group (co‐expression). Improved protective efficacy (90%) against lethal challenge by Salmonella was observed with IL‐22‐GroEL co‐expressing DNA vector as compared with plasmid encoding GroEL only (50–60%) or co‐immunization group (75–80%). This study thus shows that co‐expression of IL‐22 and GroEL genes enhances the immune responses and protective efficacy, circumventing the need of any adjuvant.

The MAPK-activator protein-1 signaling regulates changes in lung tissue of rat exposed to hypobaric hypoxia

This study reports the role of MAPKs (JNK, ERK, and p38), and activator protein‐1 (AP‐1) transcription factor in the hypobaric hypoxia induced change in lung tissue. Healthy male Sprague–Dawley rats were exposed to hypobaric hypoxia for 6, 12, 24, 48, 72, and 120 hr. Hypoxia resulted in significant increase in reactive oxygen species (ROS), vascular endothelial growth factor (VEGF) and decreased nitric oxide (NO), these act as signaling molecules for activation of MAPK and also contribute in development of vascular leakage (an indicator of pulmonary edema) as confirmed by histological studies. Our results confirmed JNK activation as an immediate early response (peaked at 6–48 hr), activation of ERKs (peaked at 24–72 hr) and p38 (peaked at 72–120 hr) as a secondary response to hypoxia. The MAPK pathway up regulated its downstream targets phospho c‐Jun (peaked at 6–120 hr), JunB (peaked at 24–120 hr) however, decreased c‐Fos, and JunD levels. DNA binding activity also confirmed activation of AP‐1 transcription factor in lung tissue under hypobaric hypoxia. Further, we analyzed the proliferative and inflammatory genes regulated by different subunits of AP‐1 to explore its role in vascular leakage. Increased expression of cyclin D1 (peaked at 12–72 hr) and p16 level (peaked at 48–120 hr) were correlated to the activation of c‐jun, c‐Fos and JunB. Administration of NFκB inhibitor caffeic acid phenethyl ester (CAPE) and SP600125 (JNK inhibitor) had no effect on increased levels of Interferon‐γ (IFN‐γ), Interleukin‐1 (IL‐1), and Tumor Necrosis Factor‐α (TNF‐α) thereby confirming the involvement of AP‐1 as well as NFκB in inflammation. Expression of c‐jun, c‐Fos were correlated with activation of proliferative genes and JunB, Fra‐1 with pro‐inflammatory cytokines. In conclusion immediate response to hypobaric hypoxia induced c‐Jun:c‐Fos subunits of AP‐1; responsible for proliferation that might cause inhomogeneous vasoconstriction leading to vascular leakage and inflammation at increased duration of hypobaric hypoxia exposure.

Rapid Acclimatization Strategies for High-Altitude Induction

High altitude (HA) is defined as elevation above 9,000 ft. At this altitude, most people develop acute mountain sickness (AMS). If untreated, this may lead to high-altitude pulmonary oedema (HAPE) or high-altitude cerebral oedema (HACE), both of which are potentially life-threatening. In emergencies/warlike conditions, rapid deployment of military personnel to high altitude frequently occurs without giving the adequate degree of altitude acclimatization, resulting in acute mountain sickness (AMS). Acclimatization to high altitude is the best strategy to prevent AMS, and this can be achieved by hypoxia preconditioning by the use of interventions like hypoxia mimetics. Efficacy of hypoxia mimetics, viz. cobalt chloride (CoCl2), ethyl 3, 4-dihydroxybenzoate (EDHB), sphingosine-1-phosphate (S1P) and other pharmacological agent nanocurmin in facilitating acclimatization to high altitude in animal model, has been discussed. An alternative approach to induce acclimatization and reduce incidence of AMS is the use of intermittent hypoxic exposure (IHE). This study was conducted to evaluate the effect of IHE exposure at sea level on incidence of AMS during acute ascent to 3,500 m altitude in Indian military personnel. The army volunteers were divided into two groups, viz. control and experimental. Experimental group of subjects were exposed to intermittent normobaric hypoxia consisting of 12 % FIO2 (altitude – air equivalent 4,350 m) for 4 h per day for 4 consecutive days. After giving IHT, the subjects were inducted to 3,500 m altitude (Leh) by air and different physiological parameters like AMS score (LLS), pulse arterial oxygen saturation (SaO2) and ventilatory parameters (V E, VO2, V T/Ti) were recorded daily. IHE-treated group showed a significant reduction in AMS at HA in comparison to control. IHE may be considered as an alternative approach to induce the altitude acclimatization at low altitude-based soldiers before their deployment to high-altitude operations in emergency-like conditions.