Shashi Bala Singh

Hypobaric hypoxia induces oxidative stress in rat brain

High altitude exposure results in decreased partial pressure of oxygen and an increased formation of reactive oxygen and nitrogen species (RONS), which causes oxidative damage to lipids, proteins and DNA. Exposure to high altitude appears to decrease the activity and effectiveness of antioxidant enzyme system. The antioxidant system is very less in brain tissue and is very much susceptible to hypoxic stress. The aim of the present study was to investigate the time dependent and region specific changes in cortex, hippocampus and striatum on oxidative stress markers on chronic exposure to hypobaric hypoxia. The rats were exposed to simulated high altitude equivalent to 6100 m in animal decompression chamber for 3 and 7 days. Results indicate an increase in oxidative stress as seen by increase in free radical production, nitric oxide level, lipid peroxidation and lactate dehydrogenase levels. The magnitude of increase in oxidative stress was more in 7 days exposure group as compared to 3 days exposure group. The antioxidant defence system such as reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and reduced/oxidized glutathione (GSH/GSSG) levels were significantly decreased in all the three regions. The observation suggests that the hippocampus is more susceptible to hypoxia than the cortex and striatum. It may be concluded that hypoxia differentially affects the antioxidant status in the cortex, hippocampus and striatum.

Neuroprotective effect of N-acetyl cysteine on hypoxia-induced oxidative stress in primary hippocampal culture

Hippocampus has received a considerable attention in the recent past due to its role in a number of important functions such as learning and memory. The effect of hypoxia on neuronal cell injury especially on hippocampal cells is not well known. The aim of the present study was to characterize the biochemical changes in primary cultured hippocampal neurons during hypoxic exposure and the protective effect of N-acetyl cysteine on hypoxia-induced cytotoxicity. The hippocampal culture grown in 24-well plates was exposed to hypoxia for 3 h in a dessicator in 95% N(2), 5% CO(2) atmosphere at 37 degrees C. Later, the cells were allowed to recover for 1 h under normoxia. It was observed that there is an appreciable increase in cytotoxicity in cells exposed to hypoxia. Further, there was a significant decrease in mitochondrial membrane potential and appreciable increase in reactive oxygen species and single-strand DNA breaks in cells exposed to hypoxia compared to control. There is a significant fall in glutathione peroxidase, glutathione reductase, reduced glutathione levels, and nitric oxide in the cells exposed to hypoxia. Significant elevation in the intracellular calcium level in the cells on exposure to hypoxia was observed. Supplementation with NAC (50 microM) resulted in a significant cytoprotection, fall in ROS generation, and higher antioxidant levels similar to that of control cells. NAC also inhibited DNA strand breaks induced by hypoxia. The study indicates that NAC has significant neuroprotective activity during hypoxia in primary hippocampal culture.

Ghrelin and leptin levels of sojourners and acclimatized lowlanders at high altitude.

Abstract The circulatory levels of two appetite regulatory hormones i.e. leptin and ghrelin were estimated in sojourners and acclimatized subjects to investigate their possible role in high altitude (HA) induced anorexia. A group of 30 lowlanders who had never visited HA were inducted to a height of 3600m by air and after 48h they were further taken to an altitude of 4300m by road. Blood samples were collected after 48h stay at 3600m and again after 48h and 7 days of stay at 4300m during 0700–0730h. There was a decrease in energy intake (850kcal/day) of sojourners, which resulted in loss of body weight by 2.12kg at HA. At an altitude of 4300m there was a significant increase in leptin over basal levels (54.9%, p <0.001) at 48h that persisted even after 7 days of stay at this altitude. Ghrelin levels of sojourners decreased by more than 30% in comparison to basal values at 48h of ascent to HA. Leptin levels of acclimatized lowlanders were also higher in comparison with control group (acclimatized group 7.6+0.6ng/ml vs. control 5.6+0.5ng/ml, p <0.01, n =50).

Bacopa monniera leaf extract ameliorates hypobaric hypoxia induced spatial memory impairment

Hypobaric hypoxia induced memory impairment has been attributed to several factors including increased oxidative stress, depleted mitochondrial bioenergetics, altered neurotransmission and apoptosis. This multifactorial response of the brain to hypobaric hypoxia limits the use of therapeutic agents that target individual pathways for ameliorating hypobaric hypoxia induced memory impairment. The present study aimed at exploring the therapeutic potential of a bacoside rich leaf extract of Bacopa monniera in improving the memory functions in hypobaric conditions. The learning ability was evaluated in male Sprague Dawley rats along with memory retrieval following exposure to hypobaric conditions simulating an altitude of 25,000 ft for different durations. The effect of bacoside administration on apoptosis, cytochrome c oxidase activity, ATP levels, and oxidative stress markers and on plasma corticosterone levels was investigated. Expression of NR1 subunit of N-methyl-d-aspartate receptors, neuronal cell adhesion molecules and was also studied along with CREB phosphorylation to elucidate the molecular mechanisms of bacoside action. Bacoside administration was seen to enhance learning ability in rats along with augmentation in memory retrieval and prevention of dendritic atrophy following hypoxic exposure. In addition, it decreased oxidative stress, plasma corticosterone levels and neuronal degeneration. Bacoside administration also increased cytochrome c oxidase activity along with a concomitant increase in ATP levels. Hence, administration of bacosides could be a useful therapeutic strategy in ameliorating hypobaric hypoxia induced cognitive dysfunctions and other related neurological disorders.

Hypobaric hypoxia damages the hippocampal pyramidal neurons in the rat brain

Hypobaric hypoxia (HH), a predisposing environmental condition at high altitude (HA), encountered by many mountaineers, jeopardizes their normal physiology like motor coordination and cognitive functions. A large body of evidence shows that HH has deleterious effect on cognitive functions. Among them the hippocampal dependent memory deficit is well known. However, our current understanding of the mechanistic details of cognitive deficits at HA remains largely unclear and hence limits a solution for this problem. Therefore, the present study was designed to investigate the temporal component of the hippocampal pyramidal neuron damage in the rat brain subjected to chronic HH exposure. Three groups (sham HH, 3 days HH and 7 days HH) of rats were exposed to simulated HH equivalent to 6100 m in an animal decompression chamber for 3 or 7 days. Later, the hippocampal (CA1 and CA3) neurons were analysed for the cell morphology, neurodegeneration and DNA fragmentation. The CA1 and CA3 neurons showed HH induced neuronal pyknosis, cell shrinkage, and consequent inter-cellular vacuolization in the CA1 and CA3 areas. In addition, the total neuron (intact) numbers and mean surface area were decreased. The number of dead neurons increased significantly following exposure to HH for 3 or 7 days. The neurodegenerative (Fluoro jade B) and apoptotic (TUNEL) markers were more positive in CA1 and CA3 neurons. The magnitude of morphological changes, neurodegeneration and apoptosis was enhanced in 7 days HH group than 3 days HH group. Our studies indicate that CA3 neurons are more vulnerable to HH than CA1 neurons, and that may destabilize the neural circuits in the hippocampus and thus cause memory dysfunction.

Differential temporal response of hippocampus, cortex and cerebellum to hypobaric hypoxia: A biochemical approach

Hypobaric hypoxia is known to cause cognitive dysfunctions and memory impairment. The present study aimed at exploring the occurrence of oxidative stress in hypobaric hypoxia and the differential temporal response of the hippocampus, cerebellum following hypobaric hypoxia. Animals were divided into control, 3 days, 7 days and 14 days exposure groups and were exposed to an altitude of 25,000 ft. Our study revealed an increase in lactate dehydrogenase activity along with increase in free radical generation and lipid peroxidation. We also noted depletion in the antioxidants and decrease in glutathione reductase and superoxide dismutase activity. There was significant decrease in reduced glutathione levels in the exposure groups when compared to the control which was accompanied by a concomitant increase in oxidized glutathione levels. Increase in glutamate dehydrogenase activity was observed coinciding with the decrease in glutathione levels which was accompanied with an increase in expression of vesicular glutamate transporter. The hippocampus was found to be more vulnerable to hypobaric hypoxia-induced oxidative stress in comparison to the cortex and cerebellum. An interesting observation was the onset of acclimatization on prolonged exposure to hypobaric hypoxia for a period of 14 days. Hypobaric hypoxia was found to affect various regions of the brain differentially and the response of each region varied as a function of time.

Acetyl-L-carnitine-mediated neuroprotection during hypoxia is attributed to ERK1/2-Nrf2-regulated mitochondrial biosynthesis

Neuronal damage in hypoxia and several neurodegenerative disorders is invariably associated with oxidative damage and mitochondrial dysfunction. Administration of acetyl‐L‐carnitine (ALCAR) on the other hand attenuates neuronal damage, prevents apoptosis, and improves energy status in hypoxic stress through less understood mechanisms. Becasue mitochondrial biogenesis could be a possible mechanism for ALCAR‐induced improvement in bioenergetics in neurons, the present study aimed at exploring signaling pathways of ALCAR‐induced neuroprotection in hypoxia and possible occurrence of mitochondrial biogenesis. To create global hypoxia, adult Sprague–Dawley rats were exposed to a simulated altitude of 7,620 m at standard temperature and humidity conditions. We here demonstrate that administration of ALCAR to hypoxic rats for a period of 2 weeks effectively protected hippocampal neurons from mitochondrial dysfunction, excitotoxicity, and neurodegeneration. ALCAR administration resulted in peroxisome proliferator‐activated receptor γ coactivator‐1α and nuclear respiratory factor‐1‐induced mitochondrial biogenesis, the expression of which was regulated by an extracellular‐related kinase‐nuclear factor erythroid 2‐related factor 2 (ERK‐Nrf2)‐mediated mechanism. Most notably, calcium buffering into nonfunctional mitochondria ameliorated excitotoxicity and improved bioenergetic status of the hippocampal neurons. Together, the data reveal the immense therapeutic potential of ALCAR for the treatment of ischemia, stroke, and other neurodegenerative disorders associated with hypoxic stress and excitotoxicity.

Nitric oxide system is involved in hypobaric hypoxia-induced oxidative stress in rat brain

Oxidative stress is involved in memory impairment at high altitude (HA). The aim of the present study was to investigate the involvement of reactive nitrogen species in hippocampus, cortex and striatum of rat brain under simulated HA conditions. Rats were exposed to hypobaric hypoxia (HH) equivalent to 6100 m of HA in an animal decompression chamber for 3, 7, 14 and 21 days. Biochemical estimation of free radicals, nitric oxide (NO) level along with immunoreactivity, reverse transcriptase polymerase chain reaction (RT-PCR) and western blot of neuronal nitric oxide synthase (nNOS), neurodegeneration and DNA fragmentation were studied after HH exposure. The free radicals, NO level, nNOS immunoreactivity (nNOS-IR), nNOS expression, neurodegeneration and DNA fragmentation were increased in hippocampus, cortex and striatum after HH exposure. After 7 and 14 days of HH exposure, the nNOS-IR, nNOS expression, free radical, NO level, neurodegeneration and DNA fragmentation were increased in comparison to 3 or 21 days of HH. The NO system may be involved in increasing oxidative stress and neurodegeneration after HH.

Ceftriaxone rescues hippocampal neurons from excitotoxicity and enhances memory retrieval in chronic hypobaric hypoxia.

Exposure to high altitude is known to cause impairment in cognitive functions in sojourners. The molecular events leading to this behavioral manifestation, however, still remain an enigma. The present study aims at exploring the nature of memory impairment occurring on chronic exposure to hypobaric hypoxia and the possible role of glutamate in mediating it. Increased ionotropic receptor stimulation by glutamate under hypobaric hypoxic conditions could lead to calcium mediated excitotoxic cell death resulting in impaired cognitive functions. Since glutamate is cleared from the synapse by the Glial Glutamate Transporter, upregulation of the transporter can be a good strategy in preventing excitotoxic cell death. Considering previous reports on upregulation of the expression of Glial Glutamate Transporter on ceftriaxone administration, the therapeutic potential of ceftriaxone in ameliorating hypobaric hypoxia induced memory impairment was investigated in male Sprague Dawley rats. Exposure to hypobaric hypoxia equivalent to an altitude of 7600 m for 14 days lead to oxidative stress, chromatin condensation and neuronal degeneration in the hippocampus. This was accompanied by delayed memory retrieval as evident from increased latency and pathlength in Morris Water Maze. Administration of ceftriaxone at a dose of 200 mg/kg for 7 days and 14 days during the exposure on the other hand improved the performance of rats in the water maze along with decreased oxidative stress and enhanced neuronal survival when compared to hypoxic group without drug administration. An increased expression of Glial Glutamate Transporter was also observed following drug administration indicating faster clearance of glutamate from the synapse. The present study not only brings to light the effect of longer duration of exposure to hypobaric hypoxia on the memory functions, but also indicates the pivotal role played by glutamate in mediating excitotoxic neuronal degeneration at high altitude. The therapeutic potential of ceftriaxone in providing neuroprotection in excitotoxic conditions by increasing Glial Glutamate Transporter expression and thereby enhancing glutamate uptake from the synapse has also been explored.

Hypoxia-induced deactivation of NGF-mediated ERK1/2 signaling in hippocampal cells: neuroprotection by acetyl-L-carnitine.

Cellular and molecular pathways underlying hypoxic neurotoxicity and cell death are multifaceted and complex. Although many potentially neuroprotective agents have been investigated, the protection conferred is often inadequate, resulting in their insufficient clinical utility. In light of the above, we investigated the therapeutic potential and mechanism of action of acetyl‐L‐carnitine (ALCAR) in protecting hippocampal neurons from hypoxia‐induced neurotoxicity and cellular death. Results showed decreased viability of hippocampal cells when exposed to hypoxia (3% O2) for 48 hr along with concomitant membrane depolarization, adenosine triphosphate depletion, DNA fragmentation, accentuated free radical production, and lactate dehydrogenase activity. Pretreatment with ALCAR significantly attenuated hypoxia‐induced cytotoxicity in a dose‐dependent manner and improved cellular glutathione levels and cytochrome c oxidase activity compared with normoxic controls. Supplementation of ALCAR also prevented apoptosis by down‐regulating caspase‐3 levels, cytochrome c release, and p‐Bcl‐2 expression. A decrease in nerve growth factor (NGF) was observed in hypoxic stress despite increased phosphorylation of ERK1/2 (extracellular signal–related kinase) and its downstream effector, Elk‐1. Supplementation of ALCAR, on the other hand, up‐regulated NGF and tyrosine kinase A expression along with concomitant increase in ERK1/2 phosphorylation, thus enhancing cell survival. ALCAR therefore provides neuroprotection by stabilizing mitochondrial membrane, restoring the cholinergic transmission, and more importantly, it stimulates NGF receptors, thus triggering cell survival pathway via ERK phosphorylation. Therefore, ALCAR may be useful as an effective therapeutic agent for hypoxic stress and associated neurodegenerative diseases.