Gurcharan Kaur

Impact of hypoglycemia and diabetes on CNS: Correlation of mitochondrial oxidative stress with DNA damage

Oxidative stress plays an important role in tissue damage caused by hypoglycemia and diabetes, which may be the result of deterioration in glucose homeostasis caused by these metabolic disorders. The present study examined the effects of insulin-induced hypoglycemia and streptozotocin induced diabetes on mitochondrial lipid peroxidation and antioxidant enzymes from different brain regions, namely, cerebral hemispheres, cerebellum, brain stem and diencephalon. In situ localization of DNA single strand breaks (SSBs) were also studied by DNA polymerase-I mediated biotin dATP labeled nick translation method after inducing hypoglycemia and diabetes. Significant decrease in mitochondrial catalase, manganese superoxide-dismutase (Mn-SOD) and reduced glutathione (GSH) content and increase in the lipid peroxidation (LPx) and glutathione peroxidase (GPx) activity was observed under these metabolic stress conditions with more pronounced effects in hypoglycemic group. We conclude that during severe energy deprivation following hypoglycemia and diabetes, mitochondrial free radicals scavenger system is down regulated, which leads to reactive oxygen species (ROS) generation. High levels of ROS in turn activate the processes leading to DNA damage. DNA SSBs, which indicates nuclear disintegration is an important feature of neuronal cell death.

Alterations in oxidative stress scavenger system in aging rat brain and lymphocytes

There is a large body of evidence indicating an age-related increase in the rate of mitochondrial O2− and H2O2 generation and huge amounts of oxidative damage leading to several neurodegenerative disorders, perhaps due to an imbalance between free radical generation and anti-oxidant defense system. The aim of the present study was to elucidate the effect of aging on free radical scavenger system profile in rat brain and lymphocytes. The enzyme activities of γ-GCS, GR, GPx, γ-GTP, GST, catalase, and SOD as well as GSH content were assayed from discrete brain areas viz., CH, CB,BS and DC along with lymphocytes from four different age group rats, namely, 1-month-oldyoung rats, 3–4-month-old young adults, 12-month-old adults and 24-month-old aged rats. Significant decline was observed in all the enzyme activities in 12- and 24-month-old rats as compared to 3–4-month-old young adult rats and also, 1-month-old rats showed lower levels of enzyme activities as compared to 3–4-month-old rats. The maximum scavenger system activity was found in the young adult rats (3–4 months) as compared to the remaining age groups. Lymphocytes and brain showed a parallel pattern of age-related alterations in the free radical scavenger system components. The analysis of such alterations is important in ultimately determining the basis of neuronal dysfunction associated with aging and also defining the nature of these changes may help to develop therapeutic means to cure not only elderly but also individuals suffering from certain organic or psychiatric disorders.

Gemini imidazolium surfactants: synthesis and their biophysiochemical study.

New gemini imidazolium surfactants 9-13 have been synthesized by a regioselective epoxy ring-opening reaction under solvent-free conditions. The surface properties of these new gemini surfactants were evaluated by surface tension and conductivity measurements. These surfactants have been found to have low critical micelle concentration (cmc) values as compared to other categories of gemini cationic surfactants and also showed the tendency to form premicellar aggregates in solution at sufficiently low concentration below their cmc values. The thermal degradation of these surfactants was determined by thermograviometry analysis (TGA). These new cationic surfactants have a good DNA binding capability as determined by agarose gel electrophoresis and ethidium bromide exclusion experiments. They have also been found to have low cytotoxicity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on the C6 glioma cell line.

Effect of the alcoholic extract of Ashwagandha leaves and its components on proliferation, migration, and differentiation of glioblastoma cells: Combinational approach for enhanced differentiation

Ashwagandha (Withania somnifera) is widely used in the Indian traditional system of medicine, Ayurveda. Although it is claimed to have a large variety of health‐promoting effects, including therapeutic effects on stress and disease, the mechanisms of action have not yet been determined. In the present study, we aimed to investigate the growth inhibition and differentiation potential of the alcoholic extract of Ashwagandha leaves (i‐Extract), its different constituents (Withaferin A, Withanone, Withanolide A) and their combinations on glioma (C6 and YKG1) cell lines. Withaferin A, Withanone, Withanolide A and i‐Extract markedly inhibited the proliferation of glioma cells in a dose‐dependent manner and changed their morphology toward the astrocytic type. Molecular analysis revealed that the i‐Extract and some of its components caused enhanced expression of glial fibrillary acidic protein, change in the immunostaining pattern of mortalin from perinuclear to pancytoplasmic, delay in cell migration, and increased expression of neuronal cell adhesion molecules. The data suggest that the i‐Extract and its components have the potential to induce senescence‐like growth arrest and differentiation in glioma cells. These assays led us to formulate a unique combination formula of i‐Extract components that caused enhanced differentiation of glial cells. (Cancer Sci 2009; 100: 1740–1747)

Neuroprotective potential of dietary restriction against kainate-induced excitotoxicity in adult male Wistar rats.

The influence that dietary factors have on the nervous system and its susceptibility to disease, is an active area of biomedical research. Recent studies have shown that dietary restriction (DR) can have profound effect on brain function and vulnerability to injury and disease and can also enhance synaptic plasticity, which may increase the ability of brain to resist aging and restore function following injury. The dietary restriction may result in neuroprotection as suggested by marked reduction in neuronal cell death of the CA3 region of hippocampus after kainate administration in our study. We examined the effects of 3 months of DR (alternate day feeding regimen) on the antioxidants and antioxidant enzymes from different brain regions such as cerebral hemispheres, diencephalon, cerebellum and brain stem after kainate-induced excitotoxicity in adult male Wistar rats. The present study reports the beneficial effects of dietary restriction on different antioxidants and antioxidant enzymes against kainate-induced excitotoxicity in different brain regions of young adult male Wistar rats. The expression of stress response protein heat shock protein 70 (HSP 70) was also studied from discrete regions of rat brain under the same set of experimental conditions. DR significantly enhanced the expression of HSP 70 in kainic acid (KA)-treated rats, whereas KA treatment of ad libitum fed rats resulted in decreased HSP 70 expression. The DR was observed to exert neuroprotection by enhancing the expression of HSP 70 in kainic acid treated rats.

Late-onset intermittent fasting dietary restriction as a potential intervention to retard age-associated brain function impairments in male rats

Lifelong dietary restriction (DR) is known to have many potential beneficial effects on brain function as well as delaying the onset of neurological diseases. In the present investigation, the effect of late-onset short-term intermittent fasting dietary restriction (IF-DR) regimen was studied on motor coordination and cognitive ability of ageing male rats. These animals were further used to estimate protein carbonyl content and mitochondrial complex I–IV activity in different regions of brain and peripheral organs, and the degree of age-related impairment and reversion by late-onset short-term IF-DR was compared with their levels in 3-month-old young rats. The results of improvement in motor coordination by rotarod test and cognitive skills by Morris water maze in IF-DR rats were found to be positively correlated with the decline in the oxidative molecular damage to proteins and enhanced mitochondrial complex IV activity in different regions of ageing brain as well as peripheral organs. The work was further extended to study the expression of synaptic plasticity-related proteins, such as synaptophysin, calcineurin and CaM kinase II to explore the molecular basis of IF-DR regimen to improve cognitive function. These results suggest that even late-onset short-term IF-DR regimen have the potential to retard age-associated detrimental effects, such as cognitive and motor performance as well as oxidative molecular damage to proteins.

Prolonged glutamate excitotoxicity: Effects on mitochondrial antioxidants and antioxidant enzymes

Glutamate, a major excitatory amino acid neurotransmitter is also an endogenous excitotoxin. The present study examined the prolonged and delayed effects of glutamate excitotoxicity on mitochondrial lipid peroxidation and antioxidant parameters in different brain regions, namely, cerebral hemisphere, cerebellum, brain stem and diencephalon. Wistar rats (male) were exposed to monosodium glutamate (MSG) (4 mg × g body wt−1, i.p.) for 6 consecutive days and sacrificed on 30th and 45th day after last MSG dose. MSG treatment markedly decreased the mitochondrial manganese superoxide-dismutase (Mn-SOD), catalase and reduced glutathione (GSH) content, and increased the lipid peroxidation (LPx), uric acid and glutathione peroxidase (GPx) activity. These results indicate that oxidative stress produced by glutamate in vulnerable brain regions may persist for longer periods and mitochondrial function impairment is an important mechanism of excitatory amino acid mediated neurotoxicity in chronic neurodegeneration.

Water Extract from the Leaves of Withania somnifera Protect RA Differentiated C6 and IMR-32 Cells against Glutamate-Induced Excitotoxicity

Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative disorders. Search for herbal remedies that may possibly act as therapeutic agents is an active area of research to combat these diseases. The present study was designed to investigate the neuroprotective role of Withania somnifera (Ashwagandha), also known as Indian ginseng, against glutamate induced toxicity in the retinoic acid differentiated rat glioma (C6) and human neuroblastoma (IMR-32) cells. The neuroprotective activity of the Ashwagandha leaves derived water extract (ASH-WEX) was evaluated. Cell viability and the expression of glial and neuronal cell differentiation markers was examined in glutamate challenged differentiated cells with and without the presence of ASH-WEX. We demonstrate that RA-differentiated C6 and IMR-32 cells, when exposed to glutamate, undergo loss of neural network and cell death that was accompanied by increase in the stress protein HSP70. ASH-WEX pre-treatment inhibited glutamate-induced cell death and was able to revert glutamate-induced changes in HSP70 to a large extent. Furthermore, the analysis on the neuronal plasticity marker NCAM (Neural cell adhesion molecule) and its polysialylated form, PSA-NCAM revealed that ASH-WEX has therapeutic potential for prevention of neurodegeneration associated with glutamate-induced excitotoxicty.

Impact of diabetes on CNS: Role of signal transduction cascade

Diabetic neuropathy is the most common secondary complication of diabetes mellitus. Several pathogenetic factors have been proposed for diabetic neuropathy. The present investigation was undertaken to study different components of signal transduction from discrete brain regions from streptozotocin-induced diabetic rats. Rats were sacrificed after 1 and 3 months of induction of diabetes, and a control group was also studied in parallel to ascertain the specificity of diabetes-associated changes. Blood glucose level and protein content of discrete brain regions were also estimated. Signal transduction cascade components like protein kinase A, protein kinase C, cAMP, phospholipase C, phospholipase A2, diacylglycerol and inositol phosphate levels were assayed in control and diabetic groups of rats. Significant attenuation in phosphoinositide metabolism along with activation of protein kinase activities were observed. These findings provide evidence to suggest a mechanism linking changes in signal transduction cascade, which is observed in 1- and 3-month diabetic rats, which ultimately leads to development of diabetic neuropathy.

Neuroendocrine plasticity in GnRH release during rat estrous cycle: correlation with molecular markers of synaptic remodeling.

Morphological changes in the gonadotropin releasing hormone (GnRH) neurons in the preoptic area (POA) and their terminals in the median eminence-arcuate (ME-ARC) region are reported to occur during ovarian cycle that may be involved in the GnRH release into the portal blood during preovulatory surge. However, the neuronal substrates participating in altered GnRH neuronal plasticity are poorly understood. The present study was designed to determine whether morphological changes occurring in the GnRH neuron cell bodies in the POA and their terminals in the ME-ARC region of hypothalamus with pulsatile GnRH release in cycling female rats are associated with expression of intrinsic determinants of neuronal plasticity. The plasticity markers studied are polysialylated neural cell adhesion molecule (PSA-NCAM), high molecular weight isoforms of NCAM, growth associated protein (GAP-43), glial fibrillary acidic protein (GFAP) and synaptophysin. Regularly cycling female rats were sacrificed at diestrous, i.e., when GnRH release is low, and at proestrous, i.e., when preovulatory GnRH surge occurs, using perfusion fixation method for immunohistochemical staining of GnRH cells. GnRH cell bodies and their terminals from the POA and ME-ARC region respectively, were localized using immunohistochemical technique in proestrous and diestrous phase of estrous cycle and our results showed a marked increase in the GnRH nerve terminals length and immunoreactivity in the ME-ARC region from proestrous phase rats as compared to diestrous rats. Immunoblot analyses of the POA and ME-ARC region of the hypothalamus revealed a significant increase in the content of PSA-NCAM, NCAM-180, NCAM-140, GAP-43 and synaptophysin from proestrous phase rats as compared to diestrous phase rats. The ME-ARC region showed more pronounced increase in the protein expression of these markers of neuronal plasticity as compared to the POA, whereas, hippocampal region did not show any significant change in the content of these markers showing specificity of the changes to the GnRH system. GFAP content was significantly decreased in the POA with a marginal increase in the GFAP level from the ME-ARC region. These results demonstrate the involvement of synaptic proteins in the dynamic plasticity of the ME-ARC region of hypothalamus, allowing GnRH nerve terminals to release the neurohormone into the pituitary portal blood on the day of proestrous.