G. Nagesh Babu

Metabolomic analysis of serum by (1) H NMR spectroscopy in amyotrophic lateral sclerosis.

BACKGROUND Amyotrophic lateral sclerosis (ALS), an invariably fatal neurological disorder shows complicated pathogenesis that poses challenges with respect to diagnosis as well as monitoring of disease progression. METHODS We investigated metabolite profiles in the serum of 30 patients with ALS, 10 patients of Hirayama disease, which served as a neurological disease control and 25 healthy controls by using (1) H NMR spectroscopy. RESULTS Compared to healthy controls, the ALS patients had higher quantities of glutamate (P<0.001), beta-hydroxybutyrate (P<0.001), acetate (P<0.01), acetone (P<0.05), and formate (P<0.001), and lower concentrations of glutamine (P<0.02), histidine (P<0.001) and N-acetyl derivatives. On the other hand, Hirayama disease patients had significantly higher median concentrations of pyruvate (P<0.05), glutamate (P<0.001), formate (P<0.05) and lower median concentrations of N-acetyl derivatives. Furthermore, we also found that serum glutamate showed a positive correlation (P<0.001, r=0.6487) whereas, histidine showed a negative correlation (P<0.001, r=-0.5641) with the duration of the disease in ALS. CONCLUSIONS Such (1) H NMR study of serum may reveal abnormal metabolite patterns, which could have the potential to serve as surrogate markers for monitoring ALS disease progression.

Oxidant-antioxidant imbalance in the erythrocytes of sporadic amyotrophic lateral sclerosis patients correlates with the progression of disease.

Free radicals are implicated in numerous disease processes including motor neuron degeneration (MND). Antioxidant defense enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the erythrocytes are capable of detoxifying reactive oxygen species produced endogenously or exogenously. In the present study, the extent of lipid peroxidation (LPO) and antioxidant defenses were evaluated in the erythrocytes of 20 sporadic amyotrophic lateral sclerosis (ALS) patients and 20 controls. We observed that lipid peroxidation in the erythrocytes of amyotrophic lateral sclerosis patients significantly increased with respect to controls (P<0.001). On the other hand, catalase activity was found to be significantly lower (P<0.001). The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione levels were also found to be significantly reduced in ALS patients compared to healthy subjects (P<0.001, P<0.01 and P<0.01, respectively). It was further observed that lipid peroxidation started to increase and catalase, glutathione reductase, glucose-6-phosphate dehydrogenase enzyme activities and glutathione levels started to decrease as amyotrophic lateral sclerosis progressed from 6 to 24 months, suggesting a correlation between these parameters and duration of amyotrophic lateral sclerosis. This study confirms the involvement of oxidative stress during the progression of amyotrophic lateral sclerosis and the need to develop specific peripheral biomarkers.

Cell death mechanisms in the early stages of acute glutamate neurotoxicity

The present study focused on the early stages of acute glutamate (L-Glu)-induced neurotoxic mechanisms, both biochemical, e.g. intracellular reactive oxygen species (ROS) and associated parameters as well as gene expression of cell survival/death pathways, i.e. Bcl-2 and caspases. Stereotactic intracortical injections of L-Glu (1micromol/1microl) resulted in decreased size of pyramidal neurons in rat after 1h. We also observed that intracellular ROS, calcium (Ca(2+)) and peroxynitrite (ONOO(-)) production were significantly elevated, whereas, mitochondrial transmembrane potential (DeltaPsim) and total glutathione were significantly decreased by L-Glu bolus. The Bcl-2/Bax ratio in the L-Glu-injected rats was found to be significantly lower than the controls. Moreover, acute L-Glu significantly induced mRNA expression of nNOS, iNOS, caspase-3 and caspase-9. It may be concluded from the present study that acute L-Glu administration, at an early stage, increases intracellular ROS accumulation, Ca(2+) levels and peroxynitrite production and decreases glutathione pool. Furthermore, it appears that decreased mitochondrial Bcl-2/Bax ratio might have upregulated the mRNA expression of caspase-3 and caspase-9 which launch cell death cascade. Regarding the chronology of the events, we presume that acute L-Glu increases ROS and decreases DeltaPsim and glutathione rapidly and it is more likely that these events precede gene expression changes, ultimately resulting in neuronal damage/death.

Proinflammatory cytokine levels in the serum and cerebrospinal fluid of tuberculous meningitis patients.

The pathophysiology underlying tuberculous meningitis (TBM), the most prominent extra pulmonary tuberculosis and a serious public health problem in developing countries is still unclear. Whereas, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) are cytokines involved in cell-mediated immune response. TNF-alpha and IFN-gamma production has earlier been shown to be associated with tissue necrosis. To see whether these cytokines have any role to play in the pathophysiology of TBM, we measured the levels of serum and cerebrospinal fluid (CSF) TNF-alpha and IFN-gamma in 31 consecutive patients of TBM by ELISA. There was a remarkable rise (P<0.001) in the levels of serum and CSF TNF-alpha and IFN-gamma levels in TBM patients with respect to 20 age and sex-matched control subjects. Furthermore, TNF-alpha and IFN-gamma levels showed a positive correlation with the severity of the disease at the end of 6 months of antibiotic therapy. Elevated TNF-alpha and IFN-gamma levels, especially in CSF, despite of these patients undergoing multidrug therapy suggests the persistence of central nervous system inflammation. We also found an associated rise (P<0.001) in the nitric oxide (NO) levels of serum and CSF but there was no correlation between NO levels and the severity of TBM. The continuous release of cytokines despite these patients undergoing anti-tubercular therapy suggests that TBM severity may result mainly from the immune response rather than the organism itself.

Effect of neonatal monosodium glutamate on lipid peroxidation in adult rat brain.

Glutamate (glu), an excitatory amino acid (EAA) abundantly present in the brain of mammals, is also a neurotoxin. We examined lipid peroxidation (LPO) potential and antioxidant parameters of midbrain region (MBR) and frontal cortex of adult rats following treatment with monosodium glutamate (MSG) during postnatal day (PND) 1 through PND 10 at a daily dose of 4 mg g-1 body weight. In PND 90 rats MSG increased LPO by 56% and altered antioxidant status of MBR. This indicates that oxidative stress produced by glu in vulnerable brain regions may persist for prolonged periods and could be one of the mechanisms of EAA neurotoxicity resulting in chronic neurodegeneration.

Blood glutamate levels in patients with motor neuron disease

This study was undertaken to evaluate the role of excitatory amino acid glutamate (Glu) in the pathophysiology of motor neuron disease (MND). It was observed that blood Glu levels were significantly higher in MND patients with respect to healthy controls. The data indicate that Glu homeostasis is altered in the patients with MND.

Single microinjection of L‐glutamate induces oxidative stress in discrete regions of rat brain

Glutamate (Glu) is a major excitatory aminoacid neurotransmitter in the mammalian brain. Under Certain Circumstances Glu can also exert toxic effects on neuronal Cells. To unravel the biochemical mechanisms of Glu‐induced acute neuronal injury, Glu 1μmol/1μl was microinjected into cerebral Cortex, striatum and hippocampus of adult rats and oxidative stress and antioxidant parameters were evaluated. The results show that the rate of lipid peroxidation was significantly increased in the above brain regions following Glu administration suggesting neuronal membrane damage and also the total and free sulfhydryl groups were significantly depleted, indicating altered red‐ox status of the cells. There was also alteration in the activity of antioxidant enzyme catalase in cerebral cortex. Some of the above Glu‐induced effects were reversed or modified by NMDA receptor antagonist MK‐801.

Inflammatory markers in the patients of Japanese encephalitis

Abstract Objectives: Japanese encephalitis (JE) is one of the commonest viral encephalitis especially prevalent in Southeast Asia. Estimated mortality rate of JE is ∼30%, with survivors undergoing severe and irreversible neurological sequelae. Although central nervous system (CNS) inflammation is imminent upon JE infection, the pathways underlying the same have not yet been clearly elucidated. However, cytokines–tumor necrosis factor-α (TNF-α) and interlukin-2 (IL-2), are small secreted proteins, which mediate and regulate immunity. Therefore, we wanted to evaluate the role, if any, of these cytokines in the pathogenesis of JE. Methods: We measured the levels of TNF-α and IL-2 in the serum and cerebrospinal fluid (CSF) of patients suffering JE, using enzyme-linked immunosorbent assay (ELISA). Results: JE infection caused a remarkable increase (p<0.0001) in the levels of TNF-α in the serum and CSF, while IL-2 levels were unaffected. Discussion: These results show that TNF-α pathway is involved in JE infection-triggered neuroinflammation.

Ornithine Decarboxylase Activity in In Vivo and In Vitro Models of Cerebral Ischemia

Ornithine decarboxylase (ODC) is considered the rate-limiting enzyme in polyamine biosynthesis, and an increase in putrescine after central nervous system (CNS) injury appears to be involved in neuronal death. Cerebral ischemia and reperfusion trigger an active series of metabolic events, which eventually lead to neuronal death. In the present study, ODC activity was evaluated following transient focal cerebral ischemia and reperfusion in rat. The middle cerebral artery (MCA) was occluded for 2 h in male rats with an intraluminal suture technique. Animals were sacrificed between 3 and 48 h of reperfusion following MCA occlusion, and ODC activity was assayed in cortex and striatum. ODC activity was also estimated in an in vitro ischemia model using primary rat cortical neuron cultures, at 6–24 h reoxygenation following 1 h oxygen-glucose deprivation (OGD). In cortex, following ischemia, ODC activity was increased at 3 h (P < .05), reached peak levels by 6–9 h (P < .001) and returned to sham levels by 48 h reperfusion. In striatum the ODC activity followed a similar time course, but returned to basal levels by 24 h. This suggests that ODC activity is upregulated in rat CNS following transient focal ischemia and its time course of activation is region specific. In vitro, ODC activity showed a significant rise only at 24 h reoxygenation following ischemic insult. The release of lactate dehydrogenase (LDH), an indicator for cell damage, was also significantly elevated after OGD. 0.25 mM α-difluoromethylornithine (DFMO) inhibited ischemia-induced ODC activity, whereas a 10-mM dose of DFMO appears to provide some neuroprotection by suppressing both ODC activity and LDH release in neuronal cultures, suggesting the involvement of polyamines in the development of neuronal cell death.

Cerebrospinal Fluid Catecholamine Levels in Japanese Encephalitis Patients with Movement Disorders

Norepinephrine and dopamine have important role in movement disorders but their role in movement disorders associated with Japanese encephalitis (JE) has not been evaluated. Therefore, in the present study, cerebrospinal fluid (CSF) catecholamine levels and its metabolites in JE patients with movement disorders were compared with those without JE. CSF was collected by lumbar puncture and analyzed by HPLC-ED. Norepinephrine, dopamine and homovanillic acid concentrations were significantly (P<0.005) lower in JE patients compared to control groups. Low levels of catecholamines in JE associated movement disorders compared to idiopathic Parkinson’s disease and other extrapyramidal symptoms may be due to severe structural damage to thalamus, basal ganglia and brainstem in JE patients as revealed by MRI findings.