An early glycolysis burst in microglia regulates mitochondrial dysfunction in oligodendrocytes under neuroinflammation.

Metabolism and energy processes governing oligodendrocyte function during neuroinflammatory disease are of great interest. However, how varied cellular environments affect oligodendrocyte activity during neuroinflammation is unknown. We demonstrate that activated microglial energy metabolism controls oligodendrocyte mitochondrial respiration and activity. Lipopolysaccharide/interferon gamma promote glycolysis and decrease mitochondrial respiration and myelin protein synthesis in rat brain glial cells. Enriched microglia showed an early burst in glycolysis. In microglia-conditioned medium, oligodendrocytes did not respire and expressed less myelin. SCENITH revealed metabolic derangement in microglia and O4-positive oligodendrocytes in endotoxemia and experimental autoimmune encephalitogenic models. The early burst of glycolysis in microglia was mediated by PDPK1 and protein kinase B/AKT signaling. We found that microglia-produced NO and itaconate, a tricarboxylic acid bifurcated metabolite, reduced mitochondrial respiration in oligodendrocytes. During inflammation, we discovered a signaling pathway in microglia that could be used as a therapeutic target to restore mitochondrial function in oligodendrocytes and induce remyelination.

Piperine ameliorates oxidative stress, inflammation and histological outcome in collagen induced arthritis.

OBJECTIVES: Piperine, a main component of Piper species, is a plant alkaloid with a long history of medical use in a variety of inflammatory disorders like rheumatoid arthritis. Due to side effects in current treatment modalities of rheumatoid arthritis, the interest in alternative, well tolerated anti-inflammatory remedies has re-emerged. The aim of this work was to evaluate the anti-inflammatory and antiarthritic effects of piperine. METHODS: Arthritis was induced in male Wistar rats by collagen induced arthritis (CIA) method. Piperine was administered at a dose of 100mgkg(-1) and indomethacin at 1mgkg(-1) body weight once daily for 21days. The effects of treatment in the rats were assessed by biochemical (articular elastase, MPO, LPO, GSH, Catalase, SOD and NO), inflammatory mediators (IL-1ß, TNF-α, IL-10 and PGE2) and histological studies in joints. RESULTS: Piperine was effective in bringing significant changes on all the parameters (articular elastase, MPO, LPO, GSH, Catalase, SOD and NO) studied. Oral administration of piperine resulted in significantly reduced the levels of pro-inflammatory mediators (IL-1ß, TNF-α and PGE2) and increased level of IL-10. The protective effects of piperine against RA were also evident from the decrease in arthritis scoring and bone histology. CONCLUSIONS: In conclusion, the fact that piperine alter a number of factors known to be involved in RA pathogenesis indicates that piperine can be used similar to indomethacin as a safe and effective therapy for CIA and may be useful in the treatment of rheumatoid arthritis.

Quercetin prevents protein nitration and glycolytic block of proliferation in hydrogen peroxide insulted cultured neuronal precursor cells (NPCs): Implications on CNS regeneration.

Survival along with optimal proliferation of neuronal precursors determines the outcomes of the endogenous cellular repair in CNS. Cellular-oxidation based cell death has been described in several neurodegenerative disorders. Therefore, this study was aimed at the identification of the potent targets of oxidative damage to the neuronal precursors and its effective prevention by a natural flavonoid, Quercetin. Neuronal precursor cells (NPCs), Nestin+ and GFAP (Glial fibrillary acidic protein)+ were isolated and cultured from adult rat SVZ (subventricular zone). These cells were challenged with a single dose of H2O2 (50µM) and/or pre-treated with different concentrations of Quercetin. H2O2 severely limited the cellular viability and expansion of the neurospheres. Cellular-oxidation studies revealed reduction in glutathione dependent redox buffering along with depletion of enzymatic cellular antioxidants that might potentiate the nitrite (NO2(-)) and superoxide anion (O2(-)) mediated peroxynitrite (ONOO(-)) formation and irreversible protein nitration. We identified depleted PK-M2 (M2 isoform of pyruvate kinase) activity and apoptosis of NPCs revealed by the genomic DNA fragmentation and elevated PARP (poly ADP ribose polymerase) activity along with increased Caspase activity initiated by severely depolarised mitochondrial membranes. However, the pre-treatment of Quercetin in a dose-response manner prevented these changes and restored the expansion of neurospheres preferably by neutralizing the oxidative conditions and thereby reducing peroxynitrite formation, protein nitration and PK-M2 depletion. Our results unravel the potential interactions of oxidative environment and respiration in the survival and activation of precursors and offer a promise shown by a natural flavonoid in the protective strategy for neuronal precursors of adult brain.

Hesperidin inhibits collagen-induced arthritis possibly through suppression of free radical load and reduction in neutrophil activation and infiltration.

Rheumatoid arthritis is a chronic inflammatory disease characterized by the destruction of articular cartilage and bone in a chronic phase. Pathology of rheumatoid arthritis suggests autoimmunity linked to inflammation. In our study, rheumatoid arthritis was induced in Wistar rats by intradermal injections of 100 µl of emulsion containing bovine type II collagen in complete Freund's adjuvant at the base of the tail. Disease developed about 13 ± 1 days after immunization and treatment with hesperidin (HES) at a dose of 160 mg kg(-1) body weight was given after onset of disease daily until 20th day. The effect of treatment in the rats was monitored by clinical scoring, biochemical parameters and histological evaluations in joints. A steady increase in the articular elastase, nitric oxide and lipid peroxidation was observed in joints of arthritic rats as compared to control, whereas a significant decrease in reduced glutathione, superoxide dismutase activity and catalase was observed in collagen-induced arthritis rats as compared to control group. The results from the present work indicate that the treatment with hesperidin was effective in bringing about significant changes on all the parameters studied in collagen-induced arthritis rats. These data confirm that erosive destruction of the joint cartilage in collagen-induced arthritis is due free radicals released by activated neutrophils and produced by other biochemical pathways. In the present study, an attempt has been made to amelioration of the disease process by a natural product. These results suggest that oral administration of HES could be effective for treating human RA patients.

Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces S-phase population in human breast cancer cells (MCF-7).

Venom of some species of scorpions induces apoptosis and arrests proliferation in cancer cells. This is an important property that can be harnessed and can lead to isolation of compounds of therapeutic importance in cancer research. Cytotoxicity was investigated using MTT reduction and confirmed with lactate dehydrogenase release following venom exposure. Apoptosis was evaluated with determination of mitochondrial membrane potential, reactive nitrogen species assay, measurement of Caspase-3 activity and DNA fragmentation analysis. To confirm that venom can inhibit DNA synthesis in proliferating breast cancer cells, immunocytochemical detection of BrdU incorporation was done. Our results demonstrated that venom of Odontobuthus doriae not only induced apoptosis but lead to the inhibition of DNA synthesis in human breast cancer cells (MCF-7). Cell viability decreased with parallel increment of LDH release in dose dependent manner after treatment with varying concentrations of venom. Moreover, venom depleted cellular antioxidants evidenced by depression of GSH and Catalases and concomitantly increased reactive nitrogen intermediates (RNI). These events were related to the depolarization of mitochondria and associated Caspase-3 activation following venom treatment in a concentration dependent manner. Finally, fragmentation of nuclear DNA following venom treatment confirmed the apoptotic property of the said venom. These results suggest that venom of O. doriae can be potential source for the isolation of effective anti-proliferative and apoptotic molecules.

Scorpion (Androctonus crassicauda) venom limits growth of transformed cells (SH-SY5Y and MCF-7) by cytotoxicity and cell cycle arrest.

The purpose of study was to examine the cytotoxic and anti-cancer properties along with addressing the plausible pathway followed by scorpion venom to reduce cell viability in SH-SY5Y and MCF-7 cells. Following exposure of cells with scorpion venom, cytotoxicity was estimated using MTT and lactate dehydrogenase assays. Apoptotic effects were measured by assessment of mitochondrial membrane potential, reactive nitrogen species, DNA fragmentation, and caspase-3 activity whereas antiproliferative effect was assayed using BrdU incorporation. Our results indicate that scorpion venom causes suppression of proliferation by arresting S-phase and induction of apoptosis through increased nitric oxide production, caspase-3 activity and depolarization of mitochondrial membrane. Induction of apoptosis and arrest of DNA synthesis are critical determinant factors for development of anti cancer drugs. These properties may lead to isolation of effective molecule(s) with potential anticancer activity from scorpion venom of Androctonus crassicauda.

Increased spontaneous apoptosis of rat primary neurospheres in vitro after experimental autoimmune encephalomyelitis.

Survival of neuronal progenitors (NPCs) is a critical determinant of the regenerative capacity of brain following cellular loss. Herein, we report for the first time, the increased spontaneous apoptosis of the first acute phase of Experimental Autoimmune Encephalomyelitis (EAE) derived neurospheres in vitro. Neuronal as well as oligodendroglial loss occurs during experimental autoimmune encephalomyelitis (EAE). This loss is replenished spontaneously by the concomitant increase in the NPC proliferation evidenced by the presence of thin myelin sheaths in the remodeled lesions. However, remyelination depends upon the survival of NPCs and their lineage specific differentiation. We observed significant increase (P < 0.001) in number of BrdU (+) cells in ependymal subventricular zone (SVZ) in EAE rats. EAE derived NPCs showed remarkable increase in S-phase population which was indeed due to the decrease in G-phase progeny suggesting activation of neuronal progenitor cells (NPCs) from quiescence. However, EAE derived neurospheres showed limited survival in vitro which was mediated by the significantly (P < 0.01) depolarized mitochondria, elevated Caspase-3 (P < 0.001) and fragmentation of nuclear DNA evidenced by single cell gel electrophoresis. Our results suggest EAE induced spontaneous apoptosis of NPCs in vitro which may increase the possibility of early stage cell death in the negative regulation of the proliferative cell number and may explain the failure of regeneration in human multiple sclerosis.

Neglected foreign body infratemporal fossa, a typical presentation: a case report.

A 25 year old male presented to our ENT OPD with the history of hyperemic swelling left zygomatic area with a pointed pustule in the centre resembling a furuncle,which progressed into a discharging sinus, complicated by trismus. The patient had a past history of trauma in the vicinity of the left zygomatic area 4-months back with a pointed stick the tip of the stick which had got lodged in the wound at that time was removed. The diagnostic dilemma was solved as neglected foreign body infratemporal fossa.

Scorpion (Odontobuthus doriae) venom induces apoptosis and inhibits DNA synthesis in human neuroblastoma cells.

Scorpion and its organs have been used to cure epilepsy, rheumatism, and male impotency since medieval times. Scorpion venom which contains different compounds like enzyme and non-enzyme proteins, ions, free amino acids, and other organic inorganic substances have been reported to posses antiproliferative, cytotoxic, apoptogenic, and immunosuppressive properties. We for the first time report the apoptotic and antiproliferative effects of scorpion venom (Odontobuthus doriae) in human neuroblastoma cells. After exposure of cells to medium containing varying concentrations of venom (10, 25, 50, 100, and 200 µg/ml), cell viability decreased to 90.75, 75.53, 55.52, 37.85, and 14.30%, respectively, after 24 h. Cells expressed morphological changes like swelling, inhibition of neurite outgrowth, irregular shape, aggregation, rupture of membrane, and release of cytosolic contents after treatment with venom. Lactate dehydrogenase (LDH) level increased in 50 and 100 µg/ml as compared to control, but there was no significant increase in LDH level at a dose of 10 and 20 µg/ml. Two concentrations viz. 50 and 100 µ/ml were selected because of the profound effect of these concentrations on the cellular health and population. Treatment with these two concentrations induced reactive nitrogen intermediates and depolarization in mitochondria. While caspase-3 activity increased in a concentration-dependent manner, only 50 µg/ml was able to fragment DNA. It was interesting to note that at higher dose, i.e., 100 µg/ml, the cells were killed, supposedly by acute necrosis. DNA synthesis evidenced by bromodeoxyuridine (BrdU) incorporation was inhibited in a concentration-dependent manner. The cells without treatment incorporated BrdU with high affinity confirming their cancerous nature whereas very less incorporation was noticed in treated cells. Our results show apoptotic and antiproliferative potential of scorpion venom (O. doriae) in human neuroblastoma cells. These properties make scorpion venom a valuable therapeutic agent in cancer research.

Cytokinetics of adult rat SVZ after EAE.

Cytokinetics regulating cell cycle division can be modulated by several endogenous factors. EAE (experimental autoimmune encephalomyelitis) increases proliferation of progenitor cells in the subventricular zone (SVZ). Using cumulative and single S phase labeling with 5-bromo-2-deoxyuridine, we examined cell cycle kinetics of neural progenitor cells in the SVZ after EAE. 20% of the SVZ cell population was proliferating in adjuvant control rats. However, EAE significantly increased them up to 27% and these cells had a cell cycle length (TC) of 15.6h, significantly (P<0.05) shorter than the 19 h TC in non EAE SVZ cells. Few TUNEL (+) cells were detected in the SVZ cells of adjuvant controls. EAE increased (P<0.05) TUNEL (+) nuclei in SVZ suggesting early stage progenitor cell death. Cell cycle phase analysis revealed that EAE substantially shortened the length of the G1 phase (9.6h) compared with the G1 phase of 12.25 h in adjuvant control SVZ cells (P<0.05). This reduction in G1 contributes to EAE-induced reduction of TC because no significant changes were detected on the length of S, G2 and M phases between the two groups. Our results show a surge in proliferating progenitor cells in the SVZ with concomitant increase in apoptotic cell death after EAE. Furthermore, increase in the SVZ proliferation contributes to EAE-induced neurogenesis and this increase is regulated by shortening the G1 phase. Our investigation suggests the activation of quiescent cells in SVZ to generate actively proliferating progenitors. Moreover, the increase in the cell death in proliferating population may contribute towards negative regulation of proliferative cell number and hence diminished regenerative capacity of CNS following EAE.

Upregulation of CSPG3 accompanies neuronal progenitor proliferation and migration in EAE.

The molecular identities of signals that regulate the CNS lesion remodeling remain unclear. Herein, we report for the first time that extracellular matrix chondroitin sulphate proteoglycan, CSPG3 (neurocan) is upregulated after primary inflammatory injury. EAE was induced using myelin oligodendrocyte glycoprotein (MOG) (35-55) which was characterized by massive polymorphonuclear cell infiltration and loss of myelin basic protein expression along with steep decrease of CNPase. Periventricular white matter (PVWM) and cortex presented with astrogliosis evidenced by increased Glial fibrillary acidic protein (GFAP) immunoreactivity 20 days post immunization (p.i). Neuronal progenitor cell (NPC) proliferation increased after first acute episode in the subventricular zone (SVZ), corpus callosum, and cortex, indicating migration of cells to structures other than rostral migration stream and olfactory bulb, which is indicative of cell recruitment for repair process and was confirmed by presence of thin myelin sheaths in the shadow plaques. Earlier CSPG3 has been demonstrated to impede regeneration. We observed neuroinflammation-induced up-regulation of the CSPG3 expression in two most affected regions viz. PVWM and cortex after proliferation and migration of NPCs. Our results show possible role of reactive astrogliosis in lesion remodeling and redefine the relation between inflammation and endogenous cellular repair which can aid in designing of newer therapeutic strategies.

Hippocampal neurodegeneration in experimental autoimmune encephalomyelitis (EAE): potential role of inflammation activated myeloperoxidase.

Experimental Autoimmune Encephalomyelitis (EAE) is a well-established animal model of human multiple sclerosis (MS). The effect of this inflammatory disease on hippocampus has not been addressed. Keeping in view the above consideration an attempt was made to delineate the effect of EAE on the hippocampus of Wistar rats. The assessment of the damage to the hippocampus was done 16 days post induction by the immunolocalization of ChAT (choline acetyl transferase). ChAT decreased remarkably after induction that revealed cholinergic neuronal degeneration in the hippocampus. Subsequently, many biochemical parameters were assessed to ascertain inflammatory activation of nitric oxide and associated oxidative damage as a putative mechanism of the cholinergic degeneration. Nitric oxide metabolites increased significantly (P < 0.05) with enhancement of MPO (Myeloperoxidase activity) (P < 0.001) in the MOG (myelin oligodendrocyte protein) group as compared to the controls. Peroxidation of biomembranes increased (P < 0.001), while reduced glutathione depleted (P < 0.001) with parallel decrease in catalase (P < 0.01) and superoxide dismutase enzyme activity (P < 0.001) in the MOG group. Our results show a strong role of peroxidase dependent oxidation of nitrite and oxidative stress in cholinergic degeneration in EAE.

Acute epiglottitis: A review of 50 patients.

We reviewed 50 patients admitted to the department of Otorhinolaryngology and Head & Neck Surgery of Govt Medical College Srinagar from September 19% to September 2002 diagnosed with acute epiglottitis. Male were more commonly involved than females in the ratio of 2.8:1 with only 6 cases younger than 10 years of age. The highest incidence was in the month of January (22%). The common symptoms of acute epiglottitis were sorethroat(92%) and odynophagia(88%). Any patient with sudden onset of these symptoms should be suspected of having acute epiglottitis and should have an indirect laryngoscopy. Blood culture was obtained in 20 cases Cultures were positive only in 5 cases, out of which 4 were positive for Hemophilus influenzae type B. Throat cultures were not obtamed The primary treatment of acute epiglottitis is intravenous antibiotics, steriods, and humidified air. Treacheostomy was needed only in 4 patients. There were no deaths.