Bacopa monnieri extracts prevent hydrogen peroxide-induced oxidative damage in a cellular model of neuroblastoma IMR32 cells / 中国天然药物

Neurodegenerative diseases are the consequences of imbalance between the production of oxidative stress and its nullification by cellular defense mechanisms. Hydrogen peroxide (HO), a precursor of deleterious reactive oxygen species, elicits oxidative stress, resulting in severe brain injuries. Bacopa monnieri is well known for its nerve relaxing and memory enhancing properties. The present study was designed to evaluate the protective effects of extracts from Bacopa monnieri against HO induced oxidative stress using a cellular model, neuroblastoma IMR32 cell line. The protective potential of methanolic, ethanolic, and water extracts of B. monnieri (BM-MEx, BM-EEx, and BM-WEx) was evaluated using MTT assay. Although, all the B. monnieri extracts were found to protect cells against HO-mediated stress but BM-MEx showed significantly greater protection. UPLC analysis of BM-MEx revealed various polyphenols, including quercetin, catechin, umbelliferone, and caffeic acid predominance. Further, BM-MEx was found to possess considerable greater neuroprotective potential in comparison to the standard polyphenols such as quercetin, catechin, umbelliferone, and caffeic acid. The levels of antioxidant enzymes were significantly elevated after the pretreatment of BM-MEx and quercetin. The expression levels of oxidative stress markers, such as NF200, HSP70, and mortalin, were significantly alleviated after the pretreatment of BM-MEx as shown by immunofluorescence and RT-PCR. In conclusion, the present study demonstrated the protective effects of BM-MEx, suggesting that it could be a candidate for the development of neuropathological therapeutics.

Effects of Fasudil on the expression of Nogo-A and NF200 in neonatal hypoxic-ischemic brain damage rats / 中华实用儿科临床杂志

Objective To study the effects of Fasudil on expression of Nogo-A and NF200 in neonatal hypoxic-ischemic brain damage(HIBD) rats.Methods One hundred and twenty 7-day-old Wistar rats were divided into 3 groups with random number table:Sham operation group (n =40),HIBD group (n =40) and Fasudil group (n =40).Sham group only separated from the common carotid artery,without ligation,direct suture the incision does not do hypoxia; HIBD group were injected with saline; Fasudil group was injected with fasudil(10 mg/kg).The rats were killed at 6 h,12 h,24 h,72 h,7 d,after administration.The pathological changes were observed by means of HE.The expression of Nogo-A and NF200 was studied with immunohistochemical staining.Results 1.Naked eye observation:Sham group bilateral symmetrical cerebral hemispheres; HIBD group of brain edema aggravated,the visible hemisphere focal necrosis; fasudil treatment group of edema than HIBD group ease.2.HE stain:the structure and shape of brain in Sham operation group were normal.In HIBD group,the cells became edema,karyopyknosis,lyse,and the inflammatory cells became more.The number of edema cells and karyopyknosis decreased in Fasudil group.3.Immunohistochemical stain:there were less expressions of Nogo-A in Sham operation group.It increased slightly after 12 h in HIBD group but decreased later.The expression of Nogo-A in Fasudil group was less than the other two groups at any time except 6 h (P ＜0.01).There was more expression in HIBD and Fasudil group compared with Sham operation group(P ＜0.01).NF200 was less expression in Sham operation group.NF200 appeared after 6 h and became less after 12 h.The expression of NF200 was at 24 h and later became more.The expression of NF200 in Fasudil group was more with HIBD group at each different time (P ＜ 0.01).The expressions of NF200 in Fasudil and HIBD group were more compared with Sham operation group.Conclusions Fasudil can rehabilitate the damaged axon and promote nerve regeneration through controlling the Rho/Rock and make the expression of NF200 increase.

Neuroprotective Effect of Convolvulus pluricaulis Methanol Extract on Hydrogen Peroxide Induced Oxidative Stress in Human IMR32 Neuroblastoma Cell Line.

Aims: The present study aimed to evaluate and ascertain the protective role of methanolic/ethanolic/water extracts of Convolvulus pluricaulis against H2O2 induced cytotoxicity in IMR32 Neuroblastoma cell line as model system and identify the factor responsible for the protective effect. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar & Department of Biotechnology, DAV College, Amritsar, PuCPab, between August 2010 and March 2012. Methodology: Firstly, cytotoxic dose of H2O2 and non-toxic dose of methanolic, ethanolic and water extracts of C. pluricaulis (CP-MEx, CP-EEx and CP-WEx respectively) was determined by MTT assay. Protective effect of CP-MEx, CP-EEx and CP-WEx was determined using quercetin as a positive control. The expression of IMR32 cytoskeletal marker, Neurofilament (NF-200) and stress markers, Heat shock protein (HSP70) and (glucose regulated protein 75, Grp75) Mortalin studied by immunofluorescence and RTPCR results. The level of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase, direct scavenger of free radicals, Glutathione and lipid peroxidation were analysed by their standard procedures. Results: The results showed that quercetin, CP-MEx, CP-EEx and CP-WEx displayed cytoprotective activity in IMR32 cells. Out of tested extracts CP-MEx significantly decreased hydrogen peroxide-induced cell death. Significant decrease in NF-200, HSP70 and Mortalin expression was observed in CP-MEx+H2O2 treated cultures as compared to H2O2 treated. Catalase, superoxide dismutase, glutathione peroxidase, Glutathione levels significantly increased in Quercetin and CP-MEx treated cultures. Lipid peroxidation was significantly decreased in both Quercetin and CP-MEx treated cultures. Conclusions: The present work establishes the protective effect of CP-MEx on IMR 32 Human Neuroblastoma cell line which is as much as by quercetin. The cytoprotective effect of CP-MEx was due to induction of antioxidant machinery of the cell hence holds therapeutic value in the treatment and/or prevention of neurodegenerative disorders of oxidative stress.

Neurochemical Properties of Dental Primary Afferent Neurons

The long belief that dental primary afferent (DPA) neurons are entirely composed of nociceptive neurons has been challenged by several anatomical and functional investigations. In order to characterize non-nociceptivepopulation among DPA neurons, retrograde transport fluorescent dye was placed in upper molars of rats and immunohistochemical detection of peripherin and neurofilament 200 in the labeled trigeminal ganglia was performed. As the results, majority ofDPA neurons were peripherin-expressing small-sized neurons, showing characteristic ofnociceptive C-fibers. However, 25.7% of DPA were stained with antibody against neurofilament 200, indicating significant portion of DPA neurons are related to large myelinated Abeta fibers. There were a small number of neurons thatexpressed both peripherin and neurofilament 200, suggestive of Adelta fibers. The possible transition of neurochemical properties by neuronal injury induced by retrograde labeling technique was ruled out by detection of minimal expression of neuronal injury marker, ATF-3. These results suggest that in addition to the large population of C-fiber-related nociceptive neurons, a subset of DPA neurons is myelinated large neurons, which is related to low-threshold mechanosensitive Abeta fibers. We suggest that these Abeta fiber-related neurons might play a role as mechanotransducers of fluid movement within dentinal tubules.

Up-regulation of PSD-95 in the Rat Hippocampal Formation after Chronic Renal Failure / 대한해부학회지

The mechanism of central nervous system (CNS) dysfunction in uremia are multifactorial and only partially characterized. Studies using hippocampal formation (HF) evaluate the relationship between the uremia and memory impairment. Immunoblots with calcium permeable NMDA (N-methyl-D-aspartate) and AMPA (2-amino-3-hydroxy-5-methylisoxazole-4-propinoic acid) receptors and their associated PSD-95 proteins after chronic renal failure (CRF) provided significant new informations. CRF rats induced by 5/6 nephrectomized had significant effects on up-regulation of PSD-95 protein rather than those of calcium permeable NMDA and AMPA receptor subunits. Up-regulation of PSD-95 after CRF might be associated with the enhanced activity of NMDA and/or AMPA receptors, thereby leads to the intracellular Ca2+ accumulation and functional neuronal cell damage subsequently. Degradation of intermediate filament 200 (NF200) in the axon after CRF may induce an impairment of intracellular transport and eventual cellular dysfunction through destruction of the neuronal cytoarchitecture. These data suggest that up-regulation of PSD-95 in CRF may increase the functional derangement between the nerve cells and ultimately lead to memory impairment.

Localization of P2X7 Receptor Immunoreactivity in the Dorsal Root Ganglia of Guinea Pig / 华中科技大学学报（医学）（英德文版）

The P2X7 receptor mRNA and proteins in guinea-pig dorsal root ganglia (DRG) were studied by using RT-PCR and immunohistochemistry. The co-localization of P2X7 receptor with four cytochemical markers, the neurofilament protein NF200, S100, substance P and isolectin B4 (IB4) binding glyco-conjugates, were also examined. It was found that P2X7 receptor immunoreactivity (P2X7R-IR) was present mostly in large- and medium-sized DRG neurons (62 %±9 % and 36 %±6 % respectively in all P2X7R-IR neurons). All the P2X7R-IR neurons were also NF200 and S100 immunopositive. However, in a small number of NF200 or S100 immunopositive neurons no P2X7R-IR was detectable. All the IB4-positive or substance P-immunopositive neurons had no P2X7R-IR. These results demonstrate that P2X7 receptors are expressed in a large subpopulation of DRG neurons and they may play a role in the transduction of specific peripheral sensory signals.

NR1/NR2B receptor Activation induces Cytoskeletal Proteolysis in Partial Ischemic Rat Hippocampal Formation / 대한해부학회지

In the rat brain, partial ischemia causes a delayed neuronal degeneration that occurs hours to days after reoxygenation. It is generally thought that the ischemic damage is initiated by neurotoxicity mediated through glutamate receptors, particulaly NMDA subtypes. Calcium entry through the NMDA receptor is responsible for the synaptic plasiticity and neuronal pathology. Degradation of MAP-2 and NF200, a major components of neuronal cytoskeleton, by Ca2+-dependent protease after NMDA receptor activation has been postulated in delayed neuronal damage. Calcium-activated protease calpain, excessive degradation of MAP-2, together with the calpain-sensitive microtubule and neurofilaments, would be expected to disrupt intracellular transport- and membrane-related functions that is vital to neurons. Changed of NR subunit 2A, 2B, MAP2 and NF200 in rat hippncampal postsynaptic density[PSD] after partial ischemic injury were investigated though immunoblot analyses. To understand the effect of Ca2+, influx through NMDA receptors on neuronal damage which is manifested by cytoskeletal disruption, morphological change was examined through immunohistochemistry and routine staining method. We found that immunoreactivity to NR2B receptor subuit in the hippocampal formation PSD was upregulated while MAP2 and NF200 was down-regulted at 18 hours after initial partial ischemic insult. On the other hand, morphological changes of neuronal cell in partial ischemic conditions were manifested as eosinophilic inclusion bodies in the cytoplasm which is progression of neuronal damage after 6 days. Calcium influx through NR1/NR2B receptor channel may activate intracellular proteases which would degrade cytoskeleton. Proteolysis of cytoskeleton leads to its reorganization and eventually damages normal function of cell membrane which cause neuronal cell death.

Up-regulation of NMDA Receptor Subunit 2B Induces Degradation of Cytoskeletons in Hypoxic Rat Cerebral Cortex / 대한해부학회지

In the rat brain, global hypoxia cause a delayed neuronal degeneration that occurs hours to days after reoxygenation. It is generally thought that the ischemic damage is initiated by neurotoxicity mediated through glutamate receptors, particulary NMDA subtypes. Calcium entry through the NMDA receptor is responsible for the synaptic plasiticity and neuronal pathology. Degradation of MAP-2 and NF200, a major components of neuronal cytoskeleton, by Ca2+-dependent protease after NMDA receptor activation has been postulated in delayed neuronal damage. Changes of NR subunit 2B, MAP2 and NF200 in rat brain postsynaptic density[PSD] after hypoxic injury were investigated through immunoblot analyses. To understand the effect of Ca2+ influx through NMDA receptors on neuronal damage which is manifested by cytoskeletal disruption, morphological change was examined through immunohistochemistry and H & E staining. We found that immunoreactivity to NR2B antibody in the cerebral cortex PSD was up-regulated while MAP2 and NF200 was down-regulated at 30 hours after initial hypoxic insult. At this time, morphological changes of neuronal cells in hypoxic conditions were manifested as down-regulation of MAP2 and NF200 immunoreactivities, hyperchromatic condensation of cytoplasm and nucleus, homogenizing cell change, expansion of perineuronal space and dispersion of chromatin. From 3 days, NR2B, MAP2, NF200 were up-regulated simultaneously. On the other hand, morphological alterations in hypoxic neurons were progress further. Our present results suggests that Calcium influx through NR1/NR2B receptor channel is effective whithin 30 hours but ineffective from 30 hours. Delayed neuronal cell death triggered by Ca2+ influx through NR1/NR2B receptor channel within 30 hours, which may activate intracellular profeases. Proteolysis of cytoskeleton by activated protease leads to its abnormal reorganization and eventually damages normal function of cell membrane which causes neuronal cell death.

Enhancement of N-methyl-D-aspartateNMDA Receptor 2B-mediated Neurotoxi-city after Hypoxia in the Rat Hippocampal Formation / 대한해부학회지

Ischemic brain hippocampal formation has been developed to understand the relationship between delayed neuronal damage and the expression of NMDA receptor subunits[NR2A, NR2B], MAP2, and NF200 in ttle conditions of hypoxia. Changes of NR subunits[NR2A, 2B], MAP2 6nd NF200 in rat brain postsynaptic density[PSD] after hypoxic injury were investigated through immunoblot analyses. To understand the effect of Ca2+ influx through NMDA receptors on neuronal damage which is manifested by morphological change, cytoskeletal disruption was examined through H & E, toluidine blue and immunohistochemical studies. The expression of NR2B was increased than normal at 30 hours after hypoxia. At this time, the expression of MAP2 and NF200 was markedly decreased and their morphology was more eosinophilic than normal and then became darker with expanded perineuronal space. Irreversible neuronal cell damage in hypoxic hippocampal formation is most prominent in CA3 region of hippocampus and the process is triggered by Ca2+ influx through NR1/MR2B receptor channel at 30 hour after initial hypoxic insult. Ca2+ influx through NR1/MR2B receptor channel may activate intracellular proteases which would degrade cytoskeleton. Proteolysis of cytoskeleton leads to its reorganization and eventually damages normal function of cell membrane which causes neuronal cell death. And, morphological changes of neuronal cells in hypoxic conditions were manifested as red neurons in the stage of reactive change, and as dark neuron in the stage of late hypoxic cell damage.