Morphine Protects Peroxynitrite-induced Cell Death in Primary Rat Neonatal Astrocytes / 대한마취과학회지

BACKGROUND: Astrocytes, representing a major non-neuronal cell population in the central nervous system (CNS), contain opioid receptors and are actively involved in several brain functions. This study is designed to evaluate the effects by which morphine contributes to cytotoxicity of nitric oxide (NO) species including NO and peroxynitrite (ONOO(-)) in primary astrocytes isolated from the cerebral cortexes of 1 - 2 day Sprague-Dawley rats. METHODS: The cultured cells were pretreated with morphine and exposed to 3-morpholinosydnonimine (SIN-1) which simultaneously generates NO and superoxide, thus possibly forming peroxynitrite. The cell damage was assessed by using an MTT (methylthizol-2-yl-2, 5-diphenyl, tetrazolium bromide) assay. Morphological nuclear changes of the cells after exposure to SIN-1 for 24 hours was evaluated by using 4', 6-diamidino-2-phenylindole (DAPI) staining. RESULTS: Morphine significantly protected primary rat astrocytes in a dose-dependent manner from the death mediated by sodium nitroprusside (SNP), a donor of nitric oxide, and SIN-1. Moreover, it was found that naloxone antagonized the protective effect of morphine on SIN-1-induced cell death, revealed as apoptosis by the occurrence of morphological nuclear changes characteristic of apoptosis. Morphine also inhibited the nuclear condensation of SIN-1-treated cells, however the action of morphine was antagonized by pretreatment of naloxone. The protective role of morphine on SIN-1-induced cytotoxicity was inhibited by DL-Buthionine-[S, R]-sulfoximine (BSO). Furthermore, the effects of morphine on SIN-1-induced cytotoxicity were blocked by pretreatment of Gi protein inhibitor, pertussis toxin, and phosphoinositide 3-kinase (PI3 kinase) inhibitors, Wortmannin and LY294002. CONCLUSIONS: These results suggest that morphine may protect primary rat astrocytes from NO species via the signaling cascades involving G-protein and PI3-kinase, and possibly regulates the anti-oxidant, glutathione (GSH).

Protective Effect of Ketamine on DNA Fragmentation in Astroglial Ischemic Model: Use of the TUNEL Assay to Determine Apoptosis / 대한마취과학회지

BACKGROUND: Ketamine, a dissociative anesthetic, was known to be beneficial in reducing neuronal or astroglial damage by anoxic or ischemic insults. Detection of DNA fragments in situ using the terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) assay is recently applied to investigate active cell death. Therefore, we tried to investigate the relationship of ketamine effect with DNA breakdown in the condition of low ATP. METHODS: Experimetal protocols are as follows: Astrocytoma cells were harvested and plated to chamber slides. Then experimental cells were divided to four groups as control group, iodoacetate (IAA)/carbonylcyanide m-chlorophenylhydrazone (CCCP) 1.5 mM/20 micrometer treated group, and IAA/ CCCP with ketamine 1 mM or 0.1 mM. In situ labelling of fragmented DNA was conducted with commercially available kits (In situ cell death detection kit, Boehringer Mannheim). Stained cells were observed by the use of light microscope. RESULTS: TUNEL-positive reaction appeared in energy depleted astrocytes. However, TUNEL-positive cells were not observed after perfusion with iodoacetate/CCCP and ketamine 0.1 mM or 1.0 mM together. CONCLUSIONS: We report here that in vitro addition of ketamine 0.1 mM or 1 mM protected DNA breakdown of astrocytes from energy depletion. These results suggest that ketamine may have potential effect in preventing ischemic damage in clinical setting.

Protective Effect of Hypothermia in Delayed Astrocyte Death after Transient Histotoxic Hypoxia in Vitro / 대한마취과학회지

BACKGROUND: It is well known that neuronal degeneration can occur after a brief deprivation of energy source. To investigate whether glial astrocyte can induce a phenomenon of delayed cell death after transient energy loss and to see how different are the effects of nifedipine, lidocaine, carnosine and hypothermia on delayed toxicity in astrocyte. METHODS: Human astrocytoma cells (U1242MG) were used in this study. To assess the astrocyte survival during post-ischemic period after transient histotoxic hypoxia, 3-[4,5-dimethylthiazol-2yl]-2,5, diphenyl tetrazolium bromide (MTT) test was used. Compared to MTT test, tryphan blue test was also used to demonstrate membrane damage of affected cells. Studies on intracellular calcium dynamics during ischemic and post-ischemic period were carried out with fluo-3 and flow cytometry system. RESULTS: The percentage survival of astrocyte during post-ischemic period was decreasing with time. Calcium channel blocker nifedipine, sodium and calcium channel blocker lidocaine and free radical scavenger carnosine could not prevent post-ischemic cell damage. But, hypothermia was only an effective method in ameliorating post-ischemic cell death. Intracellular calcium increase during ischemia and post-ischemia was dependent on extracellular calcium influx. CONCLUSIONS: Only hypothermia was effective in reducing astrocyte death during post-ischemia after transient energy depletion. Intracellular calcium alterations during post-ischemia was from extracellular space.

Regulation of Astroglial Volume by Ketamine in Glutamate Induced Cellular Volume Changes / 대한마취과학회지

BACKGROUND: Relative changes of astroglial volume constitute the major part of brain edema, which is related to delayed neuronal damage. Several factors including glutamate may contribute to astroglial swelling. Intravenous anesthetic, ketamine was known to restore neuronal damage by inhibiting NMDA receptor activity. Therefore, we decided to investigate the effect of ketamine on the astrocyte swelling by glutamate in the present study. METHODS: To analyze cell swelling in vitro, glial cell line, U1242MG was used. The effects of glutamate (1, 2, 3 mM), and glutamate with ketamine (1 mM) on the regulation of astrocyte volume were achieved by flow cytometry system. To eliminate the dead cells from experimental cell suspension and to assess cell viability, fluorescent dye propidium iodide was used. RESULTS: Glutamate addition (1, 2, 3mM) caused astroglial swelling both in calcium present and calcium absent buffer. The difference of cellular swelling dependent on glutamate concentration was only seen in calcium free buffer (p<0.05). Ketamine per se did not affect astroglial volume. However, when it was added to glutamate perfusion, 1 mM ketamine diminished cellular swelling by glutamate during first 10 minutes (p<0.05), and cellular shrinkage by glutamate after 1 hour incubation (p<0.05). CONCLUSIONS: Ketamine (1 mM) is effective in the regulation of astroglial volume alterations induced by glutamate in both short time and long time perfusion.