Role of ERK signaling protein in morphine preconditioning reducing global ischemia-reperfusion injury in isolated rat hearts / 中国药理学通报

Aim To observe the role of ERK signaling protein in morphine preconditioning reducing global ischemia-reperfusion injury in isolated rat hearts.Methods Adult male Sprague-Dawley rats were distributed into six groups (n =10 for each) using a random number table:control group (CON),ischemia-reperfusion group (I/R),ischemia preconditioning group (IPC),morphine preconditioning group at the concentration of 1 μmol · L-1 (MPC),ERK inhibitor PD98059 + MPC (MPD),and group of ERK inhibitor-PD98059 (PD).The isolated rat hearts were treated on a Langendorff perfusion apparatus system.The coronary effluent was collected at 15 min of equilibration (baseline),5 and 10 min of reperfusion for detection of the activity of LDH.Meanwhile,a water-filled balloon was inserted into the left ventricular for continuous LVDP measurement.The IS and AAR and IS/AAR ratios were observed by TTC.Western blot was used to examine the level of phosphorylated ERK in myocardium.Results As compared with the I/R group,MPC significantly decreased IS and IS/AAR ratio as well as LDH activities at 5 min and 10 min of reperfusion,but improved the LVDP at the end of reperfusion.Moreover,the phosphorylation level of ERK in myocardium was up-regulated by MPC.However,ERK inhibitor PD98059 could block the protective effects of MPC,as indicated by the increased IS and IS/AAR ratio,elevated LDH activity at the reperfusion of 5 and 10 min,and the suppressed LVDP at the end of reperfusion.Furthermore,the MPC-induced phosphorylation of ERK was also reversed by PD98059.Conclusion Morphine preconditioning may confer cardio-protection against the global ischemia-reperfusion injury in rat hearts through enhancing the phosphorylation of ERK.

Mild Hypothermia Attenuates the Excitatory Amino Acids Overflow and Diminishes the Nitric Oxide Synthase Activity during Reperfusion after Asphyxial Cardiac Arrest in Rats.

Aims: Neurotransmitter overflow into the extracellular space and activation of nitric oxide synthase were implicated in neuronal death after cerebral ischemia. A small temperature reduction induced after the insult crucially mitigated the neuronal death. To elucidate the mechanisms, dopamine and glutamate as marker of excitatory amino acid (EAA) overflow and the citrulline/arginine ratio (CAR) as marker of nitric oxide synthase were analysed. Study Design: Animal experiments in rats. Place and Duration of the Study: Laboratory of the Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Brussels between 2001 and 2003. Methodology: Striatal Glutamate and dopamine and CAR were measured by using microdialysis under normothermic and hypothermic conditions before asphyxial cardiac arrest, during the insult and resuscitation as well as during the weaning process from mechanical ventilation. Results: After the insult, the EAA overflow increased significantly in the normothermic group. In the hypothermic group, however this overflow was not significantly different from the sham group. The CAR increased up to 5-fold compared to the basal value in the normothermic group and only 2.5-fold in the hypothermic group. The brain damage was mitigated in the hypothermic group, while this increased further up 7 days after the insult in the normothermic group. Conclusion: These results suggest that the neuroprotective effect of mild hypothermia resides in attenuation of the striatal EAA overflow and diminution of the CAR and were associated with a reduction of brain damage at 24 hours and 7 days post insult.

Regulation of endothelial nitric oxide synthase by agmatine after transient global cerebral ischemia in rat brain / 대한해부학회지

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) plays a protective role in cerebral ischemia by maintaining vascular permeability, whereas NO derived from neuronal and inducible NOS is neurotoxic and can participate in neuronal damage occurring in ischemia. Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane if brain vessels to promote cell death and tissue injury. We previously reported that agmatine, synthesized from L-arginine by arginine decarboxylase (ADC) which is expressed in endothelial cells, has shown a direct increased eNOS expression and decreased MMPs expression in bEnd3 cells. But, there are few reports about the regulation of eNOS by agmatine in ischemic animal model. In the present study, we examined the expression of eNOS and MMPs by agmatine treatment after transient global ischemia in vivo. Global ischemia was induced with four vessel occlusion (4-VO) and agmatine (100 mg/kg) was administered intraperitoneally at the onset of reperfusion. The animals were euthanized at 6 and 24 hours after global ischemia and prepared for other analysis. Global ischemia led severe neuronal damage in the rat hippocampus and cerebral cortex, but agmatine treatment protected neurons from ischemic injury. Moreover, the level and expression of eNOS was increased by agmatine treatment, whereas inducible NOS (iNOS) and MMP-9 protein expressions were decreased in the brain. These results suggest that agmatine protects microvessels in the brain by activation eNOS as well as reduces extracellular matrix degradation during the early phase of ischemic insult.

Agmatine Attenuates Nitric Oxide Synthesis and Protects ER-structure from Global Cerebral Ischemia in Rats / 대한해부학회지

In ischemic strokes, apoptosis is caused by excitotoxicity, ionic imbalance, oxidative/nitrosative stress, and apoptotic-like pathways. Nitric oxide (NO), a free radical, is elevated after ischemic insult. NO, which is generated primarily by neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS), promotes neuronal damage following ischemia. Evidence obtained in recent years has demonstrated that endoplasmic reticulum (ER)-mediated cell death plays an important role in cerebral ischemia. Agmatine is an endogenous substance synthesized from L-arginine by arginine decarboxylase (ADC) and is present in mammalian brain. We had previously reported that agmatine contributes to neuroprotection against ischemic injury. In continuation of our earlier work, we intended to investigate whether agmatine protects brain from transient global ischemia, and also tried to determine the neuroprotective mechanism of agmatine. Twenty minutes of transient global ischemia was induced by 4 vessel occlusion (4-VO). Agmatine (100 mg/kg, IP) was administered simultaneously with reperfusion. Samplings of brain were done at 6, 24, 48, and 72 h after reperfusion to determine the effect of agmatine on ischemic injured hippocampus. ER-damage was also investigated using electron microscope. Results showed that agmatine treatment prevented delayed neuronal cell death in hippocampal CA1 neurons after global cerebral ischemia. It also blocked NOS expression in the rat brain. Agmatine induced the increased expression of glucose-regulated protein 78 (Grp78). These results suggest that agmatine inhibits the production of NO by decreasing the expression of nNOS and iNOS on global forebrain ischemia and the neuroprotective effect of agmatine were concerned with the ER stress-mediated condition.

The Effects of Mild Hypothermia on the Expression of the Apoptosis-related Proteins following Transient Global Ischemia in Gerbil Hippocampus / 대한구급학회지

BACKGROUND: The neuroprotective mechanisms of hypothermia remain unclear. Recently, attenuation of apoptosis by hypothermia has been suggested as one of the responsible mechanisms. The aim of this study is to investigate the effects of post-ischemic hypothermia on apoptotic neuronal death as well as expression of some apoptosis-related proteins in a gerbil transient global ischemia model. METHODS: Following 5 minutes of ischemia, normothermia (NT, 37+/-0.5degrees C) or mild hypothermia (HT, 33+/-0.5degrees C) was immediately induced and maintained for 3 hours. The hippocampal CA1 neurons were examined on day 2, 3, 4, and 7 after ischemia for the survived neuronal densities, DNA nick end labeling and immunohistochemical expressions of Bcl-2, Bax, and caspase 3 in each group. Additionally, DNA gel electrophoresis and western blot analysis for each protein in hippocampus were performed. RESULTS: The neuronal death in CA1 area on day 3, 4, and 7 was significantly reduced in HT group compared to NT group. The number of TUNEL positive cells in HT group was also significantly reduced than NT group on day 3, 4, and 7. DNA laddering of hippocampus on day 4 and 7 also reduced in HT group. Expressions of Bax on days 2, 3 and activated caspase 3 on days 3, 4 were reduced in HT group. Western blots also disclosed a decrease in the intensity of the Bax on day 2 and 3 in HT group compared to NT group. CONCLUSIONS: These results suggest that mild post-ischemic hypothermia attenuates the apoptotic neuronal death through the inhibition of the intrinsic pathway of caspase activation following transient global ischemia and these effects may be related to a reduction of pro-apoptotic events.

The protective role of erythropoietin induced by hypoxic preconditioning on cerebral injury and cognition after global cerebral ischemia / 中国中西医结合急救杂志

Objective To observe the protective effects of hypoxic preconditioning(HPC) on the improvement of the cognitive dysfunction(learning and memory) and the damage in hippocampus induced by global cerebral ischemia/reperfusion(I/R) in CA1 and CA3 for 5 days in rats,and on the regulation of expression of erythropoietin(EPO) protein to approach the mechanism of the protection.Methods One hundred and twenty adult male Sprague-Dawley(SD) rats were divided into four groups randomly: sham group,I/R group,HPC24 group(hypoxia for 24 hours before I/R) and HPC48 group(hypoxia for 48 hours before I/R).Hang(motor function),passive avoidance and Morris water maze tests were carried out on the 5th day after I/R to measure the motor and cognition functions;hematoxylin-eosin(HE) staining was used to detected histopathological changes in hippocampus tissues;and the contents of EPO were tested by immunohistochemistry at 1 hour and 4 hours after I/R from hippocampus CA1 and CA3 regions.Results Hang,passive avoidance and Morris water maze tests showed that I/R can injure rat cognition;the improvement of cognition was marked in HPC groups, and it was shown that the effects were more significant in HPC48 group than those in the HPC24 group(P

Effect of Ischemic Preconditioning and Propofol on Myocardial Protection in the Globally Ischemic-reperfused Isolated Rat Heart / 대한마취과학회지

BACKGROUND: Although ischemic preconditioning (IPC) or propofol is generally known to confer the cardioprotective effect on ischemic-reperfused hearts, especially in patients subjected to operation such as cardiopulmonary bypass and heart transplantation, the exact effects of IPC and propofol are still controversial. Furthermore, the interaction between IPC- and propofol-induced cardioprotective effects has not been studied yet. The aims of this study are to examine 1) whether IPC and propofol demonstrates the cardioprotective effect against ischemic-reperfusion injury in the isolated rat hearts, and if so, 2) whether the combination of IPC and propofol shows the additive effects. METHODS: Isolated rat hearts were subjected to 30 min global ischemia followed by 60 min of reperfusion. Four groups of hearts (n = 7 per group) were studied. Group control (no intervention); group IPC, two 2-min total coronary occlusions (ischemic preconditioning) interspersed with 5 min and 6 min of normal perfusion before global ischemia; group propofol, propofol 2microgram/ml (11.1microM) administered before global ischemia and during reperfusion; group propofol/IPC, propofol 2microgram/ml administered before IPC and during reperfusion. Left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), + dP/dtmax and - dP/dtmin were recorded and creatinine kinase (CK) in coronary effluent perfusate and coronary flow also were measured. RESULTS: There were significant differences in recovery of postischemic systolic function between control and IPC, propofol and propofol/IPC as assessed by LVDP, expressed as a percentage of preischemic value (22.2 +/-8.4 vs 59.4 +/- 6.8, 69.4 +/- 7.9 and 66.0+/- 7.1%, respectively; P <0.05) at the end of reperfusion. The propofol and propofol/IPC showed better recovery in postischemic relaxation than IPC or control as asse ssed by LVEDP (11.3 +/- 2.2 and 11.3 +/-6.1 vs 56 +/- 6.0 or, 25.2 +/- 7.6 mmHg, respectively; P < 0.05). There were significant differences in attenuation of myocardial damage between propofol/IPC and control as assessed by % change of CK (135.5 +/- 54.7 vs 602.3 +/- 225.1%, P < 0.05) and % change of coronary flow (66.6 +/- 4.0 vs 39.2 +/-5.2%, P < 0.05). CONCLUSIONS: These results suggest that ischemic preconditioning combined with propofol may not show any additive effect on IPC-and propofol-induced attenuation of postischemic ventricular dysfunction, however it show the tendency to attenuate the myocardial damage.

Expressions of the pERK1/2 and the cFos Proteins at an Early Stage of Transient Global Ischemia-reperfusion Injury in the Hippocampus of Rats

PURPOSE: This study was to evaluate temporal changes in the expressions of the phosphorylated extracellular-regulated kinase1/2 (pERK1/2), the phosphorylated MAPK/ERK kinase1/2 (pMEK1/2) and the cFos proteins in the hippocampus of rats following transient global ischemia. METHODS: Transient global ischemia was induced in the forebrains of Sprague-Dawley rats by using a 4-vessel occlusion for 20 min under anesthetic condition. Hematoxyline-eosin staining showed typical microscopic findings that represented neuronal cell death in hippocampal CA1 regions 5 days after transient global ischemia. Four-vessel occlusion-reperfusion produced ischemic injury in major forebrain structures, such as the striatum, the cortex and the hippocampus, in the finding of triphenyltetrazolium chloride (TTC) staining. RESULTS: A high density of pERK1/2 immunoreactivity existed in the pyramidal-cell layers of the CA2-3 regions and in the granular-cell layers of the dentate gyrus 5 min after ischemia. Following ischemia, expression of the pMEK1/2 protein showed temporal changes similar to that of the pERK1/2 protein. A significant expression of the cFos protein was noted in the pyramidal-cell layers of the CA2-3 regions and in the granular-cell layers of the dentate gyrus 2 hours after global ischemia. CONCLUSION: Intracellular signaling cascades of the ERK or the cFos protein take part in early cellular events in the hippocampus of rats in response to ischemic insult.

Effect of NVP-AAM077 and Ro25-6981 on hippocampal neuronal injury and brain derived neurotrophic factor expression in global cerebral ischemic mice / 中国药理学通报

Aim The aim of the present study is to in-vestigate the effect of the specific antagonist NVP-AAM077 and Ro25-6981 of NMDA receptor subunit NR2A and NR2B on global cerebral ischemia-induced neuronal injury in the CA1 region. Methods Male C57BL/6 mice subjected to global ischemia by the three-vessel occlusion( 3-VO) method,were divided into four groups: sham-operated group,ischemia/reperfusion control group,NVP-AAM077 treatment group,and Ro25-6981 treatment group. Hippocampal sections were processed for Fluoro-Jade B staining to detect degenerating neurons and for Nissl staining to identify surviving neurons. The expression of brain derived neurotrophic factor( BDNF) were measured by Western blot. Results ① Transient brain ischemia induced selective and delayed neuron death in the CA1 region of the hippocampus at 12-minute ischemia after reperfusion 3 days. NR2A subtype specific antagonist NVP-AAM077 enhanced neuronal death after transient global ischemia ( P

The Effect of Delayed Administration of Green Tea Polyphenol, (-)-pigallocatechin-3-gallate, on the Change of Putrescine Level and Hippocampal Neuronal Cell Damage after Transient Global Ischemia in Gerbil

OBJECTIVE: T(-)-Epigallocatechin gallate(EGCG) `a green tea polyphenol' is a potent antioxidant and known to reduce the free radical-induced lipid peroxidation. In our previous study, systemic administration of EGCG immediately after ischemia has been shown to inhibit the hippocampal neuronal damage in the gerbil model of global ischemia. Polyamines, especially putrescine(PU) is thought to be important in the generation of brain edema and neuronal cell damage associated with various types of excitotoxic neuronal injury. We investigate the effects of delayed administration of EGCG on the changes in polyamine levels and neuronal damage after transient global ischemia in gerbils. METHODS: To produce transient global ischemia, both common carotid arteries were occluded for 3 min with micro-clips. The gerbils were treated with EGCG(50mg/kg, i.p.) immediately or 2hr after ischemia. Putrescine levels were examined in the cerebral cortex and hippocampus 24 hours after ischemia using high performance liquid chromatography. RESULTS: PU levels in the cerebral cortex and hippocampus were increased significantly after the ischemia. The administrations of EGCG immediately after the ischemia attenuated the ischemia-induced increase of PU level, however, 2 hr delayed EGCG administration did not reduce the increase of PU level. EGCG administered immediately or 2 hr after ischemia significantly reduced neuronal damage in the hippocampal CA1 region, respectively. CONCLUSION: These findings suggest that EGCG may has a promise in the management of stroke.

Effect of Melatonin on Brain Polyamine Contents and Hippocampal Neuronal Damage after Transient Global Ischemia in Mongolian Gerbil / 대한뇌혈관학회지

OBJECTIVES: This study was designed to examine whether melatonin has a neuroprotective effect against hippocampal neuronal damage following transient global ischemia in a gerbil. Polyamine is known to play a role in the pathophysiology of ischemic neuronal damage, we evaluated the influences of melatonin on the polyamine level as well as histology. MATERIAL AND METHODS: Male Mongolian gerbils (60-80 g) were used in this study. Transient global ischemia was induced by occlusion of the bilateral common carotid arteries for 3 min with microclips. Melatonin was administered immediately after occlusion. The animals were decapitated 24 h after the occlusion for polyamine measurement by a high performance liquid chromatography (HPLC) and 4 days after the occlusion for histological evaluation (hematoxylin and eosin staining). A histological examination was performed by a blinded investigator. RESULTS: The hippocampal putrescine level increased compared to sham-operated animals and the increase of putrescine was attenuated by 20 mg/kg melatonin administration. Spermidine and spermine levels didn't show significant changes after ischemia. Hippocampal neuronal damage in the CA1 region was markedly observed in vehicle-treated animals compared to sham-operated animals. Melatonin administration (10 or 20 mg/kg) significantly inhibited hippocampal CA1 neuronal damage after ischemia compared to corresponding vehicle-treated animals (p<0.05 and p<0.01, respectively). CONCLUSION: Melatonin attenuates the putrescine level after transient global ischemia and may have putative neuroprotective effects against global ischemia induced neuronal damage.

Effect of Vascular Endothelial Growth Factor (VEGF) on Neuronal and Glial Response Following Transient Global Ischemia in Rats

OBJECTIVE: The authors present the effect of VEGF upon neuronal and glial response following transient global ischemia of the rat METHODS: We studied the effect of VEGF in 36 rats subjected to 15 minutes of transient global ischemia. Animals were devided into control group(transient global ischemia only: day-3, day-7, day-14, respectively n=6) and VEGF-treated group(transient global ischemia with intraventricular injection of 100 micro gram VEGF: day-3, day-7, day-14, respectively n=6). These animals were sacrificed at 3 days, 7 days and 14 days after induction of ischemia. Nissle stain and immunohistochemistry of GFAP(glial fibrillary acidic protein), OX-42, and ED1 were done for assessment of neuronal and glial responses. RESULTS: In the CA1 hippocampus, there was a significant reduction of pyramidal cell damage in VEGF-treated group as compared with control group in post-ischemia 3, 7, 14 days(p0.05). In the assessment of CA1 hippocampus with GFAP stained areas, there was significant reduction of reactivity in post-ischemia 3, 7 days(p0.05). In the CA3 hippocampus, reduction of GFAP reactivity was significant in post-ischemia 3, 7 days(p0.05). In the assessment of CA1 hippocampus with OX-42 stained areas, there was significant reduction of reactivity in post-ischemia 3, 7, 14 days(p<0.05). But in the CA3 hippocampus, the difference was not significant in post-ischemia 3, 7 days(p<0.05). In the assessment of of CA1 hippocampus with ED1 stained areas, there was significant reduction of reactivity in post-ischemia 3, 7, 14 days(p<0.05). But in the CA3 hippocampus, the difference was significant in post-ischemia 3 days only(p<0.05). CONCLUSION: These results suggest that VEGF can reduce neuronal damage in transient global ischemia, thus have protective effect on ischemic brain injury. In our experiment, the reduction of glial response with VEGF seems to be related to the secondary neuroprotective effect of VEGF. However, the proliferation of endothelial cells and new vessel formation take days to months, the thus neuroprotective effect of VEGF against ischemia seems to related to a certain mechanism rather than angiogenesis.

Effect of Ibuprofen on the Changes of Polyamine Level and Neuronal Cell Damage after Transient Global Ischemia in Gerbil

BACKGROUND: In brain ischemia, increased arachidonic acid metabolism can play important roles in neuronal dam-age. Ibuprofen was reported to have a protective role against neuronal damage in focal brain ischemia and reperfusion. The present study was designed to investigate whether ibuprofen can inhibit the global ischemia-induced neuronal dam-age and changes of polyamine (PA) level which is known to related to the neuronal damage, breakdown of blood brain barrier, and brain edema. METHODS: Male Mongolian gerbils were used in this study. Transient global ischemia was induced by occlusion of bilateral common carotid arteries for 3 min with microclips. Ibuprofen was administered imme-diately after ischemia. The animals were sacrificed one day after ischemia for PA measurement and sacrificed 5 days after ischemia for histological evaluation. Histological examination was performed by counting surviving neuronal cells in one mm of CA1 area in dorsal hippocampus. RESULTS: Cerebral cortex and hippocampal putrescine(PU) levels in vehicle-treated ischemic group significantly increased comparing to sham-operated animals and the increase of PU was attenuated by ibuprofen administration (50 mg/kg). Hippocampal spermine level decreased significantly after ischemia. Hippocampal neuronal cell damage in CA1 area was markedly observed in vehicle-treated animals compared to sham operated animals. Ibuprofen administration at the dose of 50 mg/kg significantly inhibited hippocampal CA1 neuronal damage compared to vehicle-treated animals. CONCLUSIONS: Ibuprofen attenuates PA response following transient glob-al ischemia and may have putative neuroprotective effect against neuronal damage induced by global ischemia.

Effect of Melatonin on the Changes of Hippocampal Polyamine Content and Neuronal Damage Following Transient Global Ischemia in Mongolian Gerbil: a Study of the Differences of Pre- and Post-ischemic Treatment / 대한마취과학회지

BACKGROUND: We designed this study to examine whether melatonin has a neuroprotective effect against hippocampal neuronal damage following transient global ischemia in a gerbil. Because polyamine is known to participate in the process of ischemic neuronal damage, we examined the influence of melatonin on the polyamine level as well as histology. In particular, we examined the difference between pre- and post-ischemic treatments of melatonin by using the above mentioned parameters. METHODS: Male Mongolian gerbils (60 - 80 g) were used in this study. Transient global ischemia was induced by occlusion of the bilateral common carotid arteries for 3 min with microclips. Melatonin was administered 1 h before or 1 h after occlusion. The animals were dissected 4 days after the occlusion for polyamine measurement by a high performance liquid chromatography (HPLC) and histological evaluation (hematoxylin and eosin staining). A histological examination was performed by a blinded investigator. RESULTS: The hippocampal putrescine (PU) level increased compared to sham-operated animals and the increase of PU was attenuated by melatonin administration (pre- or post-ischemic treatment). Spermidine (SD) and spermine (SM) levels didn't show significant changes after ischemia. Hippocampal neuronal damage in the CA1 region was markedly observed in vehicle-treated animals compared to sham- operated animals. Both pre- and post-ischemic melatonin administration significantly inhibited hippocampal CA1 neuronal damage compared to corresponding vehicle-treated animals (P < 0.01, respectively). CONCLUSIONS: Melatonin attenuates the polyamine response following transient global ischemia and may have putative neuroprotective effects against global ischemia-induced neuronal damage. There is no difference in neuroprotective effects of melatonin between pre- & post-ischemic treatments.

Effect of Fentanyl on the TNF-alpha and IL-1beta Level during Global Ischemia/reperfusion in Rats / 대한마취과학회지

BACKGROUND: To reduce surgical stress, fentanyl is frequently used for neurosurgical procedure where focal and/or global ischemia may occur. However, the effect of fentanyl on the cytokine level during ischemia/reperfusion is still uncertain. The goal of this study was to evaluate the effect of fentanyl infusion on the proinflammatory cytokine, TNF-alpha and IL-1beta, levels during global cerebral ischemia/reperfusion (I/R) in rats using the intracerebral microdialysis technique. METHODS: Forty male S-D rats weighing 280 320 g were randomly assigned to four groups. Group 1: no fentanyl infusion and only I/R, Group 2: 1.5 ng/ml of fentanyl infusion during I/R, Group 3: 3.0 ng/ml of fentanyl infusion during I/R (n = 10 in each group). Rats were anesthetized with a intraperitoneal injection of pentobarbital (50 mg/kg), intubated and ventilated with room air using an animal ventilator. Two femoral arteries and one femoral vein were cannulated with PE-50 tubing for hemorrhagic hypotension, drug infusion and hydration. Both carotid arteries were dissected and a sling was placed for brain ischemia. The head was fixed on a stereotaxic device and a small burrhole was made for probe insertion. A CMA-12 probe was inserted into the left hippocampal CA-1 region according to the guidelines. Artificial CSF was run from the inserted microdialysis probe and infused with or without fentanyl at 3 microliter/min using a microinjection syringe pump during I/R. Ischemia was induced by clamping the carotid arteries while hemorrhagic hypotension for 17 min via the femoral artery and reperfusion were accomplished by the unclamping of the sling and reinfusing the blood via the femoral artery. Nasopharyngeal and rectal temperatures were maintained within the normal range during the whole procedure. After 2 hours of stabilization, the microdialysate was collected every 17 min just before (control) and during I/R and stored at 80oC until analysis using HPLC. RESULTS: During global I/R, TNF-alpha and IL-1 beta significantly increased at reperfusion (R5) compared to the control value (P < 0.05). However, in both cases of fentanyl infusion, TNF-alpha and IL-1 beta did not increase compared to the control value. CONCLUSIONS: Fentanyl inhibited the increase of proinflammatory cytokine TNF-alpha and IL-1 beta levels during global cerebral ischemia/reperfusion in rats.

Effects of Nitric Oxide Synthase Inhibition on Hydroxyl Radical Production during Global Cerebral Ischemia/Reperfusion in Rats / 대한마취과학회지

BACKGROUND: Free radical-mediated oxidative damage has been implicated in ischemic brain injury. There are also increasing evidences that nitric oxide is involved in the mechanisms of cerebral ischemia. To elucidate the effect of nitric oxide synthesis inhibition on the hydroxyl radical formation, we used a method based on the chemical trapping of hydroxyl radical in the form of the stable adducts 2,3-DHBA following salicylate adminstration. METHODS: Sprague-Dawley rats were subjected to 15 min of global cerebral ischemia by both carotid artery occlusion plus systemic hemorrhagic hypotension (35 mmHg). Artificial CSF including salicylate (5 mM) was continuously infused through a microdiaysis probe implanted in the left hippocampus CA1. Hippocampal extracellular fluid was sampled at regular intervals before, during, and after ischemia. The levels of 2,3-DHBA were assayed by HPLC with electrochemical detection during 15 minutes of ischemia and reperfusion period. RESULTS: Cerebral blood flow was reduced to 5% level of control in ischemic period, but increased 3 or 4 times in early phase of reperfusion period, and returned to normal 50 to 60 minutes after the cessation of ischemia. Inhibition of NOS by L-NAME did not prevent ischemia-induced 2,3-DHBA elevation, but increased its level during reperfusion. This increase in 2,3-DHBA could be reversed by L-arginine. The elevated 2,3-DHBA after IR in L-NAME treated rats was not due to either changes in CBF or local blood brain barrier permeability. CONCLUSIONS: The above results indicate NO protects brain from damages by hydroxyl radical, at least less than one hour after initiation of reperfusion.

Neuroprotective Effects of Lamotrigine in Transient Global Ischemia

BACKGROUND AND PURPOSE: Current therapy for acute ischemic stroke is highly focused on neuroprotective agents, and many ion channel blockers have been challenged for experimental models. In this study, we tried to reveal the neuroprotective effect of lamotrigine, a voltage-sensitive sodium channel blocker, for transient global ischemia of Mogolian gerbil. METHODS: Lamotrigine (50mg/kg) was administered via gastric tube 30 minutes before and after global ischemia (for 10 min) under body temperature monitoring. Sham-operated and non-treated ischemia group were compared. Seven days after reperfusion, gerbils were killed with perfusion/fixation technique and representative sections were cut through the hippocampus. Hematoxylin-Eosin staining was done for microscopic examination and number of viable neurons in CA1 area was counted. RESULTS: Neuronal density was different between sham-operated (n=11), non-treated ischemic (n=11), and lamotrigine-treated (n=26) group (107.8+13.1/mm vs. 21.5+23.0/mm vs. 82.0+13.1/mm, p<0.01). Both pre-(n=17) and post-treated group (n=9) showed significant neuroprotective effect compared with non-treated group. Neuronal density of pre-treated group was slightly higher than in post-treated group, though statistically not significant (84.6+13.0/mm vs. 77.3+12.7/mm, p=0.13). CONCLUSION: These results show that lamotrigine may have some effects reducing the delayed neuronal death in transient global ischemia. Considering the mechanism of action, we suggest that activation of voltage-sensitive sodium channel and release of glutamate at early phase of ischemia may be related to the delayed neuronal death.

Experimental Studies of Hydroxyl Radical Generation in Reperfusion Injury after Cerebral Ischemia

The time course of hydroxyl radical generation in the brain and the intensity of brain hydroxyl radical(OH) generation were examined in rat during the first four hours after postischemia reperfusion. Hydroxyl radical production was measured using the salicylate trapping method in which the production of 2, 3-dihydroxybenzoic acid(DHBA) in hippocampus(CA1) 5 minutes after salicylate administration was used as an index of OH formation. The interstitial concentration changes of salicylate and 2, 3-DHBA were detected by intracerebral microdialysis following the intraperitoneal administration of salicylate(150mg/kg) using high pressure liquid chromatography-electrochemical(HPLC-EC) and -ultraviolet(-UV). Adult Sprague-Dawley rats were subjected to 20 minutes of bilateral carotid artery occlusion(BCAO) in either normotensive or hypotensive state. Serial changes of cerebral blood flow(CBF) were monitored by H2 clearance method. CBF of normotensive BCAO group(n=6) was found to be decreased only to 52% of baseline value, and OH production after reperfusion did not develop in this group. Rats in the BCAO hypotensive group(n=10) showed remarkable reduction of CBF to 27% of baseline(p<0.05) and 2~4 folds increase of 2, 3-DHBA/salicylate during the first 40 minutes of recirculation . Hydroxyl radical production in rats died(n=5) after the insult was significantly higher and lasted longer than that in rats survived(n=5)(p<0.05). Concentration of salicylate in perfusate increased during 100 minutes after the peritoneal injection and before reaching to a plateau, which lasted for 3 hours. The changes of cerebral tissue concentration of 2, 3-DHBA differed from those of salicylate. In 2, 3-DHBA, the plateau was reached rather slowly than that of salicylate and lasted for 2 hours. These data indicate that lobal cerebral ischemia could be induced by temporary BCAO only if the systemic hypotenion is accompanied, it can not be induced in normotensive group. The hydroxyl radical produced brain damage is prone to develop early in the reperfusion period and is correlated with the severity of ischemic insult.