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Effect of stobadine, U-74389G, trolox and melatonin on resistance of rat hippocampal slices to oxidative stress
Life Sciences, Vol. 65, Nos. 18/19, pp. 1%~1971, 1999 Copyright 0 1999 Elscvicz Scieacc Inc. Printed in the USA. All ri@s resewed 0024-3205/99/S-see frxmtmatter
PII soo24-3205(99)00457-9
ELSEVIER
EFFECT OF STOBADINE, U-743896, ON RESISTANCE OF RAT HH’POCAMPAL
Roman Vlkolinskjr,
TROLOX SLICES
AND MELATONIN TO OXIDATIVE STRESS
Svorad Stoic, Angela Ross*
Institute of Experimental Pharmacology, Slovak Academy of Sci., Dtibravska 9, SK-842 16 Bratislava, Slovak Republic, *Duke University Medical Center, Durham, NC 277 10, USA
Summary Reactive oxygen species have been suggested to participate in the impairment of nervous tissue by oxidative stress, induced by hypoxia (I-M’) followed by reoxygenation (ROX). Although the mechanisms of such injury are rather complex, antioxidants might exert some protective action under such circumstances. This study tested the effect of a series of compounds interfering with the generation and action of reactive oxygen species on impairment of synaptic transmission in the CA1 region of rat hippocampal slices exposed to HYP followed by ROX in vitro. Shortlasting I-M’ (typically 4.5-7.5 min under the conditions used) resulted in fast decay of the amplitude of population spikes evoked in the CA1 neurons by stimulation of Schiiffer collaterals. The impairment was mostly irreversible. However, in the presence of the antioxidants stobadine, 21 -aminosteroid U-74389G, melatonin and trolox (with optimal concentrations of lo-30 pmol/l, 10 pmol/l, 30100 l.rmol/l and 200 PmoYl, respectively), the irreversible damage of the transmission was significantly diminished. The decay of the synaptic transmission failure during I-IV was also delayed by stobadine, U-74389G and melatonin. The results demonstrated that compounds with antioxidant activity may effectively protect nervous tissue during HYP and ROX. Key Words:
hypoxia, reoxygenation, reactivate oxygen species, CA1 neurons, population spike, neuroprotection,
antioxidants
Hypoxia (HYP) and reoxygenation (ROX) impose an’oxidative stress on nervous tissue that alters its functions and may lead, among other effects, to an irreversible loss of synaptic transmission. The early changes in hypoxic brain usually develop first in the most vulnerable areas, such as hippocampus (1). Changes start early after the onset of I-M’ and continue over the period of ROX. They are at least partly mediated by reactive oxygen species (ROS) (2). HYP leads to a shift toward anaerobic metabolism, lack of ATP and acidosis. Altered mitochondrial fimctions and release of iron from binding proteins by acidosis are possible sources of excessive ROS production in early stages of HYP. ROS promote lipid peroxidation in neuronal membranes with additional inhibition of membrane-bound ATPases. Reduced ATP levels lead to pre- as well as postsynaptic cell membrane depolarization, associated with elevated extracellular concentration of K’ and glutamate. This promotes Ca” overload in postsynaptic elements and activation of enzymes participating in excessive ROS production. ROX even worsens the situation, because of the oxygen
1970
Hippccampal Resistance to Oxidative Stress
Vol. 65, Nos. 18/19, 1999
supply to the already started oxidative reactions. Antioxidant defense systems can partly modulate the resistance of nervous tissue to ROS actions. Thus, externally applied antioxidants might increase the capacity of antioxidant defense systems and improve the resistance of neurons threatened by oxidative stress. In a previous study we revealed that stobadine, a novel antioxidant with pyridoindole structure, and trolox, a congener of a-tocopherol, significantly increased the resistance of hippocampal tissue to oxidative stress, and improved the recovery of synaptic transmission after HYP (3). This study investigated the neuroprotective effect of other compounds with antioxidant activity, namely 21-aminosteroid U-74389G, and the pineal hormone melatonin, on the impairment of synaptic transmission induced by HYP and ROX in hippocampal CA1 neurons. The degree of neuroprotection was consequently compared to that induced by stobadine. Methods Ether anesthetized male Wistar rats (170-l 8Og) were decapitated, brains rapidly removed and dissected in ice-cool artificial cerebrospinal fluid (ACSF) containing glucose in concentration 10 mmol/l and equilibrated by 95% 02 + 5% CO* (pH = 7.3). Hippocampi were chopped into slices (400 pm) and incubated in gas/liquid interface chamber for at least 80 minutes before measurement. After 20 min stabilization, the slices were exposed to HYP lasting 4.5-7.5 min by switching the 95% 02 + 5% COZ to 95% Nz+ 5% CO2 and by changing the oxygenated ACSF to that equilibrated by the NZ + CO2 mixture. Moreover, the glucose concentration was diminished to 4 mmol/l. Then ROX for 20 min followed. The drugs were dissolved in ACSF and were present in the superfbsing media throughout the experiment, except a-tocopherol which was administered to the animals daily for 10 days prior to decapitation in doses of 200 mg/kg P.O. Supramaximal stimulation (5-35 V, 0.05-o. 1 ms, 0.2 Hz) of Schaffer collaterals by bipolar wire electrode elicited population spike (PoS) in CA1 neurons recorded by an extracellular glass pipette electrode. The amplitude of the PoS was measured during the experiment as a marker of synaptic transmission. In hippocampal slices, susceptibility of neurotransmission to HYP/ROX was found to be reasonably homogenous in a given series of experiments. Nevertheless, remarkable differences in this parameter occurred between sets of experiments made in different times in spite of high effort to standardize the experimental conditions. Hence, before each series duration of HYP just sufficient to induce irreversible transmission failure in approximately 50 - 80% of slices was estimated. Consequently HYP lasting 30 set longer was used resulting typically in 90% irreversibility of control preparations. Results In the first series of experiments, HYP lasting 7.5 min resulted in rapid decay of PoS amplitude with only 7.8 + 2.0% recovery during ROX in untreated preparations. Slices treated with stobadine (30 umol/l) showed significant improvement with 74.5 + 10.8% PoS recovery during ROX. Moreover, in this group the PoS decay during HYP, expressed as half-time of the decay, was delayed from tin= 1.56 + 0.11 min to t iI = 2.56 f 0.25 min. Stobadine also decreased the rate of irreversibly damaged slices from 87.2% to 13.4%. The effective concentration of stobadine spanned from 1 pmol/l to 100 umol/l. The protective effect of trolox in concentration 200 umol/l on PoS recovery was remarkably smaller, attaining approximately a quarter of the effect of stobadine. Trolox had no effect on PoS decay during HYP The iron chelator deferoxamine had no effect at all. The concentration-response curve of stobadine and trolox were bell-shaped reaching their maximum approximately at IO-30 pmol/l and 200 pmol/l, respectively. Stobadine confirmed its neuroprotective features also in another series of experiments in which HYP lasted 4.5 min. Under these conditions the 21aminosteroid U-74389G (10 pmo/l) also revealed a significant increase of PoS recovery from 9.73 I4.88% to 39.1 + 9.2% and delayed the PoS decay during HYP from
Vol. 65, Nos. 18/19, 1999
Hippocampal Resistance to Oxidative Stress
1971
tin= 1.23 + 0.08 to 1.57 + 0.15 min. A similar pattern of protective features was present also in 30 and 100 pmol/l melatonin treated groups with an even more prominent delay of PoS decay during HYP. Higher concentrations of melatonin were not effective. a-tocopherol had no remarkable protective effect under the conditions tested. Discussion
The compounds studied were able to interfere with the generation and/or action of ROS. Thus stobadine was shown to be a hydroxyl, peroxyl and alkoxyl radical scavenger, hypochloric acid scavenger and singlet oxygen quencher (4) while a-tocopherol and its water soluble congener trolox are well established antioxidants (5). The antioxidant property of the 21aminosteroid U74389G and melatonin was also described (6,7). As ROS may participate in HYP/ROX induced injury of nervous tissue (5) it is conceivable to link the observed protective effect of the compounds tested to the improved antioxidative capacity of nervous tissue following their administration. Since the concentration of a-tocopherol in the hippocampus was not measured in this study, it is not possible to conclude whether this compound is actually not able to protect the tissue against HYIYROX injury effectively or whether the failure observed was due to the insufficient bioavailability and distribution of the compound under the dosage regimen used. The delay of PoS amplitude decay during HYP occurring in the presence of stobadine, U-74389G and melatonin requires a special comment. In CA1 neurons an early extinction of PoS during I-IYP is due primarily to elevated release of adenosine (8). This might be a protective mechanism of neurons to HYP during which postsynaptic elements do not respond to presynaptic stimulation and no damage would develop after immediate tissue ROX. ROS might play a regulatory fimction in the initiation of adenosine release since antioxidants delayed its beginning. In spite of this delay, antioxidants improved the outcome of HYP. This may imply lowered needs of the challenged tissue for the initiation of the mechanism in antioxidant treated groups, possibly by shifting the oxido-redox balance of neurons to the redox site. Moreover, antioxidants might play a role also during later stages of HYP, and even during ROX, characterized by profound depolarization and Ca” dependent activation of enzymes participating in elevated ROS production. The results support the hypothesis of oxidative damage of nervous tissue by HYP and ROX and corroborate the neuroprotective potential of antioxidants in pathological states associated with oxidative stress. Ackowledgements
The authors are grateful to Dr. D.C. Zimmermann of Upjohn Co., Kalamazoo MI, USA for the sample of U-74389G. The study was supported by the Slovak Grant Agency for Science (Grants No 1018/96, No 5305/97). References
1. 2. 3. 4. 5. 6. 7. 8.
P.G. AITKEN and S.J. SCI-IIFF Neurosci. Lett. 67 92-96 (1986). L.V. DAWSON and T.M. DAWSON Death and Differentiation 3 71-78 (1996). S. STOLC and R. VLKOLINSK? Pharmacol. Res. 31 (Suppl.) 295 (1995). L. HORAKOVA and S. STOLC Gen. Pharmac. 30 627-638 (1998). Z. DURACKOVA I. BERGENDI, A. LIPTAKOVA and J. MUCHOVA Brat. Lek. Listy 94 419-434 (1993) W.C. CLARK, J.S. HAZEL and B.M. COULL Drugs 50 971-983 (1995) R.J. REITER, D. MELCHIORRI, E. SEWERYNEK, L. BARLOW-WALDEN, J. CHUANG, G.G. ORTIZ and D. ACU$IA-CASTROVIEJO J. Pineal Res. 18 l-l 1 (1995) D.J. DOOLETTE and D.I. KERR Brain Res. 667 127-137 (1997)
PII soo24-3205(99)00457-9
ELSEVIER
EFFECT OF STOBADINE, U-743896, ON RESISTANCE OF RAT HH’POCAMPAL
Roman Vlkolinskjr,
TROLOX SLICES
AND MELATONIN TO OXIDATIVE STRESS
Svorad Stoic, Angela Ross*
Institute of Experimental Pharmacology, Slovak Academy of Sci., Dtibravska 9, SK-842 16 Bratislava, Slovak Republic, *Duke University Medical Center, Durham, NC 277 10, USA
Summary Reactive oxygen species have been suggested to participate in the impairment of nervous tissue by oxidative stress, induced by hypoxia (I-M’) followed by reoxygenation (ROX). Although the mechanisms of such injury are rather complex, antioxidants might exert some protective action under such circumstances. This study tested the effect of a series of compounds interfering with the generation and action of reactive oxygen species on impairment of synaptic transmission in the CA1 region of rat hippocampal slices exposed to HYP followed by ROX in vitro. Shortlasting I-M’ (typically 4.5-7.5 min under the conditions used) resulted in fast decay of the amplitude of population spikes evoked in the CA1 neurons by stimulation of Schiiffer collaterals. The impairment was mostly irreversible. However, in the presence of the antioxidants stobadine, 21 -aminosteroid U-74389G, melatonin and trolox (with optimal concentrations of lo-30 pmol/l, 10 pmol/l, 30100 l.rmol/l and 200 PmoYl, respectively), the irreversible damage of the transmission was significantly diminished. The decay of the synaptic transmission failure during I-IV was also delayed by stobadine, U-74389G and melatonin. The results demonstrated that compounds with antioxidant activity may effectively protect nervous tissue during HYP and ROX. Key Words:
hypoxia, reoxygenation, reactivate oxygen species, CA1 neurons, population spike, neuroprotection,
antioxidants
Hypoxia (HYP) and reoxygenation (ROX) impose an’oxidative stress on nervous tissue that alters its functions and may lead, among other effects, to an irreversible loss of synaptic transmission. The early changes in hypoxic brain usually develop first in the most vulnerable areas, such as hippocampus (1). Changes start early after the onset of I-M’ and continue over the period of ROX. They are at least partly mediated by reactive oxygen species (ROS) (2). HYP leads to a shift toward anaerobic metabolism, lack of ATP and acidosis. Altered mitochondrial fimctions and release of iron from binding proteins by acidosis are possible sources of excessive ROS production in early stages of HYP. ROS promote lipid peroxidation in neuronal membranes with additional inhibition of membrane-bound ATPases. Reduced ATP levels lead to pre- as well as postsynaptic cell membrane depolarization, associated with elevated extracellular concentration of K’ and glutamate. This promotes Ca” overload in postsynaptic elements and activation of enzymes participating in excessive ROS production. ROX even worsens the situation, because of the oxygen
1970
Hippccampal Resistance to Oxidative Stress
Vol. 65, Nos. 18/19, 1999
supply to the already started oxidative reactions. Antioxidant defense systems can partly modulate the resistance of nervous tissue to ROS actions. Thus, externally applied antioxidants might increase the capacity of antioxidant defense systems and improve the resistance of neurons threatened by oxidative stress. In a previous study we revealed that stobadine, a novel antioxidant with pyridoindole structure, and trolox, a congener of a-tocopherol, significantly increased the resistance of hippocampal tissue to oxidative stress, and improved the recovery of synaptic transmission after HYP (3). This study investigated the neuroprotective effect of other compounds with antioxidant activity, namely 21-aminosteroid U-74389G, and the pineal hormone melatonin, on the impairment of synaptic transmission induced by HYP and ROX in hippocampal CA1 neurons. The degree of neuroprotection was consequently compared to that induced by stobadine. Methods Ether anesthetized male Wistar rats (170-l 8Og) were decapitated, brains rapidly removed and dissected in ice-cool artificial cerebrospinal fluid (ACSF) containing glucose in concentration 10 mmol/l and equilibrated by 95% 02 + 5% CO* (pH = 7.3). Hippocampi were chopped into slices (400 pm) and incubated in gas/liquid interface chamber for at least 80 minutes before measurement. After 20 min stabilization, the slices were exposed to HYP lasting 4.5-7.5 min by switching the 95% 02 + 5% COZ to 95% Nz+ 5% CO2 and by changing the oxygenated ACSF to that equilibrated by the NZ + CO2 mixture. Moreover, the glucose concentration was diminished to 4 mmol/l. Then ROX for 20 min followed. The drugs were dissolved in ACSF and were present in the superfbsing media throughout the experiment, except a-tocopherol which was administered to the animals daily for 10 days prior to decapitation in doses of 200 mg/kg P.O. Supramaximal stimulation (5-35 V, 0.05-o. 1 ms, 0.2 Hz) of Schaffer collaterals by bipolar wire electrode elicited population spike (PoS) in CA1 neurons recorded by an extracellular glass pipette electrode. The amplitude of the PoS was measured during the experiment as a marker of synaptic transmission. In hippocampal slices, susceptibility of neurotransmission to HYP/ROX was found to be reasonably homogenous in a given series of experiments. Nevertheless, remarkable differences in this parameter occurred between sets of experiments made in different times in spite of high effort to standardize the experimental conditions. Hence, before each series duration of HYP just sufficient to induce irreversible transmission failure in approximately 50 - 80% of slices was estimated. Consequently HYP lasting 30 set longer was used resulting typically in 90% irreversibility of control preparations. Results In the first series of experiments, HYP lasting 7.5 min resulted in rapid decay of PoS amplitude with only 7.8 + 2.0% recovery during ROX in untreated preparations. Slices treated with stobadine (30 umol/l) showed significant improvement with 74.5 + 10.8% PoS recovery during ROX. Moreover, in this group the PoS decay during HYP, expressed as half-time of the decay, was delayed from tin= 1.56 + 0.11 min to t iI = 2.56 f 0.25 min. Stobadine also decreased the rate of irreversibly damaged slices from 87.2% to 13.4%. The effective concentration of stobadine spanned from 1 pmol/l to 100 umol/l. The protective effect of trolox in concentration 200 umol/l on PoS recovery was remarkably smaller, attaining approximately a quarter of the effect of stobadine. Trolox had no effect on PoS decay during HYP The iron chelator deferoxamine had no effect at all. The concentration-response curve of stobadine and trolox were bell-shaped reaching their maximum approximately at IO-30 pmol/l and 200 pmol/l, respectively. Stobadine confirmed its neuroprotective features also in another series of experiments in which HYP lasted 4.5 min. Under these conditions the 21aminosteroid U-74389G (10 pmo/l) also revealed a significant increase of PoS recovery from 9.73 I4.88% to 39.1 + 9.2% and delayed the PoS decay during HYP from
Vol. 65, Nos. 18/19, 1999
Hippocampal Resistance to Oxidative Stress
1971
tin= 1.23 + 0.08 to 1.57 + 0.15 min. A similar pattern of protective features was present also in 30 and 100 pmol/l melatonin treated groups with an even more prominent delay of PoS decay during HYP. Higher concentrations of melatonin were not effective. a-tocopherol had no remarkable protective effect under the conditions tested. Discussion
The compounds studied were able to interfere with the generation and/or action of ROS. Thus stobadine was shown to be a hydroxyl, peroxyl and alkoxyl radical scavenger, hypochloric acid scavenger and singlet oxygen quencher (4) while a-tocopherol and its water soluble congener trolox are well established antioxidants (5). The antioxidant property of the 21aminosteroid U74389G and melatonin was also described (6,7). As ROS may participate in HYP/ROX induced injury of nervous tissue (5) it is conceivable to link the observed protective effect of the compounds tested to the improved antioxidative capacity of nervous tissue following their administration. Since the concentration of a-tocopherol in the hippocampus was not measured in this study, it is not possible to conclude whether this compound is actually not able to protect the tissue against HYIYROX injury effectively or whether the failure observed was due to the insufficient bioavailability and distribution of the compound under the dosage regimen used. The delay of PoS amplitude decay during HYP occurring in the presence of stobadine, U-74389G and melatonin requires a special comment. In CA1 neurons an early extinction of PoS during I-IYP is due primarily to elevated release of adenosine (8). This might be a protective mechanism of neurons to HYP during which postsynaptic elements do not respond to presynaptic stimulation and no damage would develop after immediate tissue ROX. ROS might play a regulatory fimction in the initiation of adenosine release since antioxidants delayed its beginning. In spite of this delay, antioxidants improved the outcome of HYP. This may imply lowered needs of the challenged tissue for the initiation of the mechanism in antioxidant treated groups, possibly by shifting the oxido-redox balance of neurons to the redox site. Moreover, antioxidants might play a role also during later stages of HYP, and even during ROX, characterized by profound depolarization and Ca” dependent activation of enzymes participating in elevated ROS production. The results support the hypothesis of oxidative damage of nervous tissue by HYP and ROX and corroborate the neuroprotective potential of antioxidants in pathological states associated with oxidative stress. Ackowledgements
The authors are grateful to Dr. D.C. Zimmermann of Upjohn Co., Kalamazoo MI, USA for the sample of U-74389G. The study was supported by the Slovak Grant Agency for Science (Grants No 1018/96, No 5305/97). References
1. 2. 3. 4. 5. 6. 7. 8.
P.G. AITKEN and S.J. SCI-IIFF Neurosci. Lett. 67 92-96 (1986). L.V. DAWSON and T.M. DAWSON Death and Differentiation 3 71-78 (1996). S. STOLC and R. VLKOLINSK? Pharmacol. Res. 31 (Suppl.) 295 (1995). L. HORAKOVA and S. STOLC Gen. Pharmac. 30 627-638 (1998). Z. DURACKOVA I. BERGENDI, A. LIPTAKOVA and J. MUCHOVA Brat. Lek. Listy 94 419-434 (1993) W.C. CLARK, J.S. HAZEL and B.M. COULL Drugs 50 971-983 (1995) R.J. REITER, D. MELCHIORRI, E. SEWERYNEK, L. BARLOW-WALDEN, J. CHUANG, G.G. ORTIZ and D. ACU$IA-CASTROVIEJO J. Pineal Res. 18 l-l 1 (1995) D.J. DOOLETTE and D.I. KERR Brain Res. 667 127-137 (1997)