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[53] Brain antioxidant activity of spin traps in Mongolian gerbils
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used to measure conjugated dienes, thus rendering that method invalid. It is worth noting that PBN also interferes with GLC analyses of fatty acids. However, PBN does not interfere with the GLC method of Kuksis et al. 31 which offers detailed information regarding the molecular species of CE and TG. The evidence presented in this study indicates that PBN is an effective antioxidant for LDL. The cardioprotective effects of PBN have previously been demonstrated. 23'34,35 PBN has been shown to reverse partially the reperfusion-induced damage in intact myocardium of dogs 23'34and to protect against reperfusion-induced arrhythmias in isolated heart models. 35 Taken together, results of these studies suggest that PBN or other spintrapping agents with similar chemical characteristics may be developed into antiatherogenic agents for use by humans. Acknowledgments The author thanks M.-m. Huang and C. Burden for skillfultechnical assistance, R. Whitmer for performingthe GLC analyses,and J. Pilcherfor typingthe manuscript. This work was supportedin part by the resourcesof the OklahomaMedicalResearchFoundation. 34R. Boll, M. O. Jeroudi, B. S. Patel, C. M. Dubose, E. K. Lai, R. Roberts, and P. B. McCay, Proc. Natl. Acad. Sci. U.S.A. 86, 4695 (1989). 35D. J. Hearse and A. Tosaki, Circ. Res. 60, 375 (1987).
[53] B r a i n A n t i o x i d a n t A c t i v i t y o f S p i n T r a p s in M o n g o l i a n G e r b i l s B y JOHN M. CARNEY and ROBERT A. FLOYD
Introduction Previous studies have demonstrated that there is a relationship between brain protein oxidation, reduction in marker enzyme activities, and behavioral dysfunction. 1 In an initial series of studies, we demonstrated that brain ischemia-reperfusion injury (IRI) results in significant increases in both salicylate hydroxylation and soluble brain protein oxidation, decreases in brain glutamine synthetase (glutamate-ammonia ligase) activ-
1 j. M. Carney, P. E. Starke-Reed, C. N. Oliver, R. W. Landum, M. S. Cheng, J. F. W u , a n d R . A . F l o y d , Proc. Natl. Acad. Sci. U.S.A. 88, 3636 (1991).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All fights of reproduction in any form reserved.
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ity, and alterations in spontaneous exploratory activity. 2,3 The IRI-mediated changes in behavior are accentuated in aged gerbils. We have discovered that the changes in IRI-mediated oxidation and behavioral dysfunction could be prevented by pretreatment with phenyl tert-butylnitrone (PBN) and related spin-trapping c o m p o u n d s ) : Based on the results obtained with PBN in IRI-mediated oxidation, we determined if age-related alterations in brain function could be the result of progressive oxidative events, as had been proposed in the p a s t ) In a separate set of studies, we demonstrated that aged gerbils have a higher level of protein oxidation, compared to young adult gerbils. 1 As a result of these initial observations, the effects of daily PBN injections were evaluated on brain protein oxidation, glutamine synthetase activity, and radial arm maze performance. Radial arm maze performance is often used as an indicator of the status of short-term temporal-spatial memory in animals. 6 We present here more details of the PBN administration schedule as well as how the animals were tested in the behavior study protocol. Animals The subjects are young adult male gerbils (3-4 months of age) and aged, retired, male breeder gerbils (18-20 months of age) obtained from Tumblebrook Farms (West Brookfield, MA). Gerbils are housed three to a cage in standard rodent cages. Animals are maintained in the University of Kentucky central animal facility under a 12-hr light-dark cycle. All experiments are conducted during the light phase of the cycle. Food and water are available ad libitum throughout the day. Methods
Young adult male (3-4 months of age) and retired male breeder gerbils (18-20 months of age) are assigned to separate groups of 18 gerbils each. One group of young adult and aged animals is assigned to the vehicle (saline) control group, and the other groups receive PBN. Animals are given intraperitoneal injections twice daily (8:00 am and 8:00 pm) for a 2 W. Cao, J. M. Carney, A. Duchon, R. A. Floyd, and M. Chevion, Neurosci. Lett. 88, 233 (1988). 3 C. N. Oliver, P. E. Starke-Reed, E. R. Stadtman, G. J. Liu, J. M. Carney, and R. A. Floyd, Proc. Natl. Acad. Sci. U.S.A. 87, 5144 (1990). 4 R. A. Floyd, Science 254, 1597 (1991). D. Harman, Proc. Natl. Acad. Sci. U.S.A. 78, 7124 (1981). 6 D. S. Olton, J. T. Becker, and G. E. Handleman, Behav. Brain Sci. 2, 313 (1979).
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I
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I
FIG. 1. Radial arm maze testing apparatus for gerbils. Subjects were placed in the central start chamber (A) and allowed to acclimate for 3 min. At the end of the acclimation period, the doors were raised and the gerbil was free to explore the arms of the maze (B).
period of 14 consecutive days. It should be noted that PBN is light sensitive and is relatively stable at neutral pH. PBN obtained from Aldrich Chemical Co. (Milwaukee, WI) is dissolved freshly in neutral saline and administered at a dose of 32 mg/kg/injection (3.2 mg/ml). In subsequent studies, substantially lower doses have been used with comparable results. At the end of the 14 days, the animals are given an additional day of no injections to allow for the elimination of any residual PBN prior to testing. Previous studies with rodents had indicated that the half-life of PBN is between 2 and 3 hr. 7 Thus the extra day allowed for essentially all of the PBN to be eliminated in the period equal to approximately 10 half-lives. After the 24hr washout period, the gerbils are tested for radial arm maze performance. Radial Arm Maze Test
An eight-arm radial maze is used for testing patrolling behavior performance (Fig. 1). The gerbils are placed one at a time in the central compartment of the sunburst maze. When the doors to the arms are raised, each gerbil is free to explore the maze. Reentry into an arm more than once before exploring all eight arms is considered an error. Arm entry is registered electronically. Animals have 15 min to explore the maze. Normal young adult gerbils made between 4 and 5 errors, while aged gerbils made 9-11 errors. After 14 days of PBN treatment, the young adult gerbils made the same number of errors as the control group.1 In contrast, when aged gerbils that had received chronic PBN were tested, 7 G. Chen, T. M. Bray, E. G. Janzen, and P. B. McCay, Free Radical Res. Commun. 9, 317 (1990).
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they made significantly fewer errors. 1 In fact, the aged gerbils made the same number of errors as did the young adult gerbils that were 14 months younger. A parallel group of gerbils are posed in an identical manner and used for biochemical determinations during the period of PBN treatment. Chronic treatment resulted in a progressive decrease in the level of oxidized protein in the soluble protein fraction of the homogenate. 1 This progressive decrease in oxidized protein was mirrored by the concomitant increase in both glutamine synthetase and neutral protease activities in the aged gerbils. There was no change in these biochemical end points in the young adult gerbils treated with PBN in the same manner as the aged gerbils. 1 At the end of the 14 days, the PBN injections are terminated. A progressive return to the original level of protein oxidation was seen in the aged control gerbils and a progressive decrease in enzyme activities. Saline injections had no effect on these measures. Therefore, it appears there is a higher level of oxidative stress on the aged brain that can be modified, but the chronic stress apparently continued even in the face of chronic PBN administration. The intracellular site of generation of the oxidizing species remains to be identified and the defect characterized. One intriguing possibility is that damaged mitochondria may give rise to these oxidizing species. Acknowledgments Research was supported in part by National Institutes of Health Grants NS23307 and AG09690 and monies from the Glenn Foundation for Medical Research.
[54] A n t i o x i d a n t A c t i v i t y o f N i t e c a p o n e a n d Its A n a l o g O R - 1 2 4 6 : E f f e c t o f S t r u c t u r a l Modification on Antioxidant Action
By LUCIA MARCOCCI, YUICHIRO J. SUZUKI, MASAHIKO TSUCHIYA, and LESTER PACKER Introduction Nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione] (Fig. 1), an effective peripherally acting inhibitor of catechol O-methylMETHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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used to measure conjugated dienes, thus rendering that method invalid. It is worth noting that PBN also interferes with GLC analyses of fatty acids. However, PBN does not interfere with the GLC method of Kuksis et al. 31 which offers detailed information regarding the molecular species of CE and TG. The evidence presented in this study indicates that PBN is an effective antioxidant for LDL. The cardioprotective effects of PBN have previously been demonstrated. 23'34,35 PBN has been shown to reverse partially the reperfusion-induced damage in intact myocardium of dogs 23'34and to protect against reperfusion-induced arrhythmias in isolated heart models. 35 Taken together, results of these studies suggest that PBN or other spintrapping agents with similar chemical characteristics may be developed into antiatherogenic agents for use by humans. Acknowledgments The author thanks M.-m. Huang and C. Burden for skillfultechnical assistance, R. Whitmer for performingthe GLC analyses,and J. Pilcherfor typingthe manuscript. This work was supportedin part by the resourcesof the OklahomaMedicalResearchFoundation. 34R. Boll, M. O. Jeroudi, B. S. Patel, C. M. Dubose, E. K. Lai, R. Roberts, and P. B. McCay, Proc. Natl. Acad. Sci. U.S.A. 86, 4695 (1989). 35D. J. Hearse and A. Tosaki, Circ. Res. 60, 375 (1987).
[53] B r a i n A n t i o x i d a n t A c t i v i t y o f S p i n T r a p s in M o n g o l i a n G e r b i l s B y JOHN M. CARNEY and ROBERT A. FLOYD
Introduction Previous studies have demonstrated that there is a relationship between brain protein oxidation, reduction in marker enzyme activities, and behavioral dysfunction. 1 In an initial series of studies, we demonstrated that brain ischemia-reperfusion injury (IRI) results in significant increases in both salicylate hydroxylation and soluble brain protein oxidation, decreases in brain glutamine synthetase (glutamate-ammonia ligase) activ-
1 j. M. Carney, P. E. Starke-Reed, C. N. Oliver, R. W. Landum, M. S. Cheng, J. F. W u , a n d R . A . F l o y d , Proc. Natl. Acad. Sci. U.S.A. 88, 3636 (1991).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All fights of reproduction in any form reserved.
524
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[53]
ity, and alterations in spontaneous exploratory activity. 2,3 The IRI-mediated changes in behavior are accentuated in aged gerbils. We have discovered that the changes in IRI-mediated oxidation and behavioral dysfunction could be prevented by pretreatment with phenyl tert-butylnitrone (PBN) and related spin-trapping c o m p o u n d s ) : Based on the results obtained with PBN in IRI-mediated oxidation, we determined if age-related alterations in brain function could be the result of progressive oxidative events, as had been proposed in the p a s t ) In a separate set of studies, we demonstrated that aged gerbils have a higher level of protein oxidation, compared to young adult gerbils. 1 As a result of these initial observations, the effects of daily PBN injections were evaluated on brain protein oxidation, glutamine synthetase activity, and radial arm maze performance. Radial arm maze performance is often used as an indicator of the status of short-term temporal-spatial memory in animals. 6 We present here more details of the PBN administration schedule as well as how the animals were tested in the behavior study protocol. Animals The subjects are young adult male gerbils (3-4 months of age) and aged, retired, male breeder gerbils (18-20 months of age) obtained from Tumblebrook Farms (West Brookfield, MA). Gerbils are housed three to a cage in standard rodent cages. Animals are maintained in the University of Kentucky central animal facility under a 12-hr light-dark cycle. All experiments are conducted during the light phase of the cycle. Food and water are available ad libitum throughout the day. Methods
Young adult male (3-4 months of age) and retired male breeder gerbils (18-20 months of age) are assigned to separate groups of 18 gerbils each. One group of young adult and aged animals is assigned to the vehicle (saline) control group, and the other groups receive PBN. Animals are given intraperitoneal injections twice daily (8:00 am and 8:00 pm) for a 2 W. Cao, J. M. Carney, A. Duchon, R. A. Floyd, and M. Chevion, Neurosci. Lett. 88, 233 (1988). 3 C. N. Oliver, P. E. Starke-Reed, E. R. Stadtman, G. J. Liu, J. M. Carney, and R. A. Floyd, Proc. Natl. Acad. Sci. U.S.A. 87, 5144 (1990). 4 R. A. Floyd, Science 254, 1597 (1991). D. Harman, Proc. Natl. Acad. Sci. U.S.A. 78, 7124 (1981). 6 D. S. Olton, J. T. Becker, and G. E. Handleman, Behav. Brain Sci. 2, 313 (1979).
[53]
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I
525
I
FIG. 1. Radial arm maze testing apparatus for gerbils. Subjects were placed in the central start chamber (A) and allowed to acclimate for 3 min. At the end of the acclimation period, the doors were raised and the gerbil was free to explore the arms of the maze (B).
period of 14 consecutive days. It should be noted that PBN is light sensitive and is relatively stable at neutral pH. PBN obtained from Aldrich Chemical Co. (Milwaukee, WI) is dissolved freshly in neutral saline and administered at a dose of 32 mg/kg/injection (3.2 mg/ml). In subsequent studies, substantially lower doses have been used with comparable results. At the end of the 14 days, the animals are given an additional day of no injections to allow for the elimination of any residual PBN prior to testing. Previous studies with rodents had indicated that the half-life of PBN is between 2 and 3 hr. 7 Thus the extra day allowed for essentially all of the PBN to be eliminated in the period equal to approximately 10 half-lives. After the 24hr washout period, the gerbils are tested for radial arm maze performance. Radial Arm Maze Test
An eight-arm radial maze is used for testing patrolling behavior performance (Fig. 1). The gerbils are placed one at a time in the central compartment of the sunburst maze. When the doors to the arms are raised, each gerbil is free to explore the maze. Reentry into an arm more than once before exploring all eight arms is considered an error. Arm entry is registered electronically. Animals have 15 min to explore the maze. Normal young adult gerbils made between 4 and 5 errors, while aged gerbils made 9-11 errors. After 14 days of PBN treatment, the young adult gerbils made the same number of errors as the control group.1 In contrast, when aged gerbils that had received chronic PBN were tested, 7 G. Chen, T. M. Bray, E. G. Janzen, and P. B. McCay, Free Radical Res. Commun. 9, 317 (1990).
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[54]
they made significantly fewer errors. 1 In fact, the aged gerbils made the same number of errors as did the young adult gerbils that were 14 months younger. A parallel group of gerbils are posed in an identical manner and used for biochemical determinations during the period of PBN treatment. Chronic treatment resulted in a progressive decrease in the level of oxidized protein in the soluble protein fraction of the homogenate. 1 This progressive decrease in oxidized protein was mirrored by the concomitant increase in both glutamine synthetase and neutral protease activities in the aged gerbils. There was no change in these biochemical end points in the young adult gerbils treated with PBN in the same manner as the aged gerbils. 1 At the end of the 14 days, the PBN injections are terminated. A progressive return to the original level of protein oxidation was seen in the aged control gerbils and a progressive decrease in enzyme activities. Saline injections had no effect on these measures. Therefore, it appears there is a higher level of oxidative stress on the aged brain that can be modified, but the chronic stress apparently continued even in the face of chronic PBN administration. The intracellular site of generation of the oxidizing species remains to be identified and the defect characterized. One intriguing possibility is that damaged mitochondria may give rise to these oxidizing species. Acknowledgments Research was supported in part by National Institutes of Health Grants NS23307 and AG09690 and monies from the Glenn Foundation for Medical Research.
[54] A n t i o x i d a n t A c t i v i t y o f N i t e c a p o n e a n d Its A n a l o g O R - 1 2 4 6 : E f f e c t o f S t r u c t u r a l Modification on Antioxidant Action
By LUCIA MARCOCCI, YUICHIRO J. SUZUKI, MASAHIKO TSUCHIYA, and LESTER PACKER Introduction Nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione] (Fig. 1), an effective peripherally acting inhibitor of catechol O-methylMETHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.