Experimental Section Gerontology 25: 185-191 (1979)
Effect of Aging on Brain Respiration and Carbohydrate Metabolism of CBF! Mice1 Michael S. Parmacek, Jacob, H. Fox, William H. Harrison, David C. Garron and Dennis Swenie Departments of Neurological Sciences, Biochemistry, Psychology, and Geriatrics, Rush Medical College and College of Health Sciences, Rush University, Chicago, 111.
Key Words. Mouse • Brain • Respiration - Hyperthermia • Metabolism • Glycolysis
The mental changes accompanying aging are not yet fully explicable in neurochemical terms (Samorajski and Ordy, 1972). Particularly lack ing is knowledge of the dynamic metabolic pro cesses in the brain associated with aging. Some elderly patients with brain dysfunction have decreased cerebral oxygen consumption (Kety and Schmidt, 1948), but it is unclear whether this results from metabolic dysfunction of the 1 Supported by NIH Grant 1 RO 3 MH 26907 and Rush-Presbyterian-St. Luke’s Grant-in-Aid No. 37123.
brain, neuronal loss, circulation dysfunction or a combination of these disorders (Himwich and Himwich, 1959; Sokoloff, 1966). Similarly, the interpretation of studies with aged animals (Reiner, 1947; Garbus, 1955; Sokoloff, 1966) are complicated by difficulty in distinguishing circulation effects from metabolic changes. We circumvented this dilemma by studying the metabolism of brain tissue slices with wellknown methods (Elliot, 1969\McIlwain, 1959, 1971), using the Syrian hamster, and comparing young (average age, 8 months) and old (average
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 6:35:36 PM
Abstract. Brain slices of mice (strain CBFt) were used to study the effect of aging on cerebral cortex respiration and metabolism. Young animals (average age 6 months) were compared with old animals (average age 34 months). Metabolism was measured at a normal temperature (37 °C) and under hyperthermic stress (40 °C). The brain slices were incubated with 14C-glucose under standard conditions with the following parameters being measured: oxygen uptake, 14C 0 2 produc tion, glucose utilization, and lactate and pyruvate formation. At the normal temperature, there were significant age-associated decreases in oxygen uptake and 14C 0 2 production but the other parameters were unchanged. At hyperthermic conditions there were significant age-associated decreases in oxygen uptake, 14C 0 2 production, lactate production, and glucose utilization. Also, in the hyperthermia study, all values were raised from control study values (37 °C) with old animals showing smaller increases in glucose utilization and lactate formation. These findings indicate the dysfunction of a number of metabolic pathways in the aged animal.
age, 18 months) animals (Fox et al., 1975a). No age-related differences were found with respect to oxygen uptake, C 0 2 production, glucose utilization, and lactate and pyruvate formation. These results did not rule out subtle age-related differences, differences in other variables, or differences which might arise in tissue function ing only under stress. We, also, could not rule out that the short survival time (18 months) of the Syrian hamster results from endemic illness rather than true ‘aging’. We have, therefore, repeated our hamster experiments with the CBF, hybrid mouse which has nearly twice the survival time (3 4-35 months) of the Syrian hamster (personal communication, Dr. Don Gibson, National Institute of Aging, NIH). We included a stress condition, hyperthermia, as a variable to check whether an age-related metab olic abnormality might be detectable only in tissue metabolizing under stress; brain slice ex periments were done with young and old mice at an elevated temperature (40 °C) as well as at a normal temperature (37 °C).
Materials and Methods Animals CBF, mice were obtained from the Charles River Breeding Laboratories (Wilmington, Mass.) from a colony maintained for the National Institute of Aging of the National Institutes of Health. All animals ap peared healthy at sacrifice. The old animals ranged from 33-35 months and the young, 6 -7 months. Tissue and Medium The mice were sacrificed by a guillotine. The brain was removed, separated into two hemispheres, and the sub-cortical structures removed. Two slices consisting wholly of grey matter, approximately 0.35 mm thick, were prepared from each hemisphere using a razor blade and a recessed guide (Mcllwain, 1951) and float ed in a Krebs-Ringer phosphate buffer pregassed with oxygen. A slice was picked up on a bent wire, drained of excess fluid on a watch glass surface, weighed on a
Parmacek/Fox/Harrison/Garron/Swenie
torsion balance to the nearest milligram, returned to the medium and then transferred to the incubation medium in the Warburg flask. The main compartment of the Warburg flask con tained a 3-ml incubation medium of NaCl, 124 mM; KCi, 4.8 mM; CaCl,, 2.6 mM; MgS04, 1.2 mM; Na; HP04, 16 mM, pH 7.4; O-glucose, 10 mM. The pH was pre-adjusted to pH 7.4 with NaOH, and the medi um was pregassed with oxygen for half an hour at 2 L/min. Two slices (total weight approximately 70 mg) were in each Warburg flask containing 3 ml medium and 1jrCi (U14C) D-glucose (specific radio activity 3 mCi/mM: (Amersham Searle Corporation, Arlington Heights, 111.). The center well of the flask had a filter paper wick and contained 0.2 ml of hy droxide of hyamine (1 molar solution in methanol) to trap the evolved CO! (Husain and Paradise, 1973). Incubation, Respiration and Tissue Extraction Incubation took place in a Warburg bath. Tempera ture was maintained at 37 ± 0.5 °C or 40 ± 0.5 °C for the hyperthermia experiments. The reading of oxygen uptake and the preparation of tissue extract involved a 15-min equilibration of the tissue in a Warburg flask containing the incubation medium of U14C-Z)-glucose followed by a 1-hour incubation. Then 0.3 ml of 50% (w/w) perchloric acid was used to stop the reaction. The tissue in the incubation medium was then re moved and homogenized at 0 °C (Fox et al., 1975b; Patel et al., 1973). Analysis and Calculations Since all our calculations were made on the basis of the tissue slice wet weight, a change in the water content of brain tissue with aging might distort results. Therefore, we weighed 30 slices (15 from young animals and 15 from old animals), and then dried them in an oven at 100 °C until a constant weight (dry weight was reached. The dry weight/wet weight ratio in the young animals was 0.142, and 0.148 in the older animals. Both figures lie between the 0.14-0.15 ratio obtained by Varon and Mcllwain (1961) for dry weight to wet weight ratio, and do not differ signifi cantly. O2 Uptake. The total oxygen consumed was con verted to micromoles per gram per hour after correct ing for thermobarometric changes. Determination o f 14CO, Production. The center well content was removed quantitatively, diluted to
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 6:35:36 PM
186
187
Aging and Brain Metabolism
Table I. Metabolic parameters of 37 and 40 °C incubations %
Age months
37 °C
40 °C
O; uptake umol/g/h ± SD
6 34
59.40 ± 7.32 (18) 1 46.94 ± 8.40 (25) J
p
Effect of Aging on Brain Respiration and Carbohydrate Metabolism of CBF! Mice1 Michael S. Parmacek, Jacob, H. Fox, William H. Harrison, David C. Garron and Dennis Swenie Departments of Neurological Sciences, Biochemistry, Psychology, and Geriatrics, Rush Medical College and College of Health Sciences, Rush University, Chicago, 111.
Key Words. Mouse • Brain • Respiration - Hyperthermia • Metabolism • Glycolysis
The mental changes accompanying aging are not yet fully explicable in neurochemical terms (Samorajski and Ordy, 1972). Particularly lack ing is knowledge of the dynamic metabolic pro cesses in the brain associated with aging. Some elderly patients with brain dysfunction have decreased cerebral oxygen consumption (Kety and Schmidt, 1948), but it is unclear whether this results from metabolic dysfunction of the 1 Supported by NIH Grant 1 RO 3 MH 26907 and Rush-Presbyterian-St. Luke’s Grant-in-Aid No. 37123.
brain, neuronal loss, circulation dysfunction or a combination of these disorders (Himwich and Himwich, 1959; Sokoloff, 1966). Similarly, the interpretation of studies with aged animals (Reiner, 1947; Garbus, 1955; Sokoloff, 1966) are complicated by difficulty in distinguishing circulation effects from metabolic changes. We circumvented this dilemma by studying the metabolism of brain tissue slices with wellknown methods (Elliot, 1969\McIlwain, 1959, 1971), using the Syrian hamster, and comparing young (average age, 8 months) and old (average
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 6:35:36 PM
Abstract. Brain slices of mice (strain CBFt) were used to study the effect of aging on cerebral cortex respiration and metabolism. Young animals (average age 6 months) were compared with old animals (average age 34 months). Metabolism was measured at a normal temperature (37 °C) and under hyperthermic stress (40 °C). The brain slices were incubated with 14C-glucose under standard conditions with the following parameters being measured: oxygen uptake, 14C 0 2 produc tion, glucose utilization, and lactate and pyruvate formation. At the normal temperature, there were significant age-associated decreases in oxygen uptake and 14C 0 2 production but the other parameters were unchanged. At hyperthermic conditions there were significant age-associated decreases in oxygen uptake, 14C 0 2 production, lactate production, and glucose utilization. Also, in the hyperthermia study, all values were raised from control study values (37 °C) with old animals showing smaller increases in glucose utilization and lactate formation. These findings indicate the dysfunction of a number of metabolic pathways in the aged animal.
age, 18 months) animals (Fox et al., 1975a). No age-related differences were found with respect to oxygen uptake, C 0 2 production, glucose utilization, and lactate and pyruvate formation. These results did not rule out subtle age-related differences, differences in other variables, or differences which might arise in tissue function ing only under stress. We, also, could not rule out that the short survival time (18 months) of the Syrian hamster results from endemic illness rather than true ‘aging’. We have, therefore, repeated our hamster experiments with the CBF, hybrid mouse which has nearly twice the survival time (3 4-35 months) of the Syrian hamster (personal communication, Dr. Don Gibson, National Institute of Aging, NIH). We included a stress condition, hyperthermia, as a variable to check whether an age-related metab olic abnormality might be detectable only in tissue metabolizing under stress; brain slice ex periments were done with young and old mice at an elevated temperature (40 °C) as well as at a normal temperature (37 °C).
Materials and Methods Animals CBF, mice were obtained from the Charles River Breeding Laboratories (Wilmington, Mass.) from a colony maintained for the National Institute of Aging of the National Institutes of Health. All animals ap peared healthy at sacrifice. The old animals ranged from 33-35 months and the young, 6 -7 months. Tissue and Medium The mice were sacrificed by a guillotine. The brain was removed, separated into two hemispheres, and the sub-cortical structures removed. Two slices consisting wholly of grey matter, approximately 0.35 mm thick, were prepared from each hemisphere using a razor blade and a recessed guide (Mcllwain, 1951) and float ed in a Krebs-Ringer phosphate buffer pregassed with oxygen. A slice was picked up on a bent wire, drained of excess fluid on a watch glass surface, weighed on a
Parmacek/Fox/Harrison/Garron/Swenie
torsion balance to the nearest milligram, returned to the medium and then transferred to the incubation medium in the Warburg flask. The main compartment of the Warburg flask con tained a 3-ml incubation medium of NaCl, 124 mM; KCi, 4.8 mM; CaCl,, 2.6 mM; MgS04, 1.2 mM; Na; HP04, 16 mM, pH 7.4; O-glucose, 10 mM. The pH was pre-adjusted to pH 7.4 with NaOH, and the medi um was pregassed with oxygen for half an hour at 2 L/min. Two slices (total weight approximately 70 mg) were in each Warburg flask containing 3 ml medium and 1jrCi (U14C) D-glucose (specific radio activity 3 mCi/mM: (Amersham Searle Corporation, Arlington Heights, 111.). The center well of the flask had a filter paper wick and contained 0.2 ml of hy droxide of hyamine (1 molar solution in methanol) to trap the evolved CO! (Husain and Paradise, 1973). Incubation, Respiration and Tissue Extraction Incubation took place in a Warburg bath. Tempera ture was maintained at 37 ± 0.5 °C or 40 ± 0.5 °C for the hyperthermia experiments. The reading of oxygen uptake and the preparation of tissue extract involved a 15-min equilibration of the tissue in a Warburg flask containing the incubation medium of U14C-Z)-glucose followed by a 1-hour incubation. Then 0.3 ml of 50% (w/w) perchloric acid was used to stop the reaction. The tissue in the incubation medium was then re moved and homogenized at 0 °C (Fox et al., 1975b; Patel et al., 1973). Analysis and Calculations Since all our calculations were made on the basis of the tissue slice wet weight, a change in the water content of brain tissue with aging might distort results. Therefore, we weighed 30 slices (15 from young animals and 15 from old animals), and then dried them in an oven at 100 °C until a constant weight (dry weight was reached. The dry weight/wet weight ratio in the young animals was 0.142, and 0.148 in the older animals. Both figures lie between the 0.14-0.15 ratio obtained by Varon and Mcllwain (1961) for dry weight to wet weight ratio, and do not differ signifi cantly. O2 Uptake. The total oxygen consumed was con verted to micromoles per gram per hour after correct ing for thermobarometric changes. Determination o f 14CO, Production. The center well content was removed quantitatively, diluted to
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 6:35:36 PM
186
187
Aging and Brain Metabolism
Table I. Metabolic parameters of 37 and 40 °C incubations %
Age months
37 °C
40 °C
O; uptake umol/g/h ± SD
6 34
59.40 ± 7.32 (18) 1 46.94 ± 8.40 (25) J
p
