368
Brain Research, 170 (1979) 36~ ~7t :(~, Elsevier/North-Holland Biomedical Pres~
Effects of aging on cerebral mmbotism following bilateral carotid ligation in male spontaneously hypertensive rats (8HR)
K1NYA TAMAKI, MASATOSHI FUJ1SHIMA, JUN OGATA, YASUO NAKATOMI, TAKAO ISHITSUKA, SEIZO SADOSHIMA and TERUO OMAE
The Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka (Japan) (Accepted March 15th, 1979)
Meyer et al. 6 have reported a progressive decrease in regional cerebral blood flow (CBF) and an increase in cerebrovascular resistance with advancing age in normal and hypertensive subjects. Shenkin et al.9. however, failed to show the significant reduction of CBF and metabolism with advancing age when patients with arteriosclerosis and hypertenston were excluded. The reduction of CBF in the aged subjects who do not manifest cerebral symptoms is considered to be due to some subclinical brain disorders. Therefore, old age per se appears to cause only a negligible diminution of the cerebral metabolism and blood flow 5. We have reported the metabolic and pathological studies of the experimental cerebral infarction produced by bilateral carotid artery occlusion in spontaneously hypertensive rats (SHR) 2-4,7,s. In the present study, to elucidate the effects of aging and blood pressure level on the susceptibility to cerebral ischemia, we measured cerebral lactate and pyruvate concentrations following bilateral carotid occlusion. Male SHR aged 1.5-15 months were anesthetized with intraperitonealamobarbital (100 mg/kg body weight). One femoral artery was cannulated for blood pressure recording and for anaerobic sampling for blood gas analysis. The animals which showed hypoxemm, hypocapnea or hypercapnea under spontaneous breathing of room air, were discarded from the present results. A plastic funnel was fitted into a skin incision over the skull bone for subsequent freezing of brain tissue. Both common carotid arteries were exposed and occluded simultaneously in 39 male S H R as an experimental group, and remained opened in another 26 mate SHR as a control group. At one hour intervals, the heads were frozen in situ by pouring liquid nitrogen into the plastic funnel, and then the entire supratentorial portion of the brain was chiselled out in the frozen state. In rapid sequence, the frozen brain was weighed and ground after the addition of cold perchloric acid. The tissue homogenate maintained at 0-4 °C was centrifuged and neutralized with potassium hydrochloride at pH between 4.5 and 5.0. Lactate and pyruvate concentrations in the tissue homogenate were determined by the standard enzymatic methods as described previouslyL
369
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22
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v
20 18
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•
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140. 120. 100. 80. 60
. . . . . I, Control
40
--o
I
2
3
4
5
6
7
8
9
IO
II
12
13
Exper,menl
14
15
A g e (months)
Fig. I. Supratentorial lactate and mean arterial pressure (MAP) with age in male S H R with ( e x p e r i and without (control) bilateral carotid occlusion. Cerebral lactate in the experimental group is increased with advancing age up to 5 months and thereafter reaches a plateau level. These changes are very similar to those in blood pressure with age. mental)
Fig. I shows changes in lactate and mean arterial pressure (MAP) with age. MAP rose progressively with age and reached a plateau at the age of 5 months. There was a slight increase in lactate at the age of 3 months, followed by a great increase at 5 months, but no further changes in older aged animals. In control SHR, cerebral lactate remained substantially unchanged with age. Changes in cerebral lactate were highly correlated with either M A P level (P < 0.005) or age (P < 0.001) in the younger animals with carotid occlusion, while in those beyond 5 months of age there was no such relationship. Mean values for MAP, arterial acid base parameter in each age group are given in Table I and for cerebral metabolites in Table [I. Significant differences in cerebral lactate were shown between the control and experimental groups of age 3 months or more and those in L/P ratio were of older than 5 months but not between the groups of younger animals. Regional CBF in SHR of either age 5 or 10 months is essentially the same as that in normotensive control rats 11. However, SHR aged over 5 months showed fibrinoid degeneration of the cerebral vessels while in the younger animals no such vascular
370 TABLE I
Mean arterial pressure ( M A P ) and arterial acid-base balanc e at resting state in male S H R with (IS) and without ( C) one hour bilateral carotid occlusion Values are mean 3_: S.E.M.
Age (months) 1.5
c E C E C E C E C E
3 5 6-8 >9
No. of rats
MAP (mmHg/
pH
5 6 3 8 4 9 6 9 9 6
124 109 153 159 171 190 166 175 178 185
7.392 7.346 7.413 7.377 7.396 7.396 7.425 7.420 7.412 7.402
changes were demonstrated.
± 8 ± 12 _~ 12 ± 9 i 7 ~ 7 i: 8 ~: 4 ~ 5 i 4
± ± ~ ± ± ± ± ± ± ±
0.015 0.039 0.026 0,015 0.018 0.011 0.010 0:017 0;033 0.013
pC02 (mmHg)
poe ~mmHg)
36.2 36.8 36.8 37.4 37.6 37.4 38.6 40.6 39.9 42.1
85~3 91.1 85.3 88.6 78.0 85.4 81.1 76.1 80.6 69.3
± 1.8 ± 2.4 i 412 ~ 1.1 ± 1.0 i 1.4 i: 2.0 ± 1.8 _£ 1.2 t:: 0.9
% 5.4 ~:: 1.4 -:E 5.4 ~ 2.9 i 1.8 :*,- A4 :+: 10.3 ± 2.9 :~- 4.7 ~ 1.4
H i g h b l o o d p r e s s u r e is n o t e d t o b e m o r e i m p o r t a n t f o r
c e r e b r o v a s c u l a r a t h e r o g e n e s i s t h a n t h e s e r u m c h o l e s t e r o l levels o r a g e 1 ° T h e p r e s e n t r e s u l t s , h o w e v e r , s h o w e d a p r o g r e s s i v e i n c r e a s e in c e r e b r a l l a c t a t e following bilateral carotid occlusion either with advancing age or with rising of blood pressure up to the age of 5 months
but no further increase in the older animals,
s u g g e s t i n g t h a t a g i n g is less i m p o r t a n t f o r a f a c t o r a f f e c t i n g t h e e x p e r i m e n t a l l y i n d u c e d cerebral ischemia in SHR. I n c o n c l u s i o n , i s c h e m i c m e t a b o l i s m o f t h e b r a i n s u c h as a n i n c r e a s e i n l a c t a t e and L/P ratio following 1 h bilateral carotid occlusion was not directly affected by
TABLE II
Cerebral metabolites in male S H R with ( E) and without ( C) one hour bilateral carotid occlusion Values are mean ± S.E.M.
Age (months) 1.5 3 5 6-8 >9
C E C E C E C E C E
No. of rats
Lactate ( mM/ kg )
Pyruvate ( mM/ kg )
LIP ratio
5 6 3 8 4 9 6 9 9 6
3.02 4.91 2.87 5.41 2.19 12.02 2.17 13.40 1.82 14.98
0.157 23 0.040 0.144 ± 0.032 0.204 ± 0.063 0.199 ± 0.021 0.168 x 0.010 0.149 ± 0.055 0,134 i 0,015 0.194 4- 0.051 0.140 + 0.070 0.163:1:0.016
18.2 43.5 17.5 28.6 13.0 92.3 17.3 76.2 15.3 75.4
~ 0.34 ± 1.00 ~ 0.47 23 0.62* ~ 0.33 ± 1.61"* ~ 0.13 :~ 1.00*** :z 0.30 ± 1.24"**
53 4.0 ~ 14.0 ± 5.6 ± 3.2 ± 1.6 ± 19.5" :~ 2.3 ± 12.2"* ~ 5.9 ± 7.5***
Statistical significance between C and E, * P < 0.05; ** P < 0.005; *** P < 0.001.
371
advancing age. The blood pressure level may contribute more to the susceptibility to cerebral ischemia. This work was supported in part by Scientific Research G r a n t 244045 from the Japanese Ministry of Education.
l Amano, S., Vascular changes in the brain of spontaneously hypertensive rats: hyaline and fibrinoid degeneration, J. Path., 121 (1977) 119 128. 2 Fujishima, M., Sugi, T., Morotomi, Y. and Omae, T., Effects of bilateral carotid artery ligation on brain lactate and pyruvate concentrations in normotensive and spontaneously hypertensive rats, Stroke, 6 (1975) 62-66. 3 Fujishima, M., Nakatomi, Y., Tamaki, K., Ogata, J. and Omae, T., Cerebral ischemia induced by bilateral carotid occlusion in spontaneously hypertensive rats, J. Neurol. Sei., 33 (1977) 1-11. 4 Fujishima, M., Ogata, J., Sugi, T. and Omae, T., Mortality and cerebral metabolism after bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats, J. Neurol. Neurosurg. Psychiat., 39 (I 976) 212-219. 5 lngvar, D. and Lassen, N., Brain Metabolism and Cerebral Disorders, Spectrum, New York, 1976, 193 pp. 6 Meyer, J. S., lshihara, N., Deshmukh, V. D., Naritomi, H., Sakai, F., Hsu, M-C. and Pollock, P., Improved method for noninvasive measurement of regional cerebral blood flow by l'~3Xenon inhalation. 1. Description of method and normal values obtained in healthy volunteers, Stroke, 9 (1978) 195 205. 7 Nakatomi, Y., Fujishima, M., Tamaki, K., lshitsuka, T., Ogata, J. and Omae, T., Influence of sex on cerebral ischemia following bilateral carotid occlusion in spontaneously hypertensive rats: a metabolic study, Stroke, (1979) in press. 8 Ogata, J., Fujishima, M,, Morotomi, Y. and Omae, T., Cerebral infarction following bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats: a pathological study, Stroke, 7 (1976) 54 60. 9 Shenkin, H. A., Novak, P., Goluboff, B., Soffe, A. M. and Bortin, L., The effects of aging, arteriosclerosis, and hypertension upon the cerebral circulation, J. olin. Invest., 32 (1953) 459-465. 10 Yamori, Y., Horie, R., Sato, M. and Fukase, M., Hypertension as an important factor for cerebrovascular atherogenesis in rats, Stroke, 7 (1976) 120-125. I 1 Yamori, Y. and Horie, R., Developmental course of hypertension and regional cerebral blood flow in stroke-prone spontaneously hypertensive rats, Stroke, 8 (1977) 456-461.
Brain Research, 170 (1979) 36~ ~7t :(~, Elsevier/North-Holland Biomedical Pres~
Effects of aging on cerebral mmbotism following bilateral carotid ligation in male spontaneously hypertensive rats (8HR)
K1NYA TAMAKI, MASATOSHI FUJ1SHIMA, JUN OGATA, YASUO NAKATOMI, TAKAO ISHITSUKA, SEIZO SADOSHIMA and TERUO OMAE
The Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka (Japan) (Accepted March 15th, 1979)
Meyer et al. 6 have reported a progressive decrease in regional cerebral blood flow (CBF) and an increase in cerebrovascular resistance with advancing age in normal and hypertensive subjects. Shenkin et al.9. however, failed to show the significant reduction of CBF and metabolism with advancing age when patients with arteriosclerosis and hypertenston were excluded. The reduction of CBF in the aged subjects who do not manifest cerebral symptoms is considered to be due to some subclinical brain disorders. Therefore, old age per se appears to cause only a negligible diminution of the cerebral metabolism and blood flow 5. We have reported the metabolic and pathological studies of the experimental cerebral infarction produced by bilateral carotid artery occlusion in spontaneously hypertensive rats (SHR) 2-4,7,s. In the present study, to elucidate the effects of aging and blood pressure level on the susceptibility to cerebral ischemia, we measured cerebral lactate and pyruvate concentrations following bilateral carotid occlusion. Male SHR aged 1.5-15 months were anesthetized with intraperitonealamobarbital (100 mg/kg body weight). One femoral artery was cannulated for blood pressure recording and for anaerobic sampling for blood gas analysis. The animals which showed hypoxemm, hypocapnea or hypercapnea under spontaneous breathing of room air, were discarded from the present results. A plastic funnel was fitted into a skin incision over the skull bone for subsequent freezing of brain tissue. Both common carotid arteries were exposed and occluded simultaneously in 39 male S H R as an experimental group, and remained opened in another 26 mate SHR as a control group. At one hour intervals, the heads were frozen in situ by pouring liquid nitrogen into the plastic funnel, and then the entire supratentorial portion of the brain was chiselled out in the frozen state. In rapid sequence, the frozen brain was weighed and ground after the addition of cold perchloric acid. The tissue homogenate maintained at 0-4 °C was centrifuged and neutralized with potassium hydrochloride at pH between 4.5 and 5.0. Lactate and pyruvate concentrations in the tissue homogenate were determined by the standard enzymatic methods as described previouslyL
369
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22
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v
20 18
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2.. ~. .... "6 . . . . . . . . . . . . . do
•
•
o 9p-----~----.-o-
o
.o
24( 22C
1
200"
•
180'
8* , - - /
fi E
%~'= % ~_..
.--'-
160,
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co
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•
.-'"
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•
-°- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
o
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oo
140. 120. 100. 80. 60
. . . . . I, Control
40
--o
I
2
3
4
5
6
7
8
9
IO
II
12
13
Exper,menl
14
15
A g e (months)
Fig. I. Supratentorial lactate and mean arterial pressure (MAP) with age in male S H R with ( e x p e r i and without (control) bilateral carotid occlusion. Cerebral lactate in the experimental group is increased with advancing age up to 5 months and thereafter reaches a plateau level. These changes are very similar to those in blood pressure with age. mental)
Fig. I shows changes in lactate and mean arterial pressure (MAP) with age. MAP rose progressively with age and reached a plateau at the age of 5 months. There was a slight increase in lactate at the age of 3 months, followed by a great increase at 5 months, but no further changes in older aged animals. In control SHR, cerebral lactate remained substantially unchanged with age. Changes in cerebral lactate were highly correlated with either M A P level (P < 0.005) or age (P < 0.001) in the younger animals with carotid occlusion, while in those beyond 5 months of age there was no such relationship. Mean values for MAP, arterial acid base parameter in each age group are given in Table I and for cerebral metabolites in Table [I. Significant differences in cerebral lactate were shown between the control and experimental groups of age 3 months or more and those in L/P ratio were of older than 5 months but not between the groups of younger animals. Regional CBF in SHR of either age 5 or 10 months is essentially the same as that in normotensive control rats 11. However, SHR aged over 5 months showed fibrinoid degeneration of the cerebral vessels while in the younger animals no such vascular
370 TABLE I
Mean arterial pressure ( M A P ) and arterial acid-base balanc e at resting state in male S H R with (IS) and without ( C) one hour bilateral carotid occlusion Values are mean 3_: S.E.M.
Age (months) 1.5
c E C E C E C E C E
3 5 6-8 >9
No. of rats
MAP (mmHg/
pH
5 6 3 8 4 9 6 9 9 6
124 109 153 159 171 190 166 175 178 185
7.392 7.346 7.413 7.377 7.396 7.396 7.425 7.420 7.412 7.402
changes were demonstrated.
± 8 ± 12 _~ 12 ± 9 i 7 ~ 7 i: 8 ~: 4 ~ 5 i 4
± ± ~ ± ± ± ± ± ± ±
0.015 0.039 0.026 0,015 0.018 0.011 0.010 0:017 0;033 0.013
pC02 (mmHg)
poe ~mmHg)
36.2 36.8 36.8 37.4 37.6 37.4 38.6 40.6 39.9 42.1
85~3 91.1 85.3 88.6 78.0 85.4 81.1 76.1 80.6 69.3
± 1.8 ± 2.4 i 412 ~ 1.1 ± 1.0 i 1.4 i: 2.0 ± 1.8 _£ 1.2 t:: 0.9
% 5.4 ~:: 1.4 -:E 5.4 ~ 2.9 i 1.8 :*,- A4 :+: 10.3 ± 2.9 :~- 4.7 ~ 1.4
H i g h b l o o d p r e s s u r e is n o t e d t o b e m o r e i m p o r t a n t f o r
c e r e b r o v a s c u l a r a t h e r o g e n e s i s t h a n t h e s e r u m c h o l e s t e r o l levels o r a g e 1 ° T h e p r e s e n t r e s u l t s , h o w e v e r , s h o w e d a p r o g r e s s i v e i n c r e a s e in c e r e b r a l l a c t a t e following bilateral carotid occlusion either with advancing age or with rising of blood pressure up to the age of 5 months
but no further increase in the older animals,
s u g g e s t i n g t h a t a g i n g is less i m p o r t a n t f o r a f a c t o r a f f e c t i n g t h e e x p e r i m e n t a l l y i n d u c e d cerebral ischemia in SHR. I n c o n c l u s i o n , i s c h e m i c m e t a b o l i s m o f t h e b r a i n s u c h as a n i n c r e a s e i n l a c t a t e and L/P ratio following 1 h bilateral carotid occlusion was not directly affected by
TABLE II
Cerebral metabolites in male S H R with ( E) and without ( C) one hour bilateral carotid occlusion Values are mean ± S.E.M.
Age (months) 1.5 3 5 6-8 >9
C E C E C E C E C E
No. of rats
Lactate ( mM/ kg )
Pyruvate ( mM/ kg )
LIP ratio
5 6 3 8 4 9 6 9 9 6
3.02 4.91 2.87 5.41 2.19 12.02 2.17 13.40 1.82 14.98
0.157 23 0.040 0.144 ± 0.032 0.204 ± 0.063 0.199 ± 0.021 0.168 x 0.010 0.149 ± 0.055 0,134 i 0,015 0.194 4- 0.051 0.140 + 0.070 0.163:1:0.016
18.2 43.5 17.5 28.6 13.0 92.3 17.3 76.2 15.3 75.4
~ 0.34 ± 1.00 ~ 0.47 23 0.62* ~ 0.33 ± 1.61"* ~ 0.13 :~ 1.00*** :z 0.30 ± 1.24"**
53 4.0 ~ 14.0 ± 5.6 ± 3.2 ± 1.6 ± 19.5" :~ 2.3 ± 12.2"* ~ 5.9 ± 7.5***
Statistical significance between C and E, * P < 0.05; ** P < 0.005; *** P < 0.001.
371
advancing age. The blood pressure level may contribute more to the susceptibility to cerebral ischemia. This work was supported in part by Scientific Research G r a n t 244045 from the Japanese Ministry of Education.
l Amano, S., Vascular changes in the brain of spontaneously hypertensive rats: hyaline and fibrinoid degeneration, J. Path., 121 (1977) 119 128. 2 Fujishima, M., Sugi, T., Morotomi, Y. and Omae, T., Effects of bilateral carotid artery ligation on brain lactate and pyruvate concentrations in normotensive and spontaneously hypertensive rats, Stroke, 6 (1975) 62-66. 3 Fujishima, M., Nakatomi, Y., Tamaki, K., Ogata, J. and Omae, T., Cerebral ischemia induced by bilateral carotid occlusion in spontaneously hypertensive rats, J. Neurol. Sei., 33 (1977) 1-11. 4 Fujishima, M., Ogata, J., Sugi, T. and Omae, T., Mortality and cerebral metabolism after bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats, J. Neurol. Neurosurg. Psychiat., 39 (I 976) 212-219. 5 lngvar, D. and Lassen, N., Brain Metabolism and Cerebral Disorders, Spectrum, New York, 1976, 193 pp. 6 Meyer, J. S., lshihara, N., Deshmukh, V. D., Naritomi, H., Sakai, F., Hsu, M-C. and Pollock, P., Improved method for noninvasive measurement of regional cerebral blood flow by l'~3Xenon inhalation. 1. Description of method and normal values obtained in healthy volunteers, Stroke, 9 (1978) 195 205. 7 Nakatomi, Y., Fujishima, M., Tamaki, K., lshitsuka, T., Ogata, J. and Omae, T., Influence of sex on cerebral ischemia following bilateral carotid occlusion in spontaneously hypertensive rats: a metabolic study, Stroke, (1979) in press. 8 Ogata, J., Fujishima, M,, Morotomi, Y. and Omae, T., Cerebral infarction following bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats: a pathological study, Stroke, 7 (1976) 54 60. 9 Shenkin, H. A., Novak, P., Goluboff, B., Soffe, A. M. and Bortin, L., The effects of aging, arteriosclerosis, and hypertension upon the cerebral circulation, J. olin. Invest., 32 (1953) 459-465. 10 Yamori, Y., Horie, R., Sato, M. and Fukase, M., Hypertension as an important factor for cerebrovascular atherogenesis in rats, Stroke, 7 (1976) 120-125. I 1 Yamori, Y. and Horie, R., Developmental course of hypertension and regional cerebral blood flow in stroke-prone spontaneously hypertensive rats, Stroke, 8 (1977) 456-461.
