Neuroscience Letters, 126 (1991) 21-24 ,c 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$03.50 ADONIS 030439409100206G
21
NSL 07724
Postischemic spontaneous hyperthermia is not a major aggravating factor for neuronal damage following repeated brief cerebral ischemia in the gerbil Hiroyuki Kato, Tsutomu Araki and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan)
(Received 16 November 1990; Revised version received 29 January 1991; Accepted 30 January 1991) Key words:
Cerebral ischemia; Repeated ischemia; Selective vulnerability; Neuronal damage; Hyperthermia; Gerbil
Brief and non-lethal cerebral ischemia produces most severe neuronal damage when such ischemia is induced repeatedly at l-h intervals. We examined whether spontaneous postischemic hyperthermia is an aggravating factor for the cumulative damage following repeated &hernia in the gerbil. We maintained body and cranial temperature at normothermia throughout the initial reperfusion period, but could not observe an amelioration of histopathological brain damage following two 2-min bilateral carotid artery occlusions at a l-h interval as compared to hyperthermic conditions. The results suggest that postischemic hyperthermia is not a major aggravating factor for the cumulative damage following repeated ischemic insults.
Brief and non-lethal cerebral ischemia can injure selectively vulnerable neurons when such ischemia is induced repeatedly [9, lo]. Two-min bilateral common carotid artery occlusion in the gerbil causes no morphological neuronal damage in the brain. However, two 2-min occlusions at a l-h interval destroy hippocampal CA1 pyramidal neurons, and 3 occlusions additionally produce moderate damage to the striatum and the thalamus. Of interest is that neuronal damage is most severe when ischemia is repeated at 1 h intervals [lo]. This phenomenon is consistent with other reports using different ischemia models [16, 171. Recently, experimental evidence indicating that differences in brain and body temperature during and after ischemia determine the extent and density of ischemic neuronal damage received a great deal of attention [2-4, 6-8, 13-15, 181.Therefore, one of the possible damaging factors for the cumulative damage following repeated ischemia at l-h intervals is the spontaneous postischemic hyperthermia because intraischemic and postischemic hyperthermia aggravate brain damage as compared to normothermic conditions [7, 13, 141. Following even 2min forebrain ischemia in the gerbil, body temperature increases, and, in consequence, secondary ischemia after
Correspondence:
nrnk nkpl.,=.=
H. Kato, Department Tnhntll
Tlnixwrcitu
Crhnd
of Neurology, nf
Mdirinp
Institute of 1-I
9,Grvn_
a l-h interval is induced at a stage of mild hyperthermia [l 11.The purpose of this study was, therefore, to examine whether the absence of postichemic hyperthermia following initial ischemic insult ameliorates neuronal damage following repeated ischemic insults. Male Mongolian gerbils (Seiwa Experimental Animals Co. Ltd., Fukuoka, Japan), aged 13-14 weeks and weighing 65-90 g were anesthetized with 2% halothane in a mixture of 3096 oxygen and 70% nitrous oxide using a semiclosed facemask. Bilateral common carotid arteries were exposed and occluded with aneurysm clips 1 min after discontinuation of anesthesia. Carotid artery blood flow was restored by releasing the clips following 2 min of occlusion. After a l-h interval, secondary occlusion was induced for 2 min. Rectal temperature was monitored for up to 2 hr after ischemic insults. Cranial temperature was also measured in eight animals under anesthesia by placing the thermoprobe under the scalp. Experiments were performed at air-conditioned room temperature at around 23°C. A total of 33 animals were divided into 5 groups. (1) Single 2-min occlusion (n = 6). (2) Two 2-min occlusions, in which anesthesia was induced only during operative procedures, therefore causing spontaneous postischemic hyperthermia (n = 6). (3) Two 2-min occlusions, in which anesthesia was induced only during operative procedures, but the animals were placed in a refrigerated room maintained at 4°C for 1 h after initial ischemia to main-
22
TABLE
I
BODY TEMPERATURE Values are expressed
(“C) OF THE ANIMALS
as mean i S.D. Before
n
During ischemia
Postischemia 10 min
60 min
30 min
I20 min
Initial ischemia anesthesia
-
normothermia
6
37.5+0.4
37.2 )O.
I
37.3 kO.3
37.4 & 0.3
37.4+0
hyperthermia
6
37.2kO.2
37.2 k 0. I
37.8 + 0.6
3x.7*0
38.2*0.3**
normothermia
9
37.3 +0.2
37.2kO.l
37.3 &O. I
hyperthermia
6
37.2kO.3
37.1 io.1
38.O;tO.2”
anesthesia
Secondary
3**
4
+ 38.b+O.
I* *
I 38.0+0.2”
37 3+0.2 *
37.3 i-0.
ischemia
anesthesia
-
normothermia
6
37.3 FO.3
37 7kO.3
38.410
2
3x l&O4
37.5kO.3
hyperthermia
6
37.X+0.3**
37.biO.S
3x.2 + 0.2
37.8 +O.?
37.b_+O.l
normothermia
9
37.2kO.l
37.1 kO.3
38.1 +o 2
38.OkO.2
37 7_+0.3
hyperthermia
6
37.9 + 0. I * *
37.3kO.2
38.2 kO.4
37.8kO.4
37.7 + 0.2
anesthesia
+
**PC 0.01 compared * *Pi
0.01 compared
Student‘s
to normothermic
animals
(unanesthetized
to normothermic
animals
(anesthetized
groups). groups).
r-test.
Two 2-min occlusions, in which anesthesia was maintained with 1% halothane in the same OI/NzO mixture for I h after initial ischemia, and the body temperature was maintained at normothermia (n = 9). (5) Two 2-min occlusions, in which anesthesia was maintained with 1% halothane for 1 h as in group 4 but the animals were warmed by external heating so that the body temperature was comparable to that in group 2 with postischemic hyperthermia (n = 6). At 7 days of survival, the animals were anesthetized with pentobarbital (50 mg/kg, i.p.), and the brains were perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 min following a brief heparinized saline flush. Paraffin sections, 5 pm in thickness, were prepared and stained with Cresyl violet and hematoxylin-eosin. The stained sections were examined with a light microscope and ischemic neuronal damage
TABLE
was graded
on a scale of 0 to 3: 0 = no damage,
4 and 5. Single 2-min ischemia caused no neuronal damage in the brain except for one animal with minimal damage. Two ischemic insults caused moderate to severe damage to the hippocampal CAI subfield, and mild damage to the CA4 subfield, the lateral part of the striatum. the
II
CRANIAL
TEMPERATURES
Values are expressed
(“C) OF THE ANIMALS
as mean + S.D. n
Anesthesia
Before
First
Postischemia
Second
ischemia
tschemia
30 min
ischemia
+
normothermia
4
36.8 +O.b
36.3&0.2
370+04
3b 5_+0.2
hyperthermia
4
36.4kO.3
36.4kO.3
17 7&0.2*
37.2&O 3**
*P
21
NSL 07724
Postischemic spontaneous hyperthermia is not a major aggravating factor for neuronal damage following repeated brief cerebral ischemia in the gerbil Hiroyuki Kato, Tsutomu Araki and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan)
(Received 16 November 1990; Revised version received 29 January 1991; Accepted 30 January 1991) Key words:
Cerebral ischemia; Repeated ischemia; Selective vulnerability; Neuronal damage; Hyperthermia; Gerbil
Brief and non-lethal cerebral ischemia produces most severe neuronal damage when such ischemia is induced repeatedly at l-h intervals. We examined whether spontaneous postischemic hyperthermia is an aggravating factor for the cumulative damage following repeated &hernia in the gerbil. We maintained body and cranial temperature at normothermia throughout the initial reperfusion period, but could not observe an amelioration of histopathological brain damage following two 2-min bilateral carotid artery occlusions at a l-h interval as compared to hyperthermic conditions. The results suggest that postischemic hyperthermia is not a major aggravating factor for the cumulative damage following repeated ischemic insults.
Brief and non-lethal cerebral ischemia can injure selectively vulnerable neurons when such ischemia is induced repeatedly [9, lo]. Two-min bilateral common carotid artery occlusion in the gerbil causes no morphological neuronal damage in the brain. However, two 2-min occlusions at a l-h interval destroy hippocampal CA1 pyramidal neurons, and 3 occlusions additionally produce moderate damage to the striatum and the thalamus. Of interest is that neuronal damage is most severe when ischemia is repeated at 1 h intervals [lo]. This phenomenon is consistent with other reports using different ischemia models [16, 171. Recently, experimental evidence indicating that differences in brain and body temperature during and after ischemia determine the extent and density of ischemic neuronal damage received a great deal of attention [2-4, 6-8, 13-15, 181.Therefore, one of the possible damaging factors for the cumulative damage following repeated ischemia at l-h intervals is the spontaneous postischemic hyperthermia because intraischemic and postischemic hyperthermia aggravate brain damage as compared to normothermic conditions [7, 13, 141. Following even 2min forebrain ischemia in the gerbil, body temperature increases, and, in consequence, secondary ischemia after
Correspondence:
nrnk nkpl.,=.=
H. Kato, Department Tnhntll
Tlnixwrcitu
Crhnd
of Neurology, nf
Mdirinp
Institute of 1-I
9,Grvn_
a l-h interval is induced at a stage of mild hyperthermia [l 11.The purpose of this study was, therefore, to examine whether the absence of postichemic hyperthermia following initial ischemic insult ameliorates neuronal damage following repeated ischemic insults. Male Mongolian gerbils (Seiwa Experimental Animals Co. Ltd., Fukuoka, Japan), aged 13-14 weeks and weighing 65-90 g were anesthetized with 2% halothane in a mixture of 3096 oxygen and 70% nitrous oxide using a semiclosed facemask. Bilateral common carotid arteries were exposed and occluded with aneurysm clips 1 min after discontinuation of anesthesia. Carotid artery blood flow was restored by releasing the clips following 2 min of occlusion. After a l-h interval, secondary occlusion was induced for 2 min. Rectal temperature was monitored for up to 2 hr after ischemic insults. Cranial temperature was also measured in eight animals under anesthesia by placing the thermoprobe under the scalp. Experiments were performed at air-conditioned room temperature at around 23°C. A total of 33 animals were divided into 5 groups. (1) Single 2-min occlusion (n = 6). (2) Two 2-min occlusions, in which anesthesia was induced only during operative procedures, therefore causing spontaneous postischemic hyperthermia (n = 6). (3) Two 2-min occlusions, in which anesthesia was induced only during operative procedures, but the animals were placed in a refrigerated room maintained at 4°C for 1 h after initial ischemia to main-
22
TABLE
I
BODY TEMPERATURE Values are expressed
(“C) OF THE ANIMALS
as mean i S.D. Before
n
During ischemia
Postischemia 10 min
60 min
30 min
I20 min
Initial ischemia anesthesia
-
normothermia
6
37.5+0.4
37.2 )O.
I
37.3 kO.3
37.4 & 0.3
37.4+0
hyperthermia
6
37.2kO.2
37.2 k 0. I
37.8 + 0.6
3x.7*0
38.2*0.3**
normothermia
9
37.3 +0.2
37.2kO.l
37.3 &O. I
hyperthermia
6
37.2kO.3
37.1 io.1
38.O;tO.2”
anesthesia
Secondary
3**
4
+ 38.b+O.
I* *
I 38.0+0.2”
37 3+0.2 *
37.3 i-0.
ischemia
anesthesia
-
normothermia
6
37.3 FO.3
37 7kO.3
38.410
2
3x l&O4
37.5kO.3
hyperthermia
6
37.X+0.3**
37.biO.S
3x.2 + 0.2
37.8 +O.?
37.b_+O.l
normothermia
9
37.2kO.l
37.1 kO.3
38.1 +o 2
38.OkO.2
37 7_+0.3
hyperthermia
6
37.9 + 0. I * *
37.3kO.2
38.2 kO.4
37.8kO.4
37.7 + 0.2
anesthesia
+
**PC 0.01 compared * *Pi
0.01 compared
Student‘s
to normothermic
animals
(unanesthetized
to normothermic
animals
(anesthetized
groups). groups).
r-test.
Two 2-min occlusions, in which anesthesia was maintained with 1% halothane in the same OI/NzO mixture for I h after initial ischemia, and the body temperature was maintained at normothermia (n = 9). (5) Two 2-min occlusions, in which anesthesia was maintained with 1% halothane for 1 h as in group 4 but the animals were warmed by external heating so that the body temperature was comparable to that in group 2 with postischemic hyperthermia (n = 6). At 7 days of survival, the animals were anesthetized with pentobarbital (50 mg/kg, i.p.), and the brains were perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 min following a brief heparinized saline flush. Paraffin sections, 5 pm in thickness, were prepared and stained with Cresyl violet and hematoxylin-eosin. The stained sections were examined with a light microscope and ischemic neuronal damage
TABLE
was graded
on a scale of 0 to 3: 0 = no damage,
4 and 5. Single 2-min ischemia caused no neuronal damage in the brain except for one animal with minimal damage. Two ischemic insults caused moderate to severe damage to the hippocampal CAI subfield, and mild damage to the CA4 subfield, the lateral part of the striatum. the
II
CRANIAL
TEMPERATURES
Values are expressed
(“C) OF THE ANIMALS
as mean + S.D. n
Anesthesia
Before
First
Postischemia
Second
ischemia
tschemia
30 min
ischemia
+
normothermia
4
36.8 +O.b
36.3&0.2
370+04
3b 5_+0.2
hyperthermia
4
36.4kO.3
36.4kO.3
17 7&0.2*
37.2&O 3**
*P
