Brain Research, 553 (1991) 238-242 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116764C

238

BRES 16764

Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects Hiroyuki Kato, Yong Liu, Tsutomu Araki and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan) (Accepted 5 February 1991)

Key words: Cerebral ischemia; Repeated ischemia; Selective vulnerability; Delayed neuronal death; Hippocampus; Gerbil

We examined the response of the gerbil brain to secondary ischemic insult following pretreatment with brief ischemia at intervals of 5 min, 1 and 6 h, 1, 2, 4, 7 and 14 days. Two minutes of bilateral carotid artery occlusion produced no histopathological brain damage, whereas 3 rain of occlusion caused a moderate to severe reduction in the number of hippocampal CA1 pyramidal cells. Two-minute occlusion followed by 3-min occlusion at 5-min, 1- and 6-h intervals resulted in almost complete destruction of CA1 neurons. Additional neuronal damage was observed in the striatum at a 1-h interval and in the thalamus and the neocortex at 1- and 6-h intervals. The neuronal damage was most severe at a l-h interval. Two-minute ischemia followed by 3-min ischemia at intervals of I, 2, 4 and 7 days, however, caused a marked protective effect, and the hippocampal CA1 neurons were preserved. The protective effect was not observed at a 14-day interval and following pretreatment with 1-min ischemia. Thus, pretreatment with brief ischemia leads to complex responses of the brain to secondary ischemic insult; cumulative damage at intervals of 1-6 h and protective effects at intervals of 1-7 days. INTRODUCTION Brief and non-lethal cerebral ischemia can produce severe neuronal d a m a g e to selectively vulnerable neurons when such ischemia is induced r e p e a t e d l y at certain intervals 3'9'1°A9. Two-minute bilateral carotid artery occlusion in the gerbil causes no neuronal damage in the brain, whereas 3 such occlusions at 1-h intervals result in almost c o m p l e t e destruction of h i p p o c a m p a l CA1 pyramidal cells and m o d e r a t e d a m a g e to the striatum and the thalamus 3,9,1°. H o w e v e r , when secondary ischemic insult is r e n d e r e d several days after p r e t r e a t m e n t with non-lethal cerebral ischemia, the result is reversed. Brain d a m a g e is ameliorated as c o m p a r e d to that from a single ischemic insult. Five minutes of bilateral carotid artery occlusion in the gerbil 1 or 2 days after 2-min occlusion produces no h i p p o c a m p a l d a m a g e in m o r e than half of the animals, although a single 5-min occlusion always results in severe neuronal d a m a g e to the h i p p o c a m p a l CA1 subfield ~6. Thus, although 2 min of bilateral carotid artery occlusion in the gerbil is non-lethal by morphological criteria, the postischemic cellular alterations m a y continue for several days. T h e purpose of this study was to

clarify the t e m p o r a l profile of the cumulative d a m a g e and the protective effects, and to d e t e r m i n e when the two reverse p h e n o m e n a t a k e place and disappear. To reveal sequential alterations in the response of the gerbil brain to secondary ischemic insult, 3 min of carotid artery occlusion, which is the minimal p e r i o d of cerebral ischemia to cause a definite b u t not c o m p l e t e hippocampal CA1 cell loss 1-3"21, was induced following pret r e a t m e n t with a non-lethal 2-min occlusion at various intervals ranging from 5 min to 14 days. This e n a b l e d us to detect both aggravation and a m e l i o r a t i o n of hippocampal damage. MATERIALS AND METHODS A total of 81 male Mongolian gerbils (Seiwa Experimental Animals Co. Ltd., Fukuoka, Japan), aged 13-14 weeks and weighing 65-95 g, were used. They were allowed free access to food and water before and after surgery. Anesthesia was induced with 2% halothane in a mixture of 30% oxygen and 70% nitrous oxide. A mid-line cervical skin incision was made, and bilateral common carotid arteries were gently exposed. The arteries were occluded with aneurysm clips 1 min after discontinuation of anesthesia, when the animals did not move spontaneously but twitehed upon stimulation. Carotid artery blood flow was restored by releasing the clips following 2 rain of occlusion. Secondary bilateral carotid artery occlusion for 3 min was then induced 5 rain, 1 and 6 h, and 1, 2, 4,

Correspondence: H. Kato, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan. Fax: (81) (22) 272-5818.

239 7 and 14 days later. The secondary ischemia was also rendered following 1 min of bilateral carotid artery occlusion at intervals of 1, 2 and 7 days. Single 2 and 3 rain of ischemia and sham-operations were also included. The isehemic insults were induced at normothermia (at around 37 °C) except for insults at a 1-h interval (the reason see below). Body temperature was monitored before, during and 10, 30, 60 and 120 min after ischemia in a number of animals. The behavior of the animals was observed, and when the behavior was not depressed during and shortly after ischemia, the animal was discarded because severe ischemia was not considered to be induced. At 7 days of survival after secondary insult, the animals were anesthetized with pentobarbital (50 mg/kg i.p.), and the brains were briefly washed by transcardiac perfusion with heparinized saline, followed by perfusion-fixation with 4% paraformaldehyde in 0.1 M phosphate buffer for 20 min. The brains were removed 1-3 h later and immersed in the same fixative for 7 days, and then they were embedded in paraffin. Paraffin sections, 5/~m in thickness, were prepared and stained with Cresyl violet and hematoxylin-eosin. The stained sections were examined by two examiners (H.K. and Y.L.) with a light microscope without the examiners knowing the experimental protocol. When the score of neuronal damage was different between the examiners, final decision was made by discussion. Neuronal density of the hippocampal CA1 subfield, i.e. the number of intact pyramidal cells per I mm linear length of CA1, was determined according to the method of Kirino et al. 15. Neuronal damage to the striatum, the thalamus, the neocortex (sensorimotor cortex) and to the hippocampal CA4 subfield was semiquantitatively graded using a 0-3 rating system with 0 = normal, 1 = a few neurons damaged, 2 = many neurons damaged, and 3 = majority of neurons damaged. The average of values of both hemispheres was considered. Each group consisted of 4-8 animals. Statistical significance was analyzed using the analysis of variance (ANOVA) and the Student's t-test, and the Kruskal-Wallis test and the Mann-Whitney U-test.

RESULTS A l l animals e x c e p t for o n e a n i m a l w h i c h was s u b j e c t e d to t w o i s c h e m i c insults at a 1-h i n t e r v a l s u r v i v e d the entire

observation

period.

Seizures

and

neurological

deficits w e r e n o t o b s e r v e d . F i v e o f 77 animals which w e r e s u b j e c t e d to i s c h e m i a w e r e c o n s i d e r e d n o t to b e r e n d e r e d severely ischemic because of the absence of behavioral depression during and some time after ischemia, and w e r e discarded. O n e o f t h e d i s c a r d e d a n i m a l s s h o w e d unilateral C A 1 d a m a g e , a n d t h e o t h e r s w e r e h i s t o p a t h o logically n o r m a l . B o d y t e m p e r a t u r e s of t h e animals are s h o w n in T a b l e I. F o l l o w i n g 2 a n d 3 m i n o f i s c h e m i a , b o d y t e m p e r a t u r e was i n c r e a s e d by a b o u t 1.5 °C, b e i n g m a x i m a l at 30 rain, and h a d r e t u r n e d to n o r m a l at 2 h. A s a c o n s e q u e n c e , s e c o n d a r y i s c h e m i c insult a f t e r a 1-h i n t e r v a l was i n d u c e d at a stage o f m i l d h y p e r t h e r m i a . T h e c h a n g e in t e m p e r a t u r e f o l l o w i n g 1 m i n o f i s c h e m i a was less r e m a r k a b l e . C h a n g e s in t e m p e r a t u r e f o l l o w i n g 3-min i s c h e m i a w e r e n o t d i f f e r e n t a m o n g groups. N e u r o n a l density o f t h e h i p p o c a m p a l C A 1 subfield a n d the score o f n e u r o n a l d a m a g e to t h e s t r i a t u m , the thalamus,

the

neocortex

and

the

hippocampal

e m i a c a u s e d n o b r a i n d a m a g e in 5 a n i m a l s but m i n i m a l d a m a g e to the m e d i a l p a r t o f the h i p p o c a m p a l C A 1 subfield in o n e

animal.

Three

minutes

of ischemia,

TABLE I Body temperatures (°C) of the animals Values are expressed as mean _+ S.D. n = 3-14. During ischemia

Postischemia 10 min

30 min

60 min

120 min

Sham-operated

37.2 + 0.2

37.1 + 0.3

37.3 + 0.2

37.5 + 0.2

37.5 + 0.2

Single ischemia 1 rain 2 rain 3 min

37.4 + 0.4 37.1 + 0.2 36.9 + 0.4

38.1 + 0.6** 38.2 + 0.6** 38.2 + 0.6*

38.1 + 0.5* 38.7 + 0.5** 38.6 + 0.2**

37.8 + 0.5 38.1 + 0.3** 38.2 + 0.4*

37.6 + 0.4 37.6 + 0.3 37.4 + 0.3

Secondary 3-min isehemia following 2-min ischemia 5-min interval 37.1 + 0.2 1-h interval 37.7 + 0.6 6-h interval 37.4 + 0.2 1-day interval 37.1 + 0.4 2-day interval 37.3 _+0.5 4-day interval 37.2 + 0.I 7-day interval 37.2 + 0.3 14-dayintervai 37.3 + 0.3

38.6 + 0.5** 38.1 + 0.6* 38.1 + 0.5* 38.0 + 0.8 38.4 + 0.6** 37.6 + 0.2* 38.3 + 0.1"* 38.4 + 0.4**

38.8 + 0.7** 38.1 + 0.4* 38.5 + 0.1"* 38.8 + 0.4** 38.7 + 0.3** 38.0 + 0.5* 38.6 + 0.2** 38.8 + 0.3**

38.2 + 0.3** 38.0 + 0.3* 38.0 + 0.5 38.5 + 0.8 38.4 + 0.4** 37.7 + 0.5 38.1 + 0.4* 38.3 + 0.5*

37.8 __+0.3 37.5 + 0.5 37.6 + 0.2 38.0 + 0.3 37.7 + 0.3 37.2 + 0.2 37.8 + 0.3 37.9 + 0.2

Secondary 3-min ischemia following 1-min ischemia 1-day interval 37.0 + 0.3 2-day interval 37.3 + 0.2 7-day interval 37.3 + 0.3

37.7 + 0.3 37.9 + 0.5* 38.1 + 0.5*

38.5 + 0.4** 38.5 + 0.2** 38.5 + 0.4**

38.1 + 0.4* 38.0 + 0.3* 37.6 + 0.6

37.6 + 0.4 37.7 + 0.5 37.5 + 0.4

*P < 0.05, **P < 0.01 compared to sham-operation.

CA4

subfield are s u m m a r i z e d in T a b l e II. T w o - m i n u t e isch-

240 TABLE II

Neuronal density (/mm) of the hippocampal CA1 subfield, and the neuronal damage (graded 0 to 3) to the hippocampal CA4 subfield, striatum, thalamus and neocortex Values are expressed as mean _+ S.D.

n

CA1

CA4

Striatum

Sham-operated 2-min ischemia 3-rain ischemia

4 6 8

221±10.4"* 216±39.3"* 50±~.2

0±0" 0.1±0.2" 0.6±0.4

0±0 0±0 0±0

2-rain + 3-min ischemia 5-min interval 1-h interval 6-h interval 1-day interval 2-day interval 4-day interval 7-day interval 14-day interval

6 5 6 5 5 5 6 5

11±4.0 5±2.1" 21±18.0 179±93.6" 207±47.0** 232±10.4"* 231±22.1"* 56±52.1

0.8±0.4 1.3±0.6 0.8±0.5 0.5±0.4 0.5±0.5 0.6±0.2 0.3±0.4 0.3±0.4

0.1±0.2 2.4±1.1 0.1±0.2 0±0 0±0 0±0 0±0 0±0

1-min + 3-rain ischemia 1-day interval 2-day interval 7-day interval

5 5 5

84±74.7 76±57.1 76±62.3

0.5±0.0 0.2±0.4 0.7±0.3

0±0 0.1±0.2 0.3±0.4

Thalamus 0±0 0±0 0±0 0±0 1.1±0.5"* 1.5±0.4"* 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0

Neocortex 0±0 0±0 0.1±0.2 0.6±0.5 1.5±1.0"* 0.8±0.4* 0.4±0.4 0±0 0±0 0±0 0.1±0.2 0.1±0.2 0±0 0.1±0.2

*P < 0.05, * *P < 0.01 compared with 3-min ischemia.

Fig. 1. Representative photomicrographs of the CA1 subfield of the hippocampus, a: 2-rain ischemia. Normal appearance, b: 3-rain ischemia. Majority of the neurons are damaged. C: 2-min ischemia followed by 3-min ischemia at a 1-h interval. Almost all CA1 pyramidal cells have been lost. d,e,f: 2-min isehemia followed by 3-min ischemia at 2-day (d), 4-day (e) and 7-day (f) intervals. CA1 pyramidal cells are preserved. Cresyl violet staining. Bar = 100/~m.

241 however, caused a moderate to severe reduction in the number of CA1 pyramidal cells and occasional neuronal damage in the CA4 subfield, but otherwise remained normal. Two-minute ischemia followed by 3-min ischemia at 5-rain, 1- and 6-h intervals caused destruction of almost all CA1 pyramidal cells, and the neuronal density at a 1-h interval was significantly lower than that after a single 3-min ischemia. In addition, moderate to severe damage to the lateral part of the striatum was observed at a 1-h interval, and mild to moderate damage was seen in the ventral part of the thalamus and in the neocortical layers 3, 5 and 6 at 1- and 6-h intervals. A small number of hippocampal CA3 neurons in the vicinity of the CA2 subfield were damaged at a 1-h interval in some animals, but there was no damage in other groups. The dentate gyrus was not damaged in any group. In contrast, CA1 pyramidal cells were preserved following 3-rain ischemia 1, 2, 4 and 7 days after pretreatment with 2-min ischemia. Other parts of the brain also showed no damage except for occasional neuronal damage in the CA4 subfield. The protective effect was not observed at a 14-day interval. Pretreatment with 1 min of ischemia also did not cause a protective effect, and the majority of the CA1 neurons were damaged. DISCUSSION The present results show that pretreatment with brief ischemia has conditioning effects on secondary ischemic insult. But the effects are complex and are dependent on the interval between two ischemic insults. Both cumulative damage and protective effects were observed. Pretreatment with 2-min ischemia aggravated neuronal damage from secondary 3-min ischemic insult at shorter intervals but protected against neuronal damage at longer intervals. These two reverse phenomena have been separately reported using different experimental design 3, 9,10,16,19. Therefore, systematized study was needed to clarify the temporal profile in detail in a series of experiments. Furthermore, the present experimental model induces both complete destruction and complete protection of CA1 pyramidal cells by changing the intervals between two ischemic insults. In consequence, this model may be useful to investigate the mechanisms of cumulative damage and protective effects following sublethal ischemia. Two minutes of bilateral carotid artery occlusion produces no, or minimal, neuronal damage as previously reported 3"9'1°'16'21. However, we observed a moderate to severe reduction in the number of hippocampal CA1 pyramidal cells after 3 min of occlusion. This result supports previous observations of ours L2,3 and of

Rudolphi et al.el, but it differs from the report of Kirino and Sano 14 in which only scattered neuronal damage was found in 1 of 5 animals. The reason for this difference may be owing to different gerbil strains and experimental conditions. Five minutes of bilateral carotid artery occlusion commonly causes destruction of almost all CA1 pyramidal cells 1'14'2~. In any way, the critical time point for the destruction of CA1 neurons lies between 2 and 3 min of occlusion. Using the 3-min occlusion which leads to moderate to severe damage to the hippocampal CA1 subfield, we could detect both aggravation of and protection against CA1 neuronal damage. Neuronal damage following 3-rain ischemia induced 5 min after pretreatment with 2-rain ischemia was not statistically different from damage after single 3-rain ischemia. However, 3-min ischemia following 2-min ischemia at 1- and 6-h intervals produced cumulative damage to the hippocampal CA1 subfield, the lateral part of the striatum, the ventral location of the thalamus and to the neocortical layers 3, 5 and 6. The damage was most severe at a 1-h interval, which is consistent with previous results of ours 1°'19 and others ~. We have reported that the cumulative damage at 1-h intervals may be produced by the following mechanisms; postischemic hypoperfusion which is maximal 1 h after ischemia 11'e3, impairment of protein synthesis which is observed for several hours after 2-rain ischemia 2, and the excitotoxic mechanism 12. Secondary ischemic insults 1 h after 2-rain ischemia are induced at a stage of mild postischemic hyperthermia ~2. The postischemic hyperthermia is considered to be the result of a direct action o f ischemia on the brain, especially on the hypothalamus 17. Recent experimental evidence indicates that a small difference in brain temperature critically determines the fate of ischemic neurons. Hyperthermia during and after ischemia aggravates ischemic brain damage 6a7. Therefore, mild hyperthermia may also be the cause of the most severe damage at a 1-h interval, but other factors such as those mentioned above are likely to play major roles because cumulative damage was also observed at a 6-h interval when the body temperature had returned to normal, and because in this series the increase in the body temperature during the secondary ischemia after a 1-h interval was not statistically significant (Table II). Two-minute ischemia followed by 3-min ischemia at longer intervals (1, 2, 4 and 7 days) showed a conspicuous protective effect. No or mild damage was found throughout the brain at these intervals. Because pretreatment with 1 min ischemia showed no protective effect, the mechanism of protection may be attributed to some cellular alterations caused by 2 min of ischemia. Obviously, the difference in body temperatures does not play a role in the protective effects because the alterations in

242 the t e m p e r a t u r e following secondary 3-min ischemia were not different from those after single 3-min ischemia. A l t h o u g h 2-min ischemia causes no morphological changes, it has a considerable impact on neurons because postischemic hypoperfusion and impairment of protein synthesis take place as m e n t i o n e d above 2'11. The present study d e m o n s t r a t e d that the protective effect is not persistent but disappears by 14 days. R e c e n t evidence indicates that selective gene expression and a subsequent induction of protein synthesis, such as heat shock proteins (HSP), take place after transient cerebral ischemia 7"8'13'2°'22'24. F u r t h e r m o r e , increase in nerve growth factor ( N G F ) contents is observed 5 days after ischemic-hypoxic insult in the rat TM. Synthesis of HSP and/or N G F m a y explain the protective effects because they are considered to ameliorate ischemic brain d a m a g e 4'5. Because the protective effects were observed REFERENCES 1 Araki, T. and Kogure, K., Prevention of delayed neuronal death in gerbil hippocampus by a novel vinca alkaloid derivative (vinconate), Mol. Chem. Neuropathol., 11 (1989) 33-43. 2 Araki, T., Kato, H., Inoue, T. and Kogure, K., Regional impairment of protein synthesis following brief cerebral ischemia in the gerbil, Acta Neuropathol., 79 (1990) 501-505. 3 Araki, T., Kato, H. and Kogure, K., Neuronal damage and calcium accumulation following brief cerebral ischemia in the gerbil, Brain Research, 588 (1990) 114-122. 4 Buchan, A.M. and Pulsinelli, W.A., Fimbria-fornix lesions: the temporal profile for protection of CA1 hippocampus against ischemic injury, J. Cereb. Blood Flow Metabol., 9 (Suppl. 1) (1989) $749. 5 Chopp, M., Chen, H., Ho, K.-L., Dereski, M.O., Brown, E., Hetzel, EW. and Welch, K.M.A., Transient hyperthermia protects against subsequent forebrain ischemia cell damage in the rat, Neurology, 39 (1989) 1396-1398. 6 Dietrich, W.D., Busto, R., Valdes, I. and Loor, Y., Effects of normothermic versus mild hyperthermic forebrain ischemia in rats, Stroke, 21 (1990) 1318-1325. 7 Jacewicz, M., Kiessling, M. and Pulsinelli, W.A., Selective gene expression in focal cerebral ischemia, J. Cereb. Blood Flow Metabol., 6 (1986) 263-272. 8 Jorgensen, M.B., Deckert, J., Wright, D.C. and Gehlert, D.R., Delayed c-los proto-oncogene expression in the rat hippocampus: an in situ hybridization study, Brain Research, 484 (1989) 393-398. 9 Kato, H., Kogure, K. and Nakano, S., Neuronal damage following repeated brief ischemia in the gerbil, Brain Research, 479 (1989) 366-370. 10 Kato, H. and Kogure, K., Neuronal damage following nonlethal but repeated cerebral ischemia in the gerbil, Acta Neuropathol., 79 (1990) 494-500. 11 Kato, H., Araki, T., Kogure, K., Murakami, M. and Uemura, K., Sequential cerebral blood flow changes in short-term cerebral ischemia in gerbils, Stroke, 21 (1990) 1346-1349. 12 Kato, H., Araki, T. and Kogure, K., Role of the excitotoxic mechanism in the development of neuronal damage following repeated cerebral ischemia in the gerbil: protective effects of

between 1 and 7 days after ischemia, a transient induction of such substances is likely to participate, the mechanism of which must be elucidated. In conclusion, we showed that the response of the brain to secondary ischemic insult following p r e t r e a t m e n t with non-lethal cerebral ischemia is complex and depends on the interval between two ischemic insults, exhibiting both cumulative d a m a g e and protective effects. Therefore, alterations in brain m e t a b o l i s m following brief and non-lethal cerebral ischemia is dynamic and continues for at least a week. The alterations m a y consist of damaging and reparative processes. Elucidation of such processes will be necessary to further clarify the p a t h o m e c h a n i s m s of ischemic brain injury. Acknowledgement. The authors gratefully acknowledge Ms. Rumiko Tanaka for her excellent technical assistance.

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Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects.

We examined the response of the gerbil brain to secondary ischemic insult following pretreatment with brief ischemia at intervals of 5 min, 1 and 6 h,...
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