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Intraischemic hypothermia during pretreatment with sublethal ischemia reduces the induction of ischemic tolerance in the gerbil hippocampus
Brain Research 746 Ž1997. 301–304
Research report
Intraischemic hypothermia during pretreatment with sublethal ischemia reduces the induction of ischemic tolerance in the gerbil hippocampus Kojiro Wada ) , Takahito Miyazawa, Hiroshi Katoh, Namiko Nomura, Akiko Yano, Katsuji Shima, Hiroo Chigasaki Department of Neurosurgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359, Japan Accepted 10 September 1996
Abstract We examined whether mild brain hypothermia during pretreatment with sublethal 2-min ischemia affected the tolerance to subsequent lethal 5-min ischemia. The neuronal densities in the hippocampal CA1 sector of gerbils preconditioned at mild brain hypothermia Ž32% of normal. were significantly lower than those in gerbils preconditioned at brain normothermia Ž70% of normal.. 72-kDa heat-shock protein immunoreactivity in the CA1 sector preconditioned at mild hypothermia was reduced. These results suggest that mild brain hypothermia during pretreatment with sublethal ischemia reduces the tolerance to subsequent lethal ischemia. Keywords: Transient global ischemia; Ischemic tolerance; Intraischemic hypothermia; Delayed neuronal death; Gerbil
1. Introduction The phenomenon that a preceding sublethal ischemic insult exhibits protective effects against delayed neuronal death in CA1 neurons of the hippocampus following subsequent lethal global forebrain ischemia in gerbils is known as ‘ischemic tolerance’ w15–17,27x. Previous studies have revealed that mild brain hypothermia during lethal ischemia markedly attenuates neuronal damage in the hippocampus w5,21,30x. Although the neuroprotective effect of mild hypothermia during lethal ischemia has been demonstrated, the effect of mild hypothermia during preceding sublethal ischemia on ischemic tolerance has not been established. Therefore, we investigated this effect to determine if this phenomenon might lead to advancements in the induction of ischemic tolerance. The mechanism of the induction of ischemic tolerance is not fully understood. Ischemic tolerance most likely relates to the synthesis of a 72-kDa heat-shock protein ŽHSP-72., because the synthesis of HSP-72 is observed in surviving CA1 pyramidal cells w16x. Furthermore, Chopp et
Abbreviations: EEG, electroencephalograph; HSP-72, 72-kDa heatshock protein ) Corresponding author. Fax: q81 Ž429. 96-5207.
al. have demonstrated that intraischemic hypothermia reduces HSP-72 expression in rat brain following transient lethal forebrain ischemia w9x. We investigated the influence of mild hypothermia on the production of HSP-72 during preceding sublethal ischemia using gerbils.
2. Materials and methods Male Mongolian gerbils ŽCharles River Japan, Yokohama, Japan., weighing 60 to 80 g, were used. Anesthesia was induced with 2% halothane and maintained with 1% halothane in a mixture of 30% oxygen and 70% nitrous oxide administered via face mask. A wire-type thermocouple temperature probe Ž0.3-mm diameter; Unique Medical Co., Tokyo, Japan. was inserted 6 mm through a small burr hole placed 2 mm lateral to the bregma at the dural surface overlying the right frontal cortex w10x. The rectal temperature, which was measured using a thermocouple probe ŽUnique Medical Co.. was inserted 6 cm into the anus. Both common carotid arteries were occluded with small vascular clip ŽKono Seisakusho Co., Tokyo, Japan.. For sublethal pretreatment ischemia carotid artery blood flow was restored by releasing the clip following 2 min of occlusion. In the normothermia pretreatment groups, the rectal temperature was maintained close to 37.58C during the procedure. Occlusion was started when the brain tem-
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 1 3 9 - 0
K. Wada et al.r Brain Research 746 (1997) 301–304
302
perature reached 36.58C. In groups of mild brain hypothermia pretreatment, mild whole body hypothermia was induced after the surgical procedure by placement on an ice pack. Occlusion was started when the brain temperature reached 328C. Immediately after ischemia, the animals were rewarmed using a feedback-controlled heating blanket ŽUnique Medical Co... Normothermic and hypothermic ischemic control groups underwent a sham operation at the appropriate brain temperatures. Two days following pretreatment, lethal cerebral ischemia was induced by simultaneous, bilateral carotid artery occlusion for 5 min. Both the brain and rectal temperatures were monitored. While the rectal temperature was regulated close to 37.58C during the procedure, the brain temperature was allowed to vary spontaneously w24,25x. The onset of ischemia in both the sublethal and lethal insults were marked by the onset of electroencephalogram ŽEEG. isoelectricity; animals with incomplete EEG suppression during either procedure were excluded. Seven days after surviving the secondary insult, the anesthetized gerbils were sacrificed by transcardiac perfusion-fixation with 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were embedded in paraffin, cut into 5-m m sections, and stained with cresyl violet and hematoxylin-eosin. The stained sections were examined with a light microscope ŽOlympus Co., Tokyo, Japan.. The neuronal density of each hippocampal CA1 subfield, i.e., the number of intact pyramidal cells per 1 mm of linear length of the CA1, was determined according to the method of Kirino et al. w16x. Statistical comparisons were made using the Kruskal-Wallis and the two-tailed Mann-Whitney U-test. Immunostaining of fixed gerbil brain tissue with a mouse monoclonal antibody against 72-kDa heat-shock protein ŽHSP-72. was performed to examine the expression of the stress response following the 2-min period of primary ischemia at brain normo- or hypothermia. Each group contained five animals. Two days following 2-min ischemia, the anesthetized gerbils were sacrificed by perfusion–fixation with 4% paraformaldehyde in 0.1 M phosphate buffer and then immersed in 30% sucrose. Ten-m mthick frozen sections, containing the dorsal hippocampi,
were prepared from these brains. The sections were stained using a mouse monoclonal antibody raised against human HSP-72-related protein ŽAmersham, Tokyo, Japan. ŽRPN 1197., essentially according to the procedure described by Vass et al. w29x.
3. Results and discussion The results of the histologic examination of CA1 neurons are summarized in Table 1. The sham-operated group Žgroup A. showed no evidence of neuronal death and had an average neuronal density of 327.9 " 28.9 Žmean " S.D... The gerbils in the normothermic ischemic control group Žgroup B. and the hypothermic ischemic control group Žgroup C. showed extensive neuronal damage in the CA1 sector, with CA1 neuronal densities of 28.5 " 14.7rmm Ž8.7% of normal. and 28.2 " 12.6rmm Ž8.6% of normal., respectively. There was no significant difference between these two groups. Animals in the normothermic inducedtolerance group Žgroup E., in contrast, showed almost complete preservation of CA1 cells, with a neuronal density of 228.5 " 20.2rmm Ž69.7% of normal.. On the other hand, a significant reduction in the neuroprotective effect was observed in animals in the hypothermic induced-tolerance group Žgroup D., with a neuronal density of 104.9 " 57.4rmm Ž32.0% of normal. Ž P - 0.01.. Brain temperatures, from 5 min prior to until 10 min following the secondary ischemia, were not significantly different in the hypothermic induced-tolerance group and the normothermic induced-tolerance group. Immunohistochemical staining for HSP-72 in CA1 pyramidal neurons revealed moderate expression in four and weak expression in one gerbil in the normothermic pretreatment group ŽFig. 1, upper two panels., but only weak staining in all five of the gerbils in the hypothermic pretreatment group ŽFig. 1, lower two panels.. There was a significant difference between these two groups Ž P - 0.05.. The results of this study suggest that mild brain hypothermia during preceding sublethal ischemia reduces the induction of ischemic tolerance. Moreover, we have demonstrated that mild hypothermia likewise reduces
Table 1 Neuronal density in CA1 according to treatment
Group A Group B Group C Group D Group E a
Animals used
Primary ischemia
Brain temperature during primary ischemia
Secondary ischemia
Neuronal density in CA1 Žrmm.
5 5 5 8 8
y y y q q
Normothermia Normothermia Hypothermia Hypothermia Normothermia
y q q q q
327.9 " 28.9 28.5 " 14.7 28.2 " 12.6 104.9 " 57.4 228.5 " 20.2
Statistically significant difference between groups D and E: P - 0.01. ys sham operation was performed; qs ischemic operation was performed.
a a
K. Wada et al.r Brain Research 746 (1997) 301–304
303
Fig. 1. Immunostaining with a monoclonal antibody against HSP-72 following the 2-min period of nonlethal ischemia. Definitive staining is visible in the normothermic pretreatment group Župper two panels., but only faint staining is visible in the hypothermic pretreatment group Žlower two panels..
HSP-72 synthesis in the gerbil brain after transient sublethal forebrain ischemia. HSP-72 is selectively expressed in neurons subjected to sublethal stress, and lethal ischemic damage can be ameliorated by preceding hyperthermia w2,8,18x, brief ischemia w16,19x, or oxidative stress w26x, all of which can induce HSP-72 synthesis. HSP-72 may exhibit a protective effect by maintaining the tertiary structure of normal or partially denatured proteins w12x. Therefore, one possible mechanism for the reduction in ischemic tolerance is the reduction of HSP-72 synthesis w16x. The signal transduction mechanisms that may lead to activation of heat-shock factors have not been fully identified, but triggering events, such as the generation of oxygen free-radicals w26x, glutamate release w3x, or the accumulation of damaged proteins w1x, have been proposed as participants in the initiation of the stress response. Brain hypothermia during lethal ischemia reduces neuronal death, possibly by altering neurotransmitter release w6,7,23x, membrane function, delaying anoxic depolarization w14x, or ion homeostasis w22x, and by reducing energy depletion w20x. Therefore, mild brain hypothermia during sublethal ischemia may attenuate the events which are required to trigger HSP-72 synthesis w28x. It could be stated, however, that the reduction of HSP-72 expression observed in the hypothermic pretreatment group
may not be causally related to the diminished neuroprotective effect, but rather may merely reflect the reduction in the severity of the sublethal ischemic insult. Another, as yet unidentified factor induced by ischemic preconditioning, may be the causal factor. Pharmacologic studies of cerebral protection against ischemic injury may have been seriously compromised by drug-induced alterations in brain temperature w4,11x. Recently, the pharmacologic effects of various agents on the induction of ischemic tolerance have been investigated to clarify mechanisms of tolerance w13x. However, few investigators have paid attention to the brain temperature during pretreatment, aside from when pharmacologic temperature alterations have been proposed. Therefore, it is necessary to monitor and maintain a constant brain temperature during sublethal as well as lethal ischemic procedures.
Acknowledgements The authors would like to thank Miss. O.J. Natsume, Miss. T. Hayashi, Miss. Y. Ichiki, Dr. A. Shimizu, Mr. M. Takahashi, and Mr. K. Murata for their technical assistance.
304
K. Wada et al.r Brain Research 746 (1997) 301–304
References w1x Ananthan, J., Goldberg, A.L. and Voellmy, R., Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes, Science, 232 Ž1986. 522–524. w2x Barbe, M.F., Tytell, M., Gower, D.J. and Welch, W.J., Hyperthermia protects against light damage in the rat retina, Science, 241 Ž1988. 1817–1820. w3x Brown, I.R., Rush, S. and Ivy, G.O., Induction of a heat shock gene at the site of tissue injury in the rat brain, Neuron, 2 Ž1985. 1635–1641. w4x Buchan, A. and Pulsinelli, W.A., Hypothermia but not the Nmethyl-D-aspartate antagonist, MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia, J. Neurosci., 10 Ž1990. 311–316. w5x Busto, R., Dietrich, W.D., Globus, M.Y. and Ginsberg, M.D., Small differences in intra-ischemic brain temperature critically determine the extent of ischemic neuronal injury, J. Cereb. Blood Flow Metab., 7 Ž1987. 729–738. w6x Busto, R., Globus, M.Y., Dietrich, W.D., Martinez, E., Valdes, I. and Ginsberg, M.D., Effect of mild hypothermia on ischemia induced release of neurotransmitters and free fatty acids in rat brain, Stroke, 20 Ž1989. 904–910. w7x Busto, R., Dietrich, W.D., Globus, M.Y. and Ginsberg, M.D., The importance of brain temperaure in cerebral ischemic injury, Stroke, 20 Ž1989. 1113–1114. w8x Chopp, M., Chen, H., Ho, K.L., Dereski, M.O., Brown, E., Hetzel, F.W. and Welch, K.M.A., Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat, Neurology, 39 Ž1989. 1396–1398. w9x Chopp, M., Li, Y., Dereski, M.O., Levine, S.R., Yoshida, Y. and Garcia, J.H., Hypothermia reduces 72-kDa heat-shock protein induction in rat brain after transient forebrain ischemia, Stroke, 23 Ž1992. 104–107. w10x Colbourne, F., Nurse, S.M. and Corbett, D., Temperature changes associated with forebrain ischemia in the gerbil, Brain Res., 602 Ž1993. 264–267. w11x Corbett, D., Evans, S., Thomas, C., Wang, D. and Jonas, R.A., MK-801 reduced cerebral ischemia injury by inducing hypothermia, Brain Res., 514 Ž1990. 300–304. w12x Gething, M.J. and Sambrook, J., Protein folding in the cell, Nature, 355 Ž1992. 33–45. w13x Kato, H., Liu, Y., Araki, T. and Kogure, K., MK801, but not anisomycin, inhibits the induction of tolerance to ischemia in the gerbil hippocampus, Neuriosci. Lett., 139 Ž1992. 118–121. w14x Katsura, K., Minamisawa, H., Ekholm, A., Folbergrova, J. and Siesjo, ¨ B.K., Changes labile metabolites during anoxia in moderately hypo- and hyperthermic rats; correlation to membrane fluxes of K, Brain Res., 590 Ž1992. 6–12. w15x Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 Ž1982. 57–69. w16x Kirino, T., Tsujita, Y. and Tamura, A., Induced tolerance to is-
w17x
w18x
w19x
w20x
w21x
w22x
w23x
w24x
w25x
w26x
w27x
w28x
w29x
w30x
chemia in gerbil hippocampal neurons, J. Cereb. Blood Flow Metab., 11 Ž1991. 299–307. Kitagawa, K., Matsumoto, M., Tagaya, M., Hata, R., Ueda, H., Niinobe, M., Handa, N., Fukunaga, R., Kimura, K., Mikoshiba, K. and Kamada, T., Ischemic tolerance phenomenon found in the brain, Brain Res., 528 Ž1990. 21–24. Kitagawa, K., Matsumoto, M., Tagaya, M., Kuwabara, K., Hata, R., Handa, N., Fukunaga, R., Kimura, K. and Kameda, T., Hyperthermia-induced neuronal protection against ischemic injury in gerbils, J. Cereb. Blood Flow Metab., 11 Ž1991. 449–452. Li, Y., Chopp, M., Zhang, Z.G., Zhang, R.L. and Garcia, J.H., Neuronal survival is associated with 72-kDa heat shock protein expression after transient middle cerebral artery occlusion in the rat, J. Neurol. Sci., 120 Ž1993. 187–194. Michenfelder, J.D. and Theye, R.A., Hypothermia; Effect on canine brain and whole body metabolism, Anesthesiology, 29 Ž1968. 1107– 1112. Minamisawa, H., Nordstrom, ¨ C.H., Smith, M.L. and Siesjo, ¨ B.K., The influence of mild body and brain hypothermia on ischemic brain damage, J. Cereb. Blood Flow Metab., 10 Ž1990. 365–374. Mitani, A., Kadoya, F. and Kataoka, K., Temperature dependence of hypoxia-induced calcium accumulation in gerbil hippocampal slices, Brain Res., 562 Ž1991. 159–163. Mitani, A. and Kataoka, K., Critical levels of extracellular glutamate mediating gerbil hippocampal delayed neuronal death during hypothermia, brain microdialysis study, Neuroscience, 42 Ž1992. 661– 670. Miyazawa, T. and Hossmann, K.A., Methodological requirement for accurate measurements of brain and body temperature during global forebrain ischemia of rat, J. Cereb. Blood Flow Metab., 12 Ž1993. 817–822. Miyazawa, T., Bonnekoh, P., Widmann, R. and Hossmann, K.A., Heating of the brain to maintain normothermia during ischemia aggravates brain injury in the rat, Acta Neuropathol., 85 Ž1993. 488–494. Ohtsuki, T., Matsumoto, M., Kuwabara, K., Kitagawa, K., Sukuki, K., Taniguchi, N. and Kamada, T., Influence of oxidative stress on induced tolerance to ischemia in gerbil hippocampal neurons, Brain Res., 599 Ž1992. 246–252. Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 Ž1982. 491–498. Shaver, E.G., Welsh, F.A., Sutton, L.N., Mora, G., Gennarelli, L.M. and Norwood, C.R., Deep hypothermia diminishes the ischemic induction of heat-shock protein-72 mRNA in piglet brain, Stroke, 26 Ž1995. 1273–1278. Vass, K., Welch, W.J. and Nowak, T.S. Jr., Localization of 70-kDa stress protein induction in gerbil brain after ischemia, Acta Neuropathol. (Berl)., 77 Ž1988. 128–135. Welsh, F.A., Sims, R.E. and Harris, V.A., Mild hypothermia prevents ischemic injury in gerbil hippocampus, J. Cereb. Blood. Flow Metab., 10 Ž1990. 557–563.
Research report
Intraischemic hypothermia during pretreatment with sublethal ischemia reduces the induction of ischemic tolerance in the gerbil hippocampus Kojiro Wada ) , Takahito Miyazawa, Hiroshi Katoh, Namiko Nomura, Akiko Yano, Katsuji Shima, Hiroo Chigasaki Department of Neurosurgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359, Japan Accepted 10 September 1996
Abstract We examined whether mild brain hypothermia during pretreatment with sublethal 2-min ischemia affected the tolerance to subsequent lethal 5-min ischemia. The neuronal densities in the hippocampal CA1 sector of gerbils preconditioned at mild brain hypothermia Ž32% of normal. were significantly lower than those in gerbils preconditioned at brain normothermia Ž70% of normal.. 72-kDa heat-shock protein immunoreactivity in the CA1 sector preconditioned at mild hypothermia was reduced. These results suggest that mild brain hypothermia during pretreatment with sublethal ischemia reduces the tolerance to subsequent lethal ischemia. Keywords: Transient global ischemia; Ischemic tolerance; Intraischemic hypothermia; Delayed neuronal death; Gerbil
1. Introduction The phenomenon that a preceding sublethal ischemic insult exhibits protective effects against delayed neuronal death in CA1 neurons of the hippocampus following subsequent lethal global forebrain ischemia in gerbils is known as ‘ischemic tolerance’ w15–17,27x. Previous studies have revealed that mild brain hypothermia during lethal ischemia markedly attenuates neuronal damage in the hippocampus w5,21,30x. Although the neuroprotective effect of mild hypothermia during lethal ischemia has been demonstrated, the effect of mild hypothermia during preceding sublethal ischemia on ischemic tolerance has not been established. Therefore, we investigated this effect to determine if this phenomenon might lead to advancements in the induction of ischemic tolerance. The mechanism of the induction of ischemic tolerance is not fully understood. Ischemic tolerance most likely relates to the synthesis of a 72-kDa heat-shock protein ŽHSP-72., because the synthesis of HSP-72 is observed in surviving CA1 pyramidal cells w16x. Furthermore, Chopp et
Abbreviations: EEG, electroencephalograph; HSP-72, 72-kDa heatshock protein ) Corresponding author. Fax: q81 Ž429. 96-5207.
al. have demonstrated that intraischemic hypothermia reduces HSP-72 expression in rat brain following transient lethal forebrain ischemia w9x. We investigated the influence of mild hypothermia on the production of HSP-72 during preceding sublethal ischemia using gerbils.
2. Materials and methods Male Mongolian gerbils ŽCharles River Japan, Yokohama, Japan., weighing 60 to 80 g, were used. Anesthesia was induced with 2% halothane and maintained with 1% halothane in a mixture of 30% oxygen and 70% nitrous oxide administered via face mask. A wire-type thermocouple temperature probe Ž0.3-mm diameter; Unique Medical Co., Tokyo, Japan. was inserted 6 mm through a small burr hole placed 2 mm lateral to the bregma at the dural surface overlying the right frontal cortex w10x. The rectal temperature, which was measured using a thermocouple probe ŽUnique Medical Co.. was inserted 6 cm into the anus. Both common carotid arteries were occluded with small vascular clip ŽKono Seisakusho Co., Tokyo, Japan.. For sublethal pretreatment ischemia carotid artery blood flow was restored by releasing the clip following 2 min of occlusion. In the normothermia pretreatment groups, the rectal temperature was maintained close to 37.58C during the procedure. Occlusion was started when the brain tem-
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 1 3 9 - 0
K. Wada et al.r Brain Research 746 (1997) 301–304
302
perature reached 36.58C. In groups of mild brain hypothermia pretreatment, mild whole body hypothermia was induced after the surgical procedure by placement on an ice pack. Occlusion was started when the brain temperature reached 328C. Immediately after ischemia, the animals were rewarmed using a feedback-controlled heating blanket ŽUnique Medical Co... Normothermic and hypothermic ischemic control groups underwent a sham operation at the appropriate brain temperatures. Two days following pretreatment, lethal cerebral ischemia was induced by simultaneous, bilateral carotid artery occlusion for 5 min. Both the brain and rectal temperatures were monitored. While the rectal temperature was regulated close to 37.58C during the procedure, the brain temperature was allowed to vary spontaneously w24,25x. The onset of ischemia in both the sublethal and lethal insults were marked by the onset of electroencephalogram ŽEEG. isoelectricity; animals with incomplete EEG suppression during either procedure were excluded. Seven days after surviving the secondary insult, the anesthetized gerbils were sacrificed by transcardiac perfusion-fixation with 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were embedded in paraffin, cut into 5-m m sections, and stained with cresyl violet and hematoxylin-eosin. The stained sections were examined with a light microscope ŽOlympus Co., Tokyo, Japan.. The neuronal density of each hippocampal CA1 subfield, i.e., the number of intact pyramidal cells per 1 mm of linear length of the CA1, was determined according to the method of Kirino et al. w16x. Statistical comparisons were made using the Kruskal-Wallis and the two-tailed Mann-Whitney U-test. Immunostaining of fixed gerbil brain tissue with a mouse monoclonal antibody against 72-kDa heat-shock protein ŽHSP-72. was performed to examine the expression of the stress response following the 2-min period of primary ischemia at brain normo- or hypothermia. Each group contained five animals. Two days following 2-min ischemia, the anesthetized gerbils were sacrificed by perfusion–fixation with 4% paraformaldehyde in 0.1 M phosphate buffer and then immersed in 30% sucrose. Ten-m mthick frozen sections, containing the dorsal hippocampi,
were prepared from these brains. The sections were stained using a mouse monoclonal antibody raised against human HSP-72-related protein ŽAmersham, Tokyo, Japan. ŽRPN 1197., essentially according to the procedure described by Vass et al. w29x.
3. Results and discussion The results of the histologic examination of CA1 neurons are summarized in Table 1. The sham-operated group Žgroup A. showed no evidence of neuronal death and had an average neuronal density of 327.9 " 28.9 Žmean " S.D... The gerbils in the normothermic ischemic control group Žgroup B. and the hypothermic ischemic control group Žgroup C. showed extensive neuronal damage in the CA1 sector, with CA1 neuronal densities of 28.5 " 14.7rmm Ž8.7% of normal. and 28.2 " 12.6rmm Ž8.6% of normal., respectively. There was no significant difference between these two groups. Animals in the normothermic inducedtolerance group Žgroup E., in contrast, showed almost complete preservation of CA1 cells, with a neuronal density of 228.5 " 20.2rmm Ž69.7% of normal.. On the other hand, a significant reduction in the neuroprotective effect was observed in animals in the hypothermic induced-tolerance group Žgroup D., with a neuronal density of 104.9 " 57.4rmm Ž32.0% of normal. Ž P - 0.01.. Brain temperatures, from 5 min prior to until 10 min following the secondary ischemia, were not significantly different in the hypothermic induced-tolerance group and the normothermic induced-tolerance group. Immunohistochemical staining for HSP-72 in CA1 pyramidal neurons revealed moderate expression in four and weak expression in one gerbil in the normothermic pretreatment group ŽFig. 1, upper two panels., but only weak staining in all five of the gerbils in the hypothermic pretreatment group ŽFig. 1, lower two panels.. There was a significant difference between these two groups Ž P - 0.05.. The results of this study suggest that mild brain hypothermia during preceding sublethal ischemia reduces the induction of ischemic tolerance. Moreover, we have demonstrated that mild hypothermia likewise reduces
Table 1 Neuronal density in CA1 according to treatment
Group A Group B Group C Group D Group E a
Animals used
Primary ischemia
Brain temperature during primary ischemia
Secondary ischemia
Neuronal density in CA1 Žrmm.
5 5 5 8 8
y y y q q
Normothermia Normothermia Hypothermia Hypothermia Normothermia
y q q q q
327.9 " 28.9 28.5 " 14.7 28.2 " 12.6 104.9 " 57.4 228.5 " 20.2
Statistically significant difference between groups D and E: P - 0.01. ys sham operation was performed; qs ischemic operation was performed.
a a
K. Wada et al.r Brain Research 746 (1997) 301–304
303
Fig. 1. Immunostaining with a monoclonal antibody against HSP-72 following the 2-min period of nonlethal ischemia. Definitive staining is visible in the normothermic pretreatment group Župper two panels., but only faint staining is visible in the hypothermic pretreatment group Žlower two panels..
HSP-72 synthesis in the gerbil brain after transient sublethal forebrain ischemia. HSP-72 is selectively expressed in neurons subjected to sublethal stress, and lethal ischemic damage can be ameliorated by preceding hyperthermia w2,8,18x, brief ischemia w16,19x, or oxidative stress w26x, all of which can induce HSP-72 synthesis. HSP-72 may exhibit a protective effect by maintaining the tertiary structure of normal or partially denatured proteins w12x. Therefore, one possible mechanism for the reduction in ischemic tolerance is the reduction of HSP-72 synthesis w16x. The signal transduction mechanisms that may lead to activation of heat-shock factors have not been fully identified, but triggering events, such as the generation of oxygen free-radicals w26x, glutamate release w3x, or the accumulation of damaged proteins w1x, have been proposed as participants in the initiation of the stress response. Brain hypothermia during lethal ischemia reduces neuronal death, possibly by altering neurotransmitter release w6,7,23x, membrane function, delaying anoxic depolarization w14x, or ion homeostasis w22x, and by reducing energy depletion w20x. Therefore, mild brain hypothermia during sublethal ischemia may attenuate the events which are required to trigger HSP-72 synthesis w28x. It could be stated, however, that the reduction of HSP-72 expression observed in the hypothermic pretreatment group
may not be causally related to the diminished neuroprotective effect, but rather may merely reflect the reduction in the severity of the sublethal ischemic insult. Another, as yet unidentified factor induced by ischemic preconditioning, may be the causal factor. Pharmacologic studies of cerebral protection against ischemic injury may have been seriously compromised by drug-induced alterations in brain temperature w4,11x. Recently, the pharmacologic effects of various agents on the induction of ischemic tolerance have been investigated to clarify mechanisms of tolerance w13x. However, few investigators have paid attention to the brain temperature during pretreatment, aside from when pharmacologic temperature alterations have been proposed. Therefore, it is necessary to monitor and maintain a constant brain temperature during sublethal as well as lethal ischemic procedures.
Acknowledgements The authors would like to thank Miss. O.J. Natsume, Miss. T. Hayashi, Miss. Y. Ichiki, Dr. A. Shimizu, Mr. M. Takahashi, and Mr. K. Murata for their technical assistance.
304
K. Wada et al.r Brain Research 746 (1997) 301–304
References w1x Ananthan, J., Goldberg, A.L. and Voellmy, R., Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes, Science, 232 Ž1986. 522–524. w2x Barbe, M.F., Tytell, M., Gower, D.J. and Welch, W.J., Hyperthermia protects against light damage in the rat retina, Science, 241 Ž1988. 1817–1820. w3x Brown, I.R., Rush, S. and Ivy, G.O., Induction of a heat shock gene at the site of tissue injury in the rat brain, Neuron, 2 Ž1985. 1635–1641. w4x Buchan, A. and Pulsinelli, W.A., Hypothermia but not the Nmethyl-D-aspartate antagonist, MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia, J. Neurosci., 10 Ž1990. 311–316. w5x Busto, R., Dietrich, W.D., Globus, M.Y. and Ginsberg, M.D., Small differences in intra-ischemic brain temperature critically determine the extent of ischemic neuronal injury, J. Cereb. Blood Flow Metab., 7 Ž1987. 729–738. w6x Busto, R., Globus, M.Y., Dietrich, W.D., Martinez, E., Valdes, I. and Ginsberg, M.D., Effect of mild hypothermia on ischemia induced release of neurotransmitters and free fatty acids in rat brain, Stroke, 20 Ž1989. 904–910. w7x Busto, R., Dietrich, W.D., Globus, M.Y. and Ginsberg, M.D., The importance of brain temperaure in cerebral ischemic injury, Stroke, 20 Ž1989. 1113–1114. w8x Chopp, M., Chen, H., Ho, K.L., Dereski, M.O., Brown, E., Hetzel, F.W. and Welch, K.M.A., Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat, Neurology, 39 Ž1989. 1396–1398. w9x Chopp, M., Li, Y., Dereski, M.O., Levine, S.R., Yoshida, Y. and Garcia, J.H., Hypothermia reduces 72-kDa heat-shock protein induction in rat brain after transient forebrain ischemia, Stroke, 23 Ž1992. 104–107. w10x Colbourne, F., Nurse, S.M. and Corbett, D., Temperature changes associated with forebrain ischemia in the gerbil, Brain Res., 602 Ž1993. 264–267. w11x Corbett, D., Evans, S., Thomas, C., Wang, D. and Jonas, R.A., MK-801 reduced cerebral ischemia injury by inducing hypothermia, Brain Res., 514 Ž1990. 300–304. w12x Gething, M.J. and Sambrook, J., Protein folding in the cell, Nature, 355 Ž1992. 33–45. w13x Kato, H., Liu, Y., Araki, T. and Kogure, K., MK801, but not anisomycin, inhibits the induction of tolerance to ischemia in the gerbil hippocampus, Neuriosci. Lett., 139 Ž1992. 118–121. w14x Katsura, K., Minamisawa, H., Ekholm, A., Folbergrova, J. and Siesjo, ¨ B.K., Changes labile metabolites during anoxia in moderately hypo- and hyperthermic rats; correlation to membrane fluxes of K, Brain Res., 590 Ž1992. 6–12. w15x Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 Ž1982. 57–69. w16x Kirino, T., Tsujita, Y. and Tamura, A., Induced tolerance to is-
w17x
w18x
w19x
w20x
w21x
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