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Cardiac arrest cerebral ischemia model in mice failed to cause delayed neuronal death in the hippocampus
Neuroscience Letters 322 (2002) 91–94 www.elsevier.com/locate/neulet
Cardiac arrest cerebral ischemia model in mice failed to cause delayed neuronal death in the hippocampus Nobutaka Kawahara a,b,*, Kensuke Kawai b,c, Tomikatsu Toyoda a,b, Hirofumi Nakatomi a,b, Kazuhide Furuya a,b, Takaaki Kirino a,b a
Department of Neurosurgery, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan CREST (Core Research for Evolutional Science and Technology), Japan Science and Technology Cooperation, Saitama 332-0012, Japan c Department of Neurosurgery, Tokyo Metropolitan Neurological Hospital, Tokyo 183-0042, Japan
b
Received 5 January 2002; received in revised form 15 January 2002; accepted 16 January 2002
Abstract Global cerebral ischemia models for genetically engineered mice are of particular importance for the study of delayed neuronal death, but have been complicated by variability of vascular anatomy. Here we developed a 5-min cardiac arrest model that was not affected by vascular anatomy, and evaluated the hippocampal neuronal injury in BL/6 and SV129 mice. Despite prolonged anoxic depolarization for approximately 7 min, however, no consistent ischemic neuronal injury was noted in the CA1 sector of the hippocampus in both strains. Thus, our observations suggested that murine hippocampal neurons are relatively resistant to ischemia compared with those in other rodents. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral ischemia; Cardiac arrest; Delayed neuronal death; Hypothermia; Anoxic depolarization; Direct current potential
Transient global cerebral ischemia causes delayed neuronal death in the CA1 sector of the hippocampus [10,16]. Though numerous studies have been undertaken to elucidate the pathophysiology of delayed neuronal death as a representative model of ischemic neuronal death, as well as a therapeutic target, its precise mechanism remains unknown. Recent progress of in vivo manipulation of genes in mice, on the other hand, has offered a new opportunity to study the in vivo effect of overexpression or targeted disruption of a specific gene. This approach has provided a powerful tool for investigation of molecular mechanisms in vivo, and demonstrated involvement of some crucial genes in the focal ischemic injury [7]. However, new gene technology in mice has contributed little to understanding of the molecular mechanism of delayed neuronal death, due to lack of reproducible global ischemia models in mice. The inconsistency of ischemic injury in this model mainly derives from the individual difference of collateral flow through the circle of Willis, even in the same strain [6,11,15,17]. To circumvent the flow-related variation, the current study was designed to address whether the cardiac arrest model, an * Corresponding author. Tel.: 1 81-3-5800-8852; fax.: 1 81-35800-8655. E-mail address: [email protected] (N. Kawahara).
already established model in rats [9], is indeed a suitable model for the investigation of molecular mechanism of delayed neuronal death in genetically engineered mice. We further provide data for anoxic depolarization and brain temperature changes, the two critical factors for ischemic injury in this small rodent model. Male C57BL/6NCrj (BL/6; Charles River Japan, Yokohama, Japan) and 129/SvEms (SV129; Jackson Laboratory, Bar Harbor, Maine) mice, aged 8–12 weeks, were used in this study. All animal-related procedures were conducted in accordance with the guidelines of the National Institutes of Health (Guide for the care and use of laboratory animals). Both BL/6 (n ¼ 15, BL/6 ischemia group) and SV129 (n ¼ 14, SV129 ischemia group) mice were subjected to cardiac arrest cerebral ischemia. After induction of anesthesia, the mice were maintained with 0.5% halothane in a 30% O2/70% N2O mixture. Following the left femoral artery and vein cannulation, atropine sulphate (0.25 mg/kg, ip), amikacin (10 mg/kg, ip), and sodium bicarbonate (0.5 mEq/kg, iv) were administered. Rectal and temporal muscle temperatures were controlled at 37 8C by a heating blanket and a heating lamp up to 30 min after ischemia. Following midline cervical-sternal incision, a bent hook was inserted into the right thoracic cavity, rotated underneath the heart, and mechanical ventilation was discontinued. The major
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 10 1- 5
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cardiac vessels were then occluded by pulling the hook and applying finger pressure from outside [8,9]. After confirming the cardiac standstill, resuscitation was initiated at 5 min with external cardiac massage and mechanical ventilation with 100% O2 under tracheostomy. Epinephrine (0.02 mg/ kg, iv) and sodium bicarbonate (0.5 mEq/kg, iv) were administered several times to maintain BP. Restoration of BP to 50 mmHg was considered recirculation. Then, the mice were weaned from the ventilator at 10–15 min and extubated. After skin closure, the animals were placed in the incubator set at 33 8C during the first 24 h. Rectal temperature was monitored at 2, 6, and 24 h post-ischemia. Arterial blood samples were analyzed before and 30 min after ischemia, by a blood gas analyzer (Model 248; Chiron Diagnostics Ltd., Essex, UK). Sham operated control animals were made in BL/6 mice (n ¼ 7, BL/6 sham group). Seven days after ischemia, the brains were perfusion fixed with 4% paraformaldehyde and embedded in paraffin. Sections (4 mm thickness) containing the dorsal hippocampi were stained with hematoxylin and eosin and the number of intact neurons in the hippocampal CA1 sector was counted in a blind fashion. Degenerated neurons with shrunken nucleus and acidophilic cell body in the CA1 sector were also counted. The mean value of the neuronal density (cells/ 0.5 mm) on each side was used for statistical analysis. In situ DNA fragmentation was detected by TUNEL method using biotinylated-dUTP and terminal transferase (Roche Diagnostics, Mannheim, Germany), as described [1]. TUNEL positive cells were counted and similarly evaluated. In another group of BL/6 mice (n ¼ 4, Temp Group), a needle microprobe (MT-26/2; Physitemp Instruments, Clifton, NJ) was inserted to the hippocampus [5] (1.9 mm posterior to the bregma, 1.2 mm left from the midline, 3.0 mm depth), and the brain temperature was monitored by a thermometer (Model BAT-12; Physitemp Instruments, Clifton, NJ) up to 6 h post-ischemia. In the fourth group (DC group, BL/6 mice, n ¼ 6), hippocampal direct current (DC) potential was measured through silver chloride wires and a glass micropipette electrode (tip diameter 10–20 mm) inserted into the left hippocampus [5] (1.9 mm posterior to the bregma, 1.2 mm left from the midline, 1.2 mm depth) with reference in the neck muscle (DC amplifier, Model AD-641G; Nihon Kohden, Tokyo, Japan) [8]. Cerebral blood flow (CBF) was also measured through a probe for laser Doppler flowmetry (Model ALF 21; Advance Co. Ltd., Tokyo, Japan) attached to the skull (1.9 mm posterior to the bregma, 1.2 mm right from the midline). All the values are expressed as mean ^ SD. Physiological parameters, and the cell counts in the CA1 sector were compared using one-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test with significance set at P , 0:05. For the analysis of temperatures, 2-factorial ANOVA with the same Bonferroni’s post-hoc test was used. Successful recirculation was achieved in all mice except one, yielding resuscitation rate of 96% in the current series. The mean ischemia time for each group ranged from 5.6 to
5.9 min without significant difference among groups. The final survival rates at 7 days were 47% and 52% for the BL/6 and SV129 ischemia groups, respectively. Concerning physiological parameters, there were no statistically significant differences among the groups except DC group. In the DC group, the mean BP (pre-ischemia 70.8 mmHg, post-ischemia 74.3 mmHg vs. 78.3–97.9 mmHg in the others) was slightly lower, and the mean PaCO2 was higher (pre-ischemia 37.6 mmHg vs. 28.0–32.4 mmHg in the others) due to longer anesthesia time in this group. In the post-ischemic period, comparison of rectal temperature by factorial analysis (time £ group) revealed a significant main effect of time (P , 0:0001), such that rectal temperature at 2 h post-ischemia (36.3 ^ 0.7 8C) was the lowest and that at 24 h post-ischemia (37.4 ^ 0.2 8C) the highest without intergroup difference. The hippocampal CA1 sector in each group did not show significant ischemic injury (Fig. 1) except one hippocampus in the BL/6 ischemia group, in which the middle third of CA1 region was segmentally injured. Cell counts (cells/0.5 mm) of surviving neurons were 117.2 ^ 9.3 in the BL/6 sham group, 114.6 ^ 19.8 in the BL/6 ischemia group, and 122.3 ^ 8.3 in the SV129 ischemia group, demonstrating no statistically significant difference (P . 0:5). Likewise, cell counts (cells/CA1 sector) of degenerating neurons revealed no significant difference among these groups (P . 0:2); 0.0 ^ 0.0 in the BL/6 sham group, 6.5 ^ 13.0 in the BL/6 ischemia group, and 0.4 ^ 0.6 in the SV129 ischemia group. In addition, TUNEL positive cells were not detected in any hippocampal CA1 sector (Fig. 1) except one in the BL/6 group (40 cells/CA1 sector), which showed segmental injury in hematoxylin and eosin staining (P ¼ 0:36 among 3 groups). Brain temperature was monitored simultaneously up to 6 h. Factorial analysis of (location £ time) demonstrated a significant main effect of location (P , 0:008). Post-hoc test revealed that the brain temperature was significantly lower than rectal temperature, reaching 0.7 8C during ischemia and 1.0 8C at 1 and 6 h post-ischemia. CBF, as demonstrated in Fig. 2, was reduced to almost zero percent (3.2 ^ 1.7%) immediately after occlusion of the aorta. Following resuscitation, early hyperperfusion (172.8 ^ 41.6% at 5 min) and delayed hypoperfusion (41.6 ^ 12.4% at 30 min) were noted. Abrupt negative shift of DC potential was also noted with a delay of 74.0 ^ 6.5 s after the circulatory arrest, which showed further negative shift, followed by slow recovery, after recirculation. Total anoxic depolarization time at half-maximal amplitude was 415.0 ^ 51 s. We demonstrated that complete circulatory arrest in mice for 5 min, which are comparable to other established global ischemia models in gerbils and rats [10,16], induced prolonged ischemic depolarization in the hippocampal CA1 sector for approximately 7 min. This depolarization time is actually longer than that for complete injury for gerbil hippocampal neurons [12]. Nevertheless, no consis-
N. Kawahara et al. / Neuroscience Letters 322 (2002) 91–94
93
Fig. 1. Histological evaluation of the hippocampal CA1 sector after 7 days. (A) BL/6 sham group. (B) BL/6 ischemia group. (C) SV129 ischemia group. No neuronal injury was noted in each group, except one hippocampus in BL/6 ischemia group (data not shown). Hematoxylin and eosin staining. (D) TUNEL staining with hematoxylin counter-staining in the ischemia group. Note no positive staining in the CA1 sector. Scale bar, 100 mm.
tent neuronal injury in this most vulnerable region was noted in two commonly used strains of mice, i.e. BL/6 and SV129. Then the question immediately arises whether murine CA1 neurons are indeed resistant to prolonged ischemia compared with those of other rodents. Several other studies demonstrating hippocampal injury also suggest that this might be the case. According to bilateral carotid artery occlusion model, ischemia longer than 10 min was required for hippocampal injury [6,11,15,17]. Crucial in these models was the residual flow through the posterior communication artery (P-com), with BL/6 strain being the most suitable due to frequent hypoplasticity of Pcom. Therefore, relatively longer ischemic threshold in mice has been ascribed to residual flow during ischemia [6]. However, recent studies using bilateral carotid artery occlusion combined with systemic hypotension showed residual flow of less than 2% in the hippocampus, which still required more than 10 min to induce neuronal injury [15,17], suggesting that murine hippocampus might be indeed resistant to ischemia. On the contrary, Bo¨ ttiger et al. [1], using similar 5-min cardiac arrest model in NMRI mice, stated that some, but limited, neuronal injury in the hippocampus. Since quantitative data was not available in this study, direct comparison with our results seems difficult. Important to note in this regard is the difference in neuronal susceptibility for insults among various strains, which is unrelated to vascular anatomy. For instance, pyramidal neurons in BL/6 and BALB/c mice are more resistant to kainic acid toxicity compared with those in SV129 and FVB mice, indicating that genetic determinant at the cellular level could affect the phenotypic outcome [14]. Thus, the difference obtained from the similar cardiac arrest models in mice might be related to strain difference.
It is well known that hypothermia is neuroprotective in gerbil and rat global ischemia, even when initiated after ischemia [2,3]. We noted slight decrease of brain temperature by 1.0 8C in the early reperfusion period, suggesting that brain temperature might have dropped to 35.3 8C for an hour or two in our experiment. However, in gerbils and rats, postischemic hypothermia less than 33 8C is required for substantial neuroprotection when its duration is less than 6 h [2–4]. Thus, it is unlikely that transient mild postischemic brain hypothermia is a major factor affecting our results. Our trial for cardiac arrest model failed to demonstrate consistent ischemic injury in the hippocampus. Although we extended circulatory arrest to 6 min, mortality reached more than 90%, showing limitation of our cardiac arrest model. We tried other reported models, such as bilateral carotid artery occlusion with either systemic hypotension [15] or basilar artery occlusion [13], which were also associated with insufficient survival rate. Nonetheless, the development of a reproducible and reliable global ischemia model in mice would contribute greatly to molecular understanding of delayed neuronal death, thus awaiting further study in this field.
Fig. 2. DC potential changes in the cardiac arrest cerebral ischemia. See text for legend.
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N. Kawahara et al. / Neuroscience Letters 322 (2002) 91–94
This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture and Technology, Japan. The authors thank Ms Reiko Matsuura for her technical assistance.
[10] [11]
[1] Bo¨ ttiger, B.W., Teschendorf, P., Krumnikl, J.J., Vogel, P., Galmbacher, R., Schmitz, B., Motsch, J., Martin, E. and Gass, P., Global cerebral ischemia due to cardiocirculatory arrest in mice causes neuronal degeneration and early induction of transcription factor genes in the hippocampus, Brain Res. Mol. Brain Res., 65 (1999) 135–142. [2] Coimbra, C. and Wieloch, T., Moderate hypothermia mitigates neuronal damage in the rat brain when initiated several hours following transient cerebral ischemia, Acta Neuropathol. (Berl., 87 (1994) 325–331. [3] Colbourne, F. and Corbett, D., Delayed and prolonged postischemic hypothermia is neuroprotective in the gerbil, Brain Res., 654 (1994) 265–272. [4] Dietrich, W.D., Busto, R. and Bethea, J.R., Postischemic hypothermia and IL-10 treatment provides long-lasting neuroprotection of CA1 hippocampus following transient global ischemia in rats, Exp. Neurol., 158 (1999) 444–450. [5] Franklin, K.B.J. and Paxinos, G., The Mouse Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1997. [6] Fujii, M., Hara, H., Meng, W., Vonsattel, J.P., Huang, Z. and Moskowitz, M.A., Strain-related differences in susceptibility to transient forebrain ischemia in SV-129 and C57Black/6 mice, Stroke, 28 (1997) 1805–1811. [7] Huang, Z., Huang, P.L., Panahian, N., Dalkara, T., Fishman, M.C. and Moskowitz, M.A., Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase, Science, 265 (1994) 1883–1885. [8] Kawahara, N., Ruetzler, C.A. and Klatzo, I., Protective effect of spreading depression against neuronal damage following cardiac arrest ischemia, Neurol. Res., 17 (1995) 9–16. [9] Kawai, K., Nitecka, L., Ruetzler, C.A., Nagashima, G., Joo, F., Mies, G., Nowak Jr, T.S., Saito, N., Lohr, J.M. and Klatzo, I.,
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Global cerebral ischemia associated with cardiac arrest in the rat: I. Dynamics of early neuronal changes, J. Cereb. Blood Flow Metab., 12 (1992) 238–249. Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57–69. Kitagawa, K., Matsumoto, M., Yang, G., Mabuchi, T., Yagita, Y., Hori, M. and Yanagihara, T., Cerebral ischemia after bilateral common carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery, J. Cereb. Blood Flow Metab., 18 (1998) 570–579. Liu, K., Adachi, N., Yanase, H., Kataoka, K. and Arai, T., Lidocaine suppresses the anoxic depolarization and reduces the increase in the intracellular Ca 21 concentration in gerbil hippocampal neurons, Anesthesiology, 87 (1997) 1470–1478. Panahian, N., Yoshida, T., Huang, P.L., Hedley-Whytes, E.T., Dalkara, T., Fishman, M.C. and Moskowitz, M.A., Attenuated hippocampal damage after global cerebral ischemia in mice mutant in neuronal nitric oxide synthase, Neuroscience, 72 (1996) 343–354. Schauwecker, P.E. and Steward, O., Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches, Proc. Natl. Acad. Sci. USA, 94 (1997) 4103–4108. Sheng, H., Laskowitz, D.T., Pearlstein, R.D. and Warner, D.S., Characterization of a recovery global ischemia model in the mouse, J. Neurosci. Methods, 88 (1999) 103– 109. Smith, M.L., Auer, R.N. and Siesjo¨, B.K., The density and distribution of ischemic brain injury in the rat following 2-10 min of forebrain ischemia, Acta Neuropathol. (Berl., 64 (1984) 319–332. Wellons, J.C., Sheng, H., Laskowitz, D.T., Mackensen, G.B., Pearlstein, R.D. and Warner, D.S., A comparison of strainrelated susceptibility in two murine recovery models of global ischemia, Brain Res., 868 (2000) 14–21.
Cardiac arrest cerebral ischemia model in mice failed to cause delayed neuronal death in the hippocampus Nobutaka Kawahara a,b,*, Kensuke Kawai b,c, Tomikatsu Toyoda a,b, Hirofumi Nakatomi a,b, Kazuhide Furuya a,b, Takaaki Kirino a,b a
Department of Neurosurgery, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan CREST (Core Research for Evolutional Science and Technology), Japan Science and Technology Cooperation, Saitama 332-0012, Japan c Department of Neurosurgery, Tokyo Metropolitan Neurological Hospital, Tokyo 183-0042, Japan
b
Received 5 January 2002; received in revised form 15 January 2002; accepted 16 January 2002
Abstract Global cerebral ischemia models for genetically engineered mice are of particular importance for the study of delayed neuronal death, but have been complicated by variability of vascular anatomy. Here we developed a 5-min cardiac arrest model that was not affected by vascular anatomy, and evaluated the hippocampal neuronal injury in BL/6 and SV129 mice. Despite prolonged anoxic depolarization for approximately 7 min, however, no consistent ischemic neuronal injury was noted in the CA1 sector of the hippocampus in both strains. Thus, our observations suggested that murine hippocampal neurons are relatively resistant to ischemia compared with those in other rodents. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral ischemia; Cardiac arrest; Delayed neuronal death; Hypothermia; Anoxic depolarization; Direct current potential
Transient global cerebral ischemia causes delayed neuronal death in the CA1 sector of the hippocampus [10,16]. Though numerous studies have been undertaken to elucidate the pathophysiology of delayed neuronal death as a representative model of ischemic neuronal death, as well as a therapeutic target, its precise mechanism remains unknown. Recent progress of in vivo manipulation of genes in mice, on the other hand, has offered a new opportunity to study the in vivo effect of overexpression or targeted disruption of a specific gene. This approach has provided a powerful tool for investigation of molecular mechanisms in vivo, and demonstrated involvement of some crucial genes in the focal ischemic injury [7]. However, new gene technology in mice has contributed little to understanding of the molecular mechanism of delayed neuronal death, due to lack of reproducible global ischemia models in mice. The inconsistency of ischemic injury in this model mainly derives from the individual difference of collateral flow through the circle of Willis, even in the same strain [6,11,15,17]. To circumvent the flow-related variation, the current study was designed to address whether the cardiac arrest model, an * Corresponding author. Tel.: 1 81-3-5800-8852; fax.: 1 81-35800-8655. E-mail address: [email protected] (N. Kawahara).
already established model in rats [9], is indeed a suitable model for the investigation of molecular mechanism of delayed neuronal death in genetically engineered mice. We further provide data for anoxic depolarization and brain temperature changes, the two critical factors for ischemic injury in this small rodent model. Male C57BL/6NCrj (BL/6; Charles River Japan, Yokohama, Japan) and 129/SvEms (SV129; Jackson Laboratory, Bar Harbor, Maine) mice, aged 8–12 weeks, were used in this study. All animal-related procedures were conducted in accordance with the guidelines of the National Institutes of Health (Guide for the care and use of laboratory animals). Both BL/6 (n ¼ 15, BL/6 ischemia group) and SV129 (n ¼ 14, SV129 ischemia group) mice were subjected to cardiac arrest cerebral ischemia. After induction of anesthesia, the mice were maintained with 0.5% halothane in a 30% O2/70% N2O mixture. Following the left femoral artery and vein cannulation, atropine sulphate (0.25 mg/kg, ip), amikacin (10 mg/kg, ip), and sodium bicarbonate (0.5 mEq/kg, iv) were administered. Rectal and temporal muscle temperatures were controlled at 37 8C by a heating blanket and a heating lamp up to 30 min after ischemia. Following midline cervical-sternal incision, a bent hook was inserted into the right thoracic cavity, rotated underneath the heart, and mechanical ventilation was discontinued. The major
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 10 1- 5
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N. Kawahara et al. / Neuroscience Letters 322 (2002) 91–94
cardiac vessels were then occluded by pulling the hook and applying finger pressure from outside [8,9]. After confirming the cardiac standstill, resuscitation was initiated at 5 min with external cardiac massage and mechanical ventilation with 100% O2 under tracheostomy. Epinephrine (0.02 mg/ kg, iv) and sodium bicarbonate (0.5 mEq/kg, iv) were administered several times to maintain BP. Restoration of BP to 50 mmHg was considered recirculation. Then, the mice were weaned from the ventilator at 10–15 min and extubated. After skin closure, the animals were placed in the incubator set at 33 8C during the first 24 h. Rectal temperature was monitored at 2, 6, and 24 h post-ischemia. Arterial blood samples were analyzed before and 30 min after ischemia, by a blood gas analyzer (Model 248; Chiron Diagnostics Ltd., Essex, UK). Sham operated control animals were made in BL/6 mice (n ¼ 7, BL/6 sham group). Seven days after ischemia, the brains were perfusion fixed with 4% paraformaldehyde and embedded in paraffin. Sections (4 mm thickness) containing the dorsal hippocampi were stained with hematoxylin and eosin and the number of intact neurons in the hippocampal CA1 sector was counted in a blind fashion. Degenerated neurons with shrunken nucleus and acidophilic cell body in the CA1 sector were also counted. The mean value of the neuronal density (cells/ 0.5 mm) on each side was used for statistical analysis. In situ DNA fragmentation was detected by TUNEL method using biotinylated-dUTP and terminal transferase (Roche Diagnostics, Mannheim, Germany), as described [1]. TUNEL positive cells were counted and similarly evaluated. In another group of BL/6 mice (n ¼ 4, Temp Group), a needle microprobe (MT-26/2; Physitemp Instruments, Clifton, NJ) was inserted to the hippocampus [5] (1.9 mm posterior to the bregma, 1.2 mm left from the midline, 3.0 mm depth), and the brain temperature was monitored by a thermometer (Model BAT-12; Physitemp Instruments, Clifton, NJ) up to 6 h post-ischemia. In the fourth group (DC group, BL/6 mice, n ¼ 6), hippocampal direct current (DC) potential was measured through silver chloride wires and a glass micropipette electrode (tip diameter 10–20 mm) inserted into the left hippocampus [5] (1.9 mm posterior to the bregma, 1.2 mm left from the midline, 1.2 mm depth) with reference in the neck muscle (DC amplifier, Model AD-641G; Nihon Kohden, Tokyo, Japan) [8]. Cerebral blood flow (CBF) was also measured through a probe for laser Doppler flowmetry (Model ALF 21; Advance Co. Ltd., Tokyo, Japan) attached to the skull (1.9 mm posterior to the bregma, 1.2 mm right from the midline). All the values are expressed as mean ^ SD. Physiological parameters, and the cell counts in the CA1 sector were compared using one-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test with significance set at P , 0:05. For the analysis of temperatures, 2-factorial ANOVA with the same Bonferroni’s post-hoc test was used. Successful recirculation was achieved in all mice except one, yielding resuscitation rate of 96% in the current series. The mean ischemia time for each group ranged from 5.6 to
5.9 min without significant difference among groups. The final survival rates at 7 days were 47% and 52% for the BL/6 and SV129 ischemia groups, respectively. Concerning physiological parameters, there were no statistically significant differences among the groups except DC group. In the DC group, the mean BP (pre-ischemia 70.8 mmHg, post-ischemia 74.3 mmHg vs. 78.3–97.9 mmHg in the others) was slightly lower, and the mean PaCO2 was higher (pre-ischemia 37.6 mmHg vs. 28.0–32.4 mmHg in the others) due to longer anesthesia time in this group. In the post-ischemic period, comparison of rectal temperature by factorial analysis (time £ group) revealed a significant main effect of time (P , 0:0001), such that rectal temperature at 2 h post-ischemia (36.3 ^ 0.7 8C) was the lowest and that at 24 h post-ischemia (37.4 ^ 0.2 8C) the highest without intergroup difference. The hippocampal CA1 sector in each group did not show significant ischemic injury (Fig. 1) except one hippocampus in the BL/6 ischemia group, in which the middle third of CA1 region was segmentally injured. Cell counts (cells/0.5 mm) of surviving neurons were 117.2 ^ 9.3 in the BL/6 sham group, 114.6 ^ 19.8 in the BL/6 ischemia group, and 122.3 ^ 8.3 in the SV129 ischemia group, demonstrating no statistically significant difference (P . 0:5). Likewise, cell counts (cells/CA1 sector) of degenerating neurons revealed no significant difference among these groups (P . 0:2); 0.0 ^ 0.0 in the BL/6 sham group, 6.5 ^ 13.0 in the BL/6 ischemia group, and 0.4 ^ 0.6 in the SV129 ischemia group. In addition, TUNEL positive cells were not detected in any hippocampal CA1 sector (Fig. 1) except one in the BL/6 group (40 cells/CA1 sector), which showed segmental injury in hematoxylin and eosin staining (P ¼ 0:36 among 3 groups). Brain temperature was monitored simultaneously up to 6 h. Factorial analysis of (location £ time) demonstrated a significant main effect of location (P , 0:008). Post-hoc test revealed that the brain temperature was significantly lower than rectal temperature, reaching 0.7 8C during ischemia and 1.0 8C at 1 and 6 h post-ischemia. CBF, as demonstrated in Fig. 2, was reduced to almost zero percent (3.2 ^ 1.7%) immediately after occlusion of the aorta. Following resuscitation, early hyperperfusion (172.8 ^ 41.6% at 5 min) and delayed hypoperfusion (41.6 ^ 12.4% at 30 min) were noted. Abrupt negative shift of DC potential was also noted with a delay of 74.0 ^ 6.5 s after the circulatory arrest, which showed further negative shift, followed by slow recovery, after recirculation. Total anoxic depolarization time at half-maximal amplitude was 415.0 ^ 51 s. We demonstrated that complete circulatory arrest in mice for 5 min, which are comparable to other established global ischemia models in gerbils and rats [10,16], induced prolonged ischemic depolarization in the hippocampal CA1 sector for approximately 7 min. This depolarization time is actually longer than that for complete injury for gerbil hippocampal neurons [12]. Nevertheless, no consis-
N. Kawahara et al. / Neuroscience Letters 322 (2002) 91–94
93
Fig. 1. Histological evaluation of the hippocampal CA1 sector after 7 days. (A) BL/6 sham group. (B) BL/6 ischemia group. (C) SV129 ischemia group. No neuronal injury was noted in each group, except one hippocampus in BL/6 ischemia group (data not shown). Hematoxylin and eosin staining. (D) TUNEL staining with hematoxylin counter-staining in the ischemia group. Note no positive staining in the CA1 sector. Scale bar, 100 mm.
tent neuronal injury in this most vulnerable region was noted in two commonly used strains of mice, i.e. BL/6 and SV129. Then the question immediately arises whether murine CA1 neurons are indeed resistant to prolonged ischemia compared with those of other rodents. Several other studies demonstrating hippocampal injury also suggest that this might be the case. According to bilateral carotid artery occlusion model, ischemia longer than 10 min was required for hippocampal injury [6,11,15,17]. Crucial in these models was the residual flow through the posterior communication artery (P-com), with BL/6 strain being the most suitable due to frequent hypoplasticity of Pcom. Therefore, relatively longer ischemic threshold in mice has been ascribed to residual flow during ischemia [6]. However, recent studies using bilateral carotid artery occlusion combined with systemic hypotension showed residual flow of less than 2% in the hippocampus, which still required more than 10 min to induce neuronal injury [15,17], suggesting that murine hippocampus might be indeed resistant to ischemia. On the contrary, Bo¨ ttiger et al. [1], using similar 5-min cardiac arrest model in NMRI mice, stated that some, but limited, neuronal injury in the hippocampus. Since quantitative data was not available in this study, direct comparison with our results seems difficult. Important to note in this regard is the difference in neuronal susceptibility for insults among various strains, which is unrelated to vascular anatomy. For instance, pyramidal neurons in BL/6 and BALB/c mice are more resistant to kainic acid toxicity compared with those in SV129 and FVB mice, indicating that genetic determinant at the cellular level could affect the phenotypic outcome [14]. Thus, the difference obtained from the similar cardiac arrest models in mice might be related to strain difference.
It is well known that hypothermia is neuroprotective in gerbil and rat global ischemia, even when initiated after ischemia [2,3]. We noted slight decrease of brain temperature by 1.0 8C in the early reperfusion period, suggesting that brain temperature might have dropped to 35.3 8C for an hour or two in our experiment. However, in gerbils and rats, postischemic hypothermia less than 33 8C is required for substantial neuroprotection when its duration is less than 6 h [2–4]. Thus, it is unlikely that transient mild postischemic brain hypothermia is a major factor affecting our results. Our trial for cardiac arrest model failed to demonstrate consistent ischemic injury in the hippocampus. Although we extended circulatory arrest to 6 min, mortality reached more than 90%, showing limitation of our cardiac arrest model. We tried other reported models, such as bilateral carotid artery occlusion with either systemic hypotension [15] or basilar artery occlusion [13], which were also associated with insufficient survival rate. Nonetheless, the development of a reproducible and reliable global ischemia model in mice would contribute greatly to molecular understanding of delayed neuronal death, thus awaiting further study in this field.
Fig. 2. DC potential changes in the cardiac arrest cerebral ischemia. See text for legend.
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N. Kawahara et al. / Neuroscience Letters 322 (2002) 91–94
This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture and Technology, Japan. The authors thank Ms Reiko Matsuura for her technical assistance.
[10] [11]
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