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Potassium channel blocker TEA prevents CA1 hippocampal injury following transient forebrain ischemia in adult rats
Neuroscience Letters 305 (2001) 83±86
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Potassium channel blocker TEA prevents CA1 hippocampal injury following transient forebrain ischemia in adult rats Hao Huang, Tian M. Gao*, Liang-Wei Gong, Zhi-Ye Zhuang, Xiaoming Li Department of Physiology, The First Military Medical University, Guangzhou 510515, P.R. China Received 27 February 2001; received in revised form 26 March 2001; accepted 30 March 2001
Abstract It has been recently reported that potassium channel increases activities in CA1 pyramidal neurons of rat hippocampus following transient forebrain ischemia. To understand the role of the enhanced potassium current in the pathogenesis of neuronal damage after ischemia, we examined the effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on the neuronal injury of CA1 region induced by 15 min forebrain ischemia using a four-vessel occlusion model. Adult rats received intracerebroventricular administration of either TEA or 4-AP after ischemia or TEA before ischemia and once each day for 7 days. In the postischemic TEA treated-rats, the neuronal injury in hippocampal CA1 region was signi®cantly less than that of the controls. In contrast, neither preischemic infusion of TEA nor postischemic treatment of 4-AP had any neuroprotective effects. The present study demonstrates that postischemic application of TEA protects hippocampal CA1 pyramidal neurons against ischemic insult, suggesting that potassium channels may play important roles in the pathogenesis of CA1 neuronal death after transient forebrain ischemia. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tetraethylammonium; Potassium channels; Cell death; Ischemia; Hippocampus; Rat
Pyramidal neurons in the CA1 ®eld of the hippocampus occur delayed neuronal death after transient forebrain ischemia [11]. Glutamate excitotoxicity has been suggested as a potential mediator of postischemic cell injury [3]. However, neuronal hyperactivity as predicted by excitotoxic hypothesis is not evident in CA1 region after ischemia. Both in vitro [13] and in vivo [5] intracellular recording studies have demonstrated that the spontaneous ®ring rate and neuronal excitability of CA1 neurons progressively decrease following reperfusion. It has been speculated that increased potassium currents may be responsible for the reduced neuronal excitability after ischemia. Indeed, recent studies reported an enhancement in activities of potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia [6,16]. Moreover, it has been shown recently that the enhancement of outward potassium current mediates apoptotic cell death in cultured cortical neurons [17]. Therefore, we hypothesized that an enhanced potassium current contributes to the pathogenesis of delayed neuronal death in hippocampal CA1 region after transient forebrain * Corresponding author. Tel.: 186-20-85148216; fax: 186-2087730321. E-mail address: tgao@®mmu.edu.cn (T.M. Gao).
ischemia. To address this question, we evaluated the effect of potassium channel blockers, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), on the neuronal damage in hippocampal CA1 ®eld following transient forebrain ischemia in rats. Male adult Wistar rats weighing 200±250 g were subjected to transient forebrain ischemia by use of the four-vessel occlusion method [6,12] with some modi®cations. Brie¯y, on the day prior to the experiment, rats were anesthetized with chloral hydrate (i.p., 40 mg/100 g weight), and an occluding device (a loop of silicone tubing) was placed loosely around each carotid artery to allow subsequent occlusion of these vessels, without interrupting carotid blood ¯ow. Both vertebral arteries were electrocauterized permanently. On the day of experiment, the fully awake rats were restrained and the carotid clasps were tightened to produce four-vessel occlusion. At the end of 15 min of ischemia, those animals that remained unresponsive to stimuli and had dilated pupils were accepted into the study. Cerebral recirculation was restored at this time and the animals were randomly assigned to receive intracerebroventricular infusions of TEA (500, 250 or 125 mmol/5 ml) or 4AP (50 mmol/5 ml) 30 min following reperfusion and once each day during the reperfusion. In some rats, TEA at a dose
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 82 1- 3
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H. Huang et al. / Neuroscience Letters 305 (2001) 83±86
of 250 mmol/5 ml was infused intracerebroventricularly 30 min before ischemic insult and once each day for 7 days. Each intracerebroventricular infusion was performed over a 5 min time period using a 10 ml syringe through a preimplanted 21 ga cannula in the left ventricle [2] (from the bregma: anteroposterior, 20.8 mm; lateral, 1.5 mm; depth, 3.5 mm). The ischemic control group and sham-operated group received volume-matched intracerebroventricular administration of arti®cial cerebrospinal ¯uid (ACSF). The rectal temperature was monitored in all animals before, during, and up to 7 days after ischemia and no signi®cant differences in rectal temperature were observed between control group and TEA or 4-AP treated groups. Seven days after ischemic insult, animals were anesthetized with an overdose of chloral hydrate and perfused transcardially with saline and then with buffered 4% paraformaldehyde. The brains were removed and processed for paraf®n embedding. Coronal sections (5 mm) were cut at the level of the dorsal hippocampus and then used for hematoxylin and eosin staining. The number of normal-appearing pyramidal cells in the stratum pyramidale within the CA1 ®eld was counted using Olympus Vanox microscope at a magni®cation of 400 £ . Histologic analysis was performed by a blinded observer and the average of the right and left survived cell numbers (neurons per 1 mm liner length) in a single section of dorsal hippocampus was calculated for each rat as reported by Kirino et al. [9]. Normal neurons were de®ned by those showing a distinct nucleus and nucleolus. The values of neuronal density were expressed as the mean value ^ the standard error of the mean (SEM). Statistical analysis was performed by analysis of variance (ANOVA, Dunnett) (n number of experiments). Fig. 1 shows representative light microscopic changes within the hippocampal CA1 ®eld for the sham-operated group, ischemic control group and postischemic TEA-treated group (250 mmol/5 ml). In sham-operated group, there were no damaged neurons in the CA1 region (Fig. 1A), and the neuronal cell density of this region was 248 ^ 7.4 (n 8). As reported previously [11], a widespread damage in the CA1 region was observed in all of the control ischemic rats (Fig. 1B). The pyramidal neurons either presented a shrunken appearance with condensed nuclei and minimal cytoplasm or, in many instances, disappeared. The density of survival neurons in the CA1 region in this group was 16 ^ 4.6 (n 8), less than 10% of that in shamoperated group. However, in postischemic TEA-treated rats except those treated with low dose of TEA, about 30±40% neurons in CA1 ®eld survived from ischemic insult (Fig. 1C). The average neuronal density was 99 ^ 9.3 (n 8) and 78 ^ 8.7 (n 5) in 250 and 500 mmol/5 ml TEA-treated group, respectively. Compared with the ACSF-treated ischemic controls, these two TEA-treated groups showed a signi®cant neuroprotective effect (P , 0:01) (Fig. 2). In contrast to postischemic administration, TEA at a dose of 250 mmol/5 ml infused before ischemia exerted no obvious neuroprotection (P . 0:05, n 6) (Fig. 2).
Another potassium channel blocker, 4-AP (50 mmol/5 ml), was also used following reperfusion, but no apparent neuroprotection was observed in CA1 region of hippocampus (P . 0:05, n 5) (Fig. 2). The present study for the ®rst time demonstrates a neuroprotective effect of postischemic TEA treatment on hippocampal CA1 pyramidal neurons against ischemic injury in vivo. Results are consistent with previous studies concerning roles for potassium channel in cell death in cultured neurons [17], lymphocyte [8] and human myeloid HL-60 cells [10]. In cultured cortical neurons, cells undergoing apoptosis exhibited an enhancement of the outward
Fig. 1. Paraf®n-embedded sections stained with hematoxylin and eosin (H&E) showing neuroprotective effects of TEA on damage of CA1 hippocampal pyramidal neurons 7 days after reperfusion in rats. (A) Sham-operated control; (B) ACSF-treated ischemic control; (C) 250 mmol/5 ml TEA administered 30 min after reperfusion. Bar 20 mm in (A, B and C).
H. Huang et al. / Neuroscience Letters 305 (2001) 83±86
Fig. 2. Neuronal density (number of intact pyramidal cells per 1 mm linear length) in the CA1 ®eld as a function of the type of treatments in rats following transient forebrain ischemia. (1) Sham-operated control (n 8); (2) ACSF-treated ischemic control (n 8); (3) (4) (5) 125 mmol/5 ml TEA (n 6), 250 mmol/5 ml TEA (n 8), and 500 mmol/5ml TEA (n 5), respectively, administered 30 min after ischemia; (6) 250 mmol/5 ml TEA administered 30 min before ischemia (n 6); (7) 50 mmol/5 ml 4AP administered 30 min after ischemia (n 5). Data represents as the mean ^ SEM. **P , 0:01 compared with ACSF control group.
potassium current before committed to die, and this apoptotic cell death could be attenuated by application of TEA [17]. Recently, a similar enhancement of potassium current [6,16] was also found long before cell death in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia. In the present study, the neuroprotective effect of TEA was maximum at 250 mmol /5 ml in the infusion solution. Since TEA was administered into the lateral ventricle, it would be diluted approximately 50-fold because the cerebrospinal ¯uid in adult rats is around 250 ml [1]. Hence, the ®nal concentration of TEA in the hippocampal tissue is assumed to be about 5 mmol/L or less. It is well known that extracellular TEA at this range of concentration has signi®cant blocking effect on potassium channels in hippocampus [14]. Taken together, it is reasonable to assume that the neuroprotection of TEA observed in the present study may be mediated by blocking potassium channels. Our study also showed that 4-AP, a transient Atype potassium channel blocker, had no apparent neuroprotection on CA1 neuronal damage after ischemia. This result indicates that transient A-type potassium channel may be not involved in the pathogenesis of postischemic neuronal injury in hippocampus. In addition, the result implies that certain other type(s) of potassium channel may mediate the neuroprotection of TEA, although further experiments are needed to ascertain the exact type of potassium channel. Blockade of K 1 channels by TEA can depolarize neuronal cells and promote Ca 21 in¯ux via voltage-dependent Ca 21 channels. Thus intracerebroventricular application of TEA before ischemia may enhance intracellular Ca 21 over-
85
load induced by ischemia, which is suggested to be responsible for neuronal death [3]. This may be one of the reasons that no neuroprotective outcome was observed in preischemic TEA-treated rats. In consistence with our results, previous study has shown that potassium channel openers administered before ischemia can prevent hippocampal CA1 neurons from ischemic cell death in rats [7]. However, when TEA applied following reperfusion, its depolarizing action on postischemic CA1 neurons would be greatly counteracted by the ischemia-induced hyperactivity of K 1 channels [6,16] and consequently its action on Ca 21 overload might be less signi®cant. In recent years, accumulating evidence has demonstrated that following transient forebrain ischemia, hippocampal CA1 neurons possess many of the biochemical and molecular characteristics needed to die by apoptosis such as activation of caspase-3 [2], release of cytochrome c from the mitochondria [15] and so on, although morphological observations either supporting [18] or opposing [4] apoptosis as the mechanism of postischemic cell death were reported. It has been shown that the ef¯ux of intracellular K 1 can promote the activation of caspase-3 [8]. Therefore, it is speculated that TEA may inhibit delayed neuronal death in the hippocampal CA1 region by blocking K 1 ef¯ux via the over-activated K 1 channels in the postischemic neurons and thereby suppressing the activity of caspase-3. In summary, the present study shows a neuroprotective effect of potassium channel blocker TEA on ischemiainduced CA1 neuronal damage in rats, suggesting that the enhancement in activities of potassium channel may, at least partially, participate in the delayed neuronal cell death after transient forebrain ischemia. We thank Dr Man-Lung Fung for critical reading and helpful comments on this paper. This research was supported by grants from National Natural Science Foundation (NNSF39970265), Natural Science Foundation of Guangdong Province (NSFGP990395) of P.R. China and Outstanding Scientists Program of PLA for medical research to Tian M. Gao. [1] Bass, N.H. and Lundberg, P., Postnatal development of bulk ¯ow in the cerebrospinal ¯uid system of the albino rat: clearance of [carboxyl-14C] insulin following intrathecal infusion, Brain Res., 52 (1973) 323±332. [2] Chen, J., Nagayama, T., Lin, K., Stetler, R.A., Zhu, R.L., Graham, S.H. and Simon, R.P., Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia, J. Neurosci., 18 (1998) 4914±4928. [3] Choi, D.W. and Rothman, S.M., The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death, Ann. Rev. Neurosci., 13 (1990) 171±182. [4] Colbourne, F., Li, H., Buchan, A.M. and Clemens, J.A., Continuing postischemic neuronal death in CA1: in¯uence of ischemia duration and cytoprotective doses of NBQX and SNK-111 in rats, Stroke, 30 (1999) 662±668.
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H. Huang et al. / Neuroscience Letters 305 (2001) 83±86
[5] Gao, T.M., Pulsinelli, W.A. and Xu, Z.C., Changes in membrane properties of CA1 pyramidal neurons after transient forebrain ischemia in vivo, Neuroscience, 90 (1999) 771±780. [6] Gong, L.W., Gao, T.M., Li, X., Huang, H. and Tong, Z., Enhancement in activities of large conductance calciumactivated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia, Brain Res., 884 (2000) 147±154. [7] Heurteaux, C., Bertaina, V., Widmann, C. and Lazdunski, M., K 1 channel openers prevent global ischemia-induced expression of c-fos, c-jun, heat shock protein, and amyloid b-protein precursor genes and neuronal death in rat hippocampus, Proc. Natl. Acad. Sci. USA, 94 (1997) 7661±7666. [8] Hughes, F.M., Bortner, C.D., Purdy, G.D. and Cidlowski, J.A., Intracellular K 1 suppresses the activation of apoptosis in lymphocytes, J. Biol. Chem., 272 (1997) 30567±30576. [9] Kirino, T., Tamura, A. and Sano, K., A reversible type of neuronal injury following ischemia in the gerbil hippocampus, Stroke, 17 (1986) 455±459. [10] McCarthy, J.V. and Cotter, T.G., Cell shrinkage and apoptosis: a role for potassium and sodium ion ef¯ux, Cell Death Differ., 4 (1997) 756±770. [11] Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal pro®le of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491±498. [12] Pulsinelli, W.A., Brierley, J.B. and Plum, F., A new model of
[13]
[14]
[15]
[16] [17]
[18]
bilateral hemispheric ischemia in the unanesthetized rat, Stroke, 10 (1979) 267±272. Shinno, K., Zhang, L., Eubanks, J.H., Carlen, P.L. and Wallace, M.C., Transient ischemia induces an early decrease of synaptic transmission in CA1 neurons of rat hippocampus: electrophysiological study in brain slice, J. Cereb. Blood Flow Metab., 17 (1997) 955±966. Storm, J.F., Potassium currents in hippocampal pyramidal cells, In J. Storm-Mathisen, J. Zimmer, O.P. Ottersen (Eds.), Progress in Brain Research, 83, Elsevier, Amsterdam, 1990, pp. 161±187. Sugawara, T., Fujimura, M., Morita-Fujimura, Y., Kawase, M. and Chan, P.H., Mitochondrial release of cytochrome c corresponds to the selective vulnerability of hippocampal CA1 neurons in rats after transient globel cerebral ischemia, J. Neurosci., 19 (1999) RC391±RC396. Xuan, C.X. and Xu, Z.C., Potassium currents in CA1 neuron of rat hippocampus increase shortly after transient cerebral ischemia, Neurosci. lett., 281 (2000) 5±8. Yu, S.P., Yeh, C.H., Sensi, S.L., Gwag, B.J., Canzoniero, L.M.T., Farhangrazi, Z.S., Ying, H.S., Tian, M., Dugan, L.L. and Choi, D.W., Mediation of neuronal apoptosis by enhancement of outward potassium current, Science, 278 (1997) 114±117. Zeng, Y.S. and Xu, Z.C., Co-existence of necrosis and apoptosis in rat hippocampus following transient forebrain ischemia, Neurosci. Res., 37 (2000) 113±125.
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Potassium channel blocker TEA prevents CA1 hippocampal injury following transient forebrain ischemia in adult rats Hao Huang, Tian M. Gao*, Liang-Wei Gong, Zhi-Ye Zhuang, Xiaoming Li Department of Physiology, The First Military Medical University, Guangzhou 510515, P.R. China Received 27 February 2001; received in revised form 26 March 2001; accepted 30 March 2001
Abstract It has been recently reported that potassium channel increases activities in CA1 pyramidal neurons of rat hippocampus following transient forebrain ischemia. To understand the role of the enhanced potassium current in the pathogenesis of neuronal damage after ischemia, we examined the effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on the neuronal injury of CA1 region induced by 15 min forebrain ischemia using a four-vessel occlusion model. Adult rats received intracerebroventricular administration of either TEA or 4-AP after ischemia or TEA before ischemia and once each day for 7 days. In the postischemic TEA treated-rats, the neuronal injury in hippocampal CA1 region was signi®cantly less than that of the controls. In contrast, neither preischemic infusion of TEA nor postischemic treatment of 4-AP had any neuroprotective effects. The present study demonstrates that postischemic application of TEA protects hippocampal CA1 pyramidal neurons against ischemic insult, suggesting that potassium channels may play important roles in the pathogenesis of CA1 neuronal death after transient forebrain ischemia. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tetraethylammonium; Potassium channels; Cell death; Ischemia; Hippocampus; Rat
Pyramidal neurons in the CA1 ®eld of the hippocampus occur delayed neuronal death after transient forebrain ischemia [11]. Glutamate excitotoxicity has been suggested as a potential mediator of postischemic cell injury [3]. However, neuronal hyperactivity as predicted by excitotoxic hypothesis is not evident in CA1 region after ischemia. Both in vitro [13] and in vivo [5] intracellular recording studies have demonstrated that the spontaneous ®ring rate and neuronal excitability of CA1 neurons progressively decrease following reperfusion. It has been speculated that increased potassium currents may be responsible for the reduced neuronal excitability after ischemia. Indeed, recent studies reported an enhancement in activities of potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia [6,16]. Moreover, it has been shown recently that the enhancement of outward potassium current mediates apoptotic cell death in cultured cortical neurons [17]. Therefore, we hypothesized that an enhanced potassium current contributes to the pathogenesis of delayed neuronal death in hippocampal CA1 region after transient forebrain * Corresponding author. Tel.: 186-20-85148216; fax: 186-2087730321. E-mail address: tgao@®mmu.edu.cn (T.M. Gao).
ischemia. To address this question, we evaluated the effect of potassium channel blockers, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), on the neuronal damage in hippocampal CA1 ®eld following transient forebrain ischemia in rats. Male adult Wistar rats weighing 200±250 g were subjected to transient forebrain ischemia by use of the four-vessel occlusion method [6,12] with some modi®cations. Brie¯y, on the day prior to the experiment, rats were anesthetized with chloral hydrate (i.p., 40 mg/100 g weight), and an occluding device (a loop of silicone tubing) was placed loosely around each carotid artery to allow subsequent occlusion of these vessels, without interrupting carotid blood ¯ow. Both vertebral arteries were electrocauterized permanently. On the day of experiment, the fully awake rats were restrained and the carotid clasps were tightened to produce four-vessel occlusion. At the end of 15 min of ischemia, those animals that remained unresponsive to stimuli and had dilated pupils were accepted into the study. Cerebral recirculation was restored at this time and the animals were randomly assigned to receive intracerebroventricular infusions of TEA (500, 250 or 125 mmol/5 ml) or 4AP (50 mmol/5 ml) 30 min following reperfusion and once each day during the reperfusion. In some rats, TEA at a dose
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 82 1- 3
84
H. Huang et al. / Neuroscience Letters 305 (2001) 83±86
of 250 mmol/5 ml was infused intracerebroventricularly 30 min before ischemic insult and once each day for 7 days. Each intracerebroventricular infusion was performed over a 5 min time period using a 10 ml syringe through a preimplanted 21 ga cannula in the left ventricle [2] (from the bregma: anteroposterior, 20.8 mm; lateral, 1.5 mm; depth, 3.5 mm). The ischemic control group and sham-operated group received volume-matched intracerebroventricular administration of arti®cial cerebrospinal ¯uid (ACSF). The rectal temperature was monitored in all animals before, during, and up to 7 days after ischemia and no signi®cant differences in rectal temperature were observed between control group and TEA or 4-AP treated groups. Seven days after ischemic insult, animals were anesthetized with an overdose of chloral hydrate and perfused transcardially with saline and then with buffered 4% paraformaldehyde. The brains were removed and processed for paraf®n embedding. Coronal sections (5 mm) were cut at the level of the dorsal hippocampus and then used for hematoxylin and eosin staining. The number of normal-appearing pyramidal cells in the stratum pyramidale within the CA1 ®eld was counted using Olympus Vanox microscope at a magni®cation of 400 £ . Histologic analysis was performed by a blinded observer and the average of the right and left survived cell numbers (neurons per 1 mm liner length) in a single section of dorsal hippocampus was calculated for each rat as reported by Kirino et al. [9]. Normal neurons were de®ned by those showing a distinct nucleus and nucleolus. The values of neuronal density were expressed as the mean value ^ the standard error of the mean (SEM). Statistical analysis was performed by analysis of variance (ANOVA, Dunnett) (n number of experiments). Fig. 1 shows representative light microscopic changes within the hippocampal CA1 ®eld for the sham-operated group, ischemic control group and postischemic TEA-treated group (250 mmol/5 ml). In sham-operated group, there were no damaged neurons in the CA1 region (Fig. 1A), and the neuronal cell density of this region was 248 ^ 7.4 (n 8). As reported previously [11], a widespread damage in the CA1 region was observed in all of the control ischemic rats (Fig. 1B). The pyramidal neurons either presented a shrunken appearance with condensed nuclei and minimal cytoplasm or, in many instances, disappeared. The density of survival neurons in the CA1 region in this group was 16 ^ 4.6 (n 8), less than 10% of that in shamoperated group. However, in postischemic TEA-treated rats except those treated with low dose of TEA, about 30±40% neurons in CA1 ®eld survived from ischemic insult (Fig. 1C). The average neuronal density was 99 ^ 9.3 (n 8) and 78 ^ 8.7 (n 5) in 250 and 500 mmol/5 ml TEA-treated group, respectively. Compared with the ACSF-treated ischemic controls, these two TEA-treated groups showed a signi®cant neuroprotective effect (P , 0:01) (Fig. 2). In contrast to postischemic administration, TEA at a dose of 250 mmol/5 ml infused before ischemia exerted no obvious neuroprotection (P . 0:05, n 6) (Fig. 2).
Another potassium channel blocker, 4-AP (50 mmol/5 ml), was also used following reperfusion, but no apparent neuroprotection was observed in CA1 region of hippocampus (P . 0:05, n 5) (Fig. 2). The present study for the ®rst time demonstrates a neuroprotective effect of postischemic TEA treatment on hippocampal CA1 pyramidal neurons against ischemic injury in vivo. Results are consistent with previous studies concerning roles for potassium channel in cell death in cultured neurons [17], lymphocyte [8] and human myeloid HL-60 cells [10]. In cultured cortical neurons, cells undergoing apoptosis exhibited an enhancement of the outward
Fig. 1. Paraf®n-embedded sections stained with hematoxylin and eosin (H&E) showing neuroprotective effects of TEA on damage of CA1 hippocampal pyramidal neurons 7 days after reperfusion in rats. (A) Sham-operated control; (B) ACSF-treated ischemic control; (C) 250 mmol/5 ml TEA administered 30 min after reperfusion. Bar 20 mm in (A, B and C).
H. Huang et al. / Neuroscience Letters 305 (2001) 83±86
Fig. 2. Neuronal density (number of intact pyramidal cells per 1 mm linear length) in the CA1 ®eld as a function of the type of treatments in rats following transient forebrain ischemia. (1) Sham-operated control (n 8); (2) ACSF-treated ischemic control (n 8); (3) (4) (5) 125 mmol/5 ml TEA (n 6), 250 mmol/5 ml TEA (n 8), and 500 mmol/5ml TEA (n 5), respectively, administered 30 min after ischemia; (6) 250 mmol/5 ml TEA administered 30 min before ischemia (n 6); (7) 50 mmol/5 ml 4AP administered 30 min after ischemia (n 5). Data represents as the mean ^ SEM. **P , 0:01 compared with ACSF control group.
potassium current before committed to die, and this apoptotic cell death could be attenuated by application of TEA [17]. Recently, a similar enhancement of potassium current [6,16] was also found long before cell death in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia. In the present study, the neuroprotective effect of TEA was maximum at 250 mmol /5 ml in the infusion solution. Since TEA was administered into the lateral ventricle, it would be diluted approximately 50-fold because the cerebrospinal ¯uid in adult rats is around 250 ml [1]. Hence, the ®nal concentration of TEA in the hippocampal tissue is assumed to be about 5 mmol/L or less. It is well known that extracellular TEA at this range of concentration has signi®cant blocking effect on potassium channels in hippocampus [14]. Taken together, it is reasonable to assume that the neuroprotection of TEA observed in the present study may be mediated by blocking potassium channels. Our study also showed that 4-AP, a transient Atype potassium channel blocker, had no apparent neuroprotection on CA1 neuronal damage after ischemia. This result indicates that transient A-type potassium channel may be not involved in the pathogenesis of postischemic neuronal injury in hippocampus. In addition, the result implies that certain other type(s) of potassium channel may mediate the neuroprotection of TEA, although further experiments are needed to ascertain the exact type of potassium channel. Blockade of K 1 channels by TEA can depolarize neuronal cells and promote Ca 21 in¯ux via voltage-dependent Ca 21 channels. Thus intracerebroventricular application of TEA before ischemia may enhance intracellular Ca 21 over-
85
load induced by ischemia, which is suggested to be responsible for neuronal death [3]. This may be one of the reasons that no neuroprotective outcome was observed in preischemic TEA-treated rats. In consistence with our results, previous study has shown that potassium channel openers administered before ischemia can prevent hippocampal CA1 neurons from ischemic cell death in rats [7]. However, when TEA applied following reperfusion, its depolarizing action on postischemic CA1 neurons would be greatly counteracted by the ischemia-induced hyperactivity of K 1 channels [6,16] and consequently its action on Ca 21 overload might be less signi®cant. In recent years, accumulating evidence has demonstrated that following transient forebrain ischemia, hippocampal CA1 neurons possess many of the biochemical and molecular characteristics needed to die by apoptosis such as activation of caspase-3 [2], release of cytochrome c from the mitochondria [15] and so on, although morphological observations either supporting [18] or opposing [4] apoptosis as the mechanism of postischemic cell death were reported. It has been shown that the ef¯ux of intracellular K 1 can promote the activation of caspase-3 [8]. Therefore, it is speculated that TEA may inhibit delayed neuronal death in the hippocampal CA1 region by blocking K 1 ef¯ux via the over-activated K 1 channels in the postischemic neurons and thereby suppressing the activity of caspase-3. In summary, the present study shows a neuroprotective effect of potassium channel blocker TEA on ischemiainduced CA1 neuronal damage in rats, suggesting that the enhancement in activities of potassium channel may, at least partially, participate in the delayed neuronal cell death after transient forebrain ischemia. We thank Dr Man-Lung Fung for critical reading and helpful comments on this paper. This research was supported by grants from National Natural Science Foundation (NNSF39970265), Natural Science Foundation of Guangdong Province (NSFGP990395) of P.R. China and Outstanding Scientists Program of PLA for medical research to Tian M. Gao. [1] Bass, N.H. and Lundberg, P., Postnatal development of bulk ¯ow in the cerebrospinal ¯uid system of the albino rat: clearance of [carboxyl-14C] insulin following intrathecal infusion, Brain Res., 52 (1973) 323±332. [2] Chen, J., Nagayama, T., Lin, K., Stetler, R.A., Zhu, R.L., Graham, S.H. and Simon, R.P., Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia, J. Neurosci., 18 (1998) 4914±4928. [3] Choi, D.W. and Rothman, S.M., The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death, Ann. Rev. Neurosci., 13 (1990) 171±182. [4] Colbourne, F., Li, H., Buchan, A.M. and Clemens, J.A., Continuing postischemic neuronal death in CA1: in¯uence of ischemia duration and cytoprotective doses of NBQX and SNK-111 in rats, Stroke, 30 (1999) 662±668.
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H. Huang et al. / Neuroscience Letters 305 (2001) 83±86
[5] Gao, T.M., Pulsinelli, W.A. and Xu, Z.C., Changes in membrane properties of CA1 pyramidal neurons after transient forebrain ischemia in vivo, Neuroscience, 90 (1999) 771±780. [6] Gong, L.W., Gao, T.M., Li, X., Huang, H. and Tong, Z., Enhancement in activities of large conductance calciumactivated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia, Brain Res., 884 (2000) 147±154. [7] Heurteaux, C., Bertaina, V., Widmann, C. and Lazdunski, M., K 1 channel openers prevent global ischemia-induced expression of c-fos, c-jun, heat shock protein, and amyloid b-protein precursor genes and neuronal death in rat hippocampus, Proc. Natl. Acad. Sci. USA, 94 (1997) 7661±7666. [8] Hughes, F.M., Bortner, C.D., Purdy, G.D. and Cidlowski, J.A., Intracellular K 1 suppresses the activation of apoptosis in lymphocytes, J. Biol. Chem., 272 (1997) 30567±30576. [9] Kirino, T., Tamura, A. and Sano, K., A reversible type of neuronal injury following ischemia in the gerbil hippocampus, Stroke, 17 (1986) 455±459. [10] McCarthy, J.V. and Cotter, T.G., Cell shrinkage and apoptosis: a role for potassium and sodium ion ef¯ux, Cell Death Differ., 4 (1997) 756±770. [11] Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal pro®le of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491±498. [12] Pulsinelli, W.A., Brierley, J.B. and Plum, F., A new model of
[13]
[14]
[15]
[16] [17]
[18]
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