ARTICLE IN PRESS

G Model NSL 27071 1–6

Neuroscience Letters xxx (2010) xxx–xxx 1

Contents lists available at ScienceDirect

Neuroscience Letters

F

journal homepage: www.elsevier.com/locate/neulet

ASICs aggravate acidosis-induced injuries during ischemic reperfusion

2

Ling Gu a , Xiaoyu Liu a , Yi Yang a , Dijun Luo b , Xiaoxiang Zheng a,∗

3

a

4

b

OO

1

Department of Biomedical Engineering, Key Laboratory of Biomedical Engineering, Ministry of Education, Zhejiang University, Zheda Road 38, 310027 Hangzhou, PR China Department of Computer Science and Engineering, University of Texas at Arlington, Arlington, TX 76019, USA

6

a r t i c l e

i n f o

a b s t r a c t

7 8 9 10 11

Article history: Received 11 April 2010 Received in revised form 3 May 2010 Accepted 7 May 2010

Although acidosis is considered as a byproduct of brain ischemia, its effect on neurons during ischemia and reperfusion remains controversial, and the exact role of acid-sensing ion channels (ASICs) is unclear. Here we investigated the effect of acidosis on hippocampal neurons and the role of ASICs during both oxygen–glucose deprivation (OGD) and reperfusion. MTT assay and annexin V/PI staining suggested that although acidosis had a negative effect during OGD, it was more detrimental during reperfusion. Furthermore, inhibition of ASICs, especially ASIC1a, protected hippocampal neurons from acidotic injuries. Data from whole-cell patch clamp recordings also indicated that acute OGD did not alter the ASIC-current amplitude and desensitization comparing with normoxia conditions, however, it delayed the recovery of ASICs from desensitization. This result partially explained the failure of amiloride and PcTx1 in protecting neurons during acidotic OGD. Collectively, our study demonstrated that the result of the severe damage caused by acidosis during reperfusion but not during OGD was partially due to the different recovery time of ASICs between reperfusion and OGD. © 2010 Published by Elsevier Ireland Ltd.

12

18

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

D

TE

17

EC

16

Acidosis is a common feature of ischemic brain. Accumulation of lactic acid as a byproduct of glycolysis and protons produced by ATP hydrolysis cause extracellular pH reduction during brain ischemia [19]. During ischemia, the brain pH typically declines to below 6.5 under normoglycemic conditions and to below 6.0 under hyperglycemic conditions [17]. During reperfusion, there is an initial normalization or alkalization of brain pH followed by a recurrent acidosis at a pH of ∼6.6 [3,18,21]. Falls of extracellular pH during ischemia and reperfusion are expected to activate heteromeric ASIC1a/ASIC2a and homomeric ASIC1a channels [8,23], which are members of ASICs family. ASICs are activated by extracellular acidosis and mainly permeate Na+ currents [8,23]. Up to now, six different ASIC isoforms, derived from four genes, have been identified: ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4 [4,7]. ASIC channels play critical roles in physiological and pathological path way in both central nervous system [5,12,24,26] and peripheral nervous system [9,10]. Therefore, ASICs are considered to be novel targets for neurological diseases [25].

RR

15

Keywords: Acidosis OGD Reperfusion Acid-sensing ion channels (ASICs) Hippocampal neuron

CO

14

UN

13

PR

5

Abbreviations: ASICs, acid-sensing ion channels; AV, annexin V; DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylene glycol tetraacetic acid; MTT, 3(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide; OGD, oxygen and glucose deprivation; PBS, phosphate-buffered saline; PcTx venom, psalmotoxin venom; PI, propidium iodide; Reper, reperfusion. ∗ Corresponding author. Tel.: +86 571 87953860; fax: +86 571 87951676. E-mail address: [email protected] (X. Zheng).

Recent studies demonstrate that ASICs serve critically in acidosis-accompanied ischemia and reperfusion [12,26]. The decrease of extracellular pH activates inward ASIC currents, resulting in membrane depolarization that may facilitate the activation of voltage-gated Ca2+ channels and NMDA receptor-gated channels [22,24]. Meanwhile, the wide spread expression of ASIC1a, which is the only ASIC channel that conducts Ca2+ , gives us a new clue to glutamate-independent intracellular Ca2+ accumulation and neuronal injury during ischemia [26,27]. Indeed, in focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene protects the brain from ischemic injury more potently than glutamate antagonism [26]. Although acidosis occurs with ischemia and reperfusion, its effect on neurons during ischemia and reperfusion remains controversial [1,16,20], and the exact role of ASICs during OGD or reperfusion is unclear. Acidosis has long been considered as one factor promoting cell death during ischemia and reperfusion [20,26]. However, recently accumulative evidence shows that the irreversible injury induced by acidosis is accelerated by the events of reperfusion [1,11,16], whereas acidosis is even protective during oxygen and glucose deprivation (OGD) [1,13,15]. However, in the investigation of mechanism of ASICs during ischemia, OGD and reperfusion are combined together [12,18,26]. Thus the effect of ASICs on individual OGD and reperfusion has not been described. In this study, we investigated the effect of acidosis on hippocampal neurons and the role of ASICs during OGD or reperfusion. We found that acidosis had a negative effect during OGD. However,

0304-3940/$ – see front matter © 2010 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2010.05.029

Please cite this article in press as: L. Gu, et al., ASICs aggravate acidosis-induced injuries during ischemic reperfusion, Neurosci. Lett. (2010), doi:10.1016/j.neulet.2010.05.029

38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

G Model NSL 27071 1–6

ARTICLE IN PRESS L. Gu et al. / Neuroscience Letters xxx (2010) xxx–xxx

OO

F

2

70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108

pipette solution contained (in mM): 130 CsCl, 1.6 MgCl2, 5 EGTA, 2 Na2 ATP, and 10 HEPES, and was adjusted to pH 7.3 using CsOH. To ensure that ASICs were sufficiently recovered from desensitization, pulses of low-pH solution were given 5 min after the formation of the whole-cell configuration with 2 min intervals till currents were stabilized. To investigate the effect of acute OGD on ASICs, OGD solution of both pH 7.3 and pH 6.0 were bubbling with N2 for at least 1 h before experiments. OGD solution with pH 7.3 was applied 10 min before pulses of pH 6.0 OGD with 2 min intervals. The OGD solution contained (in mM): 150 NaCl, 10 sucrose, 5 KCl, 2 CaCl2 , 1 MgCl2 . pH was buffered with 10 mM HEPES. The cultures were washed twice in glucose-free PBS and incubated in OGD solution. Cells were placed into the Galaxy R hypoxia incubator (RS Biotech, England) for 4 h with 5% CO2 and 95% N2 , 37 ◦ C for simulated ischemia. Then OGD solution was replaced into standard solution and maintained in normoxia incubator for 4 h. For experiments detecting the effect of ASICs during OGD/reperfusion, ASIC inhibitors were incubated 30 min before OGD or reperfusion. Matlab (Mathworks, USA) was used for data analysis. The time constant () of desensitization of ASIC current was fitted to monoexponential function: Y = p0 + p1 e−t/ , where Y was the current amplitude at time t and p0 was the residual current. To detect recovery of ASICs from desensitization, the paired pulses were applied. The time constant of recovery from desensitization was fitted to mono-exponential function: Y = p0 + p1 e−t/ , where Y was the ratio of current responses between the second pulse and the first pulse (I2 /I1 ) and t was time interval between two pulses. The data were plotted as mean ± S.D. Statistical significant differences were carried out by one-way ANOVA followed by Tukey’s post hoc tests. p < 0.05 and p < 0.01 were considered statistically significant. The time duration of OGD was set equal to the time duration of reperfusion according to our previous study [14] to determine if acidosis had effect on cell viability during OGD and reperfusion. As shown in Fig. 1A, hippocampal neurons were treated with 4 h of OGD and 4 h of reperfusion. Although the cell viability of OGD pH 6.0–Reper pH 7.3 group was not as good as OGD pH 7.3–Reper pH 7.3 group (52.7 ± 4.1% for OGD pH 7.3–Reper pH 7.3 vs. 45.5 ± 3.1% for OGD pH 6.0–Reper pH 7.3, n = 5, p < 0.05), it was also significantly better than OGD pH 7.3–Reper 6.0 (24.5 ± 3.5% for OGD pH 7.3–Reper pH 6.0, n = 5, p < 0.01 vs. OGD pH 6.0–Reper pH 7.3),

ED

69

CT

68

it significantly injured neurons and showed a more detrimental effect during reperfusion. Moreover, inhibition of ASICs, especially ASIC1a, protected hippocampal neurons from acidotic injuries. These results offer a better understanding of the underlying mechanisms of acidotic injuries in cerebral ischemia/reperfusion. Amiloride (purity above 98%) was purchased from Sigma–Aldrich (St. Louise, MO, USA). PcTx venom was purchased from Spider Pharm (Yarnell, AZ, USA). Trypsin, DMEM and NeuroBasal were purchased from Gibco (Calsbad, CA, USA). All reagents were purchased from Sigma–Aldrich (St. Louise, MO, USA) except those specifically mentioned. Hippocampal neurons from 1-day-old neonatal Sprague–Dawley rats (Zhejiang Academy of Medical Science, Hangzhou, China) were isolated as described in earlier study [14]. All procedures were conducted according to the National Institutes of Health Guide for the care and use of laboratory animals and Care Standard of the Laboratory Animal (China Ministry of Health Publication, 1998). The cells were used for studies after 8 days. Neurons in 96-well plate were treated with different solutions. 15 ␮L MTT (5 mg/mL), dissolved in PBS, was added with 100 ␮L standard solution. After 4-h incubation at 37 ◦ C, MTT was removed and 100 ␮L DMSO was added. The absorbance was measured at 490 nm wavelength and background at 650 nm wavelength was subtracted. The Vybrant Apoptosis Assay Kit (Invitrogen) was used for the detection of apoptotic and necrotic cells according to vendor’s protocol. The fluorescence was measured by a Zeiss 510 confocal microscope (Zeiss, Germany). Confocal images of green AV fluorescence were collected using 488-nm excitation light from an argon/krypton laser and a 500–550 nm bandpass filter. Images of red PI fluorescence were collected using a 543-nm excitation light from the Helium/Ne laser and a 580-nm-long pass filter. The ASIC currents were recorded by the whole-cell patch clamp at a holding potential of −60 mV at room temperature (20–24 ◦ C). Currents were amplified by an EPC10 double amplifier (HEKA, Germany) with PatchMaster (version 2.2). Signals were sampled at 10 kHz and filtered at 1000 Hz. Patch pipettes of resistance 3–5 M were made from borosilicate glass by a Sutter Instruments P-87 (Novato, USA) horizontal micropipette puller. A home-made ‘Y’-tube perfusion system was employed to achieve a rapid extracellular solution change. The standard extracellular solution contained (in mM): 150 NaCl, 10 glucose, 5 KCl, 2 CaCl2 , 1 MgCl2 . pH was buffered with 10 mM HEPES (pH 6.0 or 7.3). The

RE

67

UN CO R

66

PR

Fig. 1. MTT cleavage treated with OGD/reperfusion. (A) Cells were treated with standard solution of pH 7.3 for 8 h, pH 6.0 for 4 h and pH 7.3 for the subsequent 4 h, OGD for 4 h with pH 7.3 and reperfusion for 4 h with pH 7.3, OGD for 4 h with pH 6.0 and reperfusion for 4 h with pH 7.3, or OGD with pH 7.3 and reperfusion with pH 6.0. (B) Cells were treated with standard solution of pH 7.3 for 8 h, OGD for 4 h with pH 6.0 and reperfusion for 4 h with pH 7.3, or OGD with pH 7.3 and reperfusion with pH 6.0. Either amiloride or PcTx venom was applied during low pH. n = 5, **p < 0.01 vs. treated with pH 7.3 standard solution, # p < 0.05 vs. corresponding group, ## p < 0.01 vs., corresponding group.

Please cite this article in press as: L. Gu, et al., ASICs aggravate acidosis-induced injuries during ischemic reperfusion, Neurosci. Lett. (2010), doi:10.1016/j.neulet.2010.05.029

109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151

G Model NSL 27071 1–6

ARTICLE IN PRESS 3

UN CO R

RE

CT

ED

PR

OO

F

L. Gu et al. / Neuroscience Letters xxx (2010) xxx–xxx

Fig. 2. Neuronal death caused by acidotic OGD/reperfusion. Cells were stained with AV (green) and PI (red). (A) Neurons treated with standard solution of pH 7.3 for 8 h, were examined by confocal microscopy. (B) Neurons treated with acidic solution of pH 6.0 and standard solution of 7.3 for the subsequent 4 h. (C) Neurons treated with OGD with pH 7.3 and reperfusion with pH 7.3 for 4 h, respectively. (D) Neurons treated with OGD with pH 6.0 and reperfusion with pH 7.3 for 4 h, respectively. (E) Neurons treated Q1 with OGD with pH 7.3 and reperfusion for with pH 6.0 for 4 h, respectively. (F) Statistical analysis of neuronal death during OGD/reperfusion. Cells were stained with AV and PI. n = 5, **p < 0.01 vs. treated with pH 7.3 standard solution for AV staining, ## p < 0.01 vs. treated with pH 7.3 standard solution for PI staining. Bar: 50 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Please cite this article in press as: L. Gu, et al., ASICs aggravate acidosis-induced injuries during ischemic reperfusion, Neurosci. Lett. (2010), doi:10.1016/j.neulet.2010.05.029

G Model NSL 27071 1–6

ARTICLE IN PRESS L. Gu et al. / Neuroscience Letters xxx (2010) xxx–xxx

UN CO R

RE

CT

ED

PR

OO

F

4

Fig. 3. ASICs inhibitors protected neurons during acidotic reperfusion. Cells were stained with annexin V (green) and PI (PI). (A–C) Neurons treated with OGD at pH 6.0 and reperfusion with pH 7.3 for 4 h, respectively. Either amiloride (B) or PcTx venom (C) was applied during acidotic OGD. (D–F) Neurons treated with OGD for 4 h with pH 7.3 and reperfusion for 4 h with pH 6.0. Either amiloride (E) or PcTx venom (F) was applied during acidotic reperfusion. (G) Statistical analysis of neuronal death during OGD/reperfusion. n = 5, **p < 0.01 vs. treated with OGD pH 7.3–Reper pH 6.0 for AV staining, ## p < 0.01 vs. treated with OGD pH 7.3–Reper pH 6.0 for PI staining, && p < 0.01 vs. corresponding group. Bar: 50 ␮m.

Please cite this article in press as: L. Gu, et al., ASICs aggravate acidosis-induced injuries during ischemic reperfusion, Neurosci. Lett. (2010), doi:10.1016/j.neulet.2010.05.029

G Model NSL 27071 1–6

ARTICLE IN PRESS 5

ED

PR

OO

F

L. Gu et al. / Neuroscience Letters xxx (2010) xxx–xxx

Fig. 4. Acute OGD did not modulate amplitude and desensitization of ASICs, but slowed down recovery of ASICs from desensitization. (A) Representative traces of ASICs before (left) and after (right) OGD. (B) Statistical data of ASIC currents before and after OGD, n = 12 and 10, respectively, p > 0.05. (C) Representative traces of ASICs induced by paired pulses. (D) Recovery of ASICs from desensitization was fitted to mono-exponential function, n = 5–10.

156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186

CT

155

RE

154

which indicated that acidosis was more detrimental during reperfusion. In consistence with MTT experiments, increased cell death was detected during acidotic reperfusion (Fig. 2). Being gated by extracellular protons, the characteristics of ASICs indicated that ASICs might play a critical role in acidosisaccompanied ischemia and reperfusion. To determine the exact effect of ASICs on neurons during OGD or reperfusion, hippocampal neurons were treated with either non-selective ASICs inhibitor amiloride or PcTx venom, which contained ASIC1aspecific inhibitor PcTx1. As shown in Fig. 1B, neither amiloride nor PcTx venom protected neurons during OGD (49.3 ± 2.6% treating with amiloride 100 ␮mol/L, 44.4 ± 3.0% treating with PcTx venom 100 ng/mL, n = 5, p > 0.05 vs. OGD pH 6–Reper pH 7.3). However, both amiloride and PcTx venom significantly protected neurons from injuries during acidotic reperfusion (33.4 ± 1.8% with Amiloride and 35.2 ± 5.1% with PcTx venom, n = 5, p < 0.01 vs. OGD pH 7.3–Reper pH 6.0), suggesting that the opening of ASICs injured neurons during reperfusion rather than during OGD. In addition, both apoptotic and necrotic death significantly increased in OGD pH 7.3–Reper pH 6.0 group (AV-positive: 71.5 ± 2.45%, PI-positive: 51.6 ± 2.2 for OGD pH 7.3–Reper pH 6.0 vs. AV-positive: 36.6 ± 2.3%, PI-positive: 19.5 ± 2.2 for OGD pH 6.0–Reper pH 7.3, n = 5, p < 0.01). The cell death decreased after application of either amiloride or PcTx venom (AV-positive: 47.9 ± 3.1%, PI-positive: 26.7 ± 1.2 for application of PcTx venom 100ng/mL, and AV-positive: 47.9 ± 3.1%, PI-positive: 26.7 ± 1.2% for application of amiloride 100 ␮mol/L, n = 5, p < 0.01 vs. OGD pH 7.3–Reper pH 6.0), indicating that inhibition of ASICs during acidotic reperfusion might protect neurons from both apoptotic and necrotic death (Fig. 3). To determine if ASICs functioned differently during OGD, ASIC current during normoxia and acute OGD was detected (Fig. 4A and B). The current amplitude of ASICs during OGD was 101.1 ± 10.0% compared with standard solution, and the time constant of desensitization () during OGD was 611.2 ± 74.5 ms, while it was 582.4 ± 73.6ms induced by standard solution at pH 6 (n = 12 or

UN CO R

152 153

10, p > 0.05). This demonstrated that acute OGD did not alter the amplitude or time constant of desensitization. To further delineate the effect of OGD on ASICs, we applied paired pulses to detect recovery from desensitization. Two lowpH pulses were given for 10 s with intervals of 10, 20, 30, 40, 50, 60, 90 and 120 s. As shown in Fig. 4C, recovery time of ASICs from desensitization slowed down significantly during OGD (ASICs recovered 51.76 ± 12.93% during OGD, while 72.6 ± 16.0% perfused by standard solution with time interval of 20 s, n = 8, p < 0.05; ASICs recovered 62.6 ± 15.0% during OGD, while 86.5 ± 10.5% perfused by standard solution with time interval of 30 s, n = 5, p < 0.05). Moreover, the recovery time constant of ASICs from desensitization during OGD in hippocampal neurons was 41.00 s, significantly larger than that under normoxia conditions (19.4 s, Fig. 4D). The slower recovery of ASICs from desensitization during OGD demonstrated lower opening frequency of ASICs under OGD conditions, which might result to less injury compared with reperfusion. We could also see that 4 h of acidosis alone produced more cell death than did acidosis combined with OGD in Fig. 1A. However, using ASIC blockade, the MTT cleavage showed no difference between acidic OGD and acidosis (see supplementary Fig. 1). This surprising result indicated to us that the slower recovery from desensitization of ASIC channels might be important for the protection by OGD. In this study, we investigated the effect of acidosis on hippocampal neurons and the role of ASICs during both OGD and reperfusion. We found that although acidosis had a negative effect during OGD, its injury during reperfusion was more detrimental and irreversible. Furthermore, inhibition of ASICs, especially ASIC1a, protected hippocampal neurons from acidotic injuries. Our data also indicated that although acute OGD did not modulate amplitude and desensitization of ASICs, it delayed the recovery of ASICs from desensitization, which might partially explain the failure of amiloride and PcTx venom in protecting neurons during acidotic OGD.

Please cite this article in press as: L. Gu, et al., ASICs aggravate acidosis-induced injuries during ischemic reperfusion, Neurosci. Lett. (2010), doi:10.1016/j.neulet.2010.05.029

187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221

ARTICLE IN PRESS

G Model NSL 27071 1–6

L. Gu et al. / Neuroscience Letters xxx (2010) xxx–xxx

268

271

This work was supported by the Key Laboratory for Biomedical Engineering of the Ministry of China, the Economic and Trade Commission of Zhejiang Province, and the Key Laboratory of Chinese Medicine Screening.

272

Appendix A. Supplementary data

273 274

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2010.05.029.

275

References

228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265

269 270

276 277

CT

227

RE

226

UN CO R

225

F

Acknowledgments

224

[2] R.L. Arias, M.L. Sung, D. Vasylyev, M.Y. Zhang, K. Albinson, K. Kubek, N. Kagan, C. Beyer, Q. Lin, J.M. Dwyer, M.M. Zaleska, M.R. Bowlby, J. Dunlop, M. Monaghan, Amiloride is neuroprotective in an MPTP model of Parkinson’s disease, Neurobiol. Dis. 31 (2008) 334–341. [3] T. Back, M. Hoehn, G. Mies, E. Busch, B. Schmitz, K. Kohno, K. Hossmann, Penumbral tissue alkalosis in focal cerebral ischemia: relationship to energy metabolism, blood flow, and steady potential, Ann. Neurol. 47 (2000) 485–492. [4] C.C. Chen, S. England, A.N. Akopian, J.N. Wood, A sensory neuronspecific, proton-gated ion channel, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 10240–10245. [5] M.W Coryell, A.E. Ziemann, P.J. Westmoreland, J.M. Haenfler, Z. Kurjakovic, X.M. Zha, M. Price, M.K. Schnizler, J.A. Wemmie, Targeting ASIC1a reduces innate fear and alters neuronal activity in the fear circuit, Biol. Psychiatry 62 (2007) 1140–1148. [6] T. Cronberg, A. Rytter, F. Asztely, A. Soder, T. Wieloch, Glucose but not lactate in combination with acidosis aggravates ischemic neuronal death in vitro, Stroke 35 (2004) 753–757. [7] D.A. de la Rosa, C.M. Canessa, G.K. Fyfe, P. Zhang, Structure and regulation of amiloride-sensitive sodium channels, Annu. Rev. Physiol. 62 (2000) 573–594. [8] D.A. de la Rosa, S.R. Krueger, A. Kolar, D. Shao, R.M. Fitzsimonds, C.M. Canessa, Distribution, subcellular localization and ontogeny of ASIC1 in the mammalian central nervous system, J. Physiol. 546 (2003) 77–87. [9] E Deval, J. Noel, N. Lay, A. Alloui, S. Diochot, V. Friend, M. Jodar, M. Lazdunski, E. Lingueglia, ASIC3, a sensor of acidic and primary inflammatory pain, EMBO J. 27 (2008) 3047–3055. [10] B. Duan, L.-J. Wu, Y.-Q. Yu, Y. Ding, L. Jing, L. Xu, J. Chen, T.-L. Xu, Upregulation of acid-sensing ion channel ASIC1a in spinal dorsal horn neurons contributes to inflammatory pain hypersensitivity, J. Neurosci. 27 (2007) 11139–11148. [11] E. Froyland, F. Wibrand, R. Almaas, I. Dalen, J.K. Lindstad, T. Rootwelt, Acidosis during reoxygenation has an early detrimental effect on neuronal metabolic activity, Pediatr. Res. 57 (2005) 488–493. [12] J. Gao, B. Duan, D.G. Wang, X.H. Deng, G.Y. Zhang, L. Xu, T.L. Xu, Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death, Neuron 48 (2005) 635–646. [13] R.G. Giffard, H. Monyer, C.W. Christine, D.W. Choi, Acidosis reduces NMDA receptor activation, glutamate neurotoxicity, and oxygen–glucose deprivation neuronal injury in cortical cultures, Brain Res. 506 (1990) 339–342. [14] L Gu, Y. Yang, Y. Sun, X. Zheng, Puerarin inhibits acid-sensing ion channels and protects against neuron death induced by acidosis, Planta Med. (2009), doi:10.1055/s-0029-1240583. [15] Y. Kamada, K. Hiramatsu, T. Sakaki, Effects of acidosis on the post-hypoxic recovery of synaptic transmission in gerbil hippocampal slices, Brain Res. 868 (2000) 347–351. [16] D. Li, Z. Shao, T.L. Vanden Hoek, J.R. Brorson, Reperfusion accelerates acute neuronal death induced by simulated ischemia, Exp. Neurol. 206 (2007) 280–287. [17] M. Nedergaard, R.P. Kraig, J. Tanabe, W.A. Pulsinelli, Dynamics of interstitial and intracellular pH in evolving brain infarct, Am. J. Physiol. Regul. Integr. Comp. Physiol. 260 (1991) 581–588. [18] G. Pignataro, R.P. Simon, Z.G. Xiong, Prolonged activation of ASIC1a and the time window for neuroprotection in cerebral ischemia, Brain 130 (2007) 151–158. [19] B. Siesjö, K. Katsura, T. Kristián, Acidosis-related damage, Adv. Neurol. 71 (1996) 209–233, discussion 234–236. [20] B.K. Siesjo, Acidosis and ischemic brain damage, Neurochem. Pathol. 9 (1988) 31–88. [21] F.H. Tomlinson, R.E. Anderson, F.B. Meyer, Brain pHi, cerebral blood flow, and NADH fluorescence during severe incomplete global ischemia in rabbits, Stroke 24 (1993) 435–443. [22] M Vukicevic, S. Kellenberger, Modulatory effects of acid-sensing ion channels on action potential generation in hippocampal neurons, Am. J. Physiol.-Cell Physiol. 287 (2004) 682–690. [23] R. Waldmann, G. Champigny, F. Bassilana, C. Heurteaux, M. Lazdunski, A proton-gated cation channel involved in acid-sensing, Nature 386 (1997) 173–177. [24] J.A. Wemmie, J. Chen, C.C. Askwith, A.M. Hruska-Hageman, M.P. Price, B.C. Nolan, P.G. Yoder, E. Lamani, T. Hoshi, J.H. Freeman, M.J. Welsh, The acidactivated ion channel ASIC contributes to synaptic plasticity, Learn. Memory Neuron 34 (2002) 463–477. [25] Z.G. Xiong, G. Pignataro, M. Li, S. Chang, R.P. Simon, Acid-sensing ion channels (ASICs) as pharmacological targets for neurodegenerative diseases, Curr. Opin. Pharmacol. 8 (2008) 25–32. [26] Z.G. Xiong, X.M. Zhu, X.P. Chu, M. Minami, J. Hey, W.L. Wei, J.F. MacDonald, J.A. Wemmie, M.P. Price, M.J. Welsh, R.P. Simon, Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels, Cell 118 (2004) 687–698. [27] O. Yermolaieva, A.S. Leonard, M.K. Schnizler, F.M. Abboud, M.J. Welsh, Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 6752–6757.

OO

267

223

ED

266

Xiong et al. have reported that OGD potentiated ASIC responses in cortical neurons [26]. However, ASIC currents were recorded after 1 h reperfusion following 1 h OGD in their ischemic model, which did not distinguish the effect of OGD between that of reperfusion on ASICs, whereas in our acute ischemic model, ASICs were recorded 10 min after the initiation of OGD in hippocampal neurons. Therefore, the difference between two ischemic models might be the reason of different effect of OGD on ASICs. It has to be taken into account that viability of cells treated with OGD pH 6.0–Reper pH 7.3 was better than cells treated with normoxia solution of pH 6.0–7.3 (Figs. 1 and 2, p < 0.01 for MTT cleavage and positive stained ratio of AV and PI), suggesting that OGD might be beneficial to neurons during acidosis. Acidosis was reported to be associated with other pathological conditions such as Parkinson’s disease [2], therefore inhibition of acidotic injuries was also worth illuminating. It was demonstrated that glucose combined with acidosis aggravated ischemic neuronal death in vitro [6], nevertheless, whether glucose combined with acidosis could aggravate normoxic neuronal death remained unknown. In our study, we showed that OGD protected neurons from acidotic injuries, which might be the result of slower recovery from desensitization of ASICs. Acidosis has long been considered as a detrimental effect during ischemia and reperfusion [20,26]. It was shown that during OGD/reperfusion, acidosis activated Ca2+ -permeable ASICs, inducing glutamate receptor-independent, Ca2+ -dependent neuronal injury that could be inhibited by ASIC inhibitor [26]. However, the exact effect of acidosis and ASICs on neurons during OGD or reperfusion remained to be delineated. On the other hand, recently more and more evidence demonstrated that mild acidosis inhibited NMDA-mediated cell currents, reduced intracellular Ca2+ accumulation, and prevented the hypoxic neuronal injury during OGD [1,13,15], while the irreversible injury induced by acidosis was accelerated by the events of reperfusion [1,11,16]. In this study, we revealed that acidosis was more detrimental during reperfusion for one more reason besides inhibition of NMDA receptors: OGD slowed down recovery of ASICs from desensitization. Moreover, AV and PI data during reperfusion showed that application of amiloride or PcTx venom decreased both apoptotic and necrotic death, indicating that inhibition on ASICs also protected neurons from apoptotic and necrotic death (Fig. 3). In conclusion, our study revealed the evidence that acidosis made more severe injuries during reperfusion than during OGD partially via the acceleration of ASICs recovery from desensitization. This provided a mechanistic insight into the effect of acidosis during brain ischemia/reperfusion.

222

PR

6

[1] R. Almaas, M. Pytte, J.K. Lindstad, M. Wright, O.D. Saugstad, D. Pleasure, T. Rootwelt, Acidosis has opposite effects on neuronal survival during hypoxia and reoxygenation, J. Neurochem. 84 (2003) 1018–1027.

Please cite this article in press as: L. Gu, et al., ASICs aggravate acidosis-induced injuries during ischemic reperfusion, Neurosci. Lett. (2010), doi:10.1016/j.neulet.2010.05.029

278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355