- Email: [email protected]
Elevation of Na+–K+ ATPase immunoreactivity in GABAergic neurons in gerbil CA1 region following transient forebrain ischemia
Brain Research 977 (2003) 284–289 www.elsevier.com / locate / brainres
Short communication
Elevation of Na 1 –K 1 ATPase immunoreactivity in GABAergic neurons in gerbil CA1 region following transient forebrain ischemia Tae-Cheon Kang a , In Koo Hwang a , Seung-Kook Park a , Sung-Jin An a , Young Sam Nam b , Do-Hoon Kim c , In Se Lee b , Moo Ho Won a , * a
b
Department of Anatomy, College of Medicine, Hallym University, Kangwon-Do, Chunchon 200 -702, South Korea Department of Anatomy, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul 151 -742, South Korea c Department of Psychiatry, College of Medicine, Institute of Natural Medicine, Hallym University, Chunchon 200 -702, South Korea Accepted 24 March 2003
Abstract In a previous study, we suggested that GABAergic neurons might be resistant to ischemic insult, because of the maintenance of the GABA shunt, which is one of the ATP synthetic pathways in neurons. In the present study, we identified Na 1 –K 1 ATPase immunoreactivity in the gerbil hippocampus in order to determine whether changes in Na 1 –K 1 ATPase immunoreactivity correlate with GABA shunt following ischemic insult. At 12 h after ischemia–reperfusion, Na 1 –K 1 ATPase immunoreactivity accumulated in some neurons in the CA1 region. However, the protein content of Na 1 –K 1 ATPase was not altered. Interestingly, the density of Na 1 –K 1 ATPase immunoreactivity in neurons and the protein content in the CA1 region was intensified in the 24 h post-ischemic group. As a result of double immunofluorescence study, Na 1 –K 1 ATPase immunoreactive neurons were identified with GABAergic neurons. Therefore, our findings suggest that the increase of Na 1 –K 1 ATPase in GABAergic neurons may be able to explain the resistance of these cells to ischemic insult, and support our previous hypothesis that GABA may play an important role as a metabolite in the survival of GABAergic neurons after ischemic insult. 2003 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Na 1 –K 1 ATPase; GAD; GABA; Transient forebrain ischemia; Hippocampus; Gerbil
Brain ischemia occurs when a reduction of cerebral blood flow induces oxygen and glucose deprivation. Ischemia may be caused by a localized pathological condition, such as that caused by cardiac arrest. Brain tissue O 2 stores are so small that they sustain normal energy consumption for only a few seconds. Furthermore, a drop in cerebral blood flow results in a precipitous drop in tissue glucose, which is absolutely essential for the maintenance of cellular ATP levels. At the cellular level, ATP is used to maintain ionic gradients across the plasma membrane, and a loss of ATP results in a loss of ionic homeostasis [1]. The result is a drop in membrane potential to the point *Corresponding author. Tel.: 182-33-248-2522; fax: 182-33-2561614. E-mail address: [email protected] (M.H. Won).
where voltage-dependent Na 1 channels open, causing an influx of Na 1 , which initiates a new round of depolarizing events [1]. On the other hand, sodium–potassium adenosine triphosphatase (Na 1 –K 1 ATPase) is responsible for the restoration and maintenance of intracellular Na 1 and K 1 gradients and thereby, is intimately involved in the regulation of the ability of neurons to fire [2,7]. Accordingly, an altered Na 1 –K 1 ATPase capacity or expression induced by ischemic insult may be an important factor in the neuronal damage that follows transient forebrain ischemia. This is because the capability of Na 1 –K 1 ATPase to catalyze a substantial fraction (50–60%) of total brain ATP, is potentially impaired by a limited ATP supply [2,7]. Recently we observed increased g-aminobutyric acid
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02681-7
T.-C. Kang et al. / Brain Research 977 (2003) 284–289
(GABA) shunt enzyme expressions in the hippocampus of gerbils after transient forebrain ischemia, particularly in GABAergic neurons [4,5]. These findings suggested that GABAergic neurons might be resistant to ischemic insult, because GABA is converted to ATP via the GABA shunt [3]. Therefore, in this study, we undertook to identify Na 1 –K 1 ATPase immunoreactivity in the gerbil hippocampus associated with various sequelae of transient forebrain ischemia in order to determine whether change in Na 1 –K 1 ATPase immunoreactivity may correlate with the GABA shunt and the preservation of GABAergic neurons following transient forebrain ischemia. This study utilized the progeny of Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity and lighting conditions (2364 8C, 5565% and a 12:12 light / dark cycle with lights). Procedures involving animals and their care were conducted in conformance with our institutional guidelines that comply with currently accepted international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985). Male Mongolian gerbils weighing 55–70 g were placed under general anesthesia with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. A midline ventral incision was then made in the neck, and both common carotid arteries were isolated, freed of nerve fibers, and occluded using nontraumatic aneurysm clips. Complete interruption of blood flow was confirmed by observing the central artery in eyeballs using an ophthalmoscope. After 5 min of occlusion, the aneurysm clips were removed from both common carotid arteries. Restoration of blood flow (reperfusion) was observed directly under the ophthalmoscope. Sham-operated controls (n510) were subjected to the same surgical procedures except that common carotid arteries were not occluded. Body temperature was monitored and maintained at 3760.5 8C during the surgery and during the immediate postoperative period until the animals had recovered fully from anesthesia. At the designated reperfusion times, operated animals and sham animals were killed for immunohistochemistry [8,16] and Western blot analysis. The gerbils were anesthetized with pentobarbital sodium, and perfused via the ascending aorta with 200 ml of 4% paraformaldehyde in phosphate buffer at 30 min (n5 7), 3 h (n57), 12 h (n57), 24 h (n57), 2 days (n57) and 4 days (n57) after the surgery. The brains were removed, postfixed in the same fixative for 4 h and rinsed in PB containing 30% sucrose at 4 8C for 2 days. Thereafter the tissues were frozen and sectioned with a cryostat at 30 mm and consecutive sections were collected in six-well plates containing PBS. These free-floating sections were first incubated with 10% normal horse serum for 30 min at room temperature, and then incubated in mouse anti-Na 1 –
285
K 1 ATPase IgG (a5, diluted 1:100, Developmental Studies Hybridoma Bank, USA) in PBS containing 0.3% Triton X-100 and 2% normal horse serum overnight at room temperature. After washing three times for 10 min with PBS, the sections were incubated sequentially, in horse anti-mouse IgG (Vector, USA) and streptavidin (Vector), diluted 1:200 in the same solution as the primary antiserum. Between incubations, the tissues were washed with PBS three times for 10 min each. Sections were visualized with DAB in 0.1 M Tris buffer and mounted on the gelatin-coated slides. To confirm the neuronal type containing Na 1 –K 1 ATPase immunoreactivity, double immunofluorescent staining for both mouse anti-Na 1 –K 1 ATPase antiserum (1:25) and rabbit anti-glutamic acid decarboxylase (GAD) 67 IgG (1:100, Chemicon, USA) was also performed. Other brain tissues were incubated in a mixture of antisera overnight at room temperature. After washing three times for 10 min with PBS, the sections were incubated in a mixture of both Cy2 conjugated goat anti-rabbit IgG (1:200, Amersham, USA) and Cy3 conjugated goat antimouse IgG (1:200, Amersham) for 1 h at room temperature. Immunoreactions were observed under an Axioscope microscope (Carl Zeiss, Germany). For quantitation of data, sections (15 sections per animal) were viewed through a microscope connected via a CCD camera to a PC monitor. Images of Na 1 –K 1 ATPase immunoreactivity in the hippocampus of each animal were captured using an Applescanner. The brightness and contrast of each image file was calibrated uniformly using Adobe Photoshop version 2.4.1, and then analyzed using NIH Image 1.59 software. Values of background staining were obtained and subtracted from the immunoreactive intensities. All data obtained were analyzed using one-way ANOVA to determine statistical significance. Bonferroni’s test was used for post-hoc comparisons. P values below 0.05 or 0.01 were considered statistically significant. To clarify the alteration of Na 1 –K 1 ATPase, we conducted the Western blot study. After sacrifice of each group, the hippocampi were divided into CA1 and the other regions. The CA1 region was homogenized in 10 mM PB containing 0.1 mM EDTA, 1 mM 2-mercaptoethanol and 1 mM PMSF. The individual 25% (w / v) homogenates were centrifuged at 10,0003g for 1 h. Twenty mg of each supernatant were mixed with an equal volume of 23 sodium dodecyl sulfate (SDS) sample buffer and boiled for 3 min. For Western blotting, proteins separated by SDS gel electrophoresis were transferred to nitrocellulose membranes, and the membranes were rinsed briefly in distilled water and air-dried. After rinsing with Tris buffered saline (TBS), the blots were incubated in Na 1 –K 1 ATPase antiserum for 1 h and washed three times in TBS containing Tween 20 at 5-min intervals. Then, membrane was incubated for 1 h at 37 8C with horseradish peroxidase conjugated goat anti-mouse IgG,
286
T.-C. Kang et al. / Brain Research 977 (2003) 284–289
diluted 1:400 in TBS containing 0.05% Tween 20. Finally, the bound conjugate was identified by incubation of the membrane in substrate buffer (0.5 mg / ml 4-chloro-1-naphthol in 1:5 (v / v) methanol / TBS) and 0.015% H 2 O 2 for 5 min at room temperature. In the sham-operated group, Na 1 –K 1 ATPase immunoreactivity in the CA region was weakly detected in neuropile (Fig. 1A). Protein content of Na 1 –K 1 ATPase in the CA1 region was unaltered prior to 12 h after ischemic insult based on Western blot study (Fig. 5). However, in the immunohistochemical study, the distribution pattern of Na 1 –K 1 ATPase immunoreactivity at 12 h after ischemic insult was different from that of the shamoperated group, namely Na 1 –K 1 ATPase immunoreactivity accumulated in some neurons of the CA1 region (Figs. 1B, 2A and 4). Interestingly, the density of Na 1 –K 1 ATPase immunoreactivity in neurons and the protein content in the CA1 region was intensified in the 24 h post-ischemic group (Figs. 1C, 4 and 5). In addition, at 24 h after ischemic insult Na 1 –K 1 ATPase immunoreactivity was detected in neurons located in the strata oriens,
pyramidale, lucidum and radiatum (Figs. 2B and C). As a result of the double immunofluorescence study, Na 1 –K 1 ATPase immunoreactive neurons were identified with GABAergic neurons (Fig. 3). Moreover, 2 days after ischemia–reperfusion, Na 1 –K 1 ATPase immunoreactivity was preserved in neurons (data not shown). Four days after ischemia–reperfusion, Na 1 –K 1 ATPase immunoreactivity in neurons was reduced significantly, however, Na 1 –K 1 ATPase immunoreactivity was expressed in glial cells (Fig. 1D, 2D and 4). At this time point, the protein content of Na 1 –K 1 ATPase in the CA1 region decreased slightly as compared with 24 h post-ischemic group (Fig. 5). After ischemia–reperfusion, Na 1 –K 1 ATPase immunoreactivity in the CA2–3 regions was found to be similar in every group (data not shown). Na 1 –K 1 ATPase is a key enzyme in the generation of membrane potentials and electrocortical brain activity [6]. Furthermore, Na 1 –K 1 ATPase activity is closely related to the regulation of intracellular osmolarity, and this ion transporter plays an important role in the cerebral edema that follows brain hypoxia–ischemia [10]. In the present
Fig. 1. Immunohistochemical staining for Na 1 –K 1 ATPase in the CA1 region of the gerbil hippocampus at sham (A), 12 h (B), 24 h (C) and 4 days (D) after ischemia–reperfusion. Na 1 –K 1 ATPase immunoreactivity in the CA1 region at 12 h after ischemic insult (B) is similar to that of sham-operated group (A). The intensity of Na 1 –K 1 ATPase immunoreactivity is dramatically enhanced in this region at 24 h after ischemia–reperfusion (C). Four days after ischemia, Na 1 –K 1 ATPase immunoreactivity appears in reactive astroglial cells in the stratum oriens, lucidum and radiatum (D). Bar5100 mm.
T.-C. Kang et al. / Brain Research 977 (2003) 284–289
287
Fig. 2. Immunohistochemical staining for Na 1 –K 1 ATPase in the CA1 region of the gerbil hippocampus at 12 h (A), 24 h (B and C) and 4 days (D) after ischemia–reperfusion. In this region, Na 1 –K 1 ATPase immunoreactivity is seen in some neurons, probably GABAergic neurons at 12 h after ischemia–reperfusion (A). At 24 h after ischemic insult, Na 1 –K 1 ATPase immunoreactivity is detected in neurons located in the strata pyramidale (B) and radiatum (C). Four days after ischemia, Na 1 –K 1 ATPase immunoreactivity is detected in astroglial cells, although its immunoreactivity was reduced in the CA1 region (D). Bar525 mm.
study, protein content of Na 1 –K 1 ATPase in the hippocampal CA1 region was unaltered prior to 12 h after ischemic insult. However, at 12 h after ischemia–reperfusion, the distribution pattern of Na 1 –K 1 ATPase was altered. In addition, Na 1 –K 1 ATPase immunoreactivity was observed in the GABAergic neurons located in the CA1 region judging from their morphology and double immunofluorescent study. Moreover, at this time point, the protein content of Na 1 –K 1 ATPase increased significantly based on the Western blot study. These findings were temporally consistent with our previous study, which demonstrated that the intensity of GAD67 immunoreactivity and the GABA shunt rate markedly increased in GABAergic neurons following ischemia [9]. Therefore, our findings provide morphological evidence that ATP synthesis in GABAergic neurons may be elevated via GABA shunt, and that this selective enhancement of Na 1 – K 1 ATPase expression may play an important role in the preservation of GABAergic neurons in the hippocampus after ischemic insult [11,13].
This selective elevation of Na 1 –K 1 ATPase immunoreactivity in GABAergic neurons also suggests that GABAergic neurons in the CA1 hippocampal region may reduce neuronal sensitivity to glutamate. This is because the inhibition of the Na 1 –K 1 ATPase function markedly potentiates and prolongs glutamate depolarization, which results in the elevation of intracellular Ca 21 level [3,5]. Therefore, our finding indicates that increased Na 1 –K 1 ATPase immunoreactivity may participate in the attenuation of ion homeostasis disturbances characterized by enhanced cellular K 1 efflux and, Na 1 and Ca 21 influx and cytotoxic edema [14]. On the other hand, during hypoxia oxygen free radicals are generated and cause the lipid peroxidation of neuronal membranes [7,12,15]. Due to such changes in lipids of brain cell membranes, the functions of membrane-related enzymes and receptors may be modified after hypoxia. Previous studies have also demonstrated hypoxia-induced decreases of Na 1 –K 1 ATPase activity in neurons by free radical damage [12]. In the present study, Na 1 –K 1
288
T.-C. Kang et al. / Brain Research 977 (2003) 284–289
Fig. 3. Double immunofluorescent staining for GAD67 (green, A and C) and Na 1 –K 1 ATPase (red, B and D) in the CA1 region of the gerbil hippocampus. At 12 h after ischemic insult (A, B), strong Na 1 –K 1 ATPase immunoreactivity is colocalized with GAD67 immunoreactivity in the CA1 region (arrows). At 24 h after ischemic insult (C, D), the number of Na 1 –K 1 ATPase immunoreactive neurons and fibers are increased in this region. In addition, GAD67 immunoreactivity is also enhanced in the same neurons (arrows). Bar550 mm.
Fig. 4. The densitimetric analysis of Na 1 –K 1 ATPase immunoreactivity in the CA1 region after ischemia–reperfusion. Significant differences from the sham, *P,0.05, **P,0.01.
Fig. 5. The Western blot analysis of hippocampal CA1 region aliquot containing 20 mg total protein.
T.-C. Kang et al. / Brain Research 977 (2003) 284–289
ATPase immunoreactivity in GABAergic neurons was significantly elevated; in contrast its immunoreactivity in pyramidal cells was unaltered. Thus this finding is considered as evidence that demonstrates why GABAergic neurons are resistant to ischemic insult, unlike pyramidal cells [16]. In conclusion, the present data suggest that enhanced Na 1 –K 1 ATPase immunoreactivity in GABAergic neurons may explain why GABAergic neurons are resistant to ischemic insult. In addition, these findings support our previous hypothesis that GABA may play an important metabolic role in the survival of GABAergic neurons after ischemic insult.
Acknowledgements The monoclonal antibody developed by Douglas M. Fambrough was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA. This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01-PJ8-PG3-21301-21301-0016).
References [1] W.R. Anderson, J.E. Franck, W.L. Stahl, A.A. Maki, Na,K-ATPase is decreased in hippocampus of kainate-lesioned rats, Epilepsy Res. 17 (1994) 221–231. [2] J. Astrup, Energy-requiring cell functions in the ischemic brain. Their critical supply and possible inhibition in protective therapy, J. Neurosurg. 56 (1982) 482–497. [3] M.L. Brines, R.J. Robbins, Inhibition of alpha 2 / alpha 3 sodium pump isoforms potentiates glutamate neurotoxicity, Brain Res. 591 (1992) 94–102. [4] M. Erecinska, I.A. Silver, ATP and brain function, J. Cereb. Blood Flow Metab. 9 (1989) 2–19.
289
[5] A. Fukuda, D.A. Prince, Excessive intracellular Ca 21 inhibits glutamate-induced Na(1)–K1 pump activation in rat hippocampal neurons, J. Neurophysiol. 68 (1992) 28–35. [6] F. Groenendaal, R.A. de Graaf, G. van Vliet, K. Nicolay, Effects of hypoxia–ischemia and inhibition of nitric oxide synthase on cerebral energy metabolism in newborn piglets, Pediatr. Res. 45 (1999) 827–833. [7] B. Halliwell, Reactive oxygen species and the central nervous system, J. Neurochem. 59 (1992) 1609–1623. [8] T.-C. Kang, S.K. Park, J.H. Bahn, J.S. Chang, W.S. Koh, S.M. Jo, S.W. Cho, S.Y. Choi, M.H. Won, Elevation of the gamma-aminobutyric acid transaminase expression in the gerbil CA1 area after ischemia–reperfusion damage, Neurosci. Lett. 294 (2000) 33–36. [9] T.-C. Kang, S.K. Park, I.K. Hwang, S.J. An, S.Y. Choi, S.W. Cho, M.H. Won, Spatial and temporal alterations in the GABA shunt in the gerbil hippocampus following transient ischemia, Brain Res. 944 (2002) 10–18. [10] J. Mintorovitch, G.Y. Yang, H. Shimizu, J. Kucharczyk, P.H. Chan, P.R. Weinstein, Diffusion-weighted magnetic resonance imaging of acute focal cerebral ischemia: comparison of signal intensity with changes in brain water and Na 1 ,K 1 -ATPase activity, J. Cereb. Blood Flow Metab. 14 (1994) 332–336. [11] C. Nitsch, G. Goping, I. Klatzo, Preservation of GABAergic perikarya and boutons after transient ischemia in the gerbil hippocampal CA1 field, Brain Res. 495 (1989) 243–252. [12] M. Shadid, G. Buonocore, F. Groenendaal, R. Moison, M. Ferrali, H.M. Berger, F. van Bel, Effect of deferoxamine and allopurinol on non-protein-bound iron concentrations in plasma and cortical brain tissue of newborn lambs following hypoxia–ischemia, Neurosci. Lett. 248 (1998) 5–8. [13] K. Shinno, L. Zhang, J.H. Eubanks, P.L. Carlen, M.C. Wallace, Transient ischemia induces an early decrease of synaptic transmission in CA1 neurons of rat hippocampus: electrophysiologic study in brain slices, J. Cereb. Blood Flow Metab. 17 (1997) 955–966. [14] B.K. Siesjo, Mechanisms of ischemic brain damage, Crit. Care Med. 16 (1988) 954–963. [15] F. Van Bel, M. Shadid, R.M. Moison, C.A. Dorrepaal, J. Fontijn, L. Monteiro, M. Van De Bor, H.M. Berger, Effect of allopurinol on postasphyxial free radical formation, cerebral hemodynamics, and electrical brain activity, Pediatrics 101 (1998) 185–193. [16] M.H. Won, T.-C. Kang, S.K. Park, G.S. Jeon, Y.W. Kim, J.H. Seo, E.M. Choi, M.H. Chung, S.S. Cho, The alterations of N-methyl-Daspartate receptor expressions and oxidative DNA damage in the CA1 area at the early time after ischemia–reperfusion insult, Neurosci. Lett. 301 (2001) 139–142.
Short communication
Elevation of Na 1 –K 1 ATPase immunoreactivity in GABAergic neurons in gerbil CA1 region following transient forebrain ischemia Tae-Cheon Kang a , In Koo Hwang a , Seung-Kook Park a , Sung-Jin An a , Young Sam Nam b , Do-Hoon Kim c , In Se Lee b , Moo Ho Won a , * a
b
Department of Anatomy, College of Medicine, Hallym University, Kangwon-Do, Chunchon 200 -702, South Korea Department of Anatomy, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul 151 -742, South Korea c Department of Psychiatry, College of Medicine, Institute of Natural Medicine, Hallym University, Chunchon 200 -702, South Korea Accepted 24 March 2003
Abstract In a previous study, we suggested that GABAergic neurons might be resistant to ischemic insult, because of the maintenance of the GABA shunt, which is one of the ATP synthetic pathways in neurons. In the present study, we identified Na 1 –K 1 ATPase immunoreactivity in the gerbil hippocampus in order to determine whether changes in Na 1 –K 1 ATPase immunoreactivity correlate with GABA shunt following ischemic insult. At 12 h after ischemia–reperfusion, Na 1 –K 1 ATPase immunoreactivity accumulated in some neurons in the CA1 region. However, the protein content of Na 1 –K 1 ATPase was not altered. Interestingly, the density of Na 1 –K 1 ATPase immunoreactivity in neurons and the protein content in the CA1 region was intensified in the 24 h post-ischemic group. As a result of double immunofluorescence study, Na 1 –K 1 ATPase immunoreactive neurons were identified with GABAergic neurons. Therefore, our findings suggest that the increase of Na 1 –K 1 ATPase in GABAergic neurons may be able to explain the resistance of these cells to ischemic insult, and support our previous hypothesis that GABA may play an important role as a metabolite in the survival of GABAergic neurons after ischemic insult. 2003 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Na 1 –K 1 ATPase; GAD; GABA; Transient forebrain ischemia; Hippocampus; Gerbil
Brain ischemia occurs when a reduction of cerebral blood flow induces oxygen and glucose deprivation. Ischemia may be caused by a localized pathological condition, such as that caused by cardiac arrest. Brain tissue O 2 stores are so small that they sustain normal energy consumption for only a few seconds. Furthermore, a drop in cerebral blood flow results in a precipitous drop in tissue glucose, which is absolutely essential for the maintenance of cellular ATP levels. At the cellular level, ATP is used to maintain ionic gradients across the plasma membrane, and a loss of ATP results in a loss of ionic homeostasis [1]. The result is a drop in membrane potential to the point *Corresponding author. Tel.: 182-33-248-2522; fax: 182-33-2561614. E-mail address: [email protected] (M.H. Won).
where voltage-dependent Na 1 channels open, causing an influx of Na 1 , which initiates a new round of depolarizing events [1]. On the other hand, sodium–potassium adenosine triphosphatase (Na 1 –K 1 ATPase) is responsible for the restoration and maintenance of intracellular Na 1 and K 1 gradients and thereby, is intimately involved in the regulation of the ability of neurons to fire [2,7]. Accordingly, an altered Na 1 –K 1 ATPase capacity or expression induced by ischemic insult may be an important factor in the neuronal damage that follows transient forebrain ischemia. This is because the capability of Na 1 –K 1 ATPase to catalyze a substantial fraction (50–60%) of total brain ATP, is potentially impaired by a limited ATP supply [2,7]. Recently we observed increased g-aminobutyric acid
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02681-7
T.-C. Kang et al. / Brain Research 977 (2003) 284–289
(GABA) shunt enzyme expressions in the hippocampus of gerbils after transient forebrain ischemia, particularly in GABAergic neurons [4,5]. These findings suggested that GABAergic neurons might be resistant to ischemic insult, because GABA is converted to ATP via the GABA shunt [3]. Therefore, in this study, we undertook to identify Na 1 –K 1 ATPase immunoreactivity in the gerbil hippocampus associated with various sequelae of transient forebrain ischemia in order to determine whether change in Na 1 –K 1 ATPase immunoreactivity may correlate with the GABA shunt and the preservation of GABAergic neurons following transient forebrain ischemia. This study utilized the progeny of Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity and lighting conditions (2364 8C, 5565% and a 12:12 light / dark cycle with lights). Procedures involving animals and their care were conducted in conformance with our institutional guidelines that comply with currently accepted international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985). Male Mongolian gerbils weighing 55–70 g were placed under general anesthesia with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. A midline ventral incision was then made in the neck, and both common carotid arteries were isolated, freed of nerve fibers, and occluded using nontraumatic aneurysm clips. Complete interruption of blood flow was confirmed by observing the central artery in eyeballs using an ophthalmoscope. After 5 min of occlusion, the aneurysm clips were removed from both common carotid arteries. Restoration of blood flow (reperfusion) was observed directly under the ophthalmoscope. Sham-operated controls (n510) were subjected to the same surgical procedures except that common carotid arteries were not occluded. Body temperature was monitored and maintained at 3760.5 8C during the surgery and during the immediate postoperative period until the animals had recovered fully from anesthesia. At the designated reperfusion times, operated animals and sham animals were killed for immunohistochemistry [8,16] and Western blot analysis. The gerbils were anesthetized with pentobarbital sodium, and perfused via the ascending aorta with 200 ml of 4% paraformaldehyde in phosphate buffer at 30 min (n5 7), 3 h (n57), 12 h (n57), 24 h (n57), 2 days (n57) and 4 days (n57) after the surgery. The brains were removed, postfixed in the same fixative for 4 h and rinsed in PB containing 30% sucrose at 4 8C for 2 days. Thereafter the tissues were frozen and sectioned with a cryostat at 30 mm and consecutive sections were collected in six-well plates containing PBS. These free-floating sections were first incubated with 10% normal horse serum for 30 min at room temperature, and then incubated in mouse anti-Na 1 –
285
K 1 ATPase IgG (a5, diluted 1:100, Developmental Studies Hybridoma Bank, USA) in PBS containing 0.3% Triton X-100 and 2% normal horse serum overnight at room temperature. After washing three times for 10 min with PBS, the sections were incubated sequentially, in horse anti-mouse IgG (Vector, USA) and streptavidin (Vector), diluted 1:200 in the same solution as the primary antiserum. Between incubations, the tissues were washed with PBS three times for 10 min each. Sections were visualized with DAB in 0.1 M Tris buffer and mounted on the gelatin-coated slides. To confirm the neuronal type containing Na 1 –K 1 ATPase immunoreactivity, double immunofluorescent staining for both mouse anti-Na 1 –K 1 ATPase antiserum (1:25) and rabbit anti-glutamic acid decarboxylase (GAD) 67 IgG (1:100, Chemicon, USA) was also performed. Other brain tissues were incubated in a mixture of antisera overnight at room temperature. After washing three times for 10 min with PBS, the sections were incubated in a mixture of both Cy2 conjugated goat anti-rabbit IgG (1:200, Amersham, USA) and Cy3 conjugated goat antimouse IgG (1:200, Amersham) for 1 h at room temperature. Immunoreactions were observed under an Axioscope microscope (Carl Zeiss, Germany). For quantitation of data, sections (15 sections per animal) were viewed through a microscope connected via a CCD camera to a PC monitor. Images of Na 1 –K 1 ATPase immunoreactivity in the hippocampus of each animal were captured using an Applescanner. The brightness and contrast of each image file was calibrated uniformly using Adobe Photoshop version 2.4.1, and then analyzed using NIH Image 1.59 software. Values of background staining were obtained and subtracted from the immunoreactive intensities. All data obtained were analyzed using one-way ANOVA to determine statistical significance. Bonferroni’s test was used for post-hoc comparisons. P values below 0.05 or 0.01 were considered statistically significant. To clarify the alteration of Na 1 –K 1 ATPase, we conducted the Western blot study. After sacrifice of each group, the hippocampi were divided into CA1 and the other regions. The CA1 region was homogenized in 10 mM PB containing 0.1 mM EDTA, 1 mM 2-mercaptoethanol and 1 mM PMSF. The individual 25% (w / v) homogenates were centrifuged at 10,0003g for 1 h. Twenty mg of each supernatant were mixed with an equal volume of 23 sodium dodecyl sulfate (SDS) sample buffer and boiled for 3 min. For Western blotting, proteins separated by SDS gel electrophoresis were transferred to nitrocellulose membranes, and the membranes were rinsed briefly in distilled water and air-dried. After rinsing with Tris buffered saline (TBS), the blots were incubated in Na 1 –K 1 ATPase antiserum for 1 h and washed three times in TBS containing Tween 20 at 5-min intervals. Then, membrane was incubated for 1 h at 37 8C with horseradish peroxidase conjugated goat anti-mouse IgG,
286
T.-C. Kang et al. / Brain Research 977 (2003) 284–289
diluted 1:400 in TBS containing 0.05% Tween 20. Finally, the bound conjugate was identified by incubation of the membrane in substrate buffer (0.5 mg / ml 4-chloro-1-naphthol in 1:5 (v / v) methanol / TBS) and 0.015% H 2 O 2 for 5 min at room temperature. In the sham-operated group, Na 1 –K 1 ATPase immunoreactivity in the CA region was weakly detected in neuropile (Fig. 1A). Protein content of Na 1 –K 1 ATPase in the CA1 region was unaltered prior to 12 h after ischemic insult based on Western blot study (Fig. 5). However, in the immunohistochemical study, the distribution pattern of Na 1 –K 1 ATPase immunoreactivity at 12 h after ischemic insult was different from that of the shamoperated group, namely Na 1 –K 1 ATPase immunoreactivity accumulated in some neurons of the CA1 region (Figs. 1B, 2A and 4). Interestingly, the density of Na 1 –K 1 ATPase immunoreactivity in neurons and the protein content in the CA1 region was intensified in the 24 h post-ischemic group (Figs. 1C, 4 and 5). In addition, at 24 h after ischemic insult Na 1 –K 1 ATPase immunoreactivity was detected in neurons located in the strata oriens,
pyramidale, lucidum and radiatum (Figs. 2B and C). As a result of the double immunofluorescence study, Na 1 –K 1 ATPase immunoreactive neurons were identified with GABAergic neurons (Fig. 3). Moreover, 2 days after ischemia–reperfusion, Na 1 –K 1 ATPase immunoreactivity was preserved in neurons (data not shown). Four days after ischemia–reperfusion, Na 1 –K 1 ATPase immunoreactivity in neurons was reduced significantly, however, Na 1 –K 1 ATPase immunoreactivity was expressed in glial cells (Fig. 1D, 2D and 4). At this time point, the protein content of Na 1 –K 1 ATPase in the CA1 region decreased slightly as compared with 24 h post-ischemic group (Fig. 5). After ischemia–reperfusion, Na 1 –K 1 ATPase immunoreactivity in the CA2–3 regions was found to be similar in every group (data not shown). Na 1 –K 1 ATPase is a key enzyme in the generation of membrane potentials and electrocortical brain activity [6]. Furthermore, Na 1 –K 1 ATPase activity is closely related to the regulation of intracellular osmolarity, and this ion transporter plays an important role in the cerebral edema that follows brain hypoxia–ischemia [10]. In the present
Fig. 1. Immunohistochemical staining for Na 1 –K 1 ATPase in the CA1 region of the gerbil hippocampus at sham (A), 12 h (B), 24 h (C) and 4 days (D) after ischemia–reperfusion. Na 1 –K 1 ATPase immunoreactivity in the CA1 region at 12 h after ischemic insult (B) is similar to that of sham-operated group (A). The intensity of Na 1 –K 1 ATPase immunoreactivity is dramatically enhanced in this region at 24 h after ischemia–reperfusion (C). Four days after ischemia, Na 1 –K 1 ATPase immunoreactivity appears in reactive astroglial cells in the stratum oriens, lucidum and radiatum (D). Bar5100 mm.
T.-C. Kang et al. / Brain Research 977 (2003) 284–289
287
Fig. 2. Immunohistochemical staining for Na 1 –K 1 ATPase in the CA1 region of the gerbil hippocampus at 12 h (A), 24 h (B and C) and 4 days (D) after ischemia–reperfusion. In this region, Na 1 –K 1 ATPase immunoreactivity is seen in some neurons, probably GABAergic neurons at 12 h after ischemia–reperfusion (A). At 24 h after ischemic insult, Na 1 –K 1 ATPase immunoreactivity is detected in neurons located in the strata pyramidale (B) and radiatum (C). Four days after ischemia, Na 1 –K 1 ATPase immunoreactivity is detected in astroglial cells, although its immunoreactivity was reduced in the CA1 region (D). Bar525 mm.
study, protein content of Na 1 –K 1 ATPase in the hippocampal CA1 region was unaltered prior to 12 h after ischemic insult. However, at 12 h after ischemia–reperfusion, the distribution pattern of Na 1 –K 1 ATPase was altered. In addition, Na 1 –K 1 ATPase immunoreactivity was observed in the GABAergic neurons located in the CA1 region judging from their morphology and double immunofluorescent study. Moreover, at this time point, the protein content of Na 1 –K 1 ATPase increased significantly based on the Western blot study. These findings were temporally consistent with our previous study, which demonstrated that the intensity of GAD67 immunoreactivity and the GABA shunt rate markedly increased in GABAergic neurons following ischemia [9]. Therefore, our findings provide morphological evidence that ATP synthesis in GABAergic neurons may be elevated via GABA shunt, and that this selective enhancement of Na 1 – K 1 ATPase expression may play an important role in the preservation of GABAergic neurons in the hippocampus after ischemic insult [11,13].
This selective elevation of Na 1 –K 1 ATPase immunoreactivity in GABAergic neurons also suggests that GABAergic neurons in the CA1 hippocampal region may reduce neuronal sensitivity to glutamate. This is because the inhibition of the Na 1 –K 1 ATPase function markedly potentiates and prolongs glutamate depolarization, which results in the elevation of intracellular Ca 21 level [3,5]. Therefore, our finding indicates that increased Na 1 –K 1 ATPase immunoreactivity may participate in the attenuation of ion homeostasis disturbances characterized by enhanced cellular K 1 efflux and, Na 1 and Ca 21 influx and cytotoxic edema [14]. On the other hand, during hypoxia oxygen free radicals are generated and cause the lipid peroxidation of neuronal membranes [7,12,15]. Due to such changes in lipids of brain cell membranes, the functions of membrane-related enzymes and receptors may be modified after hypoxia. Previous studies have also demonstrated hypoxia-induced decreases of Na 1 –K 1 ATPase activity in neurons by free radical damage [12]. In the present study, Na 1 –K 1
288
T.-C. Kang et al. / Brain Research 977 (2003) 284–289
Fig. 3. Double immunofluorescent staining for GAD67 (green, A and C) and Na 1 –K 1 ATPase (red, B and D) in the CA1 region of the gerbil hippocampus. At 12 h after ischemic insult (A, B), strong Na 1 –K 1 ATPase immunoreactivity is colocalized with GAD67 immunoreactivity in the CA1 region (arrows). At 24 h after ischemic insult (C, D), the number of Na 1 –K 1 ATPase immunoreactive neurons and fibers are increased in this region. In addition, GAD67 immunoreactivity is also enhanced in the same neurons (arrows). Bar550 mm.
Fig. 4. The densitimetric analysis of Na 1 –K 1 ATPase immunoreactivity in the CA1 region after ischemia–reperfusion. Significant differences from the sham, *P,0.05, **P,0.01.
Fig. 5. The Western blot analysis of hippocampal CA1 region aliquot containing 20 mg total protein.
T.-C. Kang et al. / Brain Research 977 (2003) 284–289
ATPase immunoreactivity in GABAergic neurons was significantly elevated; in contrast its immunoreactivity in pyramidal cells was unaltered. Thus this finding is considered as evidence that demonstrates why GABAergic neurons are resistant to ischemic insult, unlike pyramidal cells [16]. In conclusion, the present data suggest that enhanced Na 1 –K 1 ATPase immunoreactivity in GABAergic neurons may explain why GABAergic neurons are resistant to ischemic insult. In addition, these findings support our previous hypothesis that GABA may play an important metabolic role in the survival of GABAergic neurons after ischemic insult.
Acknowledgements The monoclonal antibody developed by Douglas M. Fambrough was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA. This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01-PJ8-PG3-21301-21301-0016).
References [1] W.R. Anderson, J.E. Franck, W.L. Stahl, A.A. Maki, Na,K-ATPase is decreased in hippocampus of kainate-lesioned rats, Epilepsy Res. 17 (1994) 221–231. [2] J. Astrup, Energy-requiring cell functions in the ischemic brain. Their critical supply and possible inhibition in protective therapy, J. Neurosurg. 56 (1982) 482–497. [3] M.L. Brines, R.J. Robbins, Inhibition of alpha 2 / alpha 3 sodium pump isoforms potentiates glutamate neurotoxicity, Brain Res. 591 (1992) 94–102. [4] M. Erecinska, I.A. Silver, ATP and brain function, J. Cereb. Blood Flow Metab. 9 (1989) 2–19.
289
[5] A. Fukuda, D.A. Prince, Excessive intracellular Ca 21 inhibits glutamate-induced Na(1)–K1 pump activation in rat hippocampal neurons, J. Neurophysiol. 68 (1992) 28–35. [6] F. Groenendaal, R.A. de Graaf, G. van Vliet, K. Nicolay, Effects of hypoxia–ischemia and inhibition of nitric oxide synthase on cerebral energy metabolism in newborn piglets, Pediatr. Res. 45 (1999) 827–833. [7] B. Halliwell, Reactive oxygen species and the central nervous system, J. Neurochem. 59 (1992) 1609–1623. [8] T.-C. Kang, S.K. Park, J.H. Bahn, J.S. Chang, W.S. Koh, S.M. Jo, S.W. Cho, S.Y. Choi, M.H. Won, Elevation of the gamma-aminobutyric acid transaminase expression in the gerbil CA1 area after ischemia–reperfusion damage, Neurosci. Lett. 294 (2000) 33–36. [9] T.-C. Kang, S.K. Park, I.K. Hwang, S.J. An, S.Y. Choi, S.W. Cho, M.H. Won, Spatial and temporal alterations in the GABA shunt in the gerbil hippocampus following transient ischemia, Brain Res. 944 (2002) 10–18. [10] J. Mintorovitch, G.Y. Yang, H. Shimizu, J. Kucharczyk, P.H. Chan, P.R. Weinstein, Diffusion-weighted magnetic resonance imaging of acute focal cerebral ischemia: comparison of signal intensity with changes in brain water and Na 1 ,K 1 -ATPase activity, J. Cereb. Blood Flow Metab. 14 (1994) 332–336. [11] C. Nitsch, G. Goping, I. Klatzo, Preservation of GABAergic perikarya and boutons after transient ischemia in the gerbil hippocampal CA1 field, Brain Res. 495 (1989) 243–252. [12] M. Shadid, G. Buonocore, F. Groenendaal, R. Moison, M. Ferrali, H.M. Berger, F. van Bel, Effect of deferoxamine and allopurinol on non-protein-bound iron concentrations in plasma and cortical brain tissue of newborn lambs following hypoxia–ischemia, Neurosci. Lett. 248 (1998) 5–8. [13] K. Shinno, L. Zhang, J.H. Eubanks, P.L. Carlen, M.C. Wallace, Transient ischemia induces an early decrease of synaptic transmission in CA1 neurons of rat hippocampus: electrophysiologic study in brain slices, J. Cereb. Blood Flow Metab. 17 (1997) 955–966. [14] B.K. Siesjo, Mechanisms of ischemic brain damage, Crit. Care Med. 16 (1988) 954–963. [15] F. Van Bel, M. Shadid, R.M. Moison, C.A. Dorrepaal, J. Fontijn, L. Monteiro, M. Van De Bor, H.M. Berger, Effect of allopurinol on postasphyxial free radical formation, cerebral hemodynamics, and electrical brain activity, Pediatrics 101 (1998) 185–193. [16] M.H. Won, T.-C. Kang, S.K. Park, G.S. Jeon, Y.W. Kim, J.H. Seo, E.M. Choi, M.H. Chung, S.S. Cho, The alterations of N-methyl-Daspartate receptor expressions and oxidative DNA damage in the CA1 area at the early time after ischemia–reperfusion insult, Neurosci. Lett. 301 (2001) 139–142.