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Changes in Na+–K+–Cl− cotransporter immunoreactivity in the gerbil hippocampus following transient ischemia
Neuroscience Research 44 (2002) 249 /254 www.elsevier.com/locate/neures
Changes in Na K Cl cotransporter immunoreactivity in the gerbil hippocampus following transient ischemia /
/
Tae-Cheon Kang a, Sung-Jin An a, Seung-Kook Park a, In Koo Hwang a, Dae-Kun Yoon a,b, Hyo-Sun Shin a,c, Moo Ho Won a,* b
a Department of Anatomy, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, Republic of Korea Department of General Surgery, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, Republic of Korea c Shin Hyo Seon’s Plastic and Aesthetic Surgery Clinic, Seoul 136-110, Republic of Korea
Received 15 March 2002; accepted 12 July 2002
Abstract We examined alterations in Na /K /Cl cotransporter 1 (NKCC1) immunoreactivity following ischemia. Twelve hours after ischemia, NKCC1 immunoreactivity in the CA1 region and in the hilar region was significantly diminished. Twenty-four hours after ischemia, NKCC1 immunoreactivity was intensified in these hippocampal regions as well as CA2-3. Two days after ischemia, NKCC1 immunoreactivity in the CA1 and the hilar neurons had disappeared, although in the CA2-3 and the granule cell layer NKCC1 immunoreactivities had recovered to the sham level. This finding suggests that NKCC1 may play an important role in the ischemic neuronal injury induced by excitotoxicity as well as neuronal edema. # 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. Keywords: Na /K /Cl cotransporter; Ischemia; Vulnerability; Up-regulation; Gerbil; Hippocampus
Na /K /Cl cotransporter 1 (NKCC1), which carries Na , K , and Cl simultaneously and electroneutrally (1:1:2) in the same direction, has been well characterized in a wide variety of nervous tissue. In neurons, the NKCC1 regulate intracellular Cl concentration and thereby affect neuronal response to gaminobutyric acid (GABA) (Jang et al., 2001; Kanaka et al., 2001; Plotkin et al., 1997a,b; Sun and Murali, 1999; Sung et al., 2000). Ischemic injury to a neuron is due primarily to the interruption of blood flow, a lack of oxygenation, and a subsequent reoxygenation of the brain (ischemia-reperfusion). The severity of ischemic damage depends on many factors, one of which is the rate of GABA metabolism (Kang et al., 2000, 2002). However, the roles of NKCC1 in the ischemic neuronal injury has not been confirmed; Yan et al. (2001) reported that NKCC1 expression is up-regulated in the infarct zone, and this
* Corresponding author. Tel.: /82-33-240-1614; fax: /82-33-2561614 E-mail address: [email protected] (M.H. Won).
alteration evokes ischemic damage via neuronal edema. In contrast to this paper, Yamada et al. (2001) observed that the function of NKCC activity was transiently reduced by ischemic insult, in vitro. Therefore, in the present study, the immunohistochemical distribution of NKCC1 in the hippocampus of the Mongolian gerbil and its association with different sequelae of ischemic insult was investigated to identify the roles of NKCC1 in the ischemic neuronal injury. Male Mongolian gerbils (Meriones unguiculatus ) weighing 55/70 g were placed under general anesthesia using 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. A midline ventral incision was made in the neck. Both common carotid arteries were isolated, freed of nerve fibers, and occluded using non-traumatic aneurysm clips. Complete interruption of blood flow was confirmed by observing the central artery in the eyeballs using an ophthalmoscope. After 5 min of occlusion, the aneurysm clips were released, and blood flow (reperfusion) was observed directly under the microscope. Sham-operated controls (n /5) were subjected to the same surgical procedures except that the common carotid arteries were not occluded. Body temperature
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 2 ) 0 0 1 3 1 - 1
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T.-C. Kang et al. / Neuroscience Research 44 (2002) 249 /254
Fig. 1. The chronological changes in the NKCC1 immunoreactivity in the hippocampus of the sham (A, C, E) and 12 h postischemic group (B, D, F). In the sham hippocampus (A), NKCC1 immunoreactivity is observed in mostly neurons including CA1 pyramidal neurons (C), granule cells and hilar neurons (E). Twelve hours after ischemia-reperfusion, the intensity of NKCC1 immunoreactivity markedly decreases in the hippocampus except CA2 /3 regions (B). In particular, decline of NKCC1 immunoreactivity is evident in CA1 pyramidal cells (D) and hilar neurons (F). Bar /400 mm (A and B), 50 mm (C /F).
was monitored and maintained at 379/0.5 8C during surgery and during the immediate postoperative period until the animals had recovered fully from anesthesia. At the designated reperfusion time, operated animals and sham animals were killed for immunohistochemistry (Kang et al., 2000; Won et al., 2001).
All animals were anesthetized with pentobarbital sodium, and perfused transcardially with phosphatebuffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) at 30 min (n/ 7), 3 h (n /7), 6 h (n/7), 12 h (n /7), 24 h (n /7), 2 days (n /7), 3 days (n/7), and 4 days (n /7) after
T.-C. Kang et al. / Neuroscience Research 44 (2002) 249 /254
251
Fig. 2. The chronological changes in the NKCC1 immunoreactivity in the hyppocampus of 1 day (A, C, E) and 2 days postischemic group (B, D, F). One day after ischemia, NKCC1 immunoreactivity obviously increases in the CA2 /3 regions (A). These increases are also observed in CA1 pyramidal (C) and hilar neurons (E). Two days after ischemia-reperfusion, the intensity of NKCC1 immunoreactivity markedly decreases in the hippocampus and dentate gyrus (B). NKCC1 immunoreactivity is rarely detected in CA1 pyramidal cells (D) and hilar neurons (F) but that in CA2 / 3 is comparable to sham level. However, NKCC1 immunoreactivity is elevated in the astrocytes in the CA1 region (D). Bar/400 mm (A and B), 50 mm (C /F).
surgery. Brains were removed, and postfixed in the same fixative for 4 h. Brain tissues were cryoprotected by being placed in 30% sucrose overnight. Thereafter, the tissues were frozen and sectioned with a cryostat at 30 mm. Consecutive sections were collected in six-well plates containing PBS. These free-floating sections were first incubated in 10% normal goat serum for 30 min at room temperature, then incubated in mouse antiNKCC1 serum (T4, diluted 1:50, 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), followed by ABC complex (Vector, USA), diluted 1:200 in the same solution as the primary antiserum. Between incubations, the tissues were washed with three times for 10 min with PBS. Sections were visualized with 3,3?-diaminobenzidine (DAB) in 0.1 M Tris buffer and mounted on gelatin-coated slides. NKCC1 immunoreactions were
252
T.-C. Kang et al. / Neuroscience Research 44 (2002) 249 /254
observed under the Axioscope microscope (Carl Zeiss, Germany). In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of primary antibody. Immunohistochemical controls showed in the absence of immunoreactivity in any structures. All experiment procedures were performed in parallel. Sections (15 sections per animal) were viewed through a microscope connected via CCD camera to a PC monitor. At a magnification of 25 /50 /, the region was outlined on the monitor and measured their area. Images of NKCC1 immunoreactivity in the hippocampus of each animal were captured with an Applescanner. The brightness and contrast of each image file were calibrated using ADOBE PHOTOSHOP version 2.4.1, and analyzed using NIH IMAGE 1.59 software. Values of the background staining were subtracted from the immunoreactive intensities. All data obtained from the quantitative data 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 to be statistically significant. In the sham hippocampus, NKCC1 immunoreactivity was observed in the all hippocampal neurons including the granule cells of the dentate gyrus, and in particular NKCC1 immunoreactivity accumulated in the hilar neurons (Fig. 1A, C and E). Thirty minutes after ischemia, NKCC1 immunoreactivities in the CA1 and the hilar regions began to decrease, and at 12 h after the ischemic insult, NKCC1 immunoreactivity had significantly diminished in these regions. However, NKCC1 immunoreactivities in the CA2-3 regions and the granule cell layer were unaltered (Fig. 1B, D and F). Interestingly, 1 day after ischemia, a dramatic intensification of NKCC1 immunoreactivity was observed in the all hippocampal regions including the CA2-3 regions and the granule cell layer. Moreover, NKCC1 immunodensity was highest in the CA2-3 regions (Fig. 2A, C and E). Two days after ischemia, NKCC1 immunoreactivity in the CA1 and the hilar neurons had almost disappeared, although in the CA2-3 and the granule cell layer NKCC1 immunoreactivities had recovered to the sham level. In addition, NKCC1 immunoreactive astrocytes appeared in the CA1 region (Fig. 2B, D and F). This distribution pattern was maintained to 4 days after ischemia (Fig. 3). Our study demonstrates that ischemic insults evoked changes in NKCC1 immunoreactivity in the hippocampus. Twelve hours after ischemic insult, NKCC1 immunoreactivity in the both CA1 and hilar regions was significantly decreased. We postulated that it might be a compensatory reaction to protect neurons from excitotoxicity. This hypothesis is supported by previous study (Yamada et al., 2001) reporting that the NKCC1 activity was effectively suppressed by oxygen /glucose deprivation in rat cortical slices, and this decreased
Fig. 3. The densitimetric analysis of NKCC1 immunoreactivity in the hippocampus after ischemia-reperfusion. Significant differences from the sham, *, P B/0.05; **, P B/0.01.
NKCC1 immunoreactivity delayed the onset of Cl entry, which is able to produce a protective effect. However, in the present study, elevated NKCC1 immunoreactivity was detected in these regions 24 h after ischemia. This elevated NKCC1 immunoreactivity in both CA1 pyramidal cells and hilar neurons suggest that intracellular Cl concentration ([Cl ]i) may play an important role in ‘excitotoxicity’ induced by the elevation of the intracellular Ca2 concentration. In fact, GABA evoked membrane depolarization mediated by Cl efflux in immature animals, which is the result of a high [Cl ]i in the central nervous system during early postnatal life (Backus et al., 1998; Chen et al., 1996; Luhmann and Prince, 1991; Owens et al., 1996; Plotkin et al., 1997a,b; Rivera et al., 1999; Sun and Murali, 1999; Valeyev et al., 1993). This depolarization activates voltage-dependant Ca2 channels and reduces the voltage-dependent Mg2 block of NMDA channels, resulting in Ca2 influx (Flint et al., 1998; Leinekugel et al., 1997; Obrietan and van den Pol, 1997). Thus, abnormal accumulation of Ca2 accompanied by a depolarization was evoked by input fiber stimulation in the hippocampal CA1 region of gerbils 2/3 days after transient whole brain ischemia (Tsubokawa et al., 1992). Furthermore, during ischemic stage, GABA aggravates excitotoxicity by accumulation of intracellular Ca2, in the condition of high [Cl ]i (Fukuda et al., 1998). This hypothesis is also supported by previous studies, which demonstrated that GABA concentration in the CA1 area after ischemia-reperfusion was elevated (Gabriel et al., 1998; Kang et al., 2000, 2002). Interestingly, we also found that NKCC1 immunoreactivity was enhanced in CA2-3 regions, which is resistant to ischemic insult. Though not exactly an explanation of this phenomenon, it is plausible that NKCC1 in the CA2-3 regions may trigger neuronal injury, although it could not lead to cell death. Twelve to 24 h after ischemic insult, CA2-3 neurons were swollen and the position of nuclei was eccentric (reactive chromatolytic cell change; Kirino, 1982). However, in
T.-C. Kang et al. / Neuroscience Research 44 (2002) 249 /254
this time point, GABA concentration in these regions was unaltered (Kang et al., 2000, 2002). Therefore, in the CA2-3 regions, unlike CA1 region, NKCC1 may trigger or aggravate neuronal injury induced by edema (Yan et al., 2001). Likewise in the present study, elevated NKCC1 immunoreactivity without altered GABA concentration in these areas provided some morphological evidence as to why the CA2-3 regions are resistant to ischemic insult. Two days after ischemia, NKCC1 immunoreactivity in the CA1 and the hilar neurons had almost disappeared, although in the CA2 /3 and the granule cell layer NKCC1 immunoreactivities had recovered to the sham level. At this time point, the nuclear chromatin of the pyramidal cells in the CA1 regions appeared clumped, and this phenomenon indicated irreversible ischemic damage in the perikarya (Kirino, 1982). Therefore, the disappearance of NKCC1 immunoreactivity in the CA1 may demonstrate irreversible ischemic damage. In conclusion, we have demonstrated the cellular and regional distribution pattern of NKCC1 according to reperfusion time in the gerbil ischemic hippocampus, and suggest that altered NKCC1 immunoreactivity may be related to ischemic neuronal damage.
Acknowledgements This work was supported by a grant from the Korea Ministry of Science and Technology (Critical Technology 21 [M1-0016-00-0024]), and this research was also supported by the Hallym Academy of Sciences at Hallym University, Korea, 2002-1.
References Backus, K.H., Deitmer, J.W., Friauf, E., 1998. Glycine-activated currents are changed by coincident membrane depolarization in developing rat auditory brainstem neurons. J. Physiol. 507, 783 / 794. Chen, G., Trombley, P.Q., van den Pol, A.N., 1996. Excitatory actions of GABA in developing rat hypothalamic neurons. J. Physiol. 494, 451 /464. Flint, A.C., Liu, X., Kriegstein, A.R., 1998. Nonsynaptic glycine receptor activation during early neocortical development. Neuron 20, 43 /53. Fukuda, A., Muramatsu, K., Okabe, A., Shimano, Y., Hida, H., Fujimoto, I., Nishino, H., 1998. Changes in intracellular Ca2 induced by GABAA receptor activation and reduction in Cl gradient in neonatal rat neocortex. J. Neurophysiol. 79, 439 /446. Gabriel, E.M., Inglefield, J.R., Chadwick, L.E., Schwartz-Bloom, R.D., 1998. Ischemic injury and extracellular amino acid accumulation in hippocampal area CA1 are not dependent upon an intact septo-hippocampal pathway. Brain Res. 785, 279 /286.
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Jang, I.S., Jeong, H.J., Akaike, N., 2001. Contribution of the Na /K / Cl cotransporter on GABA(A) receptor-mediated presynaptic depolarization in excitatory nerve terminals. J. Neurosci. 21, 5962 /5972. Kanaka, C., Ohno, K., Okabe, A., Kuriyama, K., Itoh, T., Fukuda, A., Sato, K., 2001. The differential expression patterns of messenger RNAs encoding K /Cl cotransporters (KCC1, 2) and Na /K /2Cl cotransporter (NKCC1) in the rat nervous system. Neuroscience 104, 933 /946. Kang, T.C., Park, S.K., Bahn, J.H., Chang, J.S., Koh, W.S., Jo, S.M., Cho, S.W., Choi, S.Y., Won, M.H., 2000. Elevation of the gammaaminobutyric acid transaminase expression in the gerbil CA1 area after ischemia-reperfusion damage. Neurosci. Lett. 294, 33 /36. Kang, T.-C., Park, S.K., Hwang, I.K., An, S.J., Choi, S.Y., Cho, S.W., Won, H.W., 2002. The spatial and temporal alterations in the GABA shunt in the gerbil hippocampus following transient ischemia. Brain Res. 944, 10 /18. Kirino, T., 1982. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239, 57 /69. Leinekugel, X., Medina, I., Khalilov, I., Ben-Ari, Y., Khazipov, R., 1997. Ca2 oscillations mediated by the synergistic excitatory actions of GABA(A) and NMDA receptors in the neonatal hippocampus. Neuron 18, 243 /255. Luhmann, H.J., Prince, D.A., 1991. Postnatal maturation of the GABAergic system in rat neocortex. J. Neurophysiol. 65, 247 /263. Obrietan, K., van den Pol, A.N., 1997. GABA activity mediating cytosolic Ca2 rises in developing neurons is modulated by cAMPdependent signal transduction. J. Neurosci. 17, 4785 /4799. Owens, D.F., Boyce, L.H., Davis, M.B., Kriegstein, A.R., 1996. Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J. Neurosci. 16, 6414 /6423. Plotkin, M.D., Kaplan, M.R., Peterson, L.N., Gullans, S.R., Hebert, S.C., Delpire, E., 1997. Expression of the Na(/) /K(/) /2Cl/ cotransporter BSC2 in the nervous system. Am. J. Physiol. 272, C173 /C183. Plotkin, M.D., Snyder, E.Y., Hebert, S.C., Delpire, E., 1997. Expression of the Na /K /2Cl cotransporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA’s excitatory role in immature brain. J. Neurobiol. 33, 781 /795. Rivera, C., Voipio, J., Payne, J.A., Ruusuvuori, E., Lahtinen, H., Lamsa, K., Pirvola, U., Saarma, M., Kaila, K., 1999. The K//Cl/ co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397, 251 /255. Sun, D., Murali, S.G., 1999. Na/K/2Cl/ cotransporter in immature cortical neurons: a role in intracellular Cl/ regulation. J. Neurophysiol. 81, 1939 /1948. Sung, K.W., Kirby, M., McDonald, M.P., Lovinger, D.M., Delpire, E., 2000. Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na /K /2Cl cotransporter null mice. J. Neurosci. 20, 7531 /7538. Tsubokawa, H., Oguro, K., Robinson, H.P., Masuzawa, T., Kirino, T., Kawai, N., 1992. Abnormal Ca2 homeostasis before cell death revealed by whole cell recording of ischemic CA1 hippocampal neurons. Neuroscience 49, 807 /817. Valeyev, A.Y., Cruciani, R.A., Lange, G.D., Smallwood, V.S., Barker, J.L., 1993. Cl/ channels are randomly activated by continuous GABA secretion in cultured embryonic rat hippocampal neurons. Neurosci. Lett. 155, 199 /203. Won, M.H., Kang, T.C., Park, S., Jeon, G., Kim, Y., Seo, J.H., Choi, E., Chung, M., Cho, S.S., 2001. 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, 139 /142. Yamada, Y., Fukuda, A., Tanaka, M., Shimano, Y., Nishino, H., Muramatsu, K., Togari, H., Wada, Y., 2001. Optical imaging
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Yan, Y., Dempsey, R.J., Sun, D., 2001. Na/K/Cl/ cotransporter in rat focal cerebral ischemia. J. Cereb. Blood Flow Metab. 21, 711 / 721.
Changes in Na K Cl cotransporter immunoreactivity in the gerbil hippocampus following transient ischemia /
/
Tae-Cheon Kang a, Sung-Jin An a, Seung-Kook Park a, In Koo Hwang a, Dae-Kun Yoon a,b, Hyo-Sun Shin a,c, Moo Ho Won a,* b
a Department of Anatomy, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, Republic of Korea Department of General Surgery, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, Republic of Korea c Shin Hyo Seon’s Plastic and Aesthetic Surgery Clinic, Seoul 136-110, Republic of Korea
Received 15 March 2002; accepted 12 July 2002
Abstract We examined alterations in Na /K /Cl cotransporter 1 (NKCC1) immunoreactivity following ischemia. Twelve hours after ischemia, NKCC1 immunoreactivity in the CA1 region and in the hilar region was significantly diminished. Twenty-four hours after ischemia, NKCC1 immunoreactivity was intensified in these hippocampal regions as well as CA2-3. Two days after ischemia, NKCC1 immunoreactivity in the CA1 and the hilar neurons had disappeared, although in the CA2-3 and the granule cell layer NKCC1 immunoreactivities had recovered to the sham level. This finding suggests that NKCC1 may play an important role in the ischemic neuronal injury induced by excitotoxicity as well as neuronal edema. # 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. Keywords: Na /K /Cl cotransporter; Ischemia; Vulnerability; Up-regulation; Gerbil; Hippocampus
Na /K /Cl cotransporter 1 (NKCC1), which carries Na , K , and Cl simultaneously and electroneutrally (1:1:2) in the same direction, has been well characterized in a wide variety of nervous tissue. In neurons, the NKCC1 regulate intracellular Cl concentration and thereby affect neuronal response to gaminobutyric acid (GABA) (Jang et al., 2001; Kanaka et al., 2001; Plotkin et al., 1997a,b; Sun and Murali, 1999; Sung et al., 2000). Ischemic injury to a neuron is due primarily to the interruption of blood flow, a lack of oxygenation, and a subsequent reoxygenation of the brain (ischemia-reperfusion). The severity of ischemic damage depends on many factors, one of which is the rate of GABA metabolism (Kang et al., 2000, 2002). However, the roles of NKCC1 in the ischemic neuronal injury has not been confirmed; Yan et al. (2001) reported that NKCC1 expression is up-regulated in the infarct zone, and this
* Corresponding author. Tel.: /82-33-240-1614; fax: /82-33-2561614 E-mail address: [email protected] (M.H. Won).
alteration evokes ischemic damage via neuronal edema. In contrast to this paper, Yamada et al. (2001) observed that the function of NKCC activity was transiently reduced by ischemic insult, in vitro. Therefore, in the present study, the immunohistochemical distribution of NKCC1 in the hippocampus of the Mongolian gerbil and its association with different sequelae of ischemic insult was investigated to identify the roles of NKCC1 in the ischemic neuronal injury. Male Mongolian gerbils (Meriones unguiculatus ) weighing 55/70 g were placed under general anesthesia using 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. A midline ventral incision was made in the neck. Both common carotid arteries were isolated, freed of nerve fibers, and occluded using non-traumatic aneurysm clips. Complete interruption of blood flow was confirmed by observing the central artery in the eyeballs using an ophthalmoscope. After 5 min of occlusion, the aneurysm clips were released, and blood flow (reperfusion) was observed directly under the microscope. Sham-operated controls (n /5) were subjected to the same surgical procedures except that the common carotid arteries were not occluded. Body temperature
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 2 ) 0 0 1 3 1 - 1
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T.-C. Kang et al. / Neuroscience Research 44 (2002) 249 /254
Fig. 1. The chronological changes in the NKCC1 immunoreactivity in the hippocampus of the sham (A, C, E) and 12 h postischemic group (B, D, F). In the sham hippocampus (A), NKCC1 immunoreactivity is observed in mostly neurons including CA1 pyramidal neurons (C), granule cells and hilar neurons (E). Twelve hours after ischemia-reperfusion, the intensity of NKCC1 immunoreactivity markedly decreases in the hippocampus except CA2 /3 regions (B). In particular, decline of NKCC1 immunoreactivity is evident in CA1 pyramidal cells (D) and hilar neurons (F). Bar /400 mm (A and B), 50 mm (C /F).
was monitored and maintained at 379/0.5 8C during surgery and during the immediate postoperative period until the animals had recovered fully from anesthesia. At the designated reperfusion time, operated animals and sham animals were killed for immunohistochemistry (Kang et al., 2000; Won et al., 2001).
All animals were anesthetized with pentobarbital sodium, and perfused transcardially with phosphatebuffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) at 30 min (n/ 7), 3 h (n /7), 6 h (n/7), 12 h (n /7), 24 h (n /7), 2 days (n /7), 3 days (n/7), and 4 days (n /7) after
T.-C. Kang et al. / Neuroscience Research 44 (2002) 249 /254
251
Fig. 2. The chronological changes in the NKCC1 immunoreactivity in the hyppocampus of 1 day (A, C, E) and 2 days postischemic group (B, D, F). One day after ischemia, NKCC1 immunoreactivity obviously increases in the CA2 /3 regions (A). These increases are also observed in CA1 pyramidal (C) and hilar neurons (E). Two days after ischemia-reperfusion, the intensity of NKCC1 immunoreactivity markedly decreases in the hippocampus and dentate gyrus (B). NKCC1 immunoreactivity is rarely detected in CA1 pyramidal cells (D) and hilar neurons (F) but that in CA2 / 3 is comparable to sham level. However, NKCC1 immunoreactivity is elevated in the astrocytes in the CA1 region (D). Bar/400 mm (A and B), 50 mm (C /F).
surgery. Brains were removed, and postfixed in the same fixative for 4 h. Brain tissues were cryoprotected by being placed in 30% sucrose overnight. Thereafter, the tissues were frozen and sectioned with a cryostat at 30 mm. Consecutive sections were collected in six-well plates containing PBS. These free-floating sections were first incubated in 10% normal goat serum for 30 min at room temperature, then incubated in mouse antiNKCC1 serum (T4, diluted 1:50, 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), followed by ABC complex (Vector, USA), diluted 1:200 in the same solution as the primary antiserum. Between incubations, the tissues were washed with three times for 10 min with PBS. Sections were visualized with 3,3?-diaminobenzidine (DAB) in 0.1 M Tris buffer and mounted on gelatin-coated slides. NKCC1 immunoreactions were
252
T.-C. Kang et al. / Neuroscience Research 44 (2002) 249 /254
observed under the Axioscope microscope (Carl Zeiss, Germany). In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of primary antibody. Immunohistochemical controls showed in the absence of immunoreactivity in any structures. All experiment procedures were performed in parallel. Sections (15 sections per animal) were viewed through a microscope connected via CCD camera to a PC monitor. At a magnification of 25 /50 /, the region was outlined on the monitor and measured their area. Images of NKCC1 immunoreactivity in the hippocampus of each animal were captured with an Applescanner. The brightness and contrast of each image file were calibrated using ADOBE PHOTOSHOP version 2.4.1, and analyzed using NIH IMAGE 1.59 software. Values of the background staining were subtracted from the immunoreactive intensities. All data obtained from the quantitative data 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 to be statistically significant. In the sham hippocampus, NKCC1 immunoreactivity was observed in the all hippocampal neurons including the granule cells of the dentate gyrus, and in particular NKCC1 immunoreactivity accumulated in the hilar neurons (Fig. 1A, C and E). Thirty minutes after ischemia, NKCC1 immunoreactivities in the CA1 and the hilar regions began to decrease, and at 12 h after the ischemic insult, NKCC1 immunoreactivity had significantly diminished in these regions. However, NKCC1 immunoreactivities in the CA2-3 regions and the granule cell layer were unaltered (Fig. 1B, D and F). Interestingly, 1 day after ischemia, a dramatic intensification of NKCC1 immunoreactivity was observed in the all hippocampal regions including the CA2-3 regions and the granule cell layer. Moreover, NKCC1 immunodensity was highest in the CA2-3 regions (Fig. 2A, C and E). Two days after ischemia, NKCC1 immunoreactivity in the CA1 and the hilar neurons had almost disappeared, although in the CA2-3 and the granule cell layer NKCC1 immunoreactivities had recovered to the sham level. In addition, NKCC1 immunoreactive astrocytes appeared in the CA1 region (Fig. 2B, D and F). This distribution pattern was maintained to 4 days after ischemia (Fig. 3). Our study demonstrates that ischemic insults evoked changes in NKCC1 immunoreactivity in the hippocampus. Twelve hours after ischemic insult, NKCC1 immunoreactivity in the both CA1 and hilar regions was significantly decreased. We postulated that it might be a compensatory reaction to protect neurons from excitotoxicity. This hypothesis is supported by previous study (Yamada et al., 2001) reporting that the NKCC1 activity was effectively suppressed by oxygen /glucose deprivation in rat cortical slices, and this decreased
Fig. 3. The densitimetric analysis of NKCC1 immunoreactivity in the hippocampus after ischemia-reperfusion. Significant differences from the sham, *, P B/0.05; **, P B/0.01.
NKCC1 immunoreactivity delayed the onset of Cl entry, which is able to produce a protective effect. However, in the present study, elevated NKCC1 immunoreactivity was detected in these regions 24 h after ischemia. This elevated NKCC1 immunoreactivity in both CA1 pyramidal cells and hilar neurons suggest that intracellular Cl concentration ([Cl ]i) may play an important role in ‘excitotoxicity’ induced by the elevation of the intracellular Ca2 concentration. In fact, GABA evoked membrane depolarization mediated by Cl efflux in immature animals, which is the result of a high [Cl ]i in the central nervous system during early postnatal life (Backus et al., 1998; Chen et al., 1996; Luhmann and Prince, 1991; Owens et al., 1996; Plotkin et al., 1997a,b; Rivera et al., 1999; Sun and Murali, 1999; Valeyev et al., 1993). This depolarization activates voltage-dependant Ca2 channels and reduces the voltage-dependent Mg2 block of NMDA channels, resulting in Ca2 influx (Flint et al., 1998; Leinekugel et al., 1997; Obrietan and van den Pol, 1997). Thus, abnormal accumulation of Ca2 accompanied by a depolarization was evoked by input fiber stimulation in the hippocampal CA1 region of gerbils 2/3 days after transient whole brain ischemia (Tsubokawa et al., 1992). Furthermore, during ischemic stage, GABA aggravates excitotoxicity by accumulation of intracellular Ca2, in the condition of high [Cl ]i (Fukuda et al., 1998). This hypothesis is also supported by previous studies, which demonstrated that GABA concentration in the CA1 area after ischemia-reperfusion was elevated (Gabriel et al., 1998; Kang et al., 2000, 2002). Interestingly, we also found that NKCC1 immunoreactivity was enhanced in CA2-3 regions, which is resistant to ischemic insult. Though not exactly an explanation of this phenomenon, it is plausible that NKCC1 in the CA2-3 regions may trigger neuronal injury, although it could not lead to cell death. Twelve to 24 h after ischemic insult, CA2-3 neurons were swollen and the position of nuclei was eccentric (reactive chromatolytic cell change; Kirino, 1982). However, in
T.-C. Kang et al. / Neuroscience Research 44 (2002) 249 /254
this time point, GABA concentration in these regions was unaltered (Kang et al., 2000, 2002). Therefore, in the CA2-3 regions, unlike CA1 region, NKCC1 may trigger or aggravate neuronal injury induced by edema (Yan et al., 2001). Likewise in the present study, elevated NKCC1 immunoreactivity without altered GABA concentration in these areas provided some morphological evidence as to why the CA2-3 regions are resistant to ischemic insult. Two days after ischemia, NKCC1 immunoreactivity in the CA1 and the hilar neurons had almost disappeared, although in the CA2 /3 and the granule cell layer NKCC1 immunoreactivities had recovered to the sham level. At this time point, the nuclear chromatin of the pyramidal cells in the CA1 regions appeared clumped, and this phenomenon indicated irreversible ischemic damage in the perikarya (Kirino, 1982). Therefore, the disappearance of NKCC1 immunoreactivity in the CA1 may demonstrate irreversible ischemic damage. In conclusion, we have demonstrated the cellular and regional distribution pattern of NKCC1 according to reperfusion time in the gerbil ischemic hippocampus, and suggest that altered NKCC1 immunoreactivity may be related to ischemic neuronal damage.
Acknowledgements This work was supported by a grant from the Korea Ministry of Science and Technology (Critical Technology 21 [M1-0016-00-0024]), and this research was also supported by the Hallym Academy of Sciences at Hallym University, Korea, 2002-1.
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