C57BL/6 mice (male, 8–10 w, 20–25 g) were housed under a 12 h light/dark cycle with free access to food and water. All experiments were performed in accordance with approved animal protocols and guidelines established by the Ajou University School of Medicine Ethics Review Committee (2014-0029, AMC119).

Mice were anesthetized with intraperitoneal injections of tribromoethanol (250 mg/kg; Sigma-Aldrich, St. Louis, MO, USA), and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). Brain injury was produced by unilateral administration of ATP (500 mM, 0.8 μl; Sigma-Aldrich), a component of DAMPs produced in pathological conditions [22-25], into the striatum (from bregma: AP, +1.0 mm; ML, −1.9 mm; DV, −3.2 mm), according to stereotaxic coordinates in The Atlas of the Mouse Brain (Paxinos and Franklin, second edition), as previously described [26]. ATP was injected at a rate of 0.2 μl/min using a Hamilton syringe equipped with a 33-gauge needle attached to a syringe pump (KD Scientific, New Hope, PA, USA); the needle was left in place for an additional 10 min prior to removal to prevent leakage through the needle track.

Mice at postnatal day 7 (P7) were anesthetized, and cortical slices (400 μm thick) were prepared using a McIlwain tissue chopper (Mickle Laboratory Engineering, Goose Green, UK). Slices are immediately collected and incubated in Accumax (Millipore, Bedford, MA, USA) for 15 min at 37°C. Lysed tissues are homogenized into a single-cell suspension in culture medium by trituration with a flame-polished Pasteur pipette. Cells were resuspended in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12; WelGene, Daegu, Korea) containing 0.9 M sucrose and centrifuged to remove myelin (111 ×g for 10 min). Cells were grown in a 100-mm Petri dish with NSC growth medium (DMEM/F12) containing N-2, B27 supplement without vitamin A (Gibco-Invitrogen, Carlsbad, CA, USA) together with EGF and bFGF (20 ng/ml each; BD Bioscience, San Jose, CA, USA). For Kir4.1-inhibition experiments, cells were treated with 1 μg/ml BaCl₂ (Sigma-Aldrich) after 3 h. The size of neurospheres was measured after 1, 3, and 5 d. For differentiation, cells were collected and incubated in Accumax for 5 min. After centrifugation (180 ×g, 3 min), dissociated cells were seeded on plates coated with 0.2 mg/ml poly-L-ornithine and 1 μg/ml fibronectin (Sigma-Aldrich) in the absence of growth factors.

Forebrains from embryonic day 13.5 (E13.5) mice were freed of meninges and gently triturated several times in culture medium using a flame-polished Pasteur pipette. Cells from a brain were plated in a 100-mm Petri dish and cultured in DMEM/F12 medium supplemented with N-2 and B27 supplement without vitamin A. EGF and bFGF (20 ng/ml each) were added every 2 d. For differentiation, dissociated cells were seeded on plates coated with 0.2 mg/ml poly-L-ornithine and 1 μg/ml fibronectin in the absence of growth factors. Cells were treated with 1 μg/ml BaCl₂ 3 h after seeding.

Cells and mouse brains were lysed on ice in RIPA buffer (10 mM sodium phosphate buffer pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% sodium-deoxycholate) containing phosphate/protease inhibitor cocktails (GenDEPOT, Barker, TX, USA). Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and identified by incubating membranes with specific primary antibodies (Table 1) overnight at 4°C. After washing with phosphate-buffered saline (PBS), membranes were incubated with peroxidase-conjugated secondary antibodies and visualized using an enhanced chemiluminescence system (Daeil Lab Inc., Seoul, Korea). Band intensities were normalized to β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using Image J.

Mice were anesthetized and transcardially perfused first with saline solution containing 0.5% sodium nitrate and heparin (10 unit/ml) and then with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were obtained and post-fixed overnight at 4°C with 4% paraformaldehyde. Fixed brains were stored at 4°C in a 30% sucrose solution until they sank. Series of coronal sections (30 μm thick) were obtained with a cryostat (Leica, Wetzlar, Germany) and used for immunohistochemistry.

For 3,3’-diaminobenzidine staining, brain sections were rinsed three times with PBS, treated with 3% H₂O₂ for 5 min, and rinsed with PBS containing 0.2% Triton X-100 (PBST). After blocking non-specific binding with 1% bovine serum albumen (BSA) in PBST, sections were incubated overnight at room temperature with primary antibodies (Table 1). Sections were rinsed with PBST and incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA), then mounted on gelatin-coated slides and examined under a bright field microscope (BX51; Olympus Optical, Tokyo, Japan). For immunofluorescence staining, brain sections were fixed with 4% paraformaldehyde at room temperature for 20 min, washed with PBS, and incubated in in PBS containing 1% BSA and 0.1% Triton X-100 for 30 min. Sections were then incubated overnight with primary antibodies (Table 1) at 4°C, washed with PBS, and incubated with fluorescence-conjugated secondary antibodies (Gibco-Invitrogen) for 2 h. Finally, sections were washed, mounted using a mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and examined under a ZEISS LSM 800 confocal laser-scanning microscope (Carl Zeiss, Göttingen, Germany).

Total RNA was isolated from primary astrocytes using easy-BLUE reagent (iNtRON Biotechnology, Seoul, Korea). cDNA was synthesized from 1 μg of total RNA using a cDNA synthesis kit (iNtRON Biotechnology) under the following incubation conditions: denaturation at 75°C for 5 min, cDNA synthesis at 42°C for 60 min, and termination at 70°C for 5 min. Quantitative polymerase chain reaction (qPCR) was performed using 2x KAPA SYBR Fast Master Mix (Kapa Biosystems, Cape Town, South Africa) and an RG-6000 real-time amplification instrument (Corbett Research, Sydney, Australia). Primers for qPCR are listed in Table 2. The cycle threshold (Ct) for a given gene transcript was normalized to the average Ct for transcripts of the housekeeping gene, GAPDH. Relative quantitation of normalized transcript abundance was determined using the comparative Ct (∆∆Ct) method.

Statistical analysis was performed using the GraphPad Prism software (Prism; San Diego, CA, USA). Data were analyzed using two tailed, unpaired t-test for comparison of two groups. Two-way analysis of variance (ANOVA) was applied for comparison of multiple groups. The p-values are also indicated in the figure legends. All values are means ± SEMs of at least three independent experiments.

Both Western blotting and immunostaining showed that levels of Kir4.1 were quite low in the intact brain (Fig. 1A, B). To examine the expression of Kir4.1 in the injured brain, we induced brain injury by injecting ATP, a component of damage-associated molecular patterns (DAMP), into the striatum of mouse brains as previously reported [22,27,28]. At 3 and 7 d post-injury, levels of Kir4.1 protein as well as those of GFAP, a marker of reactive astrocytes, were significantly increased (Fig. 1A). Immunostaining also showed increased Kir4.1 expression near the damage core (*) at 3 and 7 d, and decreased at 14 d (Fig. 1B). We further found that GFAP-positive cells in the penumbra region adjacent to the damage core highly expressed Kir4.1 at 3 d (Fig. 1Ca, arrows) and 7 d (Fig. 1Cc, arrows) post-injury, with Kir4.1 staining intensity increasing with increased proximity to the damage core (Fig. 1Ca vs. Cb; Fig. 1Cc vs. Cd).

In the ATP-injured brain, Kir4.1 showed colocalization with Nestin (Fig. 1Ca–d), a marker of stem cells [29,30]. Kir4.1/GFAP/Nestin immunostaining showed that GFAP and Nestin exhibited the same pattern of expression following damage as that shown for Kir4.1 in Fig. 1B. GFAP and Nestin levels, like those of Kir4.1, were quite low or undetectable in the intact brain, as shown by Western blotting (Fig. 1C vs. Fig. 1A). Interestingly, Nestin was detectable in astrocytes that highly expressed Kir4.1 (Fig. 1C). Sox2, another stem cell marker [20], was also detectable at 3 and 7 d post injury at enhanced levels compared with its low expression in the intact brain (Fig. 1D vs. Fig. 1A). In the penumbra region adjacent to the damage core, Sox2 expression was highly increased in Kir4.1- and GFAP-positive cells (Fig. 1Da–d, arrows). In addition, there were more Sox2-positive cells in penumbra regions closer to the core: at 3 d, about 80% of Sox2/GFAP-double positive cells were Kir4.1-positive in region a, but only ~40% of such cells were Kir4.1-positive in region b (Fig. 1Da vs. Fig. 1Db). These results show that expression of both Kir4.1 and the stem cell markers Nestin and Sox2 are increased in reactive astrocytes near the damage core. Interestingly, Kir4.1 channels, which mediate K⁺ efflux out of cells and thereby hyperpolarize the membrane potential [9,10], are reported to be involved in regulating the proliferation/differentiation of cells [9,10,31,32], consistent with reports that depolarization favors cell proliferation [33]. Collectively, these observations raise the question of whether Kir4.1 is involved in the gain of stemness and/or proliferation of astrocytes in the injured brain.

Next, we examined whether there is a linkage between Kir4.1 expression and the acquisition of stemness, demonstrated by the expression of Nestin and Sox2. For this, stem cells were cultured from E13.5 or P7 mouse brains.

Stem cells from E13.5 mouse brains cultured in the presence of EGF (20 ng/ml) and FGF (20 ng/ml) formed neurospheres after 5 d in culture (Fig. 2A). During neurosphere formation, mRNA levels of Sox2, Nestin, GFAP, and Kir4.1 increased, whereas mRNA expression of β-tubulin III (Tubb3) was decreased (Fig. 2A). To examine the role of Kir4.1 channels in neurosphere formation, we cultured neurospheres in the presence of the Kir4.1 blocker, BaCl₂ (Fig. 2B). EGF/FGF increased sphere size at day 5, but BaCl₂ had little effect on the size of spheres with or without EGF/FGF (Fig. 2B). BaCl₂ likewise had little effect on Sox2, Nestin, Tubb3, or GFAP mRNA expression during neurosphere formation (Fig. 2C).

Next, we examined the effect of Kir4.1 inhibition on acquisition of stemness and expression of Nestin and Sox2 in stem cells prepared from P7 mouse brains. In the presence of EGF (20 ng/ml)/FGF (20 ng/ml), these stem cells, like those from E13.5 mouse brains, formed neurospheres after 5 d in culture (Fig. 3A). During neurosphere formation, mRNA levels of Sox2 and Nestin increased, whereas those of GFAP and Tubb3 decreased, as expected (Fig. 3A). Interestingly, in contrast to results obtained using stem cells from E13.5 mouse brains, mRNA levels of Kir4.1 decreased during formation of neurospheres from stem cells obtained from P7 mouse brains (Fig. 3A). To examine the role of Kir4.1 in neurosphere formation, we cultured neurospheres in the presence of BaCl₂, with and without EGF/FGF (Fig. 3B). In the presence of EGF/FGF, BaCl₂ significantly increased the size of spheres at day 5, an effect that became more obvious at day 8 (Fig. 3B). BaCl₂ also caused a concentration-dependent increase in sphere size in the presence of EGF/FGF (Fig. 3C). Even in the absence of EGF/FGF, BaCl₂ alone slightly increased sphere size (Fig. 3B). These loss-of-function results suggest that, under normal conditions, Kir4.1 may negatively regulate the proliferation of cells during neurosphere formation.

Next, we examined the effect of Kir4.1 inhibition on Sox2 and Nestin expression during neurosphere formation. BaCl₂ alone significantly elevated Sox2 expression at both mRNA (Fig. 3D) and protein levels (Fig. 3E), and potentiated EGF/FGF-induced Sox2 protein expression (Fig. 3E). Interestingly, the EGF/FGEF-induced increase in Sox2 mRNA was not greater than that induced by BaCl₂ alone (Fig. 3D). In contrast, BaCl₂ alone slightly reduced Nestin mRNA levels and had little effect on EGF/FGF-induced Nestin mRNA expression (Fig. 3D). BaCl₂ significantly enhanced both mRNA and protein levels of GFAP (Fig. 3D, E) and had little effect on the EGF/FGF-induced reduction in GFAP mRNA and protein levels (Fig. 3D, E). Taken together, these results suggest that blocking Kir4.1 in P7 stem cells favors neurosphere formation and expression of Sox2 and GFAP, but has little effect on Nestin expression.

Finally, we examined the effect of Kir4.1 on the differentiation of stem cells. For this, we induced differentiation of stem cells prepared from E13.5 mouse brains by withdrawal of EGF and FGF in the culture medium, and then measured levels of Sox2, Nestin, GFAP, and Tubb3. During differentiation of neural stem cells (NSCs), mRNA and/or protein levels of Sox2 and Nestin were decreased, whereas those of GFAP and Tubb3 increased (Fig. 4A, B). Kir4.1 expression was significantly increased at both mRNA and protein levels at 1 d; its expression decreased at 3 and 5 d, but was still maintained above basal levels (Fig. 4A, B). Treatment of NSCs with BaCl₂ during differentiation caused little change in expression levels of Sox2, Nestin, GFAP or Tubb3 (Fig. 4C). Collectively, these results suggest that Kir4.1 does not regulate differentiation (i.e., loss of stemness) of stem cells.