This study was performed in accordance with the approved animal use guidelines of the Catholic University of Daegu. A modification of Levine preparation was used as a model for perinatal hypoxic-ischemic brain injury as previously described.17 Seven-day-old Sprague-Dawley (SD) rat pups were prepared for surgery. The midline of the neck was incised at the longitudinal plane under ether. The left carotid artery (LCA) was permanently doubly ligated with 5-0 surgical silk. Total time of surgery never exceeded 5 min. Following a 1 hour period of recovery with the dam and feeding, the pups were placed in plastic chambers, which were submerged in a 37℃ water bath and exposed to hypoxia (mixture of 8% O₂ and 92% N₂) for 2 hours. After this hypoxic exposure, the pups were returned to their dams for the indicated time. SYM 2081, purchased from Tocris Cookson (Bristol, UK), was prepared in phosphate-buffered saline (PBS) and injected intraperitoneally with dose of 10 mg/kg at 30 minutes before hypoxic exposure.

The animals were divided into five groups. Normoxia (N, n=4) group was subjected to neither ligation nor hypoxia. Hypoxia (H, n=4) group was exposed to hypoxia without operation. Hypoxia with Sham-operation (HS, n=4) group was subjected only to open-close surgery without LCA ligation. Hypoxia with LCA ligation-operation (HO, n=6) group was subjected to hypoxia without SYM 2081 injection. HO treated with SYM 2081 (HM, n=6) group was injected with SYM 2081. Pups from each litter were randomly assigned and marked to an N, H, HS, HO, and HM groups. Pups were killed at 7 days after hypoxic exposure and then the brains were extracted. The percentages of hemispheric areas were measured using a densitometer (Multi Gauge Software, Fuji Photofilm). The area ratio of left to right hemispheres was calculated.

Cultures of dissociated cortical neuronal cells were prepared from SD rat embryos pregnant for 19 days using methods similar to those previously described.18 The fetal pups were washed in 100% ethanol and Hanks' balanced salt solution (HBSS) (GibcoBRL, Grand Island, NY, USA). Cerebral cortices were removed and dissected at 37℃ HBSS containing 1 mM sodium pyruvate and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), briefly. The dissected brain cortical tissues were then placed in 2 mL trypsin and incubated at 37℃ water bath for 1 min. After washing with 10 mL HBSS, the cell suspension was centrifuged at 1,000 rpm at 25℃ for 5 min and pellets were washed with HBSS (without phenol red). Cells were plated at approximately 2×10⁶ cells/mm² in each dish. Cells were cultured in Neurobasal media (GibcoBRL, Grand Island, NY, USA) (100 mL neurobasal, 2% B27 supplement, 0.25 mL glutamax I, 0.1 mL 25 mM glutamate, 0.1 mL 25 mM 2-mercaptoethanol) in a CO₂ chamber. A fifth of the medium was replaced with fresh feeding Neurobasal media (GibcoBRL, Grand Island, NY, USA) (100 mL neurobasal, 2% B27 supplement, 0.25 mL glutamax I) every 3 days. The cortical neuronal cells were observed using inverted microscope (TS 100-F, Nikon instruments Inc., NY, USA) under high magnification (×200).

The cultured cells were divided into three groups: NC, normoxia; HC, hypoxia; HMC, hypoxia treated with SYM2081 (10 µg/mL). The NC group was prepared in 5% CO₂ incubators while the other groups in 1% O₂ incubators (94% N2, 5% CO₂) for 16 hours.

Brain samples were rapidly removed from the left cerebral hemispheres. Tissues and cultured cells were homogenized in TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) before a 5 min incubation at room temperature and chloroform treatment (200 µL) for dissociation of nucleoprotein complexes. The tubes were shaken by hand for 15 seconds and they were incubated at room temperature for 2 min. The upper aqueous (RNA) phase was collected following centrifugation (12,00x g for 15 min at 4℃). The aqueous phase was then collected into a fresh tube then precipitated into a gel-like pellet with 500 µL isopropyl alcohol by incubating for 10 min at room temperature and centrifuging 12,000x g for 15 min at 4℃. After removal of the supernatant, the RNA pellet was washed once with 1 ml 75% ethanol by centrifugation at 7,500x g for 5 min at 4℃. The RNA pellet was dried by air for 10 min before redissolving the RNA in 100 µL RNase-free water and incubating for 10 min at 60℃. The amount and purity of extracted RNA was quantitated by GeneQuant 1,300 spectrophotometer (GeneQuant™proRNA/DNAcalculator, GE Healthcare, Milwaukee, WI, USA). The RNA was then stored at -70℃ before further processing. For real-time polymerase chain reaction (PCR, for reverse transcription), total RNA (1 µg) was reverse transcribed for 1 hour at 37℃ in a reaction mixture containing 20 U RNase inhibitor (Promega, Madison, WI, USA), 1 mM dNTP (Promega, Madison, WI, USA), 0.5 ng Oligo (dT) 15 primer (Promega, Madison, WI, USA), 1x RT buffer and 200 U M-MLV reverse transcriptase (Promega, Madison, WI, USA). The reaction mixture was then incubated at 95℃ for 5 minutes to stop the reaction. The cDNA was then stored at -20℃ before further processing.

Real-time PCR was carried out in a 20 µL volume with 10 µL of kit-supplied iQ™ SYBR Green Supermix (Bio-rad Laboratories, Hercules, CA, USA), 1 µL (2 pmol) of each primer, 7 µL RNase-free water, 1 µL cDNA. Amplification was performed in 48 well PCR plates (Mini OpticonTM Real-Time PCR System, Bio-rad Laboratories, Hercules, CA, USA) with initial denaturation for 5 min at 95℃, followed by 40 cycles of denaturation for 30 s at 95℃, annealing 30 s at temperature indicated in Table 1, and extension for 30 s at 72℃. The final extension was for 10 min at 72℃. Real-time PCR data were analyzed with LightCycler software (Bio-rad Laboratories, Hercules, CA, USA). The experiment was repeated six times in the real-time PCRs.

The data were analyzed using the SPSS version 22.0 statistical analysis package. Examined data were assessed using the Student's t-test, general linear model, and analysis of variance. In each test, the data were expressed as the mean±SD, and P<0.05 was accepted as statistically significant.

The area of the left hemisphere expressed as a percentage of that of the right hemisphere (Fig. 1). The area of left hemisphere showed similar to that of the right hemisphere in the N, H, and HS group. There was a reduction in the area of left hemisphere underwent LCA ligation compared to the contralateral, unligated side in HO group. On the other hand, under the same condition, administration of SYM 2081 before a hypoxic insult result in similar area percentages between both hemispheres in the HM group.

The cortical neuronal cells cultured in the normoxic environment (Fig. 2A) appeared normal, whereas those exposed in the hypoxic environment (Fig. 2B) appeared distinct morphological changes such as cell rounding and detachment from the growth surface. The affected cells treated with SYM 2081 in the HMc group (Fig. 2C) appeared similar to those in the Nc group.

The expressions of GluK1, GluK3, GluK4 and GluK5 were increased in the H group compared to those in the N group whereas the expression of GluK2 was not increased in the H group (Fig. 3). The expressions of GluK1, GluK3, GluK4 and GluK5 were decreased in the HO group compared to those in the H group. These decreased expressions of GluK3, GluK4 and GluK5 were inhibited in the HM group. Interestingly, the expression of GluK1 was not increased in the HM group, but rather decreased compared to that in the HO group (Fig. 3A). Unlike other KARs, the expression of GluK2 was decreased in the H group compared to that in the N group and increased in the HO group compared to that in the H group (Fig. 3B). In the HM group, the expression of GluK2 was increased compared to that in the HO group.

The expressions of all KARs except for GluK2 were decreased in the H_c group when compared to those in the N_c group, whereas increased in the HMc group compared to those in the Hc group (P<0.05) (Fig. 4). The expression of GluK2 was not decreased in the Hc group compared to that in the Nc group (Fig. 4B).