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Abstract
Objective
Several studies have demonstrated the neuroprotective effects of (+)-MK-801 hydrogen maleate (dizocilpine), in various animal models of hypoxic-ischemic (HI) brain injury. However limited data are available on the neonatal model of HI brain injury. The aim of the present study was to investigate the effects of dizocilpine and its mechanisms associated with NMDARs expression in neonatal rat model of HI brain injury.
Methods
In in vivo model, 7d-old rat pups underwent permanent unilateral carotid ligation. The animals were divided into six groups: N, normoxia; H, hypoxia without operation; HS, hypoxia with Sham operation; HO, hypoxia with operation; HV, HO treated with vehicle; HD, HO treated with dizocilpine. Dizocilpine (10 mg/kg) was administered intracerebrally to the rats 30 min before HI brain injury. Rat pups were exposed to hypoxia by placing them for 2 hours in hypoxic incubator (92% N2, 8% O2). In in vitro model, embryonic cortical neuronal cell cultures (from SD rats of embryonic days of 18) were done. The normoxia (N) group was prepared in 5% CO2 incubators. The hypoxia (H), and hypoxia treated with dizocilpine (HD) groups were placed in 1% O2 incubators (94% N2, 5% CO2) for 16 hours. In order to estimation of cell viability and growth, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was done. The degree of neuronal death was evaluated by morphometric method and the protein expression of each NMDARs was quantified by Real Time-PCR and Western blot.
Results
Both in thein vitro andin vivo models, the expressions of NMDAR subunits were lower in the hypoxia group than in the normoxia group, whereas they increased in the hypoxia treated with dizocilpine group compared to the hypoxia group. In vitro model, however, the expressions of NR1, NR2A mRNAs decreased in the H group when compared to the N group, whereas they increased a little in the HD group when compared to the H group.
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Fig. 1.
Western blots of NR2A (A; N, 100±2.1; H, 107.5±2.1; HS, 103.2±2.1; HO, 85.1±1.7; HV, 90.5±1.8; HD, 98.5±1.9) and NR2B (B; N, 100±2.0; H, 101.6±2.0; HS, 101.2±2.0; HO, 87.9±1.8; HV, 82.3±1.6; HD, 102.6± 2.1) in the neonatal hypoxic-ischemic brain injury (in vivo) (n=4). Dizocilpine was administered at 10 mg/kg. N, normoxia; H, hypoxia without operation; HS, hypoxia with Sham operation; HO, hypoxia with operation; HV, HO treated with vehicle; HD, HO treated with dizocilpine; *P<0.05, statistically significant vs. HO.
Fig. 2.
Real-time PCRs of NR1 (A; N, 100±3.0; H, 125.7±3.8; HS, 104.9±3.1; HO, 36.1±1.1; HV, 62.9±1.8; HD, 119.7±3.4), NR2A (B; N, 100±3.1; H, 137.6±4.1; HS, 123.1±3.6; HO, 43.8±1.3; HV, 41.8±1.3; HD, 85.9±2.6), NR2B (C; N, 100±3.3; H, 127.5±3.8; HS, 100.7±3.0; HO, 66.9±2.0; HV, 58.6±1.8; HD, 80.7±2.4), NR2C (D; N, 100±3.1; H, 112.5±3.4; HS, 89.5±2.6; HO, 42.3±1.3; HV, 47.9±1.4; HD, 56.6±1.7), and NR2D (E; N, 100±3.1; H, 104.2±3.1; HS, 91.4±2.7; HO, 46.0±1.3; HV, 47.9±1.4; HD, 81.8±2.4) mRNAs in the neonatal hypoxic-ischemic brain injury (in vivo) (n=6). Dizocilpine was administered at 10 mg/kg. N, normoxia; H, hypoxia without operation; HS, hypoxia with Sham operation; HO, hypoxia with operation; HV, HO treated with vehicle; HD, HO treated with dizocilpine; *P<0.05, statistically significant vs. HO.
Fig. 3.
Morphologic changes in the embryonic cortical neuronal cell culture of rat (in vitro). A, normoxia; B, hypoxia; C, hypoxia treated with dizocilpine.
Fig. 4.
Cell viability was measured by the 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Dizocilpine was administered at 10 μg/mL. N, normoxia; H, hypoxia; HD, hypoxia treated with dizocilpine; *P<0.05, statistically significant vs. H.
Fig. 5.
Western blots (A) of NR2A (B; N, 100±2.1; H, 52.5±1.5; HD, 67.1±1.6) and NR2B (C; N, 100± 2.1; H, 89.4±1.8; HD, 100.5±2.0) in the embryonic cortical neuronal cell culture (in vitro) (n=4). Dizocilpine was administered at 10 μg/mL. N, normoxia; H, hypoxia; HD, hypoxia treated with dizocilpine; *P<0.05, statistically significant vs. H.
Fig. 6.
Real-time PCRs of NR1 (A; N, 100±4.1; H, 34.0±1.4; HD, 39.5±1.6), NR2A (B; N, 100±4.0; H, 28.7±1.1; HD, 34.2±1.4), NR2B (C; N, 100±4.0; H, 47.5±1.9; HD, 56.1±2.2), NR2C (D; N, 100±4.2; H, 19.8±0.8; HD, 36.1±1.4), and NR2D (E; N, 100±4.1; H, 56.3±2.3; HD, 117.7±4.7) in the embryonic cortical neuronal cell culture (in vitro) (n=6). Dizocilpine was administered at 10 μg/mL. N, normoxia; H, hypoxia; HD, hypoxia treated with dizocilpine; *P<0.05, statistically significant vs. H.
Table 1.
Primer Pairs and Annealing Temperature for Realtime PCR
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