| The activation of N-Methyl-D-Aspartate(NMDA) receptor plays important roles in the pathogenesis of hypoxic ischemic brain damage (HIBD). The objective of this study is to explore the mechanisms of NMDA receptor 1(NR1) phosphorylation in HIBD in neonatal Sprague-Dawley(SD) rat.Method: Postnatal Day 7 Sprague-Dawley(SD) rats were randomly divided into normal control, HIBD(right carotid artery ligation plus hypoxia exposure was applied in making HIBD animal model) and NMDA microinjection groups. 2, 3, 5-triphenyltetrazolium chloride(TTC) staining method was used to assess the gross cerebral damage. The expression of NR1 and phosphorylated NR1 at Serine 897 site (phospho-NR1 S897) was studied by both fluorescent immunohistochemical(IHC) staining and western blot method. Flow cytometry method was also adapted to examine the change of mitochondrial membrane potential(Δψm) of the brain cells for the understanding of mitochondrial dysfunction. The [Ca2+]i of each group was measured by Hitacchi fluorescence spectrophometer. SPSS11.1 software was used for the statistical analysis. P＜0.05 was considered to be statistically significant.Results:(1) Normal control group: TTC staining of normal brain slice was in red color. IHC and western blot showed that the expression of NR1 in the cerebral cortex was negative, while the expression of phospho-NR1 S897 was very high. TheΔψm and [Ca2+]i of the fight hemisphere was(18.21±1.26) MFL and (559.24±52.36) nmol/L, respectively. TheΔψm and [Ca2+]i of both hemispheres were almost equal to each other.(2) HIBD group: At 12h after TTC staining, some of the brain tissue starts to turn white or pink indicating brain damage. At 24h and 48h after HI, the damage was the most severe, and the ratio of damage volume to whole brain volume was(0.172±0.012) and (0.183±0.017), respectively (each P＜0.05). IHC staining showed that the number of NR1 positive cells increased, while that of phospho-NR1 S897 positive cells decreased after HI. The location of NR1 expression decrease and phospho-NR1 S897 expression increase was correlated. At 3, 6, 12 and 24h after HI, the expression of phospho-NR1 S897 was progressively decreased, and the number of the positive cells were(64±19),(48±16),(38±15)(P＜0.05) and(21±8)(P＜0.05), respectively. Western blot results showed the similar progressive decrease of phospho-NR1 S897 expression, in which the OD at 6, 12 and 24h after HI was reduced 22.2％, 65.7％(P＜0.05) and 94.1％(P＜0.05) comparing to the normal control.Δψm of the HI damaged hemisphere had "reduce-recovery-second reduce-second recovery-third reduce" change. Three reducing was observed at 0, 4 and 24h after HI with the ispsi-lateralΔψm ratio to be(0.80±0.17), (0.70±0.22) and (0.71±0.13), respectively(each P＜0.05). The change was the most apparent at cerebral cortex. Ipsilateral hemisphere[Ca2+]i为at Oh after HI was(789.64±55.78) nmol/L(P＜0.05).(3) NMDA microinjection group: TTC staining at 0-4h after injection was normal. IHC, [Ca2+]i and Western Blot results were similar to HIBD group. IHC results showed that the number of NR1 positive cells increased, while the expression of phospho-NR1 S897 was so significantly reduced that there was almost no phospho-NR1 S897 positive cell. TheΔψm was decreased progressively. At 15min, 30min, 1h and 2h after injection, the ipsi-lateralΔψm ratio was(0.84±0.65), (0.66±0.16), (0.66±0.21) and (0.61±0.36), respectively (each P＜0.05).[Ca2+]i of the ipsilateral hemisphere was(667.52±47.78) nmol/L (P＜0.05). Western blot study showed that at hippocampus and thalamus, the expression of NR1 was significantly increased, and the expression of Phospho-NR1 S897 was significantly decreased.Conclusins:(1) The phosphorylation of NR1 at S897 site was essential to the normal function of neural cells in the cortex of neonatal SD rats. HI damage down-regulated the expression of phospho-NR1 S897 causing dysfunction of the neural cells.(2) NMDA microinjection induced similar change of the brain to HI damage, indicating, the importance of " "HI—NMDA—phospho-NR1 S897dephosphorylation—cell damage" pathway after HIBD.(3) Further study of the modulator factors in the phosphorylation of NR1 may supply novel lab evidences for reducing HI induced induced brain damage.
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