The effect of ketogenic diet on Phosphorylated CREB/CREB in the hippocampus after prolonged seizure in immature rat brain

Abstract

[한글]

목적: 미성숙 백서에서 지속적인 간질발작 유발한 후에 해마의 Phosphorylated CREB/CREB을 측정하여 케톤생성 식이요법의 인지기능에 대한 효과를 알아보고자 하였다.

대상 및 방법: Lithium-pilocarpine(Li-PC) 모델을 실험에 이용하였다. 생후 20일(P20)에 24마리에 Pilocarpine 피하 주사를 하여 지속적인 간질발작을 일으켰으며, 7마리(29%)는 죽었다. 살아남은 17마리를 정상식이(SE-ND, n=8)와 케톤생성식이(SE-KD, n=9) 2개 군으로 나누었다. Pilocarpine 대신 생리식염수를 준 대조군 16마리도 정상식이(Cont-ND, n=8)와 케톤생성식이 (Cont-KD, n=8)로 나누었다. 생후 51일(P51)에서 55일(P55) 동안 Morris water maze test를 시행하였다. serum ß-OHB 을 spectrophotometry를 이용하여 측정하였다. 해마는 적출해서 phosphorylated CREBSer-133를 분석하였다.

결과: Morris water maze 검사에서 날이 갈수록 모든 군에서 의미 있는 향상을 보였다. 첫날에는 Cont-ND가 다른 세 군에 비해 플랫폼을 찾는 시간이 유의하게 짧았으며, 세 군 간에는 차이가 없었다. 그러나 4일째에는 Cont-ND와 SE-ND만이 유의한 차이를 보였다. Probe test에서 Cont-ND, Cont-KD, SE-KD 세 군은 SE-ND에 비해 플랫폼이 있던 사분구획에서 유영하는 시간이 유의하게 길었다. 혈청 β-OH butyrate(β-OHB) level은 간질과 상관없이 정상군보다 케톤군에서 유의하게 높았다. CREBSer-133 phosphorylation은 prolonged seizure를 한 경우와 케톤생성 식이를 한 경우에 감소를 보였다.

결론: 미성숙 백서에서 지속적인 간질발작은 visuospatial learning and memory를 악화시켰으며, 케톤생성 식이로 Phosphorylated CREB/CREB은 감소하였으나 learning and memory function은 향상되었다.

[영문]Purpose: The aim of this study was to investigate the relationship between cognitive effects of ketogenic diet (KD) and phosphorylated CREB/CREB in the hippocampus after lithium-pilocarpine (Li-PC) induced prolonged seizure in immature rat brain.

Materials and Methods: Spraque-Dawley rats were subjected to Li-PC induced seizure or normal saline injection. Pilocarpine injected rats (n=24) progressed to prolonged seizure and seven rats (29%) died. On P21, the rats were assigned to four groups: prolonged seizure with normal diet (SE-ND, n=8) or KD (SE-KD, n=9), no seizure with normal diet (Cont-ND, n=8) or KD (Cont-KD, n=8). Spatial learning and memory test using the Morris water maze was performed from P51 to P55. Ketosis was assayed by spectrophotometrically measuring the serum β-OH butyrate (ß-OHB) levels. The hippocampi of sacrificed rats were treated to detect the phosphorylated CREBSer-133.

Results: In the water maze test, each group significantly had reduced escape latency during four days. At the testing day 1, Cont-ND group showed shorter latency than the other three groups, but there was no statistical differences among the others. At the day 4, however, Cont-ND group performed with shorter latency in only comparison to SE-ND group. The probe test showed that the Cont-ND, Cont-KD, and SE-KD groups spent more time in the target quadrant in comparison to SE-ND group. Serum β-OHB levels were significantly greater in KD group than in normal diet. The total CREB in hippocampus was increased by prolonged seizure (SE-ND, SE-KD) and ketogenic diet (Cont-KD), but phosphorylation of CREB was decreased in the Cont-KD, SE-ND, and SE-KD groups.

Conclusion: The Li-PC induced prolonged seizure to immature rat deteriorated the spatial learning and memory. Ketogenic diet on prolonged seizure in immature brain improved the spatial learning and memory, even though decreased the phosphorylated CREB/CREB.