The effect of acetaldehyde dehydrogenase activator, alda-1®, on the ethanol-induced brain damage in a rat of binge ethanol intoxication.

Abstract

Object: We aimed to investigate that the acetaldehyde dehydrogenase activator (Alda -1®) can improve the neuronal damage in an adolescent rat of binge ethanol exposure. Methods: The binge rats received the 25% ethanol (5g/kg) three gavages per day for four days. Thirty two adolescent male rats (130-150g) were randomly assigned into three groups, group one (n=8) received only saline (sham group), group two (n=12) received ethanol only, and group three (n= 12) received ethanol with Alda-1® (10mg/kg). The intoxication behavior score (6-point scale) was rated before each ethanol gavages. The ALDH activity of baseline and 90 min after last infusion of each group was analyzed by using ALDH activity analyzing kit (ab155893, Abcam®, Cambridge, United Kindom). The withdrawal behavior score was measured every 4 hours from 10 hours after the last infusion of ethanol dose for three days. The histopathological analysis was performed three days after behavior observation by using luxol fast blue-cresyl violet stain and immunefluorescence of glial fibrillary acidic protein (GFAP) and Iba-1. Results: 8 animals of sham group, 11 animals of ethanol only group (one was sacrificed by other animals) and 12 animals of ethanol with alda-1® group were enrolled in this study. The intoxication behavioral scores achieved between ethanol only group and ethanol with alda-1® group were not statistically significant (p=0.739). The mean baseline ALDH activity of sham group, ethanol only group and ethanol with alda-1® group were 4.33 (SD: 0.59), 4.23 (0.46) and 3.90 (0.57) mU/ml, respectively, which are not significantly different (p=0.396). After four day binge, the ALDH activity level was doubled in ethanol with alda-1® group (mean: 7.87, SD: 0.67), whereas, the levels of sham group (mean: 4.07, SD: 0.53) and ethanol only group (mean: 3.77, SD: 0.36) were slightly decreased. The mean withdrawal behavioral score in ethanol only group was significantly higher in comparison to the ethanol with alda-1® group (1.56±1.30 vs. 0.76±0.90, p=<0.001). In the histopathology analysis, the distinct shrinkage and reduced count of neurons, and lost Nissl substance of hippocampus, especially field CA1, CA3 and dentate gyrus, piriform cortex and cerebral cortex in ethanol only group were observed, whereas these were not distinct in ethanol with Alda-1® group. The reactive expression of GFAP, a marker of astrocyte, and Iba-1, a calcium binding protein expressed in miroglia were observed in the field CA3 of hippocampus, piriform cortex and cerebral cotex in ethanol only group, however, these distinct reactive astrocytosis and microgliosis were not detected in ethanol with alda-1® group. Conclusion: The administration of Alda-1® increases ALDH activity and reduces the cytotoxic damage of brain, especially hippocampus, piriform cortex and cerebral cortex in adolescent rat with acute ethanol exposure. |배경 및 목적: 폭음으로 인한 뇌 손상은 이미 널리 알려져 있으며 에탄올의 중간 분해 산물인 아세트알데하이드가 원인 물질로 알려져 있다. 아세트알데하이드는 아세트알데하이드 탈수소 효소 (acetaldehyde dehydrogenase, ALDH)에 의해 아세톤으로 변환된다. 본 연구에서는 폭음 쥐 모델에서 아세트알데하이드 탈수소 효소 활성제 (alda-1®)의 투여가 ALDH의 활성도를 높여 아세트알데하이드의 농도를 줄임으로써 뇌신경 손상을 줄일 수 있는지를 알아보고자 한다. 방법: 총 32마리의 생후 30일된 흰쥐 (130-150g)를 세 그룹으로 나누었다. (8마리의 sham 대조군, 12마리의 폭음 쥐 모델군 그리고 12마리의 폭음 쥐 모델 + alda-1®) 폭음 쥐 모델은 4일동안 25%의 에탄올을 kg 당 5g씩 8시간 간격으로 하루 3회 투여하였다. Alda-1 (10mg/kg)은 에탄올 투여 직전에 투여하였다. 에탄올 투여 첫날, 첫 번째 투여 전에 세 그룹 모두 꼬리 정맥을 통하여 혈액을 체취 하였고, 아세트알데하이드 탈수소 효소 활성도 분석 키트 (ab155893, Abcam®, Cambridge, United Kindom)를 이용하여 ALDH 활성도를 측정하였다. 실험 4일째, 마지막 에탄올 투여 90분 후 다시 혈액을 체취하여 ALDH 활성도를 측정하여 초기 활성도와 비교하였다. 마지막 에탄올 투여 10시간 후부터 72시간 동안 4시간 간격으로 금단행동을 점수로 기록하였다. 3일간의 금단행동 관찰 후, 마취 하에 관류 고정하였고 뇌를 적출하여 병리조직 분석을 위해 Luxol fast blue/cresyl violet 염색과 면역 형광 염색 (glial fibrillary acidic protein, Iba1)을 하였다. 결과: 알코올 투여군 중1마리가 희생되어 총 8마리의 샴대조군, 11마리의 폭음 쥐, 12마리의 Alda-1® 투여 쥐가 실험에 포함되었다. Alda-1®을 투여한 군에서 초기 ALDH 활성도 (3.90±0.57mU/ml)에 비해 alda-1 투여 후 (7.87±0.67mU/ml) 평균 2.02배의 활성도 증가를 보였고 에탄올만 투여한 폭음 쥐 군에서는 오히려 활성도가 감소하였다 (초기 활성도: 4.23±0.46mU/ml, 4일 후: 3.77±0.36mU/ml). 금단 행동 점수는 에탄올 투여군이 평균 1.56± 1.30, 에탄올 + Alda-1 투여군이 0.76±0.90로 기록되었고, 통계적으로 유의한 차이가 있었으며 (p<0.001), 최고 금단행동 점수 역시 통계적으로 유의하게 에탄올 투여군이 높았다 (에탄올 투여군: 3.2 ± 0.17, 에탄올 + Alda-1 투여군: 2.2 ± 0.22, p=0.008). 병리소견은 에탄올 투여군의 piriform cortex, cerebral cortex, Hippocampus CA1, CA3, 그리고 dentate gyrus에서 샴대조군과 에탄올 + Alda-1 투여군에 비해 신경원성 세포 (neurone)의 감소가 관찰 되었다. 면역 형광 염색에서는 에탄올 투여군의 piriform cortex, cerebral cortex와 Hippocampus CA3에서 astrocyte와 microglia의 발색이 다른 두 군에 비해 증가하였다. 결론: Alda-1은 ALDH 활성도를 높이며 뇌손상의 원인 물질인 아세트알데하이드의 분해를 촉진시켜 폭음 쥐 모델의 뇌 손상을 줄임을 확인하였다.; Object: We aimed to investigate that the acetaldehyde dehydrogenase activator (Alda -1®) can improve the neuronal damage in an adolescent rat of binge ethanol exposure. Methods: The binge rats received the 25% ethanol (5g/kg) three gavages per day for four days. Thirty two adolescent male rats (130-150g) were randomly assigned into three groups, group one (n=8) received only saline (sham group), group two (n=12) received ethanol only, and group three (n= 12) received ethanol with Alda-1® (10mg/kg). The intoxication behavior score (6-point scale) was rated before each ethanol gavages. The ALDH activity of baseline and 90 min after last infusion of each group was analyzed by using ALDH activity analyzing kit (ab155893, Abcam®, Cambridge, United Kindom). The withdrawal behavior score was measured every 4 hours from 10 hours after the last infusion of ethanol dose for three days. The histopathological analysis was performed three days after behavior observation by using luxol fast blue-cresyl violet stain and immunefluorescence of glial fibrillary acidic protein (GFAP) and Iba-1. Results: 8 animals of sham group, 11 animals of ethanol only group (one was sacrificed by other animals) and 12 animals of ethanol with alda-1® group were enrolled in this study. The intoxication behavioral scores achieved between ethanol only group and ethanol with alda-1® group were not statistically significant (p=0.739). The mean baseline ALDH activity of sham group, ethanol only group and ethanol with alda-1® group were 4.33 (SD: 0.59), 4.23 (0.46) and 3.90 (0.57) mU/ml, respectively, which are not significantly different (p=0.396). After four day binge, the ALDH activity level was doubled in ethanol with alda-1® group (mean: 7.87, SD: 0.67), whereas, the levels of sham group (mean: 4.07, SD: 0.53) and ethanol only group (mean: 3.77, SD: 0.36) were slightly decreased. The mean withdrawal behavioral score in ethanol only group was significantly higher in comparison to the ethanol with alda-1® group (1.56±1.30 vs. 0.76±0.90, p=<0.001). In the histopathology analysis, the distinct shrinkage and reduced count of neurons, and lost Nissl substance of hippocampus, especially field CA1, CA3 and dentate gyrus, piriform cortex and cerebral cortex in ethanol only group were observed, whereas these were not distinct in ethanol with Alda-1® group. The reactive expression of GFAP, a marker of astrocyte, and Iba-1, a calcium binding protein expressed in miroglia were observed in the field CA3 of hippocampus, piriform cortex and cerebral cotex in ethanol only group, however, these distinct reactive astrocytosis and microgliosis were not detected in ethanol with alda-1® group. Conclusion: The administration of Alda-1® increases ALDH activity and reduces the cytotoxic damage of brain, especially hippocampus, piriform cortex and cerebral cortex in adolescent rat with acute ethanol exposure.