Hydrogen sulphide and mild hypothermia activate the CREB signaling pathway and prevent ischemia-reperfusion injury.
BACKGROUND
Both hydrogen sulphide (H2S) and mild hypothermia have been reported to prevent brain damage caused by reperfusion assault through regulating the N-methyl-D-aspartate receptor (NMDAR). However, the relationship between the two treatments and how they exert neuro-protective effects through NMDARs remain to be elucidated.


METHODS
Transient cerebral ischemia was induced using the Pulsinelli four-vessel occlusion method. We used sodium hydrosulphide (NaHS) as the H2S donor. We randomly divided 100 Sprague-Dawley rats into five groups of 20: Sham operation group (Sh), normothermic (36-37 °C) ischemia group (NT), mild hypothermic (32-33 °C) ischemia group (mHT), normothermic ischemia combined with NaHS treatment group (NT + NaHS), and mild hypothermic ischemia combined with NaHS treatment group (mHT + NaHS). After 6 hrs of reperfusion, rats were decapitated and hippocampus samples were immediately collected. We measured NR2A (GluN1), NR2B (GluN2) and p-CREB protein levels using western blotting. We further analyzed BDNF mRNA expression by real-time PCR. Hematoxylin and eosin (HE) staining was used to examine pyramidal cell histology at the CA1 region. All statistical analyses were carried out by ANOVA and LSD t-test as implemented by the SPSS 13.0 software.


RESULTS
In the four test groups with ischemia-reperfusion, hippocampal H2S concentration increased following treatment, and administration of NaHS further increased H2S levels. Moreover, administration of both NaHS and mild hypothermia resulted in up-regulation of NR2A and NR2B protein expressions, as well as p-CREB protein and BDNF mRNA levels. At the cellular level, NaHS and mild hypothermia groups exhibited lower damage caused by ischemia-reperfusion in the CA1 region of the hippocampus. The strongest protective effect was observed in rats treated with combined NaHS and mild hypothermia, suggesting their effects were additive.


CONCLUSIONS
Our results support previous findings that hydrogen sulphide and mild hypothermia can prevent ischemia-reperfusion injury. Both treatments caused an up-regulation of NMDA receptors, as well as an elevation in p-CREB protein and BDNF mRNA levels. Thus, hydrogen sulphide and mild hypothermia may provide neuro-protective effect through activating the pro-survival CREB signaling pathway.
Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, No. 305 East Zhongshan Road, Jiangsu, Nanjing, 210002, China. Dai_hai_bin1@163.com.
Hai-bin
HB
Dai
Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, No. 305 East Zhongshan Road, Jiangsu, Nanjing, 210002, China. ji_xiang_jun@163.com.
Xiangjun
X
Ji
Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, No. 305 East Zhongshan Road, Jiangsu, Nanjing, 210002, China. Zhu_si_hai@163.com.
Si-hai
SH
Zhu
Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, No. 305 East Zhongshan Road, Jiangsu, Nanjing, 210002, China. Hu_yi_min@163.com.
Yi-min
YM
Hu
Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, No. 305 East Zhongshan Road, Jiangsu, Nanjing, 210002, China. Li_dong_zhang@163.com.
Li-dong
LD
Zhang
Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, No. 305 East Zhongshan Road, Jiangsu, Nanjing, 210002, China. Miao_Xiao_lei@163.com.
Xiao-lei
XL
Miao
Department of Experimental Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, China. ma_ru_meng@163.com.
Ru-Meng
RM
Ma
Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, No. 305 East Zhongshan Road, Jiangsu, Nanjing, 210002, China. Duan_man_lin@163.com.
Man-lin
ML
Duan
Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, No. 305 East Zhongshan Road, Jiangsu, Nanjing, 210002, China. Lee_wei_yan@163.com.
Wei-yan
WY
Li
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D001930:Brain Injuries / Q000378:metabolism / Q000517:prevention & control; D002545:Brain Ischemia / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004195:Disease Models, Animal; D006624:Hippocampus / Q000378:metabolism; D006862:Hydrogen Sulfide / Q000378:metabolism / Q000494:pharmacology*; D007036:Hypothermia, Induced; D008297:Male; D018696:Neuroprotective Agents / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D015427:Reperfusion Injury / Q000378:metabolism / Q000517:prevention & control*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D019208:Brain-Derived Neurotrophic Factor; D017362:Cyclic AMP Response Element-Binding Protein; D018696:Neuroprotective Agents; D006862:Hydrogen Sulfide