[Protective effect of calpain inhibitor-3 on hypoxic-ischemic brain damage of neonatal rats].
OBJECTIVE
The mechanisms of hypoxic-ischemic brain damage (HIBD) are still largely unknown. Elevation of intracellular calcium concentration and subsequent calcium-dependent proteases activation such as calpains seem to play an important role in the process of neuronal death. Calpain inhibitors showed neuroprotective effects in adult rat cerebral ischemia models. This study aimed to investigate the protective effect and associated mechanisms of calpain inhibitor-3 (MDL28170) on HIBD of neonatal rats.


METHODS
Seven-day old Sprague-Dawley rats were randomly divided into three groups: the control group (n = 18), HIBD group (n = 48) and calpain inhibitor-3 treated group (MDL group, n = 48). The mice in the latter two groups were subjected to hypoxia-ischemia (HI) insult. The puppies in MDL group were intraperitoneally injected with MDL28170 (25 mg/kg) at 0, 2 and 4 h after HI, while those in the other two groups were intraperitoneally injected with normal saline instead. All the pupies were sacrificed at 6 h, 24 h and 72 h after HI. Quantitative real-time fluorescent polymerase chain reaction was employed to detect micro-calpain gene expression, immunoblotting technique was used to measure mu-calpain and caspase-3 protein activation, apoptosis of ipsilateral cortex was detected by terminal deoxynucleotidyl transferase mediated d-UTP nick end labeling staining (TUNEL). CA1 neuronal loss was counted 24 h after HI by light microscopy.


RESULTS
After HI mu-calpain mRNA began to increase at 6 h and reached peak at 24 h compared to the control (1.805 and 4.83 vs. 1, P < 0.05); mu-calpain was activated through autolysis, the ratio of its activated fragment (76 000) vs. whole fragment (80 000) was significantly higher at 6 h (0.547 +/- 0.095) compared to the control (0.095 +/- 0.016, P < 0.05), it reached peak at 24 h (0.921 +/- 0.058, P < 0.01) and was still at a high level at 72 h (0.708 +/- 0.025, P < 0.05). Expression of activated caspase-3 protein reached peak at 24 h (3.78 +/- 0.30, P < 0.01), decreased to the same level as the control (1.56 +/- 0.07) at 72 h (1.82 +/- 0.11, P > 0.05). Apoptotic cells in the cortex ipsilateral to HI insult increased after HIBD, reached peak at 24 h (135.46 +/- 17.52/visual field) and was still markedly higher at 72 h (79.32 +/- 17.79/visual field) compared with the control (5.33 +/- 1.53/visual field, P < 0.01). At 24 h after HI CA1 neuronal loss (30.0 +/- 6.2/oil immersion lens field) in the HIBD group was significantly higher than that of the control (2.4 +/- 0.3/oil immersion lens field, P < 0.01). However, in the MDL group the expressions of mu-calpain and caspase-3 proteins were diminished, TUNEL positive cells at 6 h and 24 h were decreased and CA1 neuronal loss (18.2 +/- 2.4/oil immersion lens field, P < 0.05) was alleviated. The amount of micro-calpain mRNA was decreased in the MDL group, but there was no significant difference compared with the HIBD group.


CONCLUSIONS
mu-calpain gene and protein expressions increased after HI, which may contribute to the pathogenensis of HIBD. Calpain inhibitor-3 may intervene neural necrosis and apoptosis by diminishing expressions of mu-calpain and caspase-3 to play a protective role after HI insult of neonatal brain.
Chen
Li-na
LN
Department of Pediatrics, West China Second Hospital, Sichuan University, Chengdu 610041, China.
Yan
Bo
B
Chen
Da-peng
DP
Yao
Yu-jia
YJ
D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000378:metabolism; D002154:Calpain / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use*; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors / Q000627:therapeutic use; D005260:Female; D002534:Hypoxia, Brain / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control; D020925:Hypoxia-Ischemia, Brain / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D020287:In Situ Nick-End Labeling; D007274:Injections, Intraperitoneal; D007527:Isoenzymes / Q000627:therapeutic use*; D008297:Male; D009124:Muscle Proteins / Q000627:therapeutic use*; D009474:Neurons / Q000187:drug effects / Q000201:enzymology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009124:Muscle Proteins; D012333:RNA, Messenger; D002154:Calpain; C491372:Capn3 protein, rat; C449002:mu-calpain