Correlation between neuronal injury and Caspase-3 after focal ischemia in human hippocampus.
BACKGROUND
Cerebral ischemia is a significant clinical problem, and cerebral ischemia usually causes neuron injury such as apoptosis in various brain areas, including hippocampus. Cysteinyl aspartate-specific protease (Caspases) are fundamental factors of apoptotic mechanism. Caspase-3 inhibitors show effect in attenuating brain injury after ischemia. But all the results were from animal models in research laboratories. This study aimed at investigating the correlation between the change of ischemic neuronal injury and Caspase-3 post-ischemia in human hippocampus.


METHODS
We selected and systematized 48 post-mortem specimens from 48 patients, who died of cerebral infarction. Morphological change was firstly analyzed by observing hematoxyline/eosin-staining hippocampal sections. The expression of Caspase-3 was investigated using the methods of in situ hybridization and immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick-end labeling (TUNEL) method was used to clarify the involvement of Caspase-3 in neuron death. The loss of MAP 2 (MAP-2) was applied to judging the damaged area and degree of neuronal injury caused by ischemia.


RESULTS
In the CA1 sector of hippocampus, Caspase-3 immunostaining modestly increased at 8 hours [8.05/high-power field (hpf)], dramatically increased at 24 hours (24.85/hpf), decreased somewhat after 72 hours. Caspase-3 mRNA was detectable at 4 hours (6.75/hpf), reached a maximum at 16 hours (17.60/hpf), faded at 72 hours. TUNEL-positive cells were detectable at 24 hours (10.76/hpf), markedly increased at 48 - 72 hours. The loss of MAP-2 was obviously detected at 4 hours, progressed significantly between 24 and 72 hours; MAP-2 immunoreactivity was barely detectable at 72 hours. Before 72 hours, the Caspase-3 evolution was related with the upregulation of TUNEL and the loss of MAP-2. The positive correlation between Caspase-3 mRNA and TUNEL was significant at the 0.05 level (correlation coefficient was 0.721); the negative correlation between Caspase-3 mRNA and MAP-2 was significant at the 0.05 level (correlation coefficient is 0.857). In the early stage (before 72 hours), the staining of Caspase-3 mRNA and immunohistochemistry was predominantly present in cytoplasm; the staining of TUNEL was predominantly localized in nucleus. At 4 - 16 hours, most neurons in hippocampal CA1 areas had relatively normal morphology; at 24 - 48 hours, neurons showed apoptotic morphology; at 72 hours, most cells showed significantly pathological morphology.


CONCLUSIONS
There exist a time-dependent evolution of neuronal damage after hippocampal ischemia in human brain, which was characterized by its close correspondence to Caspase-3.
Qi
Ji-ping
JP
Department of Pathology, First Hospital of Harbin Medical University, Harbin 150001, China. qijiping2003@163.com
Wu
Ai-ping
AP
Wang
De-sheng
DS
Wang
Li-feng
LF
Li
Shu-xia
SX
Xu
Feng-lin
FL
D000328:Adult; D000368:Aged; D002545:Brain Ischemia / Q000201:enzymology / Q000473:pathology*; D053148:Caspase 3; D020169:Caspases / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D005260:Female; D006624:Hippocampus / Q000201:enzymology / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D020287:In Situ Nick-End Labeling; D008297:Male; D008869:Microtubule-Associated Proteins / Q000032:analysis; D008875:Middle Aged
C489788:MAP2 protein, human; D008869:Microtubule-Associated Proteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases