Stereotaxic microinjection of adenovirus-mediated human tissue Kallikrein gene reduces apoptosis in a rat model of middle cerebral artery occlusion

Ruiyan Lü; Lianhong Yang; Qingyu Shen; Mei Li; Xiangpen Li; Ying Peng

摘要：BACKGROUND: Several studies have demonstrated that Kallikrein gene transfer provides neuroprotection. Whether the neuroprotective effects of human tissue Kallikrein (HTK) are associated with apoptosis remains unclear.OBJECTIVE: To investigate the effects of HTK on apoptosis in the peripheral cerebral infarct region.DESIGN, TIME AND SETTING: The completely randomized grouping, gene engineered, controlled experiment was performed at the Lin Baixin Laboratory Center, the Second Affiliated Hospital of Sun Yat-sun University between September 2007 and April 2008.MATERIALS: Ninety clean, healthy, male, Sprague Dawley rats were included, pUC19-HTK plasmid was constructed in the Laboratory for Neurology, the Second Affiliated Hospital of Sun Yat-sen University,China. bcl-2, bax, caspase-3, and 13 -actin were designed and purified by Shanghai Shuiyuan Company,China.METHODS: Middle cerebral artery occlusion (MCAO) model was established in all rats. At 72 hours after MCAO model establishment, rats were randomly divided into 3 groups, with 30 rats per group: blank control,saline, and pAdCMV-HTK. The saline and pAdCMV-HTK groups were respectively stereotactically micro-injected with 5 μ L physiological saline or pAdCMV-HTK at the area surrounding the cerebral infarction region. Only puncture was performed, without any injection, in the blank control group.MAIN OUTCOME MEASURES: At 72 hours after MCAO establishment, as well as at 24 hours, 72 hours,and 7 days subsequent to treatment, exogenous HTK expression was detected by immunohistochemistry.Apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), while mRNA levels of bcl-2, bax, and caspase-3 were detected by reverse transcription-polymerase chain reaction. In addition, neurological severity scores were evaluated prior to and after treatments.RESULTS: Ninety rats were included in the final analysis. HTK was primarily detected in the cytoplasm at 24 hours after pAdCMV-HTK injection. Thereafter, HTK expression gradually increased and reached a peak level at 72 hours after injection, which was significantly different from the blank control and saline groups (P< 0.05). At 7 days, HTK expression began to decrease, but remained higher than the saline and blank control groups (P < 0.05). Apoptotic cells aggregated around the cerebral infarction region. Compared with the saline and blank control groups, the mean number of TUNEL-positive cells was notably decreased in the pAdCMV-HTK group at each time point after treatment (P < 0.05). mRNA levels of bcl-2, bax, and caspase-3 were elevated in all groups at 24 hours after treatment, peaked at 72 hours, and then gradually decreased again at 7 days. Compared with the saline and blank control groups, bcl-2 slightly increased, but was not significantly different from the pAdCMV-HTK group (P > 0.05). bax and caspase-3 mRNA levels were significantly reduced at 24 and 72 hours after treatment (P < 0.05). At 72 hours and, in particular, at 7days after treatment, neurological severity scores were significantly less in the pAdCMV-HTK group compared with the saline and blank control groups (P < 0.05-0.01).CONCLUSION: HTK could protect neural cells in the peripheral cerebral infarction region from apoptosis,which resulted in a better outcome. This may be related to modulated bcl-2 expression and reduced bax and caspase-3 expression.