| Summary: | 碩士 === 國防醫學院 === 生理學研究所 === 94 === Abstract
Reperfusion after an ischemic episode in the brain causing inflammatory reactions and leading to a delayed death of neurons and secondary brain damage. Focal cerebral ischemia-reperfusion (I/R) results in characteristic histopathological changes that manifest as a “necrotic core” in which cells die rapidly, and a surrounding “penumbra” region of variable size in which neurons die over an extended time period of days to weeks. In the present study, we have established an animal model of focal cerebral I/R with microinjection of endothelin-1 (ET-1) (120 pmol in 10μl saline) to middle cerebral artery and unilateral ligation of common carotid artery in the rat. Cerebral ischemia, as evidenced by decrease of the regional cerebral blood flow to 20~25% of the control, followed by 24 hr reperfusion (I/R24) produced stable and reproducible infarcts. Using this model we examined the effect of thaliporphine, a phenolic aporphine alkaloid identified in Chinese herbs, on ischemic injury and functional outcome. Brain infarction volume was measured by TTC staining. Our results indicated that thaliporphine at the doses of 1mg/kg (i.v.) and 2mg/kg (i.v.) did not cause vasodilation but significantly reduced infarct volume and neurodegeneration following I/R24 at the dose of 2mg/kg (i.v.). Swing test also indicated thaliporphine improved function outcome after I/R 24. The potential mechanism underlying this neuroprotective effect was further studied. Animals were subjected to I/R for various time intervals, and sacrificed after functional behavioral evaluations. Using immunocytochemical staining, we examined the cellular localization of 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (4-HNE), the markers of the attack by free radicals on cellular proteins and lipids, respectively, in penumbra regions in brain sections at 24 hr after reperfusion (I/R24). Thaliporphine reduced the number of 3-NT-positive and 4-HNE-positive cells in the penumbra region following I/R 24. The in vivo production of superoxide in penumbra region following I/R was measured by chemiluminence method. Thaliporphine also significantly reduced in vivo superoxide production as early as I/R30min to I/R60min. Tissue levels of pro-inflammatory cytokines and chemokines including IL-1β, IL-6 and MIP-2 in ischemic penumbra tissues elevated time-dependently in the I/R groups but not in the sham groups. Systemic administration of thaliporphine at the beginning of ischemia induction also reduced the I/R-induced elevation of tissue levels of pro-inflammatory cytokines (IL-1, IL-6) and chemokines (MIP-2). Histological staining of brain sections revealed that I/R caused neutrophils infiltration which was also suppressed by thaliporphine administration. Real-time reverse transcription-polymerase chain reaction (RT-PCR) indicated relative mRNA levels of pro-inflammatory cytokines (IL-1, IL-6, TNF-) and chemokines in the ipsilateral penumbra increased after I/R and reached the maximum at the 6 hr after I/R. Thaliporphine administration also reduced I/R-induced elevation of mRNA levels of pro-inflammatory cytokine (IL-1), chemokine (MIP-2) and inflammatory mediators (COX-2, iNOS) at the 4 hr, 6 hr after I/R. Taken together, these data suggests the neuroprotective effects of thaliporthine might be attributed to its anti-oxidant and anti-inflammatory mechanisms.
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