| Budget Amount *help |
¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥700,000 (Direct Cost: ¥700,000)
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| Research Abstract |
Bupivacaine hydrochloride (Marcaine^<【○!R】> ; Fujisawa ) was intramuscularly injected into extensor digitorum longus (EDL) muscles of male Wistar rats (aged 6 weeks). Isotonic saline was injected into the contralateral EDL muscles as a control. The EDL muscles were taken at respective intervals of 12h, 1, 2, 3, 4, 5, 10, and 14 days after the injection. Frozen sections were stained with H.E., macrophages using anti-rat RM-4 monoclonal antibody (a gift from Dr.M.Takeya, Dept.of Pathology, Kumamoto University), and apoptotic cells using TUNEL assay (Apop Tag^<【○!R】> ; Intergen) . Ultrathin sections were doublestained with uranyl acetate and lead citrate. reacted with TUNEL and then examined by an electron microscope. Total RNA was extracted from muscle tissues and they were converted to cDNA by revere transcriptase. Levels of monocyte chemoattractant protein-1 ( MCP-1) were examined with RT-PCR, Macrophages were increased significantly at 2 days after the injection and then decreased gradually at 4 days. Numbers of apoptotic cells were increased at 2 to 4 days and decreased thereafter. In serial sections stained immunohistochemically, some of RM-4 positive cells showed a TUNEL-positive. Electron microscope study demonstrated the macrophages undergoing apoptosis. Levels of MCP-1 mRNA showed a positive correlation with changes in macrophages. In the contralateral site received saline alone, necrotic muscular fibers and elevated numbers of macrophages were not observed. In conclusion, it was found that mechanisms on disappearance of macrophages were related with not only decreased levels of MCP-1 but also apoptosis.
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