| Project/Area Number |
15591865
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Ophthalmology
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| Research Institution | Kumamoto University |
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Principal Investigator |
MATSUMOTO Koki Kumamoto University, Graduate School of Medical Sciences Ophthalmology and Visual Science, MD, PhD., Associate Professor, 大学院・医学薬学研究部, 助教授 (70173896)
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| Co-Investigator(Kenkyū-buntansha) |
TANIHARA Hidenobu Kumamoto University, Graduate School of Medical Sciences, Ophthalmology and Visual Science, Professor, MD, PhD, 大学院・医学薬学研究部, 教授 (60217148)
HIRATA Akira Kumamoto University Hospital, Ophthalmology, Assistant Professor, MD, PhD, 医学部附属病院, 講師 (60295144)
FUKUSHIMA Mikiko Kumamoto University Hospital, Ophthalmology, Assistant Professor, MD, PhD, 医学部附属病院, 講師 (10284770)
MIYAJIMA Seiya Kumamoto University Hospital, Ophthalmology, Faculty member, MD, PhD, 医学部附属病院, 助手 (10336208)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Pseudomonas aeruginosa / corneal ulcer / matrix metalloproteinase / tissue inhibitor of MMP(TIMP) / inflammatory cytokine / zymography / immunostaining / RT-PCR / matrix metalloproteinase (MMP) / サイトカイン / プロテアーゼ / 角膜実質細胞 / 好中球 |
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| Research Abstract |
Pseudomonal keratitis was induced in New Zealand white rabbits by injecting a clinical isolate of Pseudomonas aeruginosa(1X10^4cfu). At 3,9,12,15,18,24 and 72 h post infection, the corneal lesions were observed, phptpgraphed, and the eyes were enucleated or corneas were excised for evaluation of the expression of MMP(MMP-2,MMP-9), TIMP(TIMP-1,TIMP-2) as well as inflammatory cytokines(IL-1b,IL-6,TNF-a) and chemokines(IL-8,MCP-1) using gelatin-zymography, immunohistochemistry, ELISA and RT-PCR.
Appreciable corneal lesions were observed 9 h p.i., and typical corneal ulcer accompanied with ring abscess was formed 12-24 h p.i. Morphologically the lesions were characterized with massive inflammatory cells(mainly PMN) infiltarion and liquefactive necrosis. Gelatin-zymography showed that MMP-2 was expressed constitutively even in the control eyes whereas MMP-9 was significantly expressed after pseudomonal infection, and the activated MMP-9 was also detected. Immunohistochemistry showed that MMP
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-2 was immunostained in exclusively keratocytes whereas MMP-9 was detected both in keratocytes and inflammatory cells(predominantly PMN). TIMP-1 was also immunostained in inflammatory cells. ELISA showed that the expression of TIMP-1 was enhanced with time after infection in contrast to TIMP-2 which was under the detection limit during the experiment. RT-PCR showed that mRNAs of MMP-9 and TIMP-1 were significantly expressed after pseudomonal infection.
Inflammatory cytokines and chemokines were expressed after pseudomonal infection confirmed by immunohistochemistry, ELISA and RT-PCR. The origin of the mediators was found to be inflammatory cells(PMN and macrophages) by morphological analysis. Most of these mediators are known to be chemotactic factor causing intensive infiltration of inflammatory cells in the corneas.
These results indicate that major pathogenetic factor in tissue destruction of pseudomonal keratitis is massive inflammatory cells infiltration and secondarily expression of proteases such as MMP-9. Less
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