Cloning of rabbit cyclooxygenase-2 and the time course of its induction during fungal endophthlamitis

M. S. Chang, R. Yang, D. M. O'Day, M. D. Breyer, R. N. DuBois

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Purpose. We have previously demonstrated that there is an increase in production of prostaglandins along with an induction of cyclooxygenase-2 (COX2) protein in anterior uveal tissue during fungal endophthalmitis. This study was undertaken to determine the time course of COX2 induction during fungal endophthalmitis and to obtain a full length cDNA clone of rabbit COX2. Methods. Endophthalmitis was induced in the right eye of New Zealand white albino rabbits by intravitreal injection of Candida sp. The uninfected left eye served as a control. Animals were sacrificed at 1 hr, 12 hrs, and 24 hrs following infection. Forty μg of total RNA from infected and uninfected eyes were used for Northern blot analysis; Northern blot analysis was also performed using RNA from normal eyes (no intravitreal injection) and eyes undergoing intravitreal injection with sterile normal saline. The radio-labeled probe was generated from a 236 bp rabbit COX2 partial sequence obtained from rabbit brain. PCR was performed on the same RNA samples using a primer set specific for rabbit COX2 (expected size, 236 bp). A cDNA library was constructed using anterior uveal mRNA from eyes infected for 12 hrs. The library was screened using the same Northern blot probe. Results. The Northern blot revealed a single band of approximately 4 KB in the 12 hr and 24 hr infected eyes, but not in the 1 hr infected eyes. Also, no bands were observed in the corresponding uninfected eyes, in normal eyes, and saline injected eyes. By PCR, a 236 bp band was detected in the 1 hr infected eye but not in the opposite uninfected eye. A full length clone of approximately 4 KB was obtained after tertiary screening of the cDNA library. Conclusions. Fungal endophthalmitis appears to induce COX2 expression as early as 1 hr after infection. By 24 hrs, COX2 mRNA levels have decreased to barely detectable levels. The induction of COX2 appears to be one of the initiating events in the ocular inflammatory response of fungal endophthalmitis. The full length cDNA sequence will be useful for the study of expression and regulation of COX2 in ocular inflammation.

Original languageEnglish (US)
Pages (from-to)S478
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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