EXPRESSION OF COLLAGEN-I, COLLAGEN-III, COLLAGEN-IV AND COLLAGEN-V MESSENGER-RNA IN EXCIMER WOUNDED RAT CORNEA - ANALYSIS BY SEMIQUANTITATIVE PCR

A semi-quantitative polymerase chain reaction (PCR) methodology was us ed to evaluate the kinetic changes occurring in collagens I, III, IV a nd V mRNA in rat cornea following excimer laser keratectomy. cDNA was synthesized from RNA extracted from rat cornea at various times follow ing excimer laser photoablative keratectomy. Collagen cDNA sequences w ere subsequently amplified using specific sets of oligonucleotide prim ers. Competitive PCR amplification was carried out using an internal s tandard so that a semi-quantitative analysis of message for synthesis of collagen types I, III, IV and V could be performed and time course dynamics of message for these collagens studied. There was a biphasic increase in the levels of collagens III, IV and V mRNA following excim er laser keratectomy. Collagen I mRNA levels demonstrated a more susta ined increase and were still elevated at 6 weeks following wounding. C ollagens IV and V mRNA showed the largest increase with an approximate three fold increase over controls between 4 days and 1 week. Our resu lts demonstrate that upregulation of stromal collagens I, III, and V m RNA and basement membrane collagen IV mRNA occurs in rat cornea follow ing excimer laser keratectomy.