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Purpose: This study was undertaken to evaluate for the presence of cyclooxygenase 2 (COX2) gene expression in the anterior uvea of rabbits following glaucoma filtration surgery., Methods: One of the following surgical procedures were performed on the right eye of New Zealand white albino rabbits: (1) paracentesis (2.5 mm limbal incision); (2) iridectomy through a 2.5 mm limbal incision; (3) lamellar scleral flap formation or (4) full glaucoma filtration surgery. The animals were sacrificed within 3 hours of post-surgery, and the anterior uveal tissues were isolated. Polymerase chain reaction-based techniques were employed to assay for the presence of COX2 transcript., Results: A partial coding sequence of the previously unreported rabbit COX2 gene was obtained. COX2 mRNA was detected in the operated eyes of animals that underwent either full filtration surgery or iridectomy through a limbal incision., Conclusions: In normal rabbit anterior uveal tissue, there appears to be minimal expression of COX2 message. After experimental glaucoma filtration surgery, there is rapid induction of COX2 message.

Chitin, a unique structural polysaccharide found in fungi and arthropods, is not produced by vertebrates. Thus, the potential applications of a specific and sensitive assay for chitin are numerous, including the evaluation of the extent of fungal keratitis. Chitin is a homopolymer of beta (1, 4) linked D-N-acetylglucosamine. We have developed a simple and reproducible assay for chitin and applied it to Candida albicans cultures. The assay involves homogenization of the culture and treatment with 21.1 M KOH to remove soluble materials, including proteins. This base treatment also deacetylates the chitin to the glucosamine polymer, chitosan. Chitosan is hydrolyzed by 0.5 M H2SO4 to glucosamine monomers which are then deaminated by the addition of NaNO2 to the acid solution. The resulting 2,5-anhydromannose is reduced by NaB[3H]4 to 1-[3H] 2,5-anhydromannitol. This radiolabelled sugar is isolated by paper chromatography and quantified via liquid scintillation. The sensitivity of this assay is assessed by comparison of colony forming units (CFU's) with a glucosamine standard. A typical run of the assay detects 53.1 CFU/c.p.m., and 356,000 c.p.m. per nanomole of N-acetylglucosamine. The specificity of the assay is very high because of the unique nature of chitin. This method of chitin determination may be a useful alternative method for future investigations involving the study of fungal infections in mammalian tissues.

Two different measures of response to therapy were evaluated in a model of keratitis caused by Aspergillus fumigatus in Dutch-belted rabbits. Combined pre and post-inoculation treatment with oral fluconazole 37.5 mg/kg bid or itraconazole 40 mg/kg bid was compared to post-inoculation treatment only and untreated controls using a standardized clinical disease severity score and quantitative isolate recovery techniques. For both drugs, there was no difference in isolate recovery rates among all three groups. However, a significant improvement in clinical disease was noted in the pre and post-inoculation treatment group compared to controls (p less than .01) and to the post-inoculation group (p less than .05) for fluconazole. A similar trend, though not statistically significant, was apparent with itraconazole treatment. This disparity highlights the difficulties associated with measuring responses to therapy in keratomycosis and emphasizes the need for more sensitive and specific measures.

Six patients with chronic herpes simplex keratouveitis developed a rapidly progressive ocular inflammation unresponsive to corticosteroid and antiviral therapy. Severe secondary glaucoma unresponsive to maximum medical therapy ensued and was treated by cyclocryotherapy in five patients. Ocular ischemia with secondary corneal and scleral calcification subsequently developed in all six patients. All had also received prolonged topical therapy with topical antiviral agents, corticosteroids, beta adrenergic blockers and epinephrine compounds. Three eyes eventually required enucleation for the relief of pain; one stabilized, and two others became phthisical. One of the phthisical eyes developed a secondary fungal endophthalmitis. Histopathologic examination of the three enucleated globes revealed extensive corneal, scleral and conjunctival calcification, secondary angle closure, iris and ciliary body necrosis, focal choroiditis, retinal necrosis and atrophy. The syndrome recognized in these patients appears to be a rare but devastating complication of herpes simplex keratouveitis, possibly exacerbated by the application of cyclocryotherapy and other factors.

The corneal uptake and penetration of 14C-labelled 0.15% amphotericin B and 5% natamycin were studied in Dutch-belted rabbits. Corneal levels of natamycin were substantially higher than amphotericin B. For both drugs, these levels were influenced by corneal contact time. In corneas debrided of epithelium, both agents entered the corneal stroma and levels were detected in aqueous in the therapeutic range. However, in corneas with intact epithelium, penetration was negligible for amphotericin B (0.23 microgram/gm at 2 mins). By contrast, although penetration of natamycin was greatly reduced, 7.0 micrograms/gm were present at 2 mins.

Acanthamoeba is a free-living, fresh-water protozoan that can cause severe corneal disease. Acanthamoeba keratitis can closely mimic epithelial and stromal Herpes simplex keratitis. Three cases of severe keratitis, were referred for treatment. One patient presented with a pseudodendritic epithelial lesion that gradually progressed to stromal involvement. A second patient presented with central stromal infiltrate and necrosis, while a third exhibited features of a disciform lesion with the later development of an immune ring. Acanthamoeba was recovered from the cornea in each case. The distinctive characteristics of the history and clinical findings in Acanthamoeba keratitis can aid the clinician in distinguishing between these two clinical entities. Cytopathology and special staining and culture techniques can confirm the diagnosis.

Experimental designs in ophthalmologic research frequently treat both eyes of a subject in the same fashion: e.g., therapy with a specific drug or control. In these two-eye designs, observations from the same subject are often positively correlated. Failure to account for this correlation is a serious error which overstates the precision of studies, resulting in falsely significant results. This paper reviews the statistical methods appropriate for studies where endpoints are quantitative. We present: (1) the use of analysis of variance (t-test when there are 2 treatment conditions) to estimate differences between all experimental treatments, (2) the use of contrasts to estimate differences between specific treatments, and (3) methods for analysis of data from multiple experiments. Because of the ubiquity of incorrect analysis of data from two-eye designs in the ophthalmologic research literature and the serious consequences of this error, we propose a limited statistical review of manuscripts to ascertain if the statistical analysis matched the experimental design.

We evaluated variability of isolate recovery rates in an experimental model of Candida keratitis for both heterogeneous, pigmented, outbred rabbits and homogeneous Dutch-belted rabbits. Our purpose was to achieve more precise evaluation of topical antifungal agents in our model by substantially reducing the component of variance due to inter-rabbit differences. For the homogeneous Dutch-belted rabbits, the overall variance was reduced by a factor of 2.34 (p = 0.18) vis a vis the heterogenous outbred rabbits. The greatest reduction, a factor of 3.5, was achieved for the inter-rabbit component of variance. The upper bound for the hereditability measure was reduced from 29% to 18%. These data suggest that (1) researchers using animal models should perform a components of variance analysis to identify the factors influencing experimental precision, and (2) for our model, additional reduction in variability must come from refinement of experimental technique.

The study of experimental fungal infections requires effective animal models. Two such models in the rabbit model have been developed and applied to various aspects of topical antifungal therapy. In these models, the polyenes, as a class, are superior in efficacy to the imidazoles and flucytosine, but efficacy is clearly strain related. Corticosteroids, when administered concomitantly with antifungal agents, adversely influence antifungal activity in vivo. This steroid effect is inversely proportional to the efficacy of the antifungal agent. The corneal epithelium appears to be a significant barrier to the penetration of the polyene antibiotics. Although the predictive power of in vitro susceptibility testing as a guide to in vivo efficacy is uncertain, a correlation was seen between the in vivo and in vitro effect for amphotericin B against three strains of Candida albicans.

We compared the efficacy of two polyenes, amphotericin B and natamycin, in two models of yeast infection. In one, treatment was begun immediately after inoculation, in the other it was delayed 24 hours. In each model infection with Candida albicans was established in the corneal stroma of dutch-belted rabbits and treated topically with 5% natamycin or amphotericin B 0.15% and 0.075%. Quantitative isolate recovery techniques were used to assess response after 5 days of treatment. A significant therapeutic effect was present for amphotericin B in both models. However, delayed treatment with natamycin was ineffective using treatment schedules efficacious when begun 1 hour after inoculation. A therapeutic effect was present only with administration of the drug every 1/2 hr. This altered response may reflect a difference in susceptibility between different growth phases in yeasts.

Amphotericin B, 5-flucytosine (5-FC), and ketoconazole levels were estimated in vitreous and aqueous samples taken from four patients undergoing therapeutic vitrectomy for fungal endophthalmitis. The levels of amphotericin B in the vitreous of three patients were low (.04 - .17 microgram/ml). However, 5-FC was present in a concentration of 22.2 micrograms/ml in one patient. In another case the aqueous level of ketoconazole was 0.35 microgram/ml. The vitreous in the same patient contained 0.71 microgram/ml of the drug.