Stephen P. Bartels

The effects of forskolin on cyclic AMP, intraocular pressure and aqueous humor formation in rabbits

Forskolin was used to study cyclic AMP-mediated regulation of aqueous humor dynamics in rabbits. Crystalline forskolin was solubilized in oil and its pharmacological effects were studied both in vitro and following topical ocular administration. In vitro, using cultured corneal epithelial cells, forskolin rapidly stimulated cyclic AMP production and in vivo increased cyclic AMP concentration in the aqueous humor 10-fold following topical administration. The effect of topical forskolin on intraocular pressure and aqueous humor formation was determined in vivo using pneumatonometry and fluorophotometry, respectively. Forskolin caused a prolonged reduction of intraocular pressure and decreased aqueous humor formation. The ability of forskolin to potentiate the ocular hypotensive effect of epinephrine was investigated. Forskolin in combination with epinephrine caused a decrease in intraocular pressure of longer duration than either 0.1% epinephrine or 1% forskolin administered separately. Forskolin caused a small but significant increase in the permeability of the blood-aqueous barrier at the time of maximal intraocular pressure reduction. This effect on the blood-aqueous barrier may explain the inhibitory effect of forskolin on aqueous humor formation.

Feasibility of an antiviral clinical trial requiring cross-country shipment of conjunctival adenovirus cultures and recovery of infectious virus

Purpose. Accurate and timely laboratory diagnosis of adenovirus from conjunctival cultures is essential to ensure appropriate enrollment, and detection of residual infectious virus is essential to evaluate antiviral efficacy in any multicenter national clinical trial. As part of a feasibility study, we investigated those variables that might affect the successful recovery of infectious adenovirus from patient conjunctival cultures after cross-country shipment. Materials and methods. Simulated adenovirus conjunctival cultures were prepared in viral transport media to evaluate the effect of four variables (adenovirus serotype, initial concentration, initial storage temperature [−20°C, 0°C, 20°C], and preshipment storage times [1–5 days]) on the recovery of infectious adenovirus by a central laboratory in St. Paul, MN, following air shipment from Pittsburgh, PA. Upon arrival, the internal temperatures of the containers were recorded, and the samples were cultured on A549 cells using standard tube and/or shell vial culture. Results. Overall, adenovirus was recovered in 352/354 (99.4%) of the samples when the initial titer was greater than 1.0 PFU/ml. Adenovirus serotype, initial storage temperature, and preshipment storage times had no adverse effect on virus recovery. Conclusions. Simulated conjunctival samples could successfully be shipped cross-country at ambient temperatures to a commercial laboratory for adenovirus isolation by culture. Having demonstrated feasibility, a clinical trial was subsequently carried out that confirmed the ease of shipment and recovery of infectious adenovirus from conjunctival cultures.

Aqueous to cornea fluorescein distribution ratio in normal and swollen cornea

Intravitreal sodium fluorescein was used to simulate equilibrium fluorescein kinetics, thereby allowing simple measurement of the aqueous to cornea fluorescein distribution ratio. Two groups of rabbit corneas were studied: normal corneas and corneas wounded by freezing. The aqueous to cornea fluorescein distribution ratio was approximately 0.4, was not significantly different in groups of normal or wounded eyes and little variability was noted. In addition, a comparison of in vivo and in vitro measurements of corneal fluorescein concentration in wounded eyes suggests that in vivo protein-bound fluorescein in the cornea fluoresces less efficiently than free fluorescein.

Preservation of structure and immunoreactivity at the vitreoretinal interface of the rabbit eye.

BackgroundThe vitreous body is implicated in the etiology and pathology of a variety of retinal conditions. Many such conditions are treated surgically to remove the posterior vitreous from the inner limiting lamina (ILL) of the retina, and there is current interest in the adjunct use of enzymes for this purpose. To evaluate the efficacy of these agents in future preclinical studies, improved preservation and assessment methods were developed to establish a baseline histological profile of the vitreous and retina of the rabbit, to identify and distinguish artifactual vitreoretinal separation from authentic posterior vitreous detachment, and to preserve structural integrity while maintaining antigenicity for immunohistochemical analysis.MethodsTwo pigmented rabbits each underwent perfusion with one of three fixatives, either: (1) 10% neutral buffered formalin + cetylpyridinium chloride (NBF/CPC), (2) acid-ethanolic formalin + CPC (AEF/CPC), or (3) formaldehyde-glutaraldehyde + CPC (FG/CPC). An eye fixed in NBF/CPC by immersion, from an additional rabbit, was also included for comparison. Eyes were processed whole through paraffin infiltration. Treatments were assessed by immunohistochemical labeling for retinal and cortical vitreous (CV) markers.ResultsIn contrast to immersion fixation, perfusion with either NBF/CPC or AEF/CPC maintained vitreous adherence to the ILL during histological processing. NBF/CPC proved best for highlighting intralaminar structure and for labeling type II collagen in the CV, particularly with antigen retrieval. AEF/CPC caused condensation of fibrillar elements in the CV. Collagen XVIII in the ILL was observed with AEF/CPC exclusively. Only retinal vessels near the optic nerve head were labeled for type IV collagen. The labeling of glia was useful for distinguishing between cellular and extracellular elements. GF/CPC hindered detection of collagen II and disrupted posterior segment structure. Expression of type II collagen extended from the ONH directly to CV affiliated with the central canal of Cloquet, a feature characteristic of rabbit eyes.DiscussionCareful tissue preservation and processing techniques minimize artifactual separation of the CV from the ILL. By optimizing the tissue architecture and antigenicity of the vitreoretinal complex, CV may be distinguished from the ILL immunohistochemically. The techniques described may be used to evaluate more effectively the utility of pharmacologic vitreolysis, using experimental animal models.