Bruce A. Pfeffer

Reduced myocilin expression in cultured monkey trabecular meshwork cells induced by a selective glucocorticoid receptor agonist: comparison with steroids.

PURPOSEnTo assess in vitro myocilin (MYOC) expression in trabecular meshwork (TM) cells exposed to BOL-303242-X, a selective glucocorticoid receptor (GR) agonist (SEGRA), in comparison with dexamethasone (DEX), and prednisolone acetate (PA).nnnMETHODSnAfter drug treatment of monkey TM cultures, MYOC protein in conditioned media (CM) was measured by Western blot and densitometry. MYOC mRNA levels were analyzed by qRT-PCR. RU-486 was tested for antagonism of MYOC protein expression induced by DEX and BOL-303242-X.nnnRESULTSnBaseline MYOC protein released into CM and MYOC mRNA were detected. DEX or PA elicited dose-dependent increases in MYOC in CM and also in MYOC mRNA. BOL-303242-X effects typified partial agonism, with significantly reduced MYOC protein and mRNA, compared with DEX. Maximum efficacy for BOL-303242-X was 53% of that for DEX. Mean EC(50) across all strains tested was lower, but not significantly different, for BOL-303242-X versus DEX. Compared with DEX, MYOC mRNA levels were significantly lower in BOL-303242-X-treated TM cells at the highest doses tested. EC(50)s for PA were higher than DEX, for both myocilin protein and mRNA. RU-486 displayed a dose-dependent antagonism to drug-induced increases in myocilin levels.nnnCONCLUSIONSnIn vitro quantitative assays of myocilin expression in TM cells can be used for characterizing anti-inflammatory drugs that are GR ligands. The results suggest that, compared with traditional ocular steroids, therapeutic doses of BOL-303242-X elicit a reduced myocilin expression profile in TM cells by virtue of the partial agonist properties of this compound.

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.