A photic visual cycle of rhodopsin regeneration is dependent on Rgr.

During visual excitation, rhodopsin undergoes photoactivation and bleaches to opsin and all-trans-retinal. To regenerate rhodopsin and maintain normal visual sensitivity, the all-trans isomer must be metabolized and reisomerized to produce the chromophore 11-cis-retinal in biochemical steps that constitute the visual cycle and involve the retinal pigment epithelium (RPE; refs. 3-8). A key step in the visual cycle is isomerization of an all-trans retinoid to 11-cis-retinol in the RPE (refs. 9-11). It could be that the retinochrome-like opsins, peropsin, or the retinal G protein-coupled receptor (RGR) opsin12-16 are isomerases in the RPE. In contrast to visual pigments, RGR is bound predominantly to endogenous all-trans-retinal, and irradiation of RGR in vitro results in stereospecific conversion of the bound all-trans isomer to 11-cis-retinal. Here we show that RGR is involved in the formation of 11-cis-retinal in mice and functions in a light-dependent pathway of the rod visual cycle. Mutations in the human gene encoding RGR are associated with retinitis pigmentosa.

Extracellular matrix components in retrocorneal fibrous membrane in comparison to corneal endothelium and Descemet's membrane.

PURPOSE: To investigate the extracellular matrix macromolecules found in Descemet's membrane and in retrocorneal fibrous membrane (RCFM), and to examine whether the corneal endothelium has the capacity to produce both basement and non-basement membrane phenotypes. METHODS: Rabbit corneas with and without RCFM were analyzed by immunofluorescence using antibodies to 8 different collagens (basement membrane collagens: types IV and VIII; fibrillar collagens: types I and III; interfibrillar collagens: type VI and two spliced variant forms of type XII and one anchoring fiber: type VII), proteoglycans (perlecan and decorin), (beta)ig-h3 and laminin-1. RESULTS: Normal corneal endothelium stains positively for all of the tested collagen types except type VII collagen. On the other hand, Descemet's membrane reacts positively only to the type IV collagen antibody. When non-collagenous components in normal cornea were examined, corneal endothelium stained positively for perlecan, decorin, (beta)ig-h3 and laminin, whereas Descemet's membrane staining for these proteins was negative. When collagenous components of RCFM were examined, RCFM stained positively for all of the tested collagen types except type IV collagen. When non-collagenous components of RCFM were examined, RCFM demonstrated a strong positive staining with decorin, (beta)ig-h3 and laminin, while perlecan staining was weak. CONCLUSIONS: These observations suggest that corneal endothelium is able to produce both basement membrane phenotypes and non-basement membrane, fibrillar phenotypes. This in vivo study confirms our in vitro model of endothelial mesenchymal transformation, in which corneal endothelial cells are transformed to fibroblasts that are responsible for fibrosis.

Immunohistochemical identification of androgen receptors in human lacrimal glands.

PURPOSE: Androgens are thought to play a role in the regulation of the human lacrimal gland. Androgen receptor mRNA has been isolated from human lacrimal tissue; however, it is not known which cell(s) in human lacrimal tissue may contain androgen receptors. This study is an immunohistochemical investigation of the location and distribution of androgen receptors in human lacrimal tissue. METHODS: Formalin-fixed, paraffin-embedded human lacrimal gland tissues were subjected to established antigen retrieval techniques. This was followed by routine immunohistochemical staining, employing one of two anti-human androgen receptor monoclonal antibodies, each specific for a different antigenic epitope within the receptor molecule. RESULTS: The two anti-human androgen receptor monoclonal antibodies demonstrated similar staining patterns in adjacent tissue sections from the same human lacrimal gland specimens. Specific staining for androgen receptors was observed in the nucleus and cytoplasm of lacrimal acinar cells, as well as in lacrimal duct cells. Both the intensity of staining and the number of cells demonstrating staining varied among specimens. We also observed staining for androgen receptors in interstitial and inflammatory cells distributed between lacrimal acinar units in some specimens. CONCLUSIONS: Androgen receptors are located in human lacrimal gland acinar cell nuclei as observed in other animals. However, the detection of androgen receptors in lacrimal interacinar interstitial and inflammatory cells suggests that androgens may play a role in modulating the activities of cells other than lacrimal cells within the human lacrimal gland.

Anterior segment prosthesis development: retinal function following anterior segment removal.

Replacement of the entire anterior segment of the eye is a very ambitious and complex endeavor and it is not known whether the retina remains functional when the anterior structures have been removed. We used routine histo-pathologic evaluation and electroretinographic measurements to determine the structural and functional status of rabbit retinas following surgical removal of the internal anterior structures (iris, ciliary body, and lens) and replacement of the vitreous with silicone oil. In some cases, we were able to record both a scotopic and a photopic electroretinographic response as long as 15 weeks after complete removal of the internal anterior segment structures. Although many hurdles remain and more efficacious surgical techniques and biomaterials need to be developed, our results suggest that, in the rabbit, the retina may continue to function in the absence of critical anterior segment structures.

Anterior segment prosthesis development: evaluation of expanded polytetrafluoroethylene as a sclera-attached prosthetic material.

To further the development of a sclera-attached anterior segment prosthesis we investigated the biocompatibility of expanded polytetrafluoroethylene following its surgical implantation into the rabbit sclera. Thin sheets (250 microns) of expanded polytetrafluoroethylene were cut into pieces measuring either 2 x 2, 2 x 4, 2 x 8, or 4 x 8 mm. The pieces were sterilized and placed individually within surgically prepared pockets in the sclera of rabbit eyes. Animals were sacrificed for routine histopathologic evaluation and transmission electron microscopic study of the eyes at 7 days, 14 days, 1 month, and 4 months following surgical implantation. The implants demonstrated excellent compatibility with the sclera and, by 14 days following implantation surgery, exhibited histiocytes, fibroblasts, and blood vessels infiltrating the internodal spaces of the highly porous material. The number of cells and the amount of extracellular matrix material deposited in the implants appeared to increase with time. Transmission electron microscopic studies revealed deposition of collagen within the implants. Our results suggest that expanded polytetrafluoroethylene has potential for use in the development of a sclera-attached prosthetic device.

Immunopathology of reactivation of experimental ocular histoplasmosis.

We have established a non-human primate model of experimental ocular histoplasmosis. This model has been shown to result in chronic lesions that resemble typical 'histo spots' or choroidal scars but that contain infiltrates of lymphocytes for as long as 10 yr following intracarotid injection of live Histoplasma capsulatum. Using this model, we attempted to reactive these late choroidal lesions via intracarotid challenge with specific antigen (heat-killed H. capsulatum). No clinical changes suggestive of reactivation of these lesions were observed following this antigenic challenge. However, immunopathologic analysis of choroidal lesions at 1, 3 and 7 days after antigenic challenge revealed significant increases in both the numbers of inflammatory cells and the relative percentages of the helper/inducer lymphocyte and macrophage populations. Our results demonstrate that, following antigenic challenge, a cellular change, consistent with a type IV delayed hypersensitivity, can be observed in previously active, but clinically quiescent, histoplasmosis lesions. In light of the many parallels between our primate experimental model and human ocular histoplasmosis, our findings suggest that, in the human, significant immunopathologic activity may occur subclinically in the choroid of affected individuals. It is possible that repeated bouts of subclinical reactivation may induce or enhance chronic choroiditis and, over many years, ultimately produce slow progressive damage to the Bruch's membrane/retinal pigment epithelium complex, resulting in clinically 'active' macular disease and, in selected cases, subretinal neovascularization.

Immunopathology of chronic experimental histoplasmic choroiditis in the primate.

A nonhuman primate model of ocular histoplasmosis was developed that enabled the authors to define the choroidal cellular immunopathology of both the acute and chronic phases of experimental histoplasmic choroiditis. Anti-human monoclonal antibodies were used to identify the inflammatory cell subsets and to calculate their relative percentages in the choroidal inflammatory lesions. Comparison of the acute (less than or equal to 65 days) and chronic (greater than or equal to 1 yr) phases suggested possible variations in the evolution of these lesions, resulting in the development of immunopathologically distinct chronic lesions. In this model, these late lesions could be differentiated by the presence or absence of dense lymphocytic foci, comprised predominantly of mature B-lymphocytes, located within the more diffuse inflammatory cell background. The chronic lesions containing these B-cell foci had significantly higher percentages of both mature B-cells (P less than 0.0001) and helper-inducer T-cells (P less than 0.05) than did the chronic lesions without B-cell foci. The increase in helper-inducer T-cells in the chronic lesions with B-cell foci resulted in a higher mean helper-suppressor T-cell ratio (mu = 0.60) than that seen in lesions lacking foci (mu = 0.33). These findings suggest that, even in the same eye, individual chronic histoplasmic choroidal lesions, which clinically resemble "histo spots" in humans, may have different immunopotentials.

Propionibacterium acnes-enhanced lens-induced granulomatous uveitis in the rat.

Propionibacterium acnes (Corynebacterium parvum) is being implicated more frequently as a cause of intraocular inflammation following cataract surgery. In addition to its role as an infectious agent, P. acnes also may possess adjuvant-like or adjuvant-enhancing properties. The presence of this organism in an eye with residual lens material after extracapsular cataract surgery could augment inflammation resulting from a phacoantigenic (phacoanaphylactic) response. We have modified an established rat model of lens-induced granulomatous uveitis (LIGU) to examine the adjuvant properties of P. acnes. Our results suggest that P. acnes effectively potentiates LIGU.

Chronic anaerobic bacterial endophthalmitis in pseudophakic rabbit eyes.

Experimental anaerobic bacterial endophthalmitis was produced in pseudophakic and aphakic rabbits by using anterior chamber inoculation of 2.5 x 10(6) Propionibacterium acnes organisms. Clinical inflammation was more intense and prolonged in operated eyes with an intraocular lens in place. The presence of an intraocular lens favors the development of chronic low-grade P. acnes-related inflammation.

Oval host wounds and postkeratoplasty astigmatism.

Penetrating keratoplasty frequently results in postoperative astigmatism. A rabbit model was used to investigate the relationship between the ovality of the recipient bed and the resulting postoperative astigmatism as measured by keratometry. Animals were grouped based on the extent that the recipient bed deviated from a 7.25-mm diameter circle (ovality of bed). All animals received a round 7.75-mm donor corneal button. With increasing ovality of the recipient bed, there was a corresponding increase in the postoperative astigmatism at 3 months after all sutures were removed.

Anaerobic bacterial endophthalmitis in the rabbit.

Anaerobic bacterial endophthalmitis was studied in rabbits following intravitreal injection of live Fusobacterium necrophorum. Clinical response, bacterial recovery, and histopathology were studied. An inoculum of approximately 50 organisms produced endophthalmitis in 59% of injected eyes, while 1000 or more organisms produced endophthalmitis in 100% of injected eyes. The course and severity of disease seemed to be independent of the concentration of bacteria above a minimal inoculum size. Affected eyes showed progressive endophthalmitis. Histopathologic changes corresponded to the clinical gradation of endophthalmitis, including progressive retinal necrosis.

In vivo biomicroscopy and photography of meibomian glands in a rabbit model of meibomian gland dysfunction.

The usefulness of transillumination and biomicroscopy of the lid margin in assessing meibomian gland dysfunction was studied in a rabbit model. Transillumination of rabbit lids treated with topical epinephrine for 2 to 3 months revealed plugging of the meibomian gland orifice. Plugging appeared to be correlated histopathologically with increased thickness and hyperkeratinization of the ductal epithelium at the orifice. Continued treatment resulted in microcystic changes within the duct, which were not easily discernible by routine examination without the aid of a transilluminator. Cystic changes were correlated with dilation of the duct by retained desquamated cornified cells. Biomicroscopy proved valuable in identifying and documenting early lesions associated with epinephrine-induced meibomian gland dysfunction.

Radial keratotomy in non-human primate eyes.

We performed a prospective study of the effects of radial keratotomy in the owl monkey. We compared a 16-incision and an eight-incision radial keratotomy, and followed the changes in corneal curvature, corneal thickness, endothelial cell counts, and intraocular pressure. We compared the results of the changes in these clinical factors with a histopathologic and ultrastructural analysis of the time-related changes after radial keratotomy in another group of animals. We found that the loss of initial corneal flattening following radial keratotomy corresponded with the contracture of the wound as demonstrated by histopathologic and ultrastructural study. This procedure results in a significant endothelial cell loss (14% to 15%), which is a result of the postsurgical inflammation associated with this surgery. Additionally, examination of the histopathologic structure of these corneas showed a high level of variability in the surgical incision depth, which we believe is responsible for the marked variations in the response to the surgical procedure.

Compartmentalized growth of hemopoietic stem cells within mouse Friend leukemic spleens.

A dose of 4000 rads (r) to the central portion of mouse spleens followed by Friend erythroleukemic virus infection created independent compartments where hemopoietic stem cells exhibited distinct growth kinetics. Rather than suggesting autonomous proliferation, the stem cell kinetics were indicative of the control exercised by the local microenvironment upon stem cell growth within each compartment.

Hemopoietic stem growth on a capillary stage.

A hollow fibre capillary stage was used for the maintenance and renewal of hemopoietic stem cells in extra corporeal conditions. The partial success of this technique is due to the preservation of cell-cell contacts and interactions within the tissue sections.