Lawrence Rife

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.

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.

Technique for preparation of the corneal endothelium-Descemet membrane complex for transplantation.

Purpose: Replacing diseased corneal endothelium with a preparation of Descemet membrane carrying functional endothelium and no stroma may be a feasible method for treating corneal endothelial decompensation. To obtain a viable donor of a Descemet membrane endothelium disc, we modified the Descemet membrane stripping technique and monitored the percentage of endothelial damage to the donor tissue preparation. Methods: Forty-eight human corneas were used. Cornea buttons were mounted on an artificial anterior chamber, endothelial side up. Endothelia were stained with alizarin red, examined under the microscope, and photographed at 5 different sites (microscope, ×100; digital magnification, ×2.83). A 6 × 7-mm rectangular piece of endothelium-Descemet membrane complex was obtained using a Grieshaber microsurgical knife and Kelman-McPherson forceps. Digital photographs of endothelia were analyzed with a computer, and the percentage of endothelial damage was calculated. Specimens were processed for hematoxylin-eosin staining. Results: Forty of 48 endothelium-Descemet membrane preparations (83.3%) were complete peels with minimal endothelial damage. Endothelial damage before and after the surgery was 1.57 ± 2.11% and 2.61 ± 1.77%, respectively. Eight preparations (16.7%) failed because of tearing. Multiple hematoxylin-eosin-stained sections showed the presence of endothelium with intact Descemet membrane and no stromal tissue. Conclusion: We modified the technique of Melles and obtained a sheet of Descemet membrane and endothelium with minimal endothelial damage and with no remaining stroma observed. This simple technique can be used to obtain the endothelium-Descemet membrane complex in minutes. It may be useful for corneal endothelium transplantation.

Visual Arrestin 1 Contributes to Cone Photoreceptor Survival and Light Adaptation

PURPOSE To evaluate morphologic and functional contributions of Arrestin 1 (Arr1) and Arrestin 4 (Arr4) in cone photoreceptors, the authors examined the phenotypes of visual arrestin knockout mice (Arr1(-/-), Arr4(-/-), Arr1(-/-)Arr4(-/-) [Arr-DKO]) reared in darkness. METHODS Retinal rods and cones were evaluated in wild-type (WT), Arr1(-/-), Arr4(-/-), and Arr-DKO mice using quantitative morphologic analysis, immunoblot, immunohistochemistry, TUNEL, and electroretinographic (ERG) techniques. RESULTS Compared with either Arr4(-/-) or WT, Arr1(-/-) and Arr-DKO mice had increased apoptotic nuclei in their retinal outer nuclear layer (ONL) at postnatal day (P) 22. By P60, cone density was significantly diminished, but the ONL appeared normal. After 1 minute of background illumination, cone ERG b-wave amplitudes were similar in WT and all Arr KO mice. However, by 3 minutes and continuing through 15 minutes of light adaptation, the cone b-wave amplitudes of WT and Arr4(-/-) mice increased significantly over those of the Arr1(-/-) and Arr-DKO mice, which demonstrated no cone b-wave amplitude increase. In contrast, ERG flicker analysis after the 15-minute light adaptation period demonstrated no loss in amplitude for either Arr1(-/-) or Arr4(-/-) mice, whereas Arr-DKO had significantly lower amplitudes. When Arr1 expression was restored in Arr1(-/-) mice (+p48(Arr1-/-)), normal cone density and light-adapted ERG b-wave amplitudes were observed. CONCLUSIONS In the adult dark-reared Arr1(-/-) and Arr-DKO mice, viable cones diminish over time. Arr1 expression is essential for cone photoreceptor survival and light adaptation, whereas either Arr1 or Arr4 is necessary for maintaining normal flicker responses.

Carboxypeptidase E is required for normal synaptic transmission from photoreceptors to the inner retina

Defects in the gene encoding carboxypeptidase E (CPE) in either mouse or human lead to multiple endocrine disorders, including obesity and diabetes. Recent studies on Cpe–/– mice indicated neurological deficits in these animals. As a model system to study the potential role of CPE in neurophysiology, we carried out electroretinography (ERG) and retinal morphological studies on Cpe–/– and Cpefat/fat mutant mice. Normal retinal morphology was observed by light microscopy in both Cpe–/– and Cpefat/fat mice. However, with increasing age, abnormal retinal function was revealed by ERG. Both Cpe–/– and Cpefat/fat animals had progressively reduced ERG response sensitivity, decreased b‐wave amplitude and delayed implicit time with age, while maintaining a normal a‐wave amplitude. Immunohistochemical staining showed specific localization of CPE in photoreceptor synaptic terminals in wild‐type (WT) mice, but in both Cpe–/– and Cpefat/fat mice, CPE was absent in this layer. Bipolar cell morphology and distribution were normal in these mutant mice. Electron microscopy of retinas from Cpefat/fat mice revealed significantly reduced spherule size, but normal synaptic ribbons and synaptic vesicle density, implicating a reduction in total number of vesicles per synapse in the photoreceptors of these animals. These results suggest that CPE is required for normal‐sized photoreceptor synaptic terminal and normal signal transmission to the inner retina.

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 |im) of expanded polytetrafluoroethylene were cut into pieces measuring either 2x2, 2x4, 2x8, or 4x8 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.

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.

SLOWED PHOTORESPONSE RECOVERY AND AGE-RELATED DEGENERATION IN CONES LACKING G PROTEIN-COUPLED RECEPTOR KINASE 1

The vertebrate retina is a specialized neural network that contains very sensitive signal transducers—the rod and cone photoreceptors. Rods function in near darkness (scotopic) and are responsible for dim light vision, while cones operate in bright light (photopic) and provide daytime, high acuity color vision. In the human retina, rods are the dominant photoreceptor cell type and comprise about 95% of all photoreceptor cells, while cones account for only 5% of the cells. Yet in a bright light environment, normal cone function is essential for visual perception since rods become saturated and are rendered nonfunctional.

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.

Técnica de separação da membrana de Descemet para transplante de células endoteliais da córnea: estudo experimental em coelhos

PURPOSE To evaluate the percentage of endothelial cell damage induced during a surgical technique of Descemets membrane separation containing healthy endothelium, analyze the viability and efficacy of this technique, and evaluate the percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber. METHODS The corneas from three groups of 12 New Zealand rabbits were evaluated. The Group one was used as the control, so the corneas were analyzed after collected and trephinated. The Group two was analyzed after inversion of the cornea (endothelial side up at a convex shape) mounted on an artificial anterior chamber to calculate the percentage of endothelial cell damage caused by this inversion. The Group three was evaluated after the separation between the Descemets membrane and the stroma using viscoelastic substance in corneas inverted and mounted on an artificial anterior chamber. The endothelial cell damage was analyzed by digital photographs taken under a microscope after staining the endothelium with alizarin red. Group three samples were processed for histologic evaluation. RESULTS The Group three (viscoelastic separation) showed an index of endothelial cell damage of 10.06%, the Group two showed an index of 3.58% and the control group an index of 0.18% of endothelial cell damage (p<0.05). Histological evaluation of the Group three corneas revealed that approximately a 120 microm thickness of stroma remained attached to the Descemets membrane. CONCLUSION This technique should be better investigated because it is a viable and efficient alternative of Descemets membrane separation for endothelial cells transplantation, since the percentage of induced cell damage is 10.06%. The percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber was 3.58%.