Stephen J. Ryan

Birdshot Retinochoroidopathy Associated with Hla-A29 Antigen and Immune Responsiveness to Retinal S-Antigen

Antigen HLA-A29 was present in 16 of 20 patients (80%) with birdshot retinochoroidopathy, but only in 31 of 418 controls (7.4%) (P less than .0001). The relative risk for this disease in persons possessing HLA-A29 antigen was 49.9, one of the highest reported. Of the 20 patients, 13 were also tested for evidence of an in vitro mitotic immune response to purified retinal S-antigen. Of these, 12 (92.3%) responded to the purified preparation, whereas one responded only to a crude retinal homogenate. The histopathologic and clinical findings in an eye from one of the responders to the S-antigen were similar to those in the disease induced in monkeys with the S-antigen. Our findings suggested that birdshot retinochoroidopathy has a genetic predisposition and that retinal autoimmunity, resulting from the S-antigen or other retinal antigens, plays a role in the manifestation of this disease.

Postoperative Intraocular Pressure Abnormalities in the Silicone Study: Silicone Study Report 4

BACKGROUND Chronically abnormal intraocular pressure (IOP) may follow surgery for proliferative vitreoretinopathy (PVR), using either long-acting gas or silicone oil tamponade. Its prevalence and clinical significance are unclear. METHODS In the Silicone Study, 241 eyes with severe (> or = C-3) PVR were treated with vitrectomy, randomized to perfluoropropane gas (C3F8) or silicone oil, and followed for 6 months or longer. Chronic IOP abnormalities, based on findings at two consecutive or any three postoperative visits, were defined as (1) low IOP (hypotony), 5 mmHg or less, or (2) elevated IOP, more than 25 mmHg. RESULTS Eleven (5%) eyes had chronically elevated IOP and 58 (24%) had chronic hypotony. Chronically elevated IOP was more prevalent in eyes randomized to silicone oil than in those randomized to C3F8 gas (8% versus 2%; P < 0.05). Chronic hypotony was (1) more prevalent in eyes randomized to C3F8 gas than in those randomized to silicone oil (31% versus 18%; P < 0.05); (2) more prevalent in eyes with anatomic failure (48% versus 16%; P < 0.01); and (3) correlated with poor postoperative vision (P < 0.0001), corneal opacity (P < 0.001), and retinal detachment (P < 0.001). Factors prognostic of chronic hyotony included preoperative hypotony (P < 0.01), diffuse contraction of the retina anterior to the equator (P < 0.01), rubeosis (P = 0.02), and large retinal breaks (P = 0.02). In a multivariate analysis, diffuse contraction of the retina anterior to the equator remained an independent factor prognostic of chronic hypotony (odds ratio = 4.2), regardless of whether the retina was attached postoperatively. CONCLUSION Intraocular pressure abnormalities are a common postoperative complication in eyes with PVR, and may occur with either C3F8 gas or with silicone oil. The presence of diffuse contraction of the retina anterior to the equator should alert the vitrectomy surgeon that the eye is likely to be hypotonus postoperatively.

A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.

We provide our detailed, standardized in vitro protocol for the culture and differentiation of human retinal pigment epithelial (RPE) cells into a highly polarized and functional monolayer. Disruption of the polarized RPE function plays an important role in the pathogenesis of common blinding disorders of the retina. The availability of this polarized RPE monolayer allows for reproducible evaluation of RPE function, modeling of RPE dysfunction in retinal disease and in vitro evaluation of new therapies. The protocol, which takes approximately 6 weeks to complete, describes the culture of RPE from human fetal donor eyes, the differentiation of these cells into a polarized monolayer with high transepithelial resistance and morphologic characteristics that mimic the RPE monolayer in vivo. By modifying the procedure for initial isolation of pure RPE cells and the culture conditions used in existing protocols, we have established a standardized protocol that provides highly reproducible RPE monolayers from the same donor eye.

Post-traumatic proliferative vitreoretinopathy. The epidemiologic profile, onset, risk factors, and visual outcome

PURPOSE The purpose of the study was to characterize the clinical development of proliferative vitreoretinopathy (PVR) after trauma in the human eye. METHODS A chart review was performed on the records of 1564 patients with ocular trauma seen at a large metropolitan hospital. The frequency, type of ocular trauma, time to onset, potential risk factors, and visual outcome for PVR were evaluated. RESULTS Proliferative vitreoretinopathy occurred in 71 (4%) of 1654 injured eyes. Of these 71 injured eyes, 30 (42%) resulted from rupture, 15 (21%) from penetration, 13 (18%) from perforation, and 7 (10%) from confusion. Six (9%) were associated with an intraocular foreign body (IOFB). The frequency of PVR following perforation, rupture, penetration, IOFB, and contusion was 43%, 21%, 15%, 11%, and 1%, respectively. Overall, those eyes that developed PVR had a poorer visual outcome, with PVR being the primary reason for visual loss. The time from injury to onset of PVR was shortest after perforation (median, 1.3 months), followed by rupture (2.1 months), IOFB (3.1 months), penetration (3.2 months), and contusion (5.7 months). Vitreous hemorrhage was the strongest independent predictive factor for the development of PVR. A long, posteriorly located wound and persistent intraocular inflammation were also important risk factors for PVR. CONCLUSIONS These results suggest that PVR is a common complication following a variety of ocular injuries, and that it is associated with a poor visual outcome. Its frequency, onset, and outcome are strongly dependent on the nature of the trauma. Specific high-risk groups are identified as candidates for more aggressive therapy.

METHOD OF PRODUCTION AND NATURAL HISTORY OF EXPERIMENTAL POSTERIOR PENETRATING EYE INJURY IN THE RHESUS MONKEY

We developed an experimental model for a posterior penetrating eye injury that resulted in traction retinal detachment in 21 rhesus monkey eyes. The standard injury was an incision through the pars plana with vitreous prolapse and incarceration; the wound was then carefully closed with microsurgical techniques. At one to two weeks after injury, detachment of the posterior vitreous occurred and detachment of the retina occurred between seven and 11 weeks. The development of retinal detachment was related to traction on the peripheral retina over the vitreous base; and to the contraction of epiretinal membranes on the peripheral and equatorial retina. Our study supports clinical observations on the importance of blood in the vitreous in the development of vitreous traction and traction retinal detachment after a posterior penetrating injury.

Matrix Metalloproteinase-9-Dependent Exposure of a Cryptic Migratory Control Site in Collagen is Required before Retinal Angiogenesis

Retinal neovascularization is a leading cause of human blindness. However, little is known concerning the molecular mechanisms controlling retinal neovascularization in vivo. Here we provide evidence that exposure of a collagen type IV cryptic epitope detected by monoclonal antibody (mAb) HUIV26, delineates sites of vascular bud formation and represents one of the earliest structural remodeling events required before vessel out-growth. Exposure of these cryptic sites was inhibited in matrix metalloproteinase (MMP)-9-deficient but not MMP-2-deficient mice implicating MMP-9 in their exposure. Retinal endothelial cell interactions with the HUIV26 epitopes induced endothelial cell migration, which was blocked by mAb HUIV26. Importantly, subcutaneous administration of mAb HUIV26 potently inhibited retinal angiogenesis in vivo. Taken together, these findings suggest a novel mechanism in which MMP-9 facilitates exposure of HUIV26 cryptic sites, thereby promoting retinal endothelial cell migration and neovascularization in vivo.

Novel growth factors involved in the pathogenesis of proliferative vitreoretinopathy.

Aims To determine whether hepatocyte growth factor (HGF) and connective tissue growth factor (CTGF) are expressed in human specimens of proliferative vitreoretinopathy (PVR) and to propose a model of PVR pathogenesis based upon the known activities of these growth factors.Methods Immunohistochemical methods (ABC Elite) were used to demonstrate the presence of HGF and CTGF in cryostat sections of five human PVR membranes.Results In each of the five PVR membranes, stromal cells were immunohistochemically positive for both HGF and CTGF. Based upon this information and the known actions of these growth factors, a model of PVR pathogenesis was developed. In this model, injury of the retina induces an inflammatory response that upregulates HGF expression inducing the formation of multilayered groups of migratory retinal pigment epithelial cells (RPE). These RPE, present in a provisional extracellular matrix, come in contact with vitreous containing TGF-β. The TGF-β is activated, upregulating expression of CTGF. Under the influence of TGF-β and CTGF, RPE become myofibroblastic and fibrosis ensues. Retinal traction induces further detachment continuing the cycle of retinal injury.Conclusions HGF and CTGF are expressed in PVR membranes and may play important roles in the pathogenesis of PVR. The expression and function of these growth factors should be critically examined in human PVR specimens, in in vitro cultures of RPE, and in animal models of PVR.

Histology of wound, vitreous, and retina in experimental posterior penetrating eye injury in the rhesus monkey.

We performed a histologic study to support our clinical observations on the mechanisms responsible for traction retinal detachment after a penetrating injury in the rhesus monkey eye. The monkey eyes (40 eyes; 40 monkeys) were characterized by intraocular fibrosis with the formation of a cyclitic membrane and epiretinal and subretinal membranes. The progression to a fibrous ingrowth from the wound occurred only in eyes with blood in the vitreous. The intravitreal fibroblastic proliferation had its origin mainly from the stroma of the ciliary body and choroid at the wound but probably also from the nonpigmented ciliary epithelium. A fibroblastic response was present within the vitreous as early as four days after injury, and had progressed to form a cyclitic membrane by six weeks. Epiretinal membranes were identified as early as four weeks after injury. They were most prominent over the peripheral retina anterior to the equator. It is likely that they are derived from multiple cellular sources including the fibrous ingrowth from the wound but they were also connected to the surface of the retina by bridges of tissue indicating a glial origin. The subretinal membranes appeared to be derived from both retinal pigment epithelium cells and glial cells.

αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells

αB Crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether αB crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular αB crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that αB crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of αB crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced αB crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, αB crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that αB crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of αB crystallin to the basolateral side. In normal mouse retinal sections, αB crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous αB crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. αB Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for αB crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of αB crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for αB crystallin in ocular diseases.

Formation of Drusen in the Human Eye

Light and electron microscopy of drusen formation in the human eye showed yellow-white spots in the fundus with two morphologic patterns: that of typical drusen and a nodular accumulation of cellular components beneath the retinal pigment epithelial cells. By electron microscopy, the progression of drusen formation could be classified into four stages. Stage I showed budding or evagination of retinal pigment epithelial cells into the subpigment epithelial space. This evaginated portion was connected to the retinal pigment epithelial cell cytoplasm and was surrounded by its basement membrane. In Stage II the evaginated portion of the cell was completely separate from the cytoplasm of its parent retinal pigment epithelial cell. In Stage III, the evaginated portion showed degeneration and disintegration. Finally, in Stage IV, an accumulation of vesicular, granular, tubular, and linear material was seen free within the nodular space beneath the retinal pigment epithelial cell.