Eleut Hernandez

5-fluorouracil: new applications in complicated retinal detachment for an established antimetabolite.

Long-term reattachment of the retina following the development of proliferative vitreoretinopathy is often prevented by the occurrence of cellular reproliferation. 5-fluorouracil, a synthetic pyrimidine analog, is a potent inhibitor of fibroblast proliferation in cell culture and an animal model of tractional retinal detachment. Doses of up to 1.0 mg, when administered intravitreally to rabbits, result in no demonstrable retinal toxicity by microscopic and electrophysiologic criteria. The first 22 consecutive patients with advanced forms of proliferative vitreoretinopathy were treated with a combination of periocular and intraocular 5-fluorouracil, in addition to scleral buckling and vitrectomy. Retinal reattachment was achieved in 60% of patients at 6 months postoperatively. No serious systemic or ocular complications were observed although delayed healing of corneal epithelial defects occurred in 18% of cases and subtle subepithelial scarring in 31.8%. In combination with standard vitrectomy techniques, post-operative fluid gas exchange, and photocoagulation, periocular and subconjunctival 5-fluorouracil appears to improve the prognosis for longterm retinal reattachment following the development of proliferative vitreoretinopathy.

Accommodation of an Endocapsular Silicone Lens (Phaco-Ersatz ) in the Nonhuman Primate

In owl monkeys, as a model of endocapsular cataract extraction, the lens material was removed through a small hole in the capsule and was replaced with a silicone polymeric gel. The ability of this endocapsular implant (Phaco-Ersatz) to accommodate was documented when pilocarpine produced an increase in the curvature of the lens surface and a decrease in the anterior chamber depth, just as occurs with the untouched natural lens. Refinements of the surgical technique are required to achieve a controlled degree of filling of the capsular bag and inhibit postoperative epithelial proliferation on the inner capsular surface.

Inactivation of astroglial NF‐κB promotes survival of retinal neurons following ischemic injury

Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF‐κB, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer (GCL) after retinal ischemia‐reperfusion in wild‐type (WT) and in GFAP‐IκBα‐dn transgenic mice, where the NF‐κB classical pathway is suppressed specifically in astrocytes. The GFAP‐IκBα‐dn mice showed significantly increased survival of neurons in the GCL following ischemic injury as compared with WT littermates. Neuroprotection was associated with significantly reduced expression of pro‐inflammatory genes, encoding Tnf‐α, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO‐synthase in the retinas of GFAP‐IκBα‐dn mice. These data suggest that certain NF‐κB‐regulated pro‐inflammatory and redox‐active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke.

Tissue plasminogen activator treatment of experimental subretinal hemorrhage.

Previous investigators have suggested that subretinal blood damages the retina in part because of its solid fibrin meshwork. The role of fibrinolysis in facilitating the clearance of subretinal hemorrhage and preventing degeneration of the overlying retina was studied. Autologous whole blood (0.1 ml) was injected into the subretinal space of 20 rabbits. Twenty-four hours later, the animals were randomized to subretinal treatment with 2.5 µg of tissue plasminogen activator or a similar volume of physiologic saline. Mean subretinal hemorrhage thickness 3 days after treatment had decreased to 42% of pretreatment thickness in treated eyes and remained unchanged in control eyes (P < 0.0005). By 7 days mean clot thickness was 9% in treated eyes and 60% in controls (P = 0.005). Light microscopy revealed severe progressive retinal degeneration in both groups. No histologic evidence of retinal toxicity was found in cat retina after subretinal injection of tissue plasminogen activator (50 (µg/ml). Although treatment with tissue plasminogen activator accelerated the clearance of subretinal hemorrhage, it failed to prevent secondary retinal degeneration in this rabbit model.

Tissue Plasminogen Activator Thrombolysis during Surgical Evacuation of Experimental Subretinal Hemorrhage

To assess the role for intraoperative thrombolysis during surgical evacuation of massive subretinal hemorrhage, the authors studied the ability of tissue plasminogen activator (tPA) to facilitate removal of an experimental subretinal blood clot through a small drainage retinotomy. In rabbit eyes, a single subretinal injection of tissue plasminogen activator in concentrations of up to 250 micrograms/ml failed to produce significant (greater than 50%) clot dissolution during a 3-hour period. However, repetitive subretinal lavage and aspiration with tPA (50 micrograms/ml) resulted in progressive intraoperative clot dissolution in rabbits and allowed complete evacuation of blood through a small drainage retinotomy in 6 (100%) of 6 cat eyes. Repetitive vigorous subretinal irrigation with saline solution had no discernible effect on clot size in rabbit eyes. Histopathologic examination of cat eyes following tPA-assisted surgical evacuation of subretinal blood showed preservation of the outer retina in 2 eyes and severe atrophy of the outer retina in 4 eyes.

Induction of Conjunctival Transdifferentiation on Vascularized Corneas by Photothrombotic Occlusion of Corneal Neovascularization

Previous studies have established that conjunctival transdifferentiation (transformation into cornea-like morphology) is inhibited by corneal vascularization. Conversely, occlusion of corneal vessels may induce conjunctival transdifferentiation on vascularized corneas. To test this hypothesis, the corneal epithelia of New Zealand albino rabbits were debrided 3 mm beyond the limbus with n-heptanol. Sixteen corneas healed by conjunctival epithelium, with vascularization persisting for 20 months, were used in this study. Photochemically induced occlusion of the corneal vessels was achieved by intravenous administration of rose bengal-saline solution (40 mg/kg body weight) with subsequent argon laser irradiation of the vessels (514.5 nm, 130 mW, 63 micron and 0.2 sec). The treated vessels remained occluded in an 18-week study, as confirmed by corneal fluorescein angiography. Corneal clarity and epithelial integrity were improved after treatment. Goblet cell loss and morphologic transformation into a cornea-like epithelium were verified by flat-mount preparations, histology, impression cytology, and immunofluorescence studies using a mucin-specific monoclonal antibody. These results indicate that conjunctival transdifferentiation can be induced on vascularized corneas after occlusion of corneal vessels by photothrombosis.

Tissue Plasminogen Activator Treatment of Postoperative Intraocular Fibrin

Intraocular recombinant tissue-type plasminogen activator was used for treatment of postoperative intraocular fibrin clots. Using a rabbit vitrectomy and cyclocryotherapy model of postoperative fibrin, rabbits were randomized on the first postoperative day to receive either an anterior chamber injection of lactated Ringers solution or 25,000, 50,000, or 100,000 IU tissue plasminogen activator (tPA). An intraocular dose of 25,000 IU of tPA produced marked resolution of the fibrin clot 24 hours later. A 50,000 and 100,000 IU dose produced slightly more resolution of the fibrin. No ophthalmoscopic or histologic evidence of intraocular toxicity was seen.

Retinal tumor imaging and volume quantification in mouse model using spectral-domain optical coherence tomography

We have successfully imaged the retinal tumor in a mouse model using an ultra-high resolution spectral-domain optical coherence tomography (SD-OCT) designed for small animal retinal imaging. For segmentation of the tumor boundaries and calculation of the tumor volume, we developed a novel segmentation algorithm. The algorithm is based on parametric deformable models (active contours) and is driven by machine learning-based region classification, namely a Conditional Random Field. With this algorithm we are able to obtain the tumor boundaries automatically, while the user can specify additional constraints (points on the boundary) to correct the segmentation result, if needed. The system and algorithm were successfully applied to studies on retinal tumor progression and monitoring treatment effects quantitatively in a mouse model of retinoblastoma.

The high-mobility group box-1 nuclear factor mediates retinal injury after ischemia reperfusion.

PURPOSE High-mobility group protein B1 (Hmgb1) is released from necrotic cells and induces an inflammatory response. Although Hmgb1 has been implicated in ischemia/reperfusion (IR) injury of the brain, its role in IR injury of the retina remains unclear. Here, the authors provide evidence that Hmgb1 contributes to retinal damage after IR. METHODS Retinal IR injury was induced by unilateral elevation of intraocular pressure and the level of Hmgb1 in vitreous humor was analyzed 24 hours after reperfusion. To test the functional significance of Hmgb1 release, ischemic or normal retinas were treated with the neutralizing anti-Hmgb1 antibody or recombinant Hmgb1 protein respectively. To elucidate in which cell type Hmgb1 exerts its effect, primary retinal ganglion cell (RGC) cultures and glia RGC cocultures were treated with Hmgb1. To clarify the downstream signaling pathways involved in Hmgb1-induced effects in the ischemic retina, receptor for advanced glycation end products (Rage)-deficient mice (RageKO) were used. RESULTS Hmgb1 is accumulated in the vitreous humor 24 hours after IR. Inhibition of Hmgb1 activity with neutralizing antibody significantly decreased retinal damage after IR, whereas treatment of retinas or retinal cells with Hmgb1 induced a loss of RGCs. The analysis of RageKO versus wild-type mice showed significantly reduced expression of proinflammatory genes 24 hours after reperfusion and significantly increased survival of ganglion cell layer neurons 7 days after IR injury. CONCLUSIONS These results suggest that an increased level of Hmgb1 and signaling via the Rage contribute to neurotoxicity after retinal IR injury.

Neuronal NAD(P)H Oxidases Contribute to ROS Production and Mediate RGC Death after Ischemia

PURPOSE To study the role of neuronal nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase-dependent reactive oxygen species (ROS) production in retinal ganglion cell (RGC) death after ischemia. METHODS Ischemic injury was induced by unilateral elevation of intraocular pressure via direct corneal cannulation. For in vitro experiments, RGCs isolated by immunopanning from retinas were exposed to oxygen and glucose deprivation (OGD). The expression levels of NAD(P)H oxidase subunits were evaluated by quantitative PCR, immunocytochemistry, and immunohistochemistry. The level of ROS generated was assayed by dihydroethidium. The NAD(P)H oxidase inhibitors were then tested to determine if inhibition of NAD(P)H oxidase altered the production of ROS within the RGCs and promoted cell survival. RESULTS It was reported that RGCs express catalytic Nox1, Nox2, Nox4, Duox1, as well as regulatory Ncf1/p47phox, Ncf2/p67phox, Cyba/p22phox, Noxo1, and Noxa1 subunits of NAD(P)H oxidases under normal conditions and after ischemia. However, whereas RGCs express only low levels of catalytic Nox2, Nox4, and Duox1, and regulatory Ncf1/p47, Ncf2/p67 subunits, they exhibit significantly higher levels of catalytic subunit Nox1 and the subunits required for optimal activity of Nox1. It was observed that the nonselective NAD(P)H oxidase inhibitors VAS-2870, AEBSF, and the Nox1 NAD(P)H oxidase-specific inhibitor ML-090 decreased the ROS burst stimulated by OGD, which was associated with a decreased level of RGC death. CONCLUSIONS The findings suggest that NAD(P)H oxidase activity in RGCs renders them vulnerable to ischemic death. Importantly, high levels of Nox1 NAD(P)H oxidase subunits in RGCs suggest that this enzyme could be a major source of ROS in RGCs produced by NAD(P)H oxidases.