In situ generation of iminodiacetic acid groups on nanoporous alumina for the reversible immobilization of enzymes and other biomolecules.

Nanoporous alumina membranes were silanized with aminopropylsilane and iminodiacetic acid (IDA) groups were generated in situ by reaction with iodoacetate. The membranes were mounted in standard filter holders, connected to a HPLC system and saturated with selected metal ions. Cu(II) allowed the capture of chicken muscle lactate dehydrogenase with such stability, repeatability and reproducibility that Michaelis-Menten kinetics could be studied. The IDA surface was stable for months and could be depleted and regenerated with metal ions multiple times without appreciable loss of capacity. The binding of lactate dehydrogenase influenced the backpressure to the extent that could be expected for a monolayer according to Poiseuilles law.

Anatomic alterations in aging and age-related diseases of the eye.

PURPOSE: We described anatomic age-related changes in the human eye to determine potential areas of investigation that may lead to identifying eyes at risk for age-related disease. METHODS: A descriptive review of anatomic changes in the eye related to aging was performed in the context of current areas of investigation. The review was performed specifically for differing anatomic ocular structures, including cornea, trabecular meshwork, lens, uveal tract, Bruch's membrane, retina, RPE, vitreous, sclera, and optic nerve. RESULTS: Age-related changes occur in all ocular tissues. The cornea flattens and there is an attrition of endothelial cells. The shape of the trabecular meshwork changes and there is a loss of trabecular endothelium. The lens grows and becomes cataractous. The ciliary body becomes collagenized, there are choroidal vascular changes, and Bruch's membrane thickens. Retinal vessels become hyalinized and there is a loss of rods before cones in the macula. RPE morphometric changes occur with aging. The vitreous becomes liquefied and there is a loss of vitreous compartmentalization. The sclera becomes rigid and may become calcified. The optic nerve exhibits structural changes with age. CONCLUSIONS: There are numerous anatomic age-related changes in the human eye. Current areas of investigation related to these changes include adaptive optics scanning laser ophthalmoscopy imaging of the RPE mosaic in the context of aging, and drug delivery devices that overcome age-related alterations to retinal and macular perfusion.

Subretinal delivery and electroporation in pigmented and nonpigmented adult mouse eyes.

Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, Müller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumulating to high concentrations because there is no dilution due to transport processes or diffusion. Dilution in the interphotoreceptor space (IPS) is minimal because the IPS volume is only 10-20 µl in the human eye and less than 1 µl in the mouse eye. For gene delivery purposes, we wished to transfect the cells adjacent to the IPS in adult mouse eyes. Others transfect these cells in neonatal rats to study the development of the retina. In both neonates and adults, electroporation is found to be effective. Here we describe the optimization of electroporation conditions for RPE cells in the adult mouse eye with naked plasmids. However, both techniques, subretinal injection and electroporation, present some technical challenges that require skill on the part of the surgeon to prevent untoward damage to the eye. Here we describe methods that we have used for the past 10 years (Johnson et al. Mol Vis 14: 2211-2226, 2008).

Animal models of choroidal and retinal neovascularization.

There have been numerous types of animal models of choroidal neovascularization (CNV) and retinal neovascularization (RNV). Understanding the pathobiology of CNV and RNV is important when evaluating and utilizing these models. Both CNV and RNV are dynamic processes. A break or defect in Bruchs' membrane is necessary for CNV to develop. This may be induced with a laser, mechanically via surgery, or in the setting of transgenic mice. Some of the transgenic mouse models spontaneously develop RNV and/or retinal angiomatous proliferation (RAP)-like lesions. The pathogenesis of RNV is well-known and is generally related to ischemic retinopathy. Models of oxygen-induced retinopathy (OIR) closely resemble retinopathy of prematurity (ROP). The streptozotocin (STZ) rat model develops features similar to diabetic retinopathy. This review summarizes general categories and specific examples of animal models of CNV and RNV. There are no perfect models of CNV or RNV and individual investigators are encouraged to choose the model that best suits their needs.

Modulation of choroidal neovascularization by subretinal injection of retinal pigment epithelium and polystyrene microbeads.

PURPOSE: The study was conducted to create a rapidly developing and reproducible animal model of subretinal choroidal neovascularization (CNV) that allows a time-dependent evaluation of growth dynamics, histopathologic features, and cytokine expression. METHODS: C57BL/6 and chemoattractant leukocyte protein-2 deficient (DeltaCcl-2) mice were studied. Mice received single or combined subretinal injections of cultured retinal pigment epithelium (RPE; C57BL/6-derived), polystyrene microbeads, or phosphate buffer solution (PBS). Fluorescence angiograms were performed over a period of 3 weeks. Mice were euthanized on post inoculation day 3, 7, 10, 14, or 21, and their eyes were evaluated by light, confocal, and electron microscopy. RESULTS: CNV membranes occurred in all study groups with an overall incidence of 94.3%. They extended in the subretinal space through central breaks in Bruch's membrane. CNV lesions were characterized by dynamic changes such as initiation, active inflammatory, and involution stages. CNV thickness peaked around PI day 7 and was greater in mice that received combined injections of RPE and microbeads or RPE cells alone. Small lesions developed in the control groups (microbeads or PBS only), in DeltaCcl-2, and old C57BL/6 mice. Variable expression of cytokines and growth factors was detected within the membranes. CONCLUSIONS: Our murine model represents a reliable approach inducing CNV growth by subretinal injection of either RPE cells alone or RPE cells and microbeads. The development of CNV lesions is a dynamic process that relies in part on macrophage trafficking and age.

Technical brief: subretinal injection and electroporation into adult mouse eyes.

PURPOSE: Our goal was to improve and standardize the procedure for subretinal injection of mouse eyes. Also, we wished to optimize conditions for electroporation of retinal pigment epithelium (RPE) cells in the mouse eye with naked plasmids. METHODS: Mouse eyes were injected subretinally with reporter plasmid DNA, nanobeads, or buffer. A blunt needle was introduced across the cornea, through the pupil, into the vitreous, until it made contact with the neural retina. Gentle pressure was applied to the needle, forcing it to puncture the retina and enter the subretinal space. Fluid was injected subretinally, raising large blebs evident on the mouse fundus. Subretinal injection surgery and outcomes were monitored and evaluated by video recording. Vidisic aided in fundus examination of the blebs. Pores in RPE cells, across which the plasmid in the fluid could diffuse, were created by several short electrical bursts. Expression of the delivered gene, tdTomato, in the plasmid was examined under fluorescence microscopy to identify targeted cells. Electroporation conditions were varied from 0 to 200 V, 5 to 10 pulses, 0.1 ms to 100 ms duration of each pulse, and a space of 1.5 to 2 mm between electrodes on the cornea and sclera. RESULTS: A critical sign of surgical success was the appearance and persistence of three large blebs in the mouse eye. This was illustrated by video recordings from two different systems. Application of Vidisic to the cornea made immediate examination of the fundus possible at the end of the surgery. An 80% success rate was readily achieved by following this method. Once a successful subretinal injection technique was established, electroporation conditions were evaluated. Parameters of 50 V, 1 ms pulse duration, 5-10 pulses, 1 s apart and electrodes spaced 1.5-2 mm apart with the anode placed on the sclera in the vicinity of the blebs produced a tight pattern of RPE transfection at approximately 30% efficiency. CONCLUSIONS: A standardized surgical method and a fast distinct indicator of successful surgery were essential to establishing a gene delivery system based on subsequent electroporation. With the surgery better controlled, electroporation was adequate to transfect a substantial number of RPE cells in a defined position in the globe.

Expression of JAM-A in the human corneal endothelium and retinal pigment epithelium: localization and evidence for role in barrier function.

PURPOSE: Junctional adhesion molecules (JAMs) are a family of adhesion proteins found in intercellular junctions. Evidence suggests that JAM-A is important for the regulation of tight junction assembly and epithelial barrier function. The authors recently reported that JAM-A is expressed in rabbit corneal endothelium and that antibody to JAM-A produces corneal swelling. In the present study, they investigate JAM-A expression in the human corneal endothelium and retinal pigment epithelium (RPE) and examine the effect of a function-blocking antibody to JAM-A on the permeability of cultured RPE cell monolayers. METHODS: Expression of JAM-A in human corneal endothelium, human RPE tissue, and cultured ARPE-19 monolayers was assessed by immunofluorescence confocal microscopy. Localization of JAM-A was compared with the tight junction-associated protein zonula occludens-1 (ZO-1). To investigate JAM-A function in ARPE-19 cells, ARPE-19 monolayers were subjected to a calcium switch protocol to disrupt cell junctions and treated with a function-blocking antibody to JAM-A or an isotype-matched control. Dextran flux assays were performed to assess the effect of JAM-A antibody on ARPE-19 monolayer permeability. RESULTS: Expression of JAM-A was observed in human corneal endothelium, and its distribution correlated with the tight junction-associated protein ZO-1. In addition, expression of JAM-A was observed in human RPE and in intercellular junctions of ARPE-19 monolayers. The localization pattern of JAM-A in the RPE and ARPE-19 monolayers was similar to that of ZO-1. ARPE-19 monolayers treated with antibody to JAM-A demonstrated a 33% increase in permeability to 10,000 MWt dextran compared with monolayers treated with control antibody. CONCLUSIONS: Results of this study provide new information about JAM-A expression in tight junctions of the human corneal endothelium and human RPE. The observation that antibodies to JAM-A increase ARPE-19 monolayer permeability is consistent with previous findings of JAM-A function in epithelial tight junctions and suggests JAM-A may have a role in the regulation of RPE barrier function.

Preoperative sub-Tenon's capsule injection of ropivacaine in conjunction with general anesthesia in retinal detachment surgery.

OBJECTIVE: To evaluate the effects of preoperative sub-Tenon's capsule injection of ropivacaine on intraoperative hemodynamics, postoperative pain, nausea, and recovery in patients undergoing scleral buckling surgery under general anesthesia (GA). DESIGN: Randomized double-masked controlled clinical trial. PARTICIPANTS: Ninety-eight patients with primary rhegmatogenous retinal detachment undergoing scleral buckling surgery under GA. METHODS: Random allocation to either preoperative sub-Tenon's capsule injection of 3 ml of 0.75% ropivacaine or sub-Tenon's capsule injection of 3 ml of saline (controls) immediately before a scleral buckling procedure under GA. Intraoperative monitoring of hemodynamic parameters, need of analgesia with sevoflurane and alfentanil, time in the recovery unit, measurements of pain and nausea on the visual analog scale (VAS) up to 12 hours postoperatively, and consumption of analgesics and antiemetics was recorded. MAIN OUTCOME MEASURES: Intraoperative systolic blood pressure (BP); bradycardia; minimum alveolar concentration (MAC) of sevoflurane; maximum postoperative VAS scores of pain and nausea; time in recovery unit; and total need of alfentanil, ketobemidone, dextropropoxyphene, and dixyrazine. RESULTS: Ninety-seven patients were analyzed (48 in the ropivacaine group and 49 controls). A significantly lower intraoperative systolic BP (104+/-6 vs. 112+/-7 mmHg; P = 0.004), less need of sevoflurane (1.33+/-0.19 vs. 1.56+/-0.23; P = 0.03), and shorter time in the recovery unit (67+/-9 vs. 76+/-16 minutes; P = 0.01) were observed in the ropivacaine group. Maximum VAS pain scores were 50+/-21 in the control group and 36+/-25 in the ropivacaine group (P = 0.05), with a significantly lower consumption of opioids (ketobemidone) in the ropivacaine group (3.6+/-3.5 vs. 1.3+/-2.0 mg). No significant difference was observed regarding nausea or need of dixyrazine or dextropropoxyphene postoperatively. CONCLUSIONS: Preoperative sub-Tenon's capsule injection of ropivacaine in scleral buckling surgery under GA lowers the intraoperative systolic BP, reduces the amount of inhalable sevoflurane needed, and enhances postoperative vigilance through reduction of pain and need of opioids.

Detection of PEDF in subretinal fluid of retinal detachment: possible role in the prevention of subretinal neovascularization. preliminary results.

UNLABELLED: PEDF (pigment-epithelium-derived factor) is a member of the serpin family of protease inhibitors. It is considered to be an important regulator of human eye disease and is known to inhibit angiogenesis. We have therefore investigated the presence of PEDF in the subretinal fluid of patients with retinal detachment. METHODS: Eighteen samples from SRF were collected from patients during retinal detachment surgery. Specific ELISA analysis was performed with specific IgG against human PEDF. RESULTS: PEDF was detected in the subretinal fluid of all cases. The mean concentration of PEDF was 33.9 ng/ml (SD 23.7 ng/ml; range 5.3-74.7 ng/ml). The majority of samples had however a concentration of more than 22 ng PEDF/ml fluid. CONCLUSION: PEDF appears to be a constant component of the fluid accumulating in the subretinal space after retinal detachment. The known effects of PEDF, however, suggest that it may be involved in physiological processes of wound healing in the subretinal space.

Choroidal biopsies for intraocular tumors of indeterminate origin.

PURPOSE: To evaluate the role of pars plana vitrectomy-assisted incisional biopsies in the management of choroidal tumors of unclear origin. DESIGN: Retrospective, noncomparative, consecutive interventional case series. METHODS: Ten consecutive patients with indeterminate choroidal tumors underwent a standardized three-port pars-plana vitrectomy-assisted subretinal biopsy using a bimanual approach with standard intraocular forceps and a diamond knife. Specimens were fixed in formaldehyde embedded in paraffin and further subjected to histopathological and immunohistochemical analyses. RESULTS: A histologic diagnosis was obtained in all (10 of 10) cases including choroidal melanoma (five of 10), metastasis (two of 10), subretinal hemorrhage (two of 10), and nodular scleritis (one of 10). Five eyes were enucleated as a result of the histologic diagnosis. Three cases of postoperative complications were seen in three patients (newly formed rhegmatogenous retinal detachment, increased serous retinal detachment, and vitreous hemorrhage). No cases of intra- or extraocular tumor spread were detected through follow-up periods ranging from 3 to 29 months. CONCLUSIONS: Pars plana vitrectomy-assisted incisional biopsy is a valuable diagnostic procedure for cases of choroidal tumors of unknown origin in selected patients. However, the relatively high frequency of postoperative complications noted in the present study and the potential risk of dissemination of tumor cells underscores the importance of rigorous case selection.

Detection of connective tissue growth factor in subretinal Fluid following retinal detachment: possible contribution to subretinal scar formation, preliminary results.

UNLABELLED: Connective tissue growth factor (CTGF) has been shown to be substantially involved in various processes of fibrosis. Herein we report on the presence of CTGF in the subretinal fluid (SRF) of patients with retinal detachment. METHODS: Samples of SRF were collected from 10 patients during retinal detachment surgery. Specific ELISA analysis was performed with goat IgG against human CTGF. RESULTS: CTGF was above the detection limit of the assay in all samples. On average the concentration of CTGF in SRF was 10 ng/ml (SD 3.7, range 3.7-15.7 ng/ml). There was an increase in the CTGF concentration with time between the diagnosis of retinal detachment and surgery (correlation r = 0.67). CONCLUSION: CTGF appears to be a constant component of the fluid accumulating in the subretinal space after retinal detachment. The origin of subretinal CTGF and its physiological importance is still unclear. The known effects of CTGF, however, suggest that it may be involved in both the physiological processes of wound healing in the subretinal space and also in the pathological events such as subretinal fibrosis. The observed increase in CTGF concentration with time suggest that CTGF plays a role in the pathophysiology of subretinal scarring in cases of delayed retinal surgery.

Subthreshold transpupillary thermotherapy reduces experimental choroidal neovascularization in the mouse without collateral damage to the neural retina.

PURPOSE: Transpupillary thermotherapy (TTT) is currently being evaluated for treatment of choroidal neovascularization (CNV) in age-related macular degeneration. To optimize TTT for CNV, the effect was analyzed of invisible (subthreshold) or visible (threshold) doses of TTT on the normal mouse retina and on experimental CNV. METHODS: TTT was delivered to the normal retina of 42 mice with a diode laser at increasing power settings (50, 60, 70, or 80 mW), to obtain thermal lesions ranging from invisible (subthreshold) to visible (threshold) burns. CNV was induced in 53 mice by krypton laser photocoagulation of the fundus, after which the CNV lesions were treated with TTT (50, 60, or 80 mW). Eyes were enucleated 7 days after TTT and prepared for histology, and the CNV complex was evaluated on hematoxylin-eosin stained serial sections by measuring the maximum height of the CNV lesions. Ultrastructural changes were examined by transmission electron microscopy. RESULTS: Increasing the TTT laser power yielded gradually more visible effects. At 50 mW, which induced subthreshold burns, no damage was seen in the neural retina, retinal pigment epithelium (RPE), or choroid at any time point. By contrast, eyes treated with higher power exhibited progressively more damage to the neural retina, including a complete disruption of the outer nuclear layer. When TTT was applied to the laser-induced CNV lesions, the height of lesions was significantly reduced (P < 0.001) in response to all three power settings at 7 days after treatment. The mean relative thickness of the CNV lesion was 3.29 +/- 0.89 in untreated mice, whereas in TTT-treated mice it was 1.69 +/- 0.35, 1.69 +/- 0.41 and 1.70 +/- 0.17 at power settings of 50, 60, and 80 mW, respectively. The overlying neural retina showed no apparent damage with the 50- or 60-mW settings, whereas outer nuclear layer disruption occurred with a power of 80 mW. Electron microscopy confirmed the presence of vascular occlusion at 1 day and a fibrotic scar at 7 days after TTT. CONCLUSIONS: Subthreshold TTT can effectively occlude newly formed vessels and cause regression of the experimental CNV complex without damaging the neural retina. The results demonstrate the importance of using subthreshold laser power in experimental and clinical evaluation of TTT.

Reduced choroidal neovascular membrane formation in matrix metalloproteinase-2-deficient mice.

PURPOSE: Findings in studies have suggested a role for matrix metalloproteinase (MMP)-2 in angiogenesis, including choroidal neovascularization (CNV). To investigate further, the current study was conducted to observe the formation of experimental CNV in MMP-2-deficient mice. METHODS: CNV was induced in wild-type and MMP-2-deficient mice by krypton laser photocoagulation of the fundus. The time-course of expression of MMP-2 mRNA after laser treatment was determined by in situ hybridization with anti-sense and sense cRNA probes. MMP-2 protein distribution was determined by immunohistochemistry. Ten days after treatment, the extent of CNV was evaluated on hematoxylin-eosin stained serial sections. The maximum height of the CNV lesions was calculated by image analysis of digitized histologic images. RESULTS: Expression of MMP-2 mRNA was detected in the CNV lesions at day 3 after laser treatment and peaked at day 5, after which it slowly declined. MMP-2 mRNA expression appeared to be highest at the margins of the membrane. Immunostaining for MMP-2 confirmed the presence of MMP-2 protein in the CNV lesions. The CNV lesions of MMP-2-deficient mice showed that relative thickness was reduced by 31% compared with wild-type mice (P = 0.006). CONCLUSIONS: The present study demonstrated that MMP-2 mRNA and protein are upregulated during experimental CNV in the mouse. The marked difference in thickness of the CNV membrane between wild-type and MMP-2-deficient mice shows that MMP-2 is involved in the formation of experimental CNV in the mouse. These results suggest that pharmacologic targeting of MMPs, including MMP-2, may reduce formation of CNV in conditions such as age-related macular degeneration.