Eye Perfusion Research Articles

Consensus Recommendations for Studies of Outflow Facility and Intraocular Pressure Regulation Using Ex Vivo Perfusion Approaches.

Intraocular pressure (IOP) elevation is the primary risk factor and currently the main treatable factor for progression of glaucomatous optic neuropathy. In addition to direct clinical and living animal in vivo studies, ex vivo perfusion of anterior segments and whole eyes is a key technique for studying conventional outflow function as it is responsible for IOP regulation. We present well-tested experimental details, protocols, considerations, advantages, and limitations of several ex vivo model systems for studying IOP regulation. These include: (1) perfused whole globes, (2) stationary anterior segment organ culture, (3) perfused human anterior segment organ culture, (4) perfused animal anterior segment organ culture, (5) perfused human corneal rims, and (6) perfused human anterior segment wedges. These methods, with due consideration paid to their strengths and limitations, comprise a set of very strong tools for extending our understanding of IOP regulation.

Evaluation of Macular Perfusion Analyzed by Optical Coherence Tomography Angiography after Uncomplicated Phacoemulsification

Abstract Background Age related cataract is one of the commonest causes of diminution of vision worldwide. Phacoemulsification is one of the most common surgical procedures in the world1. Changes in arterial blood pressure, position of blood vessels, venous return of blood and CO2 levels all influence eye perfusion during cataract surgery. The exact impact of phacoemulsification on macular perfusion is unknown2&3. Purpose To investigate the changes of macular perfusion parameters after uncomplicated phacoemulsification using an optical coherence tomography angiography (OCT-A). Subjects and Methods Thirty eyes of 30 cataractous patients, who had uneventful phacoemulsification with implantation of IOL in the bag, were enrolled in this cohort study. Participants underwent a complete ophthalmologic assessment and measurement of macular perfusion parameters using optical coherence tomography angiography preoperatively and one week, one month, and three months postoperatively. Results There was statistically significant increase in the level of parafoveal retinal thickness and perifoveal retinal thickness in postoperative visits than preoperative visits. We have also found statistically significant increase in the level of grid central vessel density in both superficial and deep capillary plexuses in postoperative visits than preoperative visits. Conclusion Phacoemulsification positively increases retinal thickness particularly at parafovea and increases vascularity in the center of fovea which makes phacoemulsification beneficial even beyond visual restoration.

Systemic Arterial Hypertension and Ophthalmohypertension as Independent Risk Factors for Poor Response to Antiangiogenic Therapy with Line 1 Drugs in Neovascular Age-related Macular Degeneration

Purpose: to assess hypertension as a risk factor for a poor response to antiangiogenic therapy.Patients and methods. Systemic blood pressure was studied in 84 patients (92 eyes) with age-related macular degeneration who were treated with intravitreal injections of Eilea in a fixed mode.Results. It was found that significantly more often a poor response to treatment in the form of partial non-resposing or progression of the disease, despite treatment, is associated with elevated diastolic blood pressure (DBP, p = 0.01). An increase in systolic (SBP) blood pressure in patients with arterial hypertension and AMD is accompanied by an increase in eye perfusion pressure (p < 0.01), which apparently worsens the absorption of angiostatics and causes a poor response to treatment. According to the results of the study, the most favorable corridor of SBP values associated with ideal response is in the range of values of 104–140 mm Hg, and DBP is in the range of 68–80 mm Hg st., which should be taken as the recommended parameters of blood pressure in patients with nVMD receiving a course of treatment for Eylea. Intraocular pressure (IOP) has demonstrated itself as a second modifiable independent and independent risk factor for poor response to treatment with nVMD with line 1 anti-VEGF therapy drugs. Intraocular pressure (IOP) has demonstrated itself as a second modifiable independent and independent risk factor for poor response to nVMD treatment with line 1 anti-VEGF therapy drugs. The biomarker associated with the ideal response was — 12.6 mm Hg, and the corridor of recommended values — 11–21 mm Hg. An increase in ophthalmotonus with the output of personalized values beyond this corridor seems to worsen the outcome of treatment.Conclusion. The identification of modifiable risk factors is extremely important in practical ophthalmology, as it opens up the possibility of increasing the patient’s chances of a better treatment outcome. Modifiable risk factors are valuable and powerful tools that replenish our arsenal. Information about them is important not only in the treatment of AMD, but can also be the patient’s motivation for switching to a healthy lifestyle and reducing the risk of developing the disease.

Rapidly Dissolving Trans-scleral Microneedles for Intraocular Delivery of Cyclosporine A

Cyclosporine A (CsA) is a cyclic peptide immunosuppressant drug that is beneficial in the treatment of various ocular diseases. However, its ocular bioavailability in the posterior eye is limited due to its poor aqueous solubility. Conventional CsA formulations such as a solution or emulsion permeate poorly across the eye due to various static and dynamic barriers of the eye. Dissolvable microneedle (MN)-based patches can be used to overcome barrier properties and, thus, enhance the ocular bioavailability of CsA in the posterior eye. CsA-loaded dissolvable MN patches were fabricated using polyvinylpyrrolidone (PVP) and characterized for MN uniformity and sharpness using SEM. Further characterization for its failure force, penetration force, and depth of penetration were analyzed using a texture analyzer. Finally, the dissolution time, ex vivo permeation, and ocular distribution of cyclosporine were determined in isolated porcine eyes. PVP MNs were sharp, uniform with good mechanical properties, and dissolved within 5 min. Ocular distribution of CsA in a whole porcine eye perfusion model showed a significant increase of CsA levels in various posterior segment ocular tissues as compared to a topically applied ophthalmic emulsion (Restasis®) (P < 0.001). Dissolving MNs of CsA were prepared, and the MN arrays can deliver CsA to the back of the eye offering potential for treating various inflammatory diseases.

Continuous Optimisation of Experimental Models - an Example from Glaucoma Research

There is a great demand for suitable models to test novel surgical and therapeutic approaches in glaucoma therapy. To address this need and to provide further alternatives to in vivo animal models, we aimed at modifying an established in vitro porcine eye perfusion model. Two weaknesses of the previously established porcine anterior segment model include media leakage during perfusion and setup disintegration due to mechanical instability. To overcome these, we slightly modified the previously used custom-made perfusion dishes and incorporated new components into the model setup. To prevent fluid leakage, we secured the anterior segments more firmly to the perfusion trays using a compression ring, steel screws, and nuts. Customised mounts were used to stabilise the perfusion dish and pressure transducer as a single unit. The mounts were made of polylactide (PLA) and printed using a 3D printer. The use of steel screws and nuts allowed tighter clamping of the anterior segments and prevented medium leakage. Our PLA custom mounts stabilised the entire assembly and facilitated handling during experiments and improved comparability between tested eyes. They also prevented accidental detachment of the pressure transducers, which resulted in more stable pressure curves. Our PLA mounts tolerated incubation temperatures of up to 37 °C and disinfection with enzymatic detergents and 70% ethanol without showing signs of deformation or degradation after four months of regular usage. Modifications introduced to an established in vitro perfusion model improved its efficacy and reproducibility. Our adjusted model is an example of how many models can be optimised through critical analysis, thereby saving resources and providing reliable results in the long run.

Ex vivo ocular perfusion model to study vascular physiology in the mouse eye

Several hypotheses have been tested to understand whole organ regulation in other organs such as the brain and kidney, but no such hypothesis has yet been proposed for ocular circulations. To some extent resolve this deficit our ex vivo mouse eye perfusion model takes the first step in elucidating the mechanisms controlling the individual components of the ocular circulation. Various isolated ocular vascular preparations have been utilized in studies of ocular vascular biology, physiology, and pharmacology, including studies on both normal and pathological conditions. However, there is still significant potential for further studies to improve our understanding of ocular circulation and its regulation. The choroid specifically is inaccessible to direct visualization due to the retina's high metabolic requirement with a transparency that cannot be compromised by an overly rich vascular network on the inner retinal side hindering the visualization of the choroid. In this technical paper, we provide a detailed description of all the steps to be followed from the enucleation of mouse eyes to cannulation of the ophthalmic artery and perfusion and ex vivo confocal microscopy imaging of the dynamic nature of the choroid circulation.

Early Structural and Vascular Changes after Within-24 Hours Vitrectomy for Recent Onset Rhegmatogenous Retinal Detachment Treatment: A Pilot Study Comparing Bisected Macula and Not Bisected Macula.

Background: In this study we aimed at investigating macular perfusion/anatomical changes in eyes with early onset rhegmatogenous retinal detachment (RRD) after prompt surgery within 24 hours, comparing a bisected macula and not bisected macula RRD. Methods: In this prospective observational study, 14 eyes of 14 patients who underwent within-24 hours vitreoretinal surgery for early onset RRD were enrolled. Patients were further divided into two subgroups: the not bisected macula group (NBM group) and the bisected macula group (BM group). At baseline and 3-month follow up, macular architecture and vessel analysis were assessed using optical coherence tomography angiography (OCTA) imaging. In detail, quantitative and qualitative analyses of the macular area were performed to quantify topographical retinal perfusion changes after surgery, calculating the foveal avascular zone (FAZ), vessel density (VD) and vessel length density (VLD) at the superficial capillary plexus (SCP) and deep capillary plexus (DCP). Results: Most cases (43%) were superotemporal RRD. Primary retinal reattachment was obtained in all cases, without recurrences within 3-month follow up. After surgery, a significant FAZ enlargement was observed at both the SCP and DCP level (p < 0.001; p < 0.05), with a significant effect of time noted between the two time points in the NBM and BM subanalysis (F = 3.68; p < 0.017). An excellent functional outcome was maintained for the whole follow-up. On the other hand, after surgery, perfusion parameters did not change significantly apart from the vessel density of the inferior macular sector at the DCP level (p = 0.03). Conclusions: Our findings suggest that the macular perfusion of eyes with RRD is still preserved if the surgery is performed really promptly, thus highlighting the great importance of a correct timing for surgery. OCTA analysis allows for a better understanding of the pathophysiological mechanisms underneath early vascular microarchitecture modifications of the posterior pole in retinal detachment, differentiating the two types of RRD not completely involving the fovea (BM and NBM).

MiR-21-5p: A viable therapeutic strategy for regulating intraocular pressure

Lowering intraocular pressure (IOP) is the most effective treatment of glaucoma, however most of the current available glaucoma drugs target a single molecule. MicroRNAs (miRNAs) are noncoding RNAs that target a network of molecules. This study aims to investigate the role of miR-21-5p in regulating IOP and the mechanism of function. miR-21-5p mimics was topically applied to C57/BL6 mouse eyes, which significantly increased miR-21-5p expression in the conventional outflow tissue and reduced IOP by a maximum of 17.77% at 24 h after treatment. The conventional outflow facility measured by ex vivo moue eye perfusion of miR-21-5p was significantly increased by 60.14%. Moreover, miR-21-5p overexpression significantly reduced the transendothelial electrical resistance in porcine angular aqueous plexus cells. Transcriptome analysis and further quantification by Western blot and PCR revealed that SMAD7 and FGF18 might be the downstream target of miR-21-5p in regulating aqueous humor outflow. The predicted functional pathways PTEN/eNOS, RhoB/pMLC and TIMP3/MMP9 were significantly altered after miR-21-5p transfection. Dual luciferase assay verified the direct targets of miR-21-5p. In conclusion, miR-21-5p seems to regulate IOP by modulating multiple genes that are associated with aqueous humor outflow, including genes those regulating cell adhesion, cytoskeletal dynamics and extracellular matrix turnover. Thus, miR-21-5p represents a new therapeutic strategy for glaucoma and a viable alternative to existing multidrug regimens.

Application of Cell Impedance as a Screening Tool to Discover Modulators of Intraocular Pressure.

Purpose: To identify new targets and compounds involved in mediating cellular contractility or relaxation in trabecular meshwork (TM) cells and test their efficacy in an ex vivo model measuring outflow facility. Methods: A low-molecular weight compound library composed of 3,957 compounds was screened for cytoskeletal changes using the Acea xCelligence impedance platform in immortalized human NTM5 TM cells. Hits were confirmed by 8-point concentration response and were subsequently evaluated for impedance changes in 2 primary human TM strains, as well as cross-reactivity in bovine primary cells. A recently described bovine whole eye perfusion system was used to evaluate effects of compounds on aqueous outflow facility. Results: The primary screen conducted was robust, with Z' values >0.5. Fifty-two compounds were identified in the primary screen and confirmed to have concentration-dependent effects on impedance in NTM5 cells. Of these, 9 compounds representing distinct drug classes were confirmed to modulate impedance in both human primary TM cells and bovine cells. One of these compounds, wortmannin, an inhibitor of phosphoinositide 3-kinase, increased outflow facility by 11%. Conclusions: A robust phenotypic assay was developed that enabled identification of contractility modulators in immortalized TM cells. The screening hits were translatable to primary TM cells and modulated outflow facility in an ex vivo perfusion assay.

In Situ Gel Formulation for Enhanced Ocular Delivery of Nepafenac

Nepafenac is a water-insoluble nonsteroidal antiinflammatory drug that is available as an ophthalmic suspension (Nevanac®). Suspensions are undesirable for 2 reasons: they tend to cause foreign body sensation and lacrimation, which could limit residence time and drug bioavailability. This decreases the amount of time the drug has to reach the site of action, the cornea. Previously, we improved the solubility and ocular permeability of nepafenac by complexing the drug with hydroxypropyl-β-cyclodextrin. In this study, we used the complex to formulate an ion-activated in situ gel system using sodium alginate, Protanal PH 1033, to increase the residence time and to reduce repeat eye drop instillation. Rheological properties of the formulations revealed that the viscosity of the optimized formulation was increased 30-fold when exposed to the simulated tear fluid (35°C). Permeation studies showed that the drug concentration of the in situ formulations were approximately 10 times higher than the commercial product, Nevanac® (p < 0.001). In addition, the in situ gel formulations had 5-fold higher concentrations of nepafenac retained in the cornea when compared to Nevanac® (p <0.001). Finally, ex vivo drug distribution studies in the porcine eye perfusion model revealed a higher drug retention in various ocular tissues such as cornea, sclera, retina, as compared to Nevanac®.

Improved Ocular Delivery of Nepafenac by Cyclodextrin Complexation.

Nepafenac is a nonsteroidal anti-inflammatory drug (NSAID), currently only available as 0.1% ophthalmic suspension (Nevanac®). This study utilized hydroxypropyl-β-cyclodextrin (HPBCD) to increase the water solubility and trans-corneal permeation of nepafenac. The nepafenac-HPBCD complexation in the liquid and solid states were confirmed by phase solubility, differential scanning calorimetry (DSC), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance spectroscopy (NMR) analyses. Nepafenac 0.1% ophthalmic solution was formulated using HPBCD (same pH and osmolality as that of Nevanac®) and pig eye trans-corneal permeation was studied versus Nevanac®. Furthermore, nepafenac content in cornea, sclera, iris, lens, aqueous humor, choroid, ciliary body, retina, and vitreous humor was studied in a continuous isolated pig eye perfusion model in comparison to the suspension and Nevanac®. Permeation studies using porcine corneas revealed that the solution formulation had a permeation rate 18 times higher than Nevanac®. Furthermore, the solution had 11 times higher corneal retention than Nevanac®. Drug distribution studies using porcine eyes revealed that the solution formulation enables detectable levels in various ocular tissues while the drug was undetectable by Nevanac®. The ocular solution formulation had a significantly higher drug concentration in the cornea compared to the suspension or Nevanac®.

Outflow enhancement by three different ab interno trabeculectomy procedures in a porcine anterior segment model.

To evaluate three different microincisional ab interno trabeculectomy procedures in a porcine eye perfusion model. In perfused porcine anterior segments, 90° of trabecular meshwork (TM) was ablated using the Trabectome (T; n = 8), Goniotome (G; n = 8), or Kahook device (K; n = 8). After 24h, additional 90° of TM was removed. Intraocular pressure (IOP) and outflow facility were measured at 5 and 10μl/min perfusion to simulate an elevated IOP. Structure and function were assessed with canalograms and histology. At 5μl/min infusion rate, T resulted in a greater IOP reduction than G or K from baseline (76.12% decrease versus 48.19% and 47.96%, P = 0.013). IOP reduction between G and K was similar (P = 0.420). Removing another 90° of TM caused an additional IOP reduction only in T and G but not in K. Similarly, T resulted in the largest increase in outflow facility at 5μl/min compared with G and K (first ablation, 3.41 times increase versus 1.95 and 1.87; second ablation, 4.60 versus 2.50 and 1.74) with similar results at 10μl/min (first ablation, 3.28 versus 2.29 and 1.90 (P = 0.001); second ablation, 4.10 versus 3.01 and 2.01 (P = 0.001)). Canalograms indicated circumferential flow beyond the ablation endpoints. T, G, and K significantly increased the outflow facility. In this model, T had a larger effect than G and K.

Contrast-enhanced ultrasonography for evaluation of blood perfusion in normal canine eyes.

This study was performed to evaluate ocular structures using contrast-enhanced ultrasonography (CEUS) in dogs to assess the feasibility of CEUS for investigating the blood perfusion of canine eyes. Eight purpose-bred beagles were used. Blood perfusion and vascularity of the right eye were evaluated using color Doppler, power Doppler, and CEUS with Sonazoid® . Vascular changes were quantitatively evaluated by measuring peak intensity, time to initial upslope, and time to peak from the ciliary body, iris, choroid, retina, and the retrobulbar region by CEUS. On CEUS images, all parts of the examined ocular structures were markedly enhanced and clearly identified from the adjacent region. After injection, the contrast agent initially flowed to the choroid and retina at 14.2seconds, then to the ciliary body and iris at 20seconds. The blood signal reached its peak intensity in the ciliary body at 27.2seconds (47.4±10.63), in the iris at 31.6seconds (74.00±41.85), and in the retrobulbar region at 23.4seconds (149±24.59). The optic nerve was clearly distinguished from the retrobulbar region over 5minutes after the initiation of CEUS. Significantly, more vascular signals were detected in the ciliary body and iris by CEUS than by color and power Doppler. Blood perfusion of the intraocular structures and the retrobulbar region can be quantitatively and qualitatively analyzed by CEUS. CEUS may be a useful, noninvasive, and sensitive tool for the evaluation of blood perfusion in ocular diseases.

Експериментальна оцінка впливу нарізного введення мелатоніну, цитиколіну, корвітину та тіотриазоліну на мікроциркуляцію в судинах циліарного тіла кролів після контузії ока за даними лазерної доплерівської флоуметрії

Introduction. Neuroretinoprotective activity of melatonin is the basis for further comprehensive assessment of possible mechanisms of its protective action in order to reasonable implementation of the drug in ophthalmic practice as a remedy with neuroretinoprotective action for the treatment of traumatic lesions of eye.The aim of the study – to learn Laser Doppler Flowmetry method to investigate the effect of melatonin and reference drugs (citicoline, corvitin and thiotriazoline) on completeness of recovery of blood flow in the vessels of the ciliary body in dynamic of its contusion as a possible mechanism of their neuroretinoprotective action.Research Methods. Model of eye contusion is a blank shot into the center of the cornea closely with carbon dioxide under pressure. Therapy is separate intravenous administration of drugs (melatonin 10 mg / kg, corvitin 10 mg / kg citicoline 250 mg / kg and thiotriazoline 100 mg / kg) twice a day during 7 days, the first application 1 h after injury. Effect of drugs on microcirculation in the eye ciliary body in this condition was studied with using Laser Doppler Flowmetry module BIOPAC (USA).Results and Discussion. Therapeutic application of all test substances amortized rapid deterioration of blood supply to the eye. Melatonin is the leader among the selected drugs for the ability to improve the circulation in the ciliary body of eye during the first week after its contusion.Conclusion. Restoration of eye perfusion caused by contusion on the background of application of melatonin, citicoline, corvitin and thiotriazoline is one of the leading mechanisms of neuroretinoprotective action of these drugs.

A Compact Whole-Eye Perfusion System to Evaluate Pharmacologic Responses of Outflow Facility.

To discover novel therapies that lower IOP by increasing aqueous humor outflow facility, ex vivo ocular perfusion systems provide a valuable tool. However, currently available designs are limited by their throughput. Here we report the development of a compact, scalable perfusion system with improved throughput and its validation using bovine and porcine eyes. At a fixed IOP of 6 mm Hg, flow rate was measured by flow sensors. We validated the system by measuring the outflow responses to Y-39983 (a Rho kinase inhibitor), endothelin-1 (ET-1), ambrisentan (an antagonist for endothelin receptor A [ETA]), sphigosine-1-phosphate (S1P), JTE-013 (antagonist for S1P receptor 2 [S1P2]), S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide [NO] donor), and 3-Morpholino-sydnonimine (SIN-1, another NO donor). The instrument design enabled simultaneous measurements of 20 eyes with a footprint of 1 m2. Relative to vehicle control, Y-39983 increased outflow by up to 31% in calf eyes. On the contrary, ET-1 decreased outflow by up to 79%, a response that could be blocked by pretreatment with ambrisentan, indicating a role for ETA receptors. Interestingly, the effect of ET-1 was also inhibited by up to 70% to 80% by pretreatment with NO donors, SNAP and SIN-1. In addition to testing in calf eyes, similar effects of ET-1 and ambrisentan were observed in adult bovine and porcine eyes. The compact eye perfusion platform provides an opportunity to efficiently identify compounds that influence outflow facility and may lead to the discovery of new glaucoma therapies.

Comparison of stability between a modular intraocular lens system and a single-piece hydrophobic acrylic intraocular lens

To compare the movement of a modular intraocular lens (IOL) with that of a standard single-piece hydrophobic acrylic IOL in a human cadaver eye perfusion model. Department of Ophthalmology, University of Colorado, Aurora, Colorado, USA. Experimental study. Eight phakic human donor eyes of 4 patients had standard phacoemulsification with lens removal. One of 2 IOLs was then implanted in the capsular bag: a modular IOL (Harmoni) or a standard single-piece IOL (Acrysof SN60). Each globe was connected to a programmable perfusion pump with an in-line pressure transducer. Ultrasound biomicroscopy (UBM) was used to evaluate the anterior chamber depth (ACD) in each eye, measuring from the posterior cornea to the anterior surface of the optic at an intraocular pressure (IOP) of 5mm Hg, 10mm Hg, 20mm Hg, and 30mm Hg. Five consecutive measurements were recorded for all eyes at each pressure, and the results were averaged. There was significantly less movement in eyes with the modular IOL than in eyes with the single-piece IOL. The mean position of the modular IOL varied from a minimum of 0.03mm to a maximum of 0.07mm, and the mean position of the single-piece IOL varied from a minimum of 0.26mm to a maximum of 0.87mm(P=.002). The modular IOL showed less movement with changes in IOP than a standard single-piece IOL. Improved IOL stability might allow more accuracy in determining the effective lens position and hence improve the predictability of the refractive target. Proprietary or commercial disclosures are listed after the references.

Characterization of micro-invasive trabecular bypass stents by ex vivo perfusion and computational flow modeling

Micro-invasive glaucoma surgery with the Glaukos iStent® or iStent inject® (Glaukos Corporation, Laguna Hills, CA, USA) is intended to create a bypass through the trabecular meshwork to Schlemm’s canal to improve aqueous outflow through the natural physiologic pathway. While the iStent devices have been evaluated in ex vivo anterior segment models, they have not previously been evaluated in whole eye perfusion models nor characterized by computational fluid dynamics. Intraocular pressure (IOP) reduction with the iStent was evaluated in an ex vivo whole human eye perfusion model. Numerical modeling, including computational fluid dynamics, was used to evaluate the flow through the stents over physiologically relevant boundary conditions. In the ex vivo model, a single iStent reduced IOP by 6.0 mmHg from baseline, and addition of a second iStent further lowered IOP by 2.9 mmHg, for a total IOP reduction of 8.9 mmHg. Computational modeling showed that simulated flow through the iStent or iStent inject is smooth and laminar at physiological flow rates. Each stent was computed to have a negligible flow resistance consistent with an expected significant decrease in IOP. The present perfusion results agree with prior clinical and laboratory studies to show that both iStent and iStent inject therapies are potentially titratable, providing clinicians with the opportunity to achieve lower target IOPs by implanting additional stents.

Preclinical Investigation of Ab Interno Trabeculectomy Using a Novel Dual-Blade Device

To evaluate the effects of a novel ab interno trabeculectomy device on human trabecular meshwork (TM). Laboratory evaluation. The TM from human cadaveric corneal rim tissue was incised using 3 instruments: (1) novel dual-blade device; (2) microvitreoretinal (MVR) blade; and (3) Trabectome. Tissue samples underwent histologic processing and comparative analyses. Subsequently, human eye perfusion studies were performed to evaluate intraocular pressure (IOP)-lowering effects of each device. Main outcome measures were degree of TM removal by histology and IOP in a perfusion model. The MVR blade exhibited minimal removal of TM and obvious injury to the adjacent sclera. The Trabectome removed a large portion of the central TM, but leaflets of residual tissue remained and thermal injury was noted in all samples. The dual-blade device achieved a more complete removal of TM without injury to surrounding tissues. All devices resulted in statistically significant lowering of IOP during perfusion model studies. MVR blade treatment across 170.0 ± 14.1 degrees of TM resulted in a decrease of IOP from 18.5 ± 1.9 mm Hg to 12.8 ± 2.2 mm Hg (P < .01). Trabectome treatment across 117.5 ± 12.6 degrees resulted in a decrease of IOP from 18.8 ± 1.7 mm Hg to 11.3 ± 1.0 mm Hg (P < .01). Dual-blade device treatment across 157.5 ± 26.3 degrees resulted in a decrease of IOP from 18.3 ± 3.0 mm Hg to 11.0 ± 2.2 mm Hg (P < .01). The novel dual-blade device demonstrated a more complete removal of TM without residual TM leaflets or damage to surrounding tissues and significantly reduced IOP in a human eye perfusion model.

Passage of low-density lipoproteins through Bruch’s membrane and choroid

Plasma lipoproteins are thought to transport cholesterol, vitamins and carotenoids to the retinal pigment epithelium (RPE) for ultimate use by the photoreceptors. However, to reach the RPE, these lipoprotein particles must cross Bruch’s membrane. We examined the reflection coefficient of Bruch’s membrane (BrM) to low-density lipoprotein (LDL). Bruch’s membrane and choroid were removed from 47 bovine eyes. Specimens were placed in a Ussing chamber and perfused with phosphate-buffered saline (PBS) with (31 specimens) or without (16 specimens) fluorescent low-density lipoproteins (DiI-LDL). The hydraulic conductivity of the tissue was determined for both calf and cow eyes. In the perfusions with DiI-LDL, the fluorescence intensity emitted by DiI-LDL in the efflux was measured and the reflection coefficient of BrM/choroid preparations to DiI-LDL determined. Leakage tests were done to confirm tissue integrity. Several specimens were examined using scanning electron microscopy (SEM) to examine tissue integrity before and after perfusion. Leak testing confirmed that BrM was intact both before and after perfusion. The average hydraulic conductivity of BrM/choroid perfusion of calf eyes with PBS alone was 1.42 ± 0.55 × 10−9 m/s/Pa (mean ± SD, n = 11). The average hydraulic conductivity of the cow eyes was 4.94 ± 1.48 × 10−10 m/s/Pa (n = 5), nearly a 3-fold decrease with age. While the flow rate remained constant during the PBS perfusions, it decreased as a function of time during perfusion with DiI-LDLs. Our major finding was of fluorescence in the effluent collected in all perfusions with DiI-LDLs, demonstrating passage of LDL through the tissue. The average reflection coefficient of calf BrM/choroid preparations to DiI-LDL was 0.58 ± 0.25 (n = 23); a similar distribution of reflection coefficients was seen in tissue from cow eyes (0.51 ± 0.33, n = 8). Our data suggested that the DiI-LDL was modestly hindered and/or captured by the tissue. This might explain the progressive decrease of hydraulic conductivity with continued perfusion of DiI-LDL.

3D visualization of aqueous humor outflow structures in-situ in humans

Aqueous humor (AH) exiting the eye via the trabecular meshwork and Schlemm’s canal (SC) passes through the deep and intrascleral venous plexus (ISVP) or directly through aqueous veins. The purpose of this study was to visualize the human AH outflow system 360° in three dimensions (3D) during active AH outflow in a virtual casting. The conventional AH outflow pathways of 7 donor eyes were imaged with a modified Bioptigen spectral-domain optical coherence tomography system (Bioptigen Inc, USA; SuperLum LTD, Ireland) at a perfusion pressure of 20 mmHg ( N = 3), and 10 mmHg ( N = 4). In all eyes, 36 scans (3 equally distributed in each clock hour), each covering a 2 × 3 × 2 mm volume (512 frames, each 512 × 1024 pixels), were obtained. All image data were black/white inverted, and the background subtracted (ImageJ 1.40 g, http://rsb.info.nih.gov/ij/). Contrast was adjusted to isolate the ISVP. SC, collector channels, the deep and ISVP, and episcleral veins were observed throughout the limbus. Aqueous veins could be observed extending into the episcleral veins. Individual scan ISVP castings were rendered and assembled in 3D space in Amira 4.1 (Visage Imaging Inc. USA). A 360-degree casting of the ISVP was obtained in all perfused eyes. The ISVP tended to be dense and overlapping in the superior and inferior quadrants, and thinner in the lateral quadrants. The human AH outflow pathway can be imaged using SD-OCT. The more superficial structures of the AH outflow pathway present with sufficient contrast as to be optically isolated and cast in-situ 360° in cadaver eye perfusion models. This approach may be useful as a model in future studies of human AH outflow.