Anterior chamber inflammation grading methods: A critical review.

Assessing anterior chamber inflammation is highly subjective and challenging. Although various grading systems attempt to offer objectivity and standardization, the clinical assessment has high interobserver variability. Traditional techniques, such as laser flare meter and fluorophotometry, are not widely used since they are time-consuming. With the development of optical coherence tomography with high sensitivity, direct imaging offers an excellent alternative to assess objectively inflammation with the potential for automated analysis. We describe various anterior chamber inflammation grading methods and discuss their utility, advantages, and disadvantages.

Grading the Severity of Damage to the Perijunctional Actomyosin Ring and Zonula Occludens-1 of the Corneal Endothelium by Ensemble Learning Methods.

Purpose: In many epithelia, including the corneal endothelium, intracellular/extracellular stresses break down the perijunctional actomyosin ring (PAMR) and zonula occludens-1 (ZO-1) at the apical junctions. This study aims to grade the severity of damage to PAMR and ZO-1 through machine learning. Methods: Immunocytochemical images of PAMR and ZO-1 were drawn from recent studies on the corneal endothelium subjected to hypothermia and oxidative stress. The images were analyzed for their morphological (e.g., Hu moments) and textural features (based on gray-level co-occurrence matrix [GLCM] and Gabor filters). The extracted features were ranked by SHapley analysis and analysis of variance. Then top features were used to grade the severity of damage using a suite of ensemble classifiers, including random forest, bagging classifier (BC), AdaBoost, extreme gradient boosting, and stacking classifier. Results: A partial set of features from GLCM, along with Hu moments and the number of hexagons, enabled the classification of damage to PAMR into Control, Mild, Moderate, and Severe with the area under the receiver operating characteristics curve (AUC) = 0.92 and F1 score = 0.77 with BC. In contrast, a bank of Gabor filters provided a partial set of features that could be combined with Hu moments, branch length, and sharpness for the classification of ZO-1 images into four levels with AUC = 0.95 and F1 score of 0.8 with BC. Conclusions: We have developed a workflow that enables the stratification of damage to PAMR and ZO-1. The approach can be applied to similar data during drug discovery or pathophysiological studies of epithelia.

Experimental Oxidative Stress Breaks Down the Barrier Function of the Corneal Endothelium.

Purpose: The fluid pump and barrier functions of the corneal endothelium maintain stromal deturgescence required for corneal transparency. The effect of oxidative stress, a hallmark of Fuchs endothelial corneal dystrophy (FECD), on the endothelial barrier function has been investigated. Methods: The endothelium of porcine corneas ex vivo was exposed to (1) membrane permeable oxidants (H2O2, 100 µM, 1 h; tert-butyl-hydroperoxide, 100 µM, 1 h), or (2) ultraviolet A (UVA) with photosensitizers for 15 min, riboflavin (50 µM) or tryptophan (Trp) (100 µM). The effects on the apical junction complex were analyzed by (1) immunostaining the perijunctional actomyosin ring (PAMR) and ZO-1 and (2) assessment of paracellular flux of fluorescein isothiocyanate (FITC)-avidin across cultured endothelial cells grown on biotinylated-gelatin film. The extent of oxidative stress was quantified by changes in intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in addition to lipid peroxidation and release of lactate dehydrogenase (LDH). Results: Both methods of oxidative stress led to the disruption of PAMR and ZO-1 concurrent with changes in ROS levels, depolarization of MMP, increased lipid peroxidation, elevated LDH release, and increased permeability of FITC-avidin. The effects of direct oxidants were opposed by SB-203580 [p38 mitogen-activating protein (MAP) kinase inhibitor; 10 µM]. The damage by UVA+photosensitizers was blocked by extracellular catalase (10,000 U/mL). Conclusions: (1) Acute oxidative stress breaks down the barrier function through destruction of PAMR in a p38 MAP kinase-dependent manner. (2) UVA+photosensitizers elicit the breakdown of PAMR via type I reactions, involving H2O2 release. (3) Blocking the oxidative stress prevents loss of barrier function, which could be helpful in the therapeutics of FECD.

Nanoliposomes for Sensing Local Osmolarity of the Tear Film on the Corneal Surface.

Purpose: Local hotspots of elevated tear hyperosmolarity (exceeding 900 mOsM) are predicted in dry eye disease (DED) but have not been measured. This study aims to develop, characterize, and evaluate the suitability of fluorescent nanoliposomes for noninvasive sensing of the local osmolarity of the tear film. Methods: Fluorescent nanoliposomes, loaded with calcein (susceptible to self-quenching; sensor dye) and sulforhodamine 101 (SR101; reference dye), were produced by the thin-film hydration method. Results: Dynamic light scattering measurements and Cryo-TEM showed that liposomes were negatively charged (-23.7 ± 1.5 mV), spherical (diameter = 117.9 ± 6.4 nm), and uniform in size (polydispersity index = 0.15 ± 0.02). These properties were unaffected by cold storage (4°C; 14 days), but dye leakage was significant after 3 days. Swelling and shrinkage of the liposomes by exposure to hypoosmotic and hyperosmotic media led to rapid dequenching and quenching of calcein fluorescence (FGreen), with no effect on SR101 fluorescence (FRed). The ratio FGreen/FRed decreased with increasing osmolarity and vice versa, obeying the Boyle van't Hoff relationship. When liposomes were dispersed in a gelatin film with dynamic radial sucrose gradients, local FGreen/FRed decreased with increasing hyperosmolarity as predicted. When instilled on the hydrophilic surface of contact lenses or ex vivo corneas, nanoliposomes dispersed evenly as thin films. Importantly, the measured FGreen/FRed declined continuously with evaporation and consequent increase in their osmolarities. Conclusions: The study provides proof of principle for noninvasive measurement of local tear film osmolarity based on osmosensitive fluorescent nanoliposomes. The strategy can potentially advance our understanding of the pathophysiology of DED.

Role of Oxidative Stress in the Disruption of the Endothelial Apical Junctional Complex During Corneal Cold Storage.

Purpose: To characterize the impact of corneal cold storage (CS) on the endothelial apical junctional complex (AJC). Methods: Porcine corneas were held in CS (4°C; 1-7 days) with Cornisol™ preservation medium supplemented with epothilone B (EpoB; microtubule stabilizer; 100 nM), SB-203580 (p38 mitogen-activated protein [MAP] kinase inhibitor; 20 µM), or antioxidants (quercetin, 100 µM; vitamin E, 1 mM; deferoxamine, an iron chelator, 10 mM). After CS termination, the damage to endothelial AJC was characterized by imaging perijunctional actomyosin ring (PAMR) and zonula occludens (ZO-1). The effects of EpoB and SB-203580 were characterized by imaging microtubules. The loss in the barrier function was assessed in cultured cells grown on biotin-coated gelatin by permeability to fluorescein isothiocyanate (FITC)-avidin. The accumulation of reactive oxygen species (ROS), altered mitochondrial membrane potential (MMP), lipid peroxidation, and lactate dehydrogenase (LDH) release were also determined in response to CS. Results: CS led to the loss of microtubules, destruction of PAMR, and breakdown of ZO-1 in the endothelium. The severity of damage increased when CS was prolonged. Although rewarming of the tissue increased the damage, the effect was marginal. CS also induced accumulation of ROS, alteration in MMP, lipid peroxidation, enhanced LDH release, and increased permeability to FITC-avidin. These changes were opposed by EpoB, SB-203580, and antioxidants. Conclusion: Corneal CS destroys AJC of the endothelium, leading to loss of its barrier function. The effects were surmounted by microtubule stabilization, p38 MAP kinase inhibition, and antioxidants. Thus, there is potential for reformulation of the preservation medium to maintain the health of the donor corneal endothelium before transplantation.

Double functionalized haemocompatible silver nanoparticles control cell inflammatory homeostasis.

Infection, trauma, and autoimmunity trigger tissue inflammation, often leading to pain and loss of function. Therefore, approaches to control inflammation based on nanotechnology principles are being developed in addition to available methods. The metal-based nanoparticles are particularly attractive due to the ease of synthesis, control over physicochemical properties, and facile surface modification with different types of molecules. Here, we report curcumin conjugated silver (Cur-Ag) nanoparticles synthesis, followed by their surface functionalization with isoniazid, tyrosine, and quercetin, leading to Cur-AgINH, Cur-AgTyr, and Cur-AgQrc nanoparticles, respectively. These nanoparticles possess radical scavenging capacity, haemocompatibility, and minimal cytotoxicity to macrophages. Furthermore, the nanoparticles inhibited the secretion of pro-inflammatory cytokines such as interleukin-6, tumor necrosis factor-α, and interleukin-1ß from macrophages stimulated by lipopolysaccharide (LPS). The findings reveal that the careful design of surface corona of nanoparticles could be critical to increasing their efficacy in biomedical applications.

Oxidative Stress Induces a Breakdown of the Cytoskeleton and Tight Junctions of the Corneal Endothelial Cells.

Purpose: To investigate the impact of oxidative stress, which is a hallmark of Fuchs dystrophy, on the barrier function of the corneal endothelial cells. Methods: Experiments were carried out with cultured bovine and porcine corneal endothelial cells. For oxidative stress, cells were supplemented with riboflavin (Rf) and exposed to UV-A (15-30 min) to induce Type-1 photochemical reactions that release H2O2. The effect of the stress on the barrier function was assayed by transendothelial electrical resistance (TER) measurement. In addition, the associated changes in the organization of the microtubules, perijunctional actomyosin ring (PAMR), and ZO-1 were evaluated by immunocytochemistry, which was also repeated after direct exposure to H2O2 (100 µM, 1 h). Results: Exposure to H2O2 led to the disassembly of microtubules and the destruction of PAMR. In parallel, the contiguous locus of ZO-1 was disrupted, marking a loss of barrier integrity. Accordingly, a sustained loss in TER was induced when cells in the Rf-supplemented medium were exposed to UV-A. However, the addition of catalase (7,000 U/mL) to rapidly decompose H2O2 limited the loss in TER. Furthermore, the adverse effects on microtubules, PAMR, and ZO-1 were suppressed by including catalase, ascorbic acid (1 mM; 30 min), or pretreatment with p38 MAP kinase inhibitor (SB-203580; 10 µM, 1 h). Conclusions: Acute oxidative stress induces microtubule disassembly by a p38 MAP kinase-dependent mechanism, leading to the destruction of PAMR and loss of barrier function. The response to oxidative stress is reminiscent of the (TNF-α)-induced breakdown of barrier failure in the corneal endothelium.

Reliability of Aqueous Flare Measurements During Uveitis by a Spot Fluorometer.

Purpose: To evaluate the reliability of intensity of light scatter (ILS) for diagnosing mild-to-moderate uveitis, which is difficult to score by the subjective standardized uveitis nomenclature (SUN) grading. Methods: ILS (in millivolts) was measured in healthy subjects and uveitis patients by a custom-made Spot fluorometer. The reliability was assessed by noise analysis of the instrument using turbid samples and intra- and interobserver variabilities. Results: ILS increased with the severity of inflammation: healthy (0.1498 ± 0.0564) and SUN0 (0.1590 ± 0.0564) < SUN1+ (0.2050 ± 0.0887) < SUN2+ (0.4283 ± 0.1895). The increase in ILS was more significant between healthy and SUN1+ (P < 0.0001) compared to the difference between SUN0 and SUN1+ (P = 0.0006). The instrument noise was small (σ = 0.002). Intraobserver measurements revealed no variability (P > 0.98) between measurements and predicted repeatability coefficients of 0.0754, 0.0522, and 0.0532 in healthy, SUN0, and SUN1+, respectively. Interobserver measurements also showed no variability (P > 0.88) and predicted intraclass correlation coefficient > 0.63 in healthy and uveitis subjects. Conclusions: The fluorometer enables precise measurement of ILS with negligible instrument noise, and the measurements are unaffected by intra- and interobserver variabilities. However, the difference in mean ILS between SUN0 and SUN1+ is greater than the instrument noise and comparable to intra- and interobserver variabilities. Thus, ILS measurements are likely impacted by blinking, eye movements, and focusing jitter. The impact of these factors must be reduced before using the fluorometer to distinguish mild-to-moderate uveitis.

Clinical, Morphological, and Optical Correlates of Visual Function in Patients With Fuchs Endothelial Corneal Dystrophy.

PURPOSE: The purpose of this article was to study the clinical, optical, and morphological correlates of visual function in patients with Fuchs endothelial corneal dystrophy (FECD). METHODS: The case records were analyzed for patients diagnosed with FECD between September 2019 and March 2020. The best-corrected visual acuity (BCVA) was recorded as decimal visual acuity and converted to the logarithm of the minimum angle of resolution units. Contrast sensitivity was measured with the Pelli-Robson contrast sensitivity test. Corneal alterations, including central corneal thickness, depression of the posterior cornea, and corneal densitometry values, were evaluated using Scheimpflug images. Corneal epithelial thickness was measured by spectral-domain optical coherence tomography. RESULTS: A total of 107 eyes of 61 patients (18 male and 43 female) with FECD were retrospectively investigated. The Spearman rank correlation coefficient showed moderate correlation between BCVA and contrast sensitivity (ρ = -0.66, P < 0.001), with some patients maintaining relatively good BCVA but having reduced contrast sensitivity. Logistic regression analysis demonstrated that age, central corneal thickness, depression of the posterior cornea, and epithelial thickening were negatively associated with contrast sensitivity but not with BCVA. CONCLUSIONS: Contrast sensitivity is a useful tool for assessing visual dysfunction and should be incorporated into the assessment protocol of patients with FECD. Alterations in the cornea, including central corneal thickness, depression of the posterior cornea, and epithelial thickening, might be objective parameters that can help the clinician in grading the severity of the disease and tracking its progression.

Automated Image Segmentation of the Corneal Endothelium in Patients With Fuchs Dystrophy.

Purpose: To perform segmentation of specular microscopy (SM) images of the corneal endothelium for comparing average perimeter length (APL) between Fuchs endothelial corneal dystrophy (FECD) patients and healthy subjects. Methods: A retrospective review of clinical records of FECD patients and those with healthy endothelium was carried out to collect images of the endothelium. The images were segmented by modified U-Net, a deep learning architecture, followed by the Watershed algorithm to resolve merged cell borders (<5%). The segmented images were analyzed for endothelial cell density (ECDUW) and APL. Results: The combination of the U-Net and Watershed algorithm, referred to as the UW approach, enabled a complete segmentation of the endothelium. In healthy, ECDUW was close to estimates by SM and manual segmentation (31 subjects; P > 0.1). However, in FECD, ECDUW was closer to estimates by manual segmentation but not by SM (27 patients; P < 0.001). ECDUW in FECD (2547 ± 499 cells/mm2; 60 patients) was smaller compared to that in the healthy (2713 ± 401 cells/mm2; 70 subjects) (P < 0.001). APL in the healthy was 66.87 ± 7.68 µm/cell (70 subjects), but it increased with %Guttae in FECD (56.60-195.30 µm/cell; 60 patients) (P < 0.0001). Conclusions: The UW approach is precise for the segmentation of SM images from the healthy and FECD. Our analysis has revealed that APL increases with %Guttae. Translational Relevance: The average perimeter length of the corneal endothelium, which represents the length of the paracellular pathway for fluid flux into the stroma, is increased in Fuchs dystrophy.

Microtubule Stabilization Protects Hypothermia-Induced Damage to the Cytoskeleton and Barrier Integrity of the Corneal Endothelial Cells.

Purpose: To determine the impact of hypothermia on the barrier function of donor corneal endothelium, thereby enhancing the success of corneal transplantation. Methods: Primary cultures of porcine endothelial cells were subjected to hypothermia (15 h; 4°C). The impact on microtubule assembly, peri-junctional actomyosin ring (PAMR), and ZO-1 was assessed by immunocytochemistry with and without pretreatment with a microtubule-stabilizing agent (Epothilone B; EpoB; 100 nM) and a p38 MAP kinase inhibitor (SB-203580; 20 µM). In addition, EpoB-loaded PLGA nanoparticles (ENPs) prepared by nanoprecipitation technique and coated with poly-L-lysine (PLL-ENPs) were administered one-time for sustained intracellular delivery of EpoB. Results: Exposure to hypothermia led to microtubule disassembly concomitant with the destruction of PAMR and the displacement of ZO-1 at the cellular periphery, suggesting a loss in barrier integrity. These adverse effects were attenuated by pretreatment with EpoB or SB-203580. PLL-ENPs possessed a zeta potential of ∼26 mV and a size of ∼110 nm. Drug loading and entrapment efficiency were 5% (w/w) and ∼87%, respectively, and PLL-ENPs showed a biphasic release in vitro: burst phase (1 day), followed by a sustained phase (∼4 weeks). Pretreatment with PLL-ENPs (0.4 mg/mL) for 24 h stabilized the microtubules and opposed the hypothermia-induced damage to PAMR and the redistribution of ZO-1. Conclusions: Hypothermia induces microtubule disassembly via activation of p38 MAP kinase and subsequently breaks down the barrier function of the endothelium. Sustained intracellular delivery of EpoB using nanoparticles has the potential to overcome endothelial barrier failure during prolonged cold storage of donor cornea.

Analysis of Immune Cells on Donor Corneal Endothelium After Corneal Transplantation Using the HRT-Rostock Cornea Module.

PURPOSE: The aim of this study was to investigate the immune cells on corneal endothelium of the graft in patients who underwent penetrating keratoplasty (PK), Descemet-stripping endothelial keratoplasty (DSEK), and Descemet membrane endothelial keratoplasty (DMEK). METHODS: A total of 43 eyes of 43 patients who underwent PK (17 eyes), DSEK (13 eyes), and DMEK (13 eyes) and who did not show any sign of graft rejection were recruited for the study. Patients who underwent cataract surgery (26 eyes) served as controls. Immune cells on the corneal endothelium were examined with laser in vivo confocal microscopy. The associations between the corneal endothelial cell density, type of keratoplasty, aqueous flare, repeated keratoplasty, and time after surgery versus the density of immune cells were investigated. RESULTS: In vivo confocal microscopy visualized similar numbers of immune cells on the corneal endothelium in the PK, DSEK, and DMEK groups, whereas no immune cells were observed in any of the control patients. The numbers of immune cells tended to be higher in regraft eyes in the PK group (P = 0.00221) and in the DSEK group (P = 0.168) than those in the primary graft eyes. No significant association was found between the density of immune cells and corneal endothelial cell density in the PK, DSEK, and DMEK groups. CONCLUSIONS: Immune cells were observed to a similar extent in the eyes of PK, DSEK, and DMEK subjects even in the absence of any clinical sign of immune rejection. A further prospective longitudinal study will evaluate the effect of immune cells on long-term graft survival and the risk for graft rejection.

A surfactant polymer wound dressing protects human keratinocytes from inducible necroptosis.

Chronic wounds show necroptosis from which keratinocytes must be protected to enable appropriate wound re-epithelialization and closure. Poloxamers, a class of synthetic triblock copolymers, are known to be effective against plasma membrane damage (PMD). The purpose of this study is to evaluate the efficacy of a specific poloxamer, surfactant polymer dressing (SPD), which is currently used clinically as wound care dressing, against PMD in keratinocytes. Triton X-100 (TX100) at sub-lytic concentrations caused PMD as demonstrated by the efflux of calcein and by the influx of propidium iodide and FM1-43. TX100, an inducer of necroptosis, led to mitochondrial fragmentation, depletion of nuclear HMGB1, and activation of signaling complex associated with necroptosis (i.e., activation of RIP3 and phosphorylation of MLKL). All responses following exposure of human keratinocytes to TX100 were attenuated by pre- or co-treatment with SPD (100 mg/ml). The activation and translocation of phospho-MLKL to the plasma membrane, taken together with depletion of nuclear HMGB1, characterized the observed cell death as necroptosis. Thus, our findings show that TX100-induced plasma membrane damage and death by necroptosis were both attenuated by SPD, allowing keratinocyte survival. The significance of such protective effects of SPD on keratinocytes in wound re-epithelialization and closure warrant further studies.

Highly efficient and selective antimicrobial isonicotinylhydrazide-coated polyoxometalate-functionalized silver nanoparticles.

With the rapidly approaching post-antibiotic era, new and effective combinations of antibiotics are imperative to combat multiple drug resistance (MDR). We have synthesized multimodal antimicrobials that integrate the antibiotic isonicotinylhydrazide (INH), silver nanoparticles (AgNPsINH), and two different polyoxometalates (POMs) namely, phosphotungstic acid (PTA) and phosphomolybdic acid (PMA) to prepare AgNPsINH@PTA and AgNPsINH@PMA, respectively. AgNPsINH have peroxidase-like (nanozyme) activity and very high antibacterial potential toward S. aureus, which was further enhanced upon modification with POMs. The selectivity of these functional nanozymes was evaluated with m5S mouse fibroblasts using WST-8, LDH viability, in vitro reactive oxygen species (ROS) generation assays, and crystal violet morphological studies. These investigations showed very low cytotoxicity for the nanoparticles compared to free metal ions (Ag+), pristine POMs and INH, demonstrating the ability of multifunctional materials to provide efficient and selective antimicrobials.

Penetration of Nile red-loaded nanostructured lipid carriers (NLCs) across the porcine cornea.

Nile Red-loaded nanostructured lipid carriers (NR-NLCs), prepared by high-pressure homogenization technique, have been investigated for their transcorneal penetration using a confocal scanning microfluorometer (CSMF). Topical exposure of NR-NLCs led to their penetration into the epithelium and anterior stroma. The NR-NLC-40 (NR-NLCs of 40 nm) showed faster penetration compared to NR-NLC-150 (NR-NLCs of 150 nm). The surface modification of NR-NLC-40 with polyethylene glycol 400 (NR-NLC-PEG) and stearylamine (NR-NLC-SA), although did not cause any significant effect on size, resulted in an increased penetration into the epithelium concomitant with a reduced penetration into the stroma compared to the NR-NLC-40. Ex vivo mucoadhesion assay revealed that NR-NLC-PEG and NR-NLC-SA adhered more strongly to the porcine corneal surface compared to NR-NLC-40. Flow cytometry experiments with porcine corneal epithelial cells showed that NR-NLC-40 was internalized better than NR-NLC-PEG and NR-NLC-SA. These results, taken together, suggest that NLCs are potentially useful for lipophilic drug delivery to the corneal epithelium and anterior stroma without any surface modifications. However, surface modifications with polyethylene glycol 400 or stearylamine could be useful to treat ocular surface disorders.

Impact of Nanostructured Lipid Carriers as an Artificial Tear Film in a Rabbit Evaporative Dry Eye Model.

PURPOSE: To characterize formulations of nanostructured lipid carriers (NLCs) as an artificial tear film and evaluate their efficacy in protecting the ocular surface epithelial cells from desiccating stress in vivo. METHODS: The physicochemical properties of NLCs, produced with components similar to the tear film such as phosphatidylcholine and squalene, were determined. In vitro cytotoxicity of NLCs was evaluated by a short-time exposure test in porcine corneal epithelial cells using a methyl thiazol diphenyl-tetrazolium bromide assay. The residence time of NLCs in rabbit eyes and the efficacy of NLCs eye drops in protecting the rabbit corneal epithelium from desiccating stress were assessed. RESULTS: Nanosized NLCs with a mean size of ∼39 ± 5 nm and a zeta potential of -30 mV could be produced and formulated into eye drop with a pH of 6.90 ± 0.01, osmolarity of 273 ± 1 mOsm/L, and surface tension of 39 ± 1 mN/m (for air interface). Eye drop formulations of NLCs were nontoxic to porcine corneal epithelial cells. NLCs drops showed higher ocular surface retention and formed a stable corneal film compared with a saline solution. Moreover, NLCs eye drops showed greater efficacy in protecting the corneal surface against desiccating stress compared with a polymer-based commercial artificial tear. CONCLUSIONS: NLCs eye drops are biocompatible in rabbit eyes and show potential as a tear replacement vehicle for the treatment of dry eye disease.

Corneal epithelial permeability to fluorescein in humans by a multi-drop method.

PURPOSE: The permeability of the corneal epithelium to fluorescein Pdc is an indicator of the health of the ocular surface. It can be measured in a clinical setting by determining the accumulation of fluorescein in the stroma following administration of the dye on the ocular surface. Here we demonstrate a new multi-drop method for the measurement of Pdc by a spot fluorometer. METHODS: Twenty-nine healthy participants were recruited for this study. First, a probe-drop of fluorescein (0.35%, 2 µL) was instilled on the conjunctiva. The clearance of the dye from the tears was immediately measured using the fluorometer. Following this, two loading drops (2%; 6 µL each) were administered 10 min apart. Fifteen minutes later, the ocular surface was washed and fluorescence from the stroma Fs was measured. Permeability was calculated using Pdc = (Q x Fs)/ (2 x AUC), where Q is the stromal thickness and AUC is the area under the fluorescence vs. time curve for the loading drops. RESULTS: After the probe drop, the tear fluorescence followed an exponential decay (elimination rate constant; kd = 0.41 ± 0.28 per min; 49 eyes of 29 subjects), but the increase in Fs was negligible. However, after the loading drops, the measured Fs was ~ 20-fold higher than the autofluorescence and could be recorded at a high signal to noise ratio (SNR > 40). The intra-subject variability of kd was insignificant. Since fluorescein undergoes concentration quenching at > 0.5%, the value of AUC for the loading drops was estimated by scaling the AUC of the probe drop. The calculated Pdc was 0.54 ± 0.54 nm/sec (n = 49). A Monte Carlo simulation of the model for the multi-drop protocol confirmed the robustness of the estimated Pdc. CONCLUSIONS: The new multi-drop method can be used in place of the single-drop approach. It can overcome a lack of sensitivity in fluorometers of high axial resolution. The Pdc estimated by the multi-drop method is ~ 11-fold higher than previously reported but closer to the value reported for other drugs with equivalent octanol/water partition coefficient.

Ocular Spot Fluorometer Equipped With a Lock-In Amplifier for Measurement of Aqueous Flare.

PURPOSE: To evaluate a custom-made ocular fluorometer for detection of intensity of light scatter (ILS) from the anterior chamber (A/C) as an objective measure of aqueous flare. METHODS: The fluorometer, equipped with a lock-in amplifier, was employed in the scatter mode to detect ILS from A/C. Measurements were performed with two illumination slit widths of 0.5 and 0.25 mm. The axial resolution at these slit widths were 80 and 200 µm, respectively. Healthy and pseudophakic eyes, with grade 0 Standardization of Uveitis Nomenclature (SUN) score, were employed as control subjects. ILS was also recorded in a cohort of patients who had undergone phacoemulsification and showed grades 1+ or 2+ on postoperative days 1 and 4. RESULTS: The inter- and intraobserver variabilities in the measurement of ILS were not significant. In cataract patients, ILS was significantly higher on postoperative day 1 relative to healthy eyes. By day 4, ILS decreased significantly and was only marginally different from ILS in quiet pseudophakic eyes or healthy eyes. Eyes with higher SUN scores showed proportionately increased ILS. The receiver-operator characteristic analysis indicated no advantage in using the smaller slit width in discriminating ILS at different SUN scores although it provided higher axial resolution. CONCLUSIONS: The lock-in-based spot fluorometer is reliable for measurement of ILS with high precision and accuracy.The measured ILS correlates linearly with SUN scores and can be used to provide a higher granularity for recording aqueous flare. TRANSLATIONAL RELEVANCE: The instrument can be used in the clinical management of uveitis and drug development toward uveitis.

Penetration of mucoadhesive chitosan-dextran sulfate nanoparticles into the porcine cornea.

Topical application of drugs to the eyes suffers from poor bioavailability at the ocular surface and in the anterior chamber. This is due to rapid clearance of the drug because of tear secretion and outflow. This study has investigated mucoadhesive and penetration characteristics of chitosan-dextran sulfate nanoparticles (CDNs), prepared by polyelectrolyte complexation technique, following topical administration to the ocular surface. Topical FITC-labeled CDNs (FCDNs; mean size of 400nm and a surface charge of +48mV) were retained on the porcine ocular surface for more than 4h. Topical FCDNs were partially endocytosed into porcine corneal epithelial cells via a clathrin-dependent pathway. After 6h of topical FCDNs, particles accumulated in the corneal epithelium but not found in the corneal stroma. When epithelium was removed, FCDNs penetrated the stroma. Thus, CDNs are potentially useful for drug/gene delivery to the ocular surface and to stroma when epithelium is damaged.

Crosslinked chitosan-dextran sulfate nanoparticle for improved topical ocular drug delivery.

PURPOSE: To examine the benefits of chitosan-dextran sulfate nanoparticles (CDNs) as a topical ocular delivery system with lutein as a model drug. METHODS: CDNs were developed by polyelectrolyte complexation of positively charged chitosan (CS) and negatively charged dextran sulfate (DS). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and polyethylene glycol 400 (PEG400) were used as co-crosslinking and stabilizing agents, respectively. The influence of these on the properties of CDNs, including drug release and mucoadhesiveness, was examined. The chemical stability of lutein in CDNs (LCDNs) was also examined. RESULTS: Typically, LCDNs showed a spherical shape, possessing a mean size of ~400 nm with a narrow size distribution. The entrapment efficiency of lutein was in the range of 60%-76%. LCDNs possessing a positive surface charge (+46 mV) were found to be mucoadhesive. The release profile of LCDNs followed Higuchi's square root model, suggesting drug release by diffusion from the polymer matrix. Lutein in LCDNs showed increased chemical stability during storage compared to its solution form. CONCLUSIONS: These characteristics of CDNs make them suitable for drug delivery to the ocular surface.