Basil Rigas

Background: The symptoms of dry eye disease (DED) result from activation of ocular sensory nerves and constitute the dominant component of its clinical presentation. We assessed the effect of phosphosulindac (PS), a small molecule efficacious in the treatment of DED in preclinical models, on corneal sensitivity (CS). Methods: CS was determined with a Cochet-Bonnet esthesiometer in New Zealand white (NZW) and Dutch-belted black (DBB) rabbits. DED was induced by Concanavalin A injections into the rabbits’ lacrimal glands. Changes in CS, tear osmolarity, tear break up time (TBUT) and Schirmer tear test were measured before and after DED induction. PS in various formulations (emulsions, nanoparticles and hydrogels) and other ocular drugs were applied in eye drop form to normal rabbits and those with DED. Results: nduction of DED caused a decrease in the CS, TBUT time as measured by fluorescein dye, in tear production as measured by Schirmer’s tear test and an increase in tear osmolarity. PS markedly restored the suppressed CS in dry eyes. The effect was immediate, fully reversible, lasted ∼26 h and appeared dissociated from its anti-inflammatory properties. The most efficacious formulation was a Carbopol-based hydrogel; cyclodextrin-based and emulsion formulations were also effective. The most optimal dose of PS was 0.2% and the optimal pH was 6.2. None of 7 compounds structurally related to PS affected CS nor did cyclosporine, lifitegrast and artificial tears. PS does not have an anesthetic effect on the cornea. In normal eyes, PS suppressed CS and this effect was concentration-, formulation-, and pH-dependent. Two non-steroidal anti-inflammatory drugs (NSAIDs), ketorolac and bromfenac, and lidocaine suppressed CS in normal but not in dry eyes. Conclusion: PS restores the suppressed CS in dry eyes possiblely by a direct effect on corneal nerves. This effect appears unique to PS, distinct from all tested compounds including the two currently approved drugs for DED. PS, in addition to affecting CS of DED, may improve its symptoms and merits further evaluation for the treatment of DED.

Purpose: To perform a comparative analysis of 13 over the counter artificial tear formulations and perflourohexyloctane assessing performance using objective, non-invasive imaging. The data provides a rationale to make better informed therapeutic decisions regarding their use. Patients and Methods: Prospective, sequential study of 13 drop formulations in participants over 18 years of age. Tear meniscus height, non-invasive keratographic break up time, and central lipid layer thickness were measured before and 5 and 15 minutes after drop administration. Outcomes were compared using linear mixed models. Predictors included group (between-subjects), time (within-subjects), and the group by time interaction. Results: One hundred and thirty-seven participants, mean age 67 years (54.3% female), were evaluated. Most drops showed increased tear meniscus height at 5 and 15 minutes compared to baseline. Changes in non-invasive keratographic break up time and lipid layer thickness were more variable. A few artificial tears demonstrated better performance with regards to tear meniscus height, non-invasive keratographic break up time or lipid layer thickness; but no single drop outperformed others across all three measured endpoints. Perflourohexyloctane did not show better performance in any test parameter. Conclusion: The performance of various over-the-counter artificial tears can be determined with non-invasive imaging technologies. Certain drops can outperform others with regard to individual test parameters suggesting that personalization or optimization of therapy may be possible. Both active and inactive ingredients contribute to the efficacy of artificial tears. Further work is needed to determine if differences observed here translate into meaningful clinical differences for dry eye disease treatment. This report highlights the complexity of dry eye disease and the need for ongoing work to find optimal ways to treat the ocular surface.

Experimental studies of Dry Eye Disease (DED) using animal models are hampered by the lack of reliable, easy-to-use assays that can adequately diagnose disease or monitor effects of novel treatments. The Oculus Keratograph 5 M, an advanced keratography unit (AKU), has shown promise, enjoying recent clinical use. We assessed whether this AKU could be used in DED studies in the rabbit, perhaps the ideal experimental animal for this disease. All measures were made in strictly controlled temperature and humidity spaces. A panel of AKU parameters was measured in 15 New Zealand White rabbits at baseline, after Concanavalin A induced DED, and following recovery. Eyelid aperture and corneal irregularity were also measured. A subset of these parameters was measured in patients and compared with those from rabbits. AKU parameters in both humans and rabbits showed similar patterns and coefficients of variation (CV). Measurements of tear and eyelid architecture were more reproducible than tear film function in both species. The CV for most parameters were less than the observed changes in the respective parameters after DED induction. In rabbits, all parameters improved returning close to baseline following DED recovery. In the rabbit, additional measures (eyelid aperture and corneal irregularity) not traditionally associated with DED, also demonstrated changes that evolved over the development and recovery of DED. AKU technology can effectively detect changes in multiple parameters during the evolution and resolution of DED in rabbits. DED parameters showed similar patterns for most variables in both humans and rabbits demonstrating great potential of this device in translational research. The AKU can also follow additional parameters evaluating the responses of the lacrimal functional unit. Our findings document the applicability of this technology for translational studies of DED and underscores its potential to further refine understanding of the disease pathophysiology.

Background: Chemotherapy-induced peripheral neuropathy (CIPN), a side effect of chemotherapy, is particularly difficult to treat. We explored whether phosphosulindac (PS), a modified NSAID, could treat CIPN. Methods: CIPN was induced in male C57BL/6 J mice by paclitaxel, vincristine or oxaliplatin. Mechanical allodynia was measured with the von Frey test and cold allodynia with the acetone test. To determine the preventive effect of PS, it was administered 2 days before the induction of CIPN. Mouse Lewis lung carcinoma xenografts were used to determine if PS altered the chemotherapeutic efficacy of paclitaxel. Cultured cell lines were used to evaluate the effect of PS on neuroinflammation. Results: Treatment with each of the three chemotherapeutic agents used to induce CIPN lowered the mechanical allodynia scores by 56 to 85% depending on the specific agent. PS gel was applied topically 3x/day for 16–22 days to the hind paws of mice with CIPN. This effect was dose-dependent. Unlike vehicle, PS returned mechanical allodynia scores back to pre-CIPN levels. PS had a similar effect on paclitaxel-induced CIPN cold allodynia. Sulindac, a metabolite of PS, had no effect on CIPN. PS significantly prevented CIPN compared to vehicle. Given concomitantly with paclitaxel to mice with lung cancer xenografts, PS relieved CIPN without affecting the anticancer effect of paclitaxel. The enantiomers of PS were equally efficacious against CIPN, suggesting the therapeutic suitability of the racemate PS. There were no apparent side effects of PS. PS suppressed the levels of IL-6, IL-10, CXCL1, and CXCL2 induced by paclitaxel in a neuroblastoma cell line, and macrophage activation to the M1 proinflammatory phenotype. Conclusion: Topically applied PS demonstrated broad therapeutic and preventive efficacy against CIPN, preserved the anticancer effect of paclitaxel, and was safe. Its anti-CIPN effect appears to be mediated, in part, by suppression of neuroinflammation. These data support further evaluation of topical PS for the control of CIPN. Chemotherapy-induced peripheral neuropathy (CIPN), a side effect of chemotherapy, has a prevalence as high as 85%, depending on its duration and the agent used: 72.3% with oxaliplatin, 42.2% with cisplatin, 70.8% with paclitaxel, 19.6% with vincristine (Seretny et al., 2014; Flatters et al., 2017). CIPN pain is caused by damage to nerve structures such as peripheral nerve endings, which become extremely sensitive to stimulation and can generate pulses even in the absence of stimulation (Staff et al., 2017). Patients diagnosed with CIPN may report sharp pains, dull aches, paresthesia, altered sensitivity to cold, loss of proprioception, difficulty in using the hands or in walking (Kolb et al., 2016), numbness, and loss of pain sensation. Hands and feet are most typically affected. The neuropathic pain of CIPN is particularly difficult to treat (Hou et al., 2018; Loprinzi et al., 2020). With the exception of tetrodotoxin, several agents have failed to show efficacy when subjected to the rigors of a clinical trial (Quintao et al., 2019; Huerta et al., 2023). It is currently managed with oral antidepressants (e.g., duloxetine) and/or antiepileptics (e.g., gabapentin and pregabalin), which provide unsatisfactory pain control and induce major side effects and poor treatment adherence. The only approved drug (duloxetine) is generally considered ineffective. A recent ASCO guideline states that “no agents are recommended for the prevention of CIPN” and that “duloxetine is the only agent that has appropriate evidence to support its use for patients with established painful CIPN. Nonetheless, the amount of benefit from duloxetine is limited” (Loprinzi et al., 2020). CIPN adversely affects cancer patients: First, CIPN can have a profound negative impact on long-term quality of life (Park et al., 2013; Ewertz et al., 2015). Second, CIPN increases the cost of care (Pike et al., 2012). Third, symptoms can be so severe that patients deem CIPN so unacceptable that they choose to discontinue treatment despite the potential for decreased survival (Miltenburg and Boogerd, 2014). The mechanism of CIPN appears complex and may vary depending on the causative agent (Eldridge et al., 2020; Kocot-Kępska et al., 2021). Possible mechanisms include the following: effects on mitochondria, which lead to oxidative stress (Doyle and Salvemini, 2021); effects on ion channels, which may alter nerve signal conduction (Aromolaran and Goldstein, 2017); disruption of microtubules, which could impact axonal transport of proteins, RNA and mitochondria along the nerve axon (Chua et al., 2022); and neuroinflammation which could enhance neuronal excitability and pain hypersensitivity (Makker et al., 2017). Neuroinflammation is emerging as a key component in the pathophysiology of CIPN, with innate and adaptive immune responses contributing to its symptoms, while macrophages and glial cells help maintain the neuroinflammatory process (Fumagalli et al., 2021). Macrophages, forming dense infiltrates in CIPN affected nerves (Ma et al., 2022), may significantly contribute to peripheral neuropathies, including CIPN (Msheik et al., 2022). Interestingly, interferon-γ can enhance macrophage activation, inducing high levels of proinflammatory cytokines and low levels of anti-inflammatory cytokines (Schroder et al., 2004). The resultant secretion of pro-inflammatory mediators may lead to some of the symptoms of CIPN. In particular, pro-inflammatory cytokines released upon treatment are considered a key trigger of neuroinflammation in the sensory nervous system leading to sensitization of nociceptors and mechanical hypersensitivity (Brandolini et al., 2019). Among them, IL-6 plays a large role in the inflammatory process following nerve injury and has been implicated in the initiation and maintenance of neuropathic pain (De Jongh et al., 2003). It was shown, for example, that women with breast cancer and CIPN had significantly higher levels of IL-6 compared to controls (Starkweather, 2010). Anti-inflammatory cytokines such as interleukin IL-10, can reduce the neuropathic pain of CIPN. CXCL-1 and CXCL-2, both pro-inflammatory cytokines known to mediate the infiltration of neutrophils and monocytes/macrophages (Lee et al., 2018), seem to also play a significant role in CIPN (Biber and Boddeke, 2014). For example, their expression is altered in CIPN, and receptor antagonists attenuate neuropathic pain (Zhou et al., 2020). The relationship between changes in cytokine expression and CIPN symptoms has been explored in animal models and indirectly in patients with CIPN. For example, intraperitoneally injected IL-6 induced nociceptive hypersensitivity (Cunha et al., 2005) while IL-6 knockout mice demonstrated lowered mechanical allodynia following vincristine treatment (Kiguchi et al., 2008). Animal data align with clinical studies demonstrating increased IL-6 serum levels in CIPN-affected women following chemotherapy, indicating the importance of assessing IL-6 as a potential mediator of CIPN (Starkweather, 2010). Another example is provided by CCL2, which is upregulated in injured DRG neurons implying a role as neuron-migroglia signaling factor participating in the pathogenesis of neuropathic pain (Zhang et al., 2007). Finally, IL-10 gene therapy in rats (injected with an IL-10-encoding plasmid) prevents and reverses paclitaxel-induced mechanical allodynia (Schroder et al., 2004; Ledeboer et al., 2007). Given the lack of efficacious treatments for CIPN, we explored whether phosphosulindac (PS, OXT-328) could be efficacious in its prevention and treatment. PS consists of conventional sulindac to which a diethylphosphate group is covalently attached through a butane spacer. PS, extensively studied for its anticancer and anti-inflammatory properties (Sun and Rigas, 2008; Mackenzie et al., 2011; Huang et al., 2011a,b; Cheng et al., 2012, 2013; Mattheolabakis et al., 2013), consistently displayed higher efficacy than sulindac and lacked side effects. Its anticancer efficacy is significant and broad, encompassing colon, lung and skin cancer (applied topically in the latter (Cheng et al., 2012). PS was also efficacious in animal models of arthritis (Huang et al., 2011a; Mattheolabakis et al., 2013), essentially reversing joint inflammation and edema. An important feature of PS is its chirality, deriving from the chiral center at its methyl sulfoxide moiety (Brunell et al., 2011). Chirality can shape the pharmacology of a given drug (Silvestri and Colbon, 2021), and differences in efficacy and toxicity can exist between enantiomers. Several NSAIDs are chiral molecules, and some are marketed as a single enantiomer. Thus, we separated the enantiomers of PS and determined their efficacy in an animal model of paclitaxel-induced peripheral neuropathy (PN). Three murine cell lines, Neuro-2a (neuroblastoma), RAW264.7 (macrophage), and Lewis lung carcinoma (LL/2), were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown as monolayers in the culture medium and under conditions suggested by ATCC. All cell lines were passaged in our laboratory for less than 6 months after receipt. Phosphosulindac was a gift of Medicon Pharmaceuticals, Inc., Setauket, NY. We separated the two enantiomers of PS by supercritical fluid chromatography using an AD-H (25×0.46 cm) column (3,5-dimethylphenylcarbamate bound to amylose supported on silica gel; Analytics-Shop USA LP, Stockbridge GA) and purified several grams of each. PS and its two enantiomers were each formulated in a gel based on hydroxypropyl-methylcellulose as the gelling agent and xanthan gum as a phase stabilizer. Briefly, PS was solubilized in a 4:1 ethanol: triethanolamine mixture, and this was followed by the addition of water containing the perservative benzalkonium chloride 0.05% w/v. After proper mixing, 15 mg of both xanthan gum and hydroxypropyl methylcellulose were added and the mixture was sonicated. This was followed by addition of 100 μL of glycerol and thorough mixing. Finally, the pH of the gel was adjusted to 7.5 with sodium citrate. Paclitaxel, oxaliplatin, and vincristine were purchased from MilliporeSigma (St. Louis, MO). Paclitaxel was dissolved in a mixture of 1 volume ethanol/1 volume Cremophor EL (EMD Millipore Corp, Burlington, MA)/18 volumes distilled water. Oxaliplatin was dissolved in ddH₂O. Vincristine was dissolved in PBS. All injections were administered intraperitoneally (i.p.) in a volume of 1 mL/100 g body weight. The strain, sex and age of the mice to be used in CIPN studies are critical determinants of the degree of allodynia that can be induced by specific chemotherapeutic agents [reviewed in Höke and Ray (2014), Gadgil et al. (2019)]. Younger male mice appear to display more robust allodynia in response to chemotherapy. Preliminary work with several strains of mice and other parameters led us to conduct our studies using adult male C57BL/6 J mice, 8 weeks of age at the beginning of the experiments and weighing 20–30 g (The Jackson Laboratory, Bar Harbor, ME). Mice were housed in an AAALAC-accredited facility in groups of four. Food and water were available ad libitum. The mice in each cage were randomly allocated to treatment groups. All studies were conducted by experimenters blinded to the identity of the treatment groups. Experiments were performed during the light cycle (7:00 am to 7:00 pm) and animals were euthanized with CO₂ asphyxiation. Studies were approved by the Institutional Animal Care and Use Committee of Stony Brook University and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010). Adult male C57BL/6 J mice as above were inoculated subcutaneously into each flank with 1.0 × 10⁶ murine Lewis lung carcinoma cells in PBS (final volume 100 μL). Once the tumor reached approximately 180 mm³, animals were randomized into the control group, which received the solvent of paclitaxel, and the three treatment groups, which received only paclitaxel or paclitaxel plus vehicle gel or 5% PS gel (n = 8 per group). Paclitaxel 12 mg/kg was administered intraperitoneally once daily for 5 consecutive days. The vehicle or PS gel was applied topically onto both hind paws of the mice 3x/day, 7 days/week for 14 days. Tumor volume was calculated as [length × width × (length + width/2) × 0.56]. CIPN was induced in mice with chemotherapeutic agents using established protocols (Carozzi et al., 2010; Currie et al., 2019; Eldridge et al., 2020). Dosing regiments for each of the three chemotherapeutics were as follows. Paclitaxel: Four i.p. injections of 8 mg/kg paclitaxel every other day, resulting in a cumulative dose of 32 mg/kg. Oxaliplatin: Oxaliplatin 3 mg/kg was injected i.p. daily for 5 days, followed by 5 days of no treatment, which was followed by another 5-day period of daily oxaliplatin i.p. injections as previously for 10 injections leading to a cumulative dose of 30 mg/kg. Vincristine: Two i.p. injections of vincristine 1.5 mg/kg were made within one week for a total cumulative dose of 3 mg/kg. We used one protocol for the treatment of established CIPN and a second for its prevention. Treatment protocol: Once CIPN was established, documented by reduced mechanical allodynia threshold, PS 8% or vehicle control (gel) was applied three times daily to the hind paws of the mice for the indicated period. Prevention protocol: Administration of PS 8% or vehicle to the hind paws of mice as above started two days before initiating the administration of paclitaxel as above. Mechanical allodynia was measured before the administration of PS and on day 10 after initiation of PS treatment. In these mice, we determined both mechanical and cold allodynia, as described below. Mechanical allodynia thresholds were determined using von Frey filaments according to an established method (Chaplan et al., 1994; Bagdas et al., 2015). Briefly, mice were placed in a quiet room for 30 min and then put in a Plexiglas cage with mesh metal flooring where they were allowed to acclimate for 15 min before testing. A series of calibrated von Frey filaments (Stoelting, Wood Dale, IL) with incremental stiffness were applied perpendicularly to the paw with sufficient force to cause slight bending and held 2–3 s. This process was repeated at each level of stiffness 5 times, a few seconds apart. Paw withdrawal, licking or shaking were considered positive responses. The mechanical threshold or the paw withdrawal threshold (PWT) expressed in g, indicates the force of the von Frey filament to which the animal reacted. This was performed according to published methods (Otrubova et al., 2013; Toma et al., 2017). After placing the mice in a quiet room and acclimating them as above, we applied 10 μL of acetone held in the end of a pipette to the plantar surface of each hind paw by a blast of air applied to the other end of the pipette. Total time spent licking, lifting and/or shaking of the hind paw during a 1-min test period was recorded as the cold allodynia score, which was measured in seconds. Neuro-2A and RAW 264.7 cells were co-cultured in the presence and absence of paclitaxel and/or PS. Cells were grown in basic growth media including 1:1 mixture of DMEM (Gibco, 11,995,065) and EMEM (ATCC, 30–2003) with 10% heat-inactivated fetal bovine serum (Sigma Aldrich, F9665), 1% penicillin/streptomycin and 2 mM glutamine. Cells were split 1:10–1:20 using DPBS (Sigma, D8537) and 0.05% trypsin–EDTA (Fisher Scientific, 10,779,413) at 70–80% confluence. Only cells below passage 15 were used. For co-culture, 0.3×10⁶ cells of each cell line were seeded in 6-well plates (ThermoFisher Scientific, 165,218) and incubated overnight. Immediately before treatment, the culture medium was supplemented with DMSO (Sigma-Aldrich, D2650) for controls, or paclitaxel (Sigma-Aldrich,T7402), PS or their combination. Stock solution in DMSO (Sigma-Aldrich, D2650) of paclitaxel 10 mM and PS 100 mM were used to provide the final concentration of 1 μM and 20 μM, respectively (Stage et al., 2020). Control samples were treated with DMSO. After 24 h, media were collected, centrifuged and frozen at −80\u00B0C until analyzed. The levels of cytokines in the cell culture medium were determined using the magnetic bead-based immunoassay kit (Luminex 200; Luminex Corp., Austin, TX) and the MILLPLEX Mouse Cytokine/chemokine Magnetic Panel (#MPXMCYTO-70 K, Millipore, Billerica, MA). Cell supernatant samples were incubated with antibody-coated capture beads overnight at 4°C. Washed beads were further incubated with biotin-labeled anti-human cytokine antibodies, followed by streptavidin– phycoerythrin incubation. Standard curves of known concentrations of recombinant mouse cytokines/chemokines were used to convert fluorescence units to concentrations (μg/mL). To calculate the molecular concentration of cytokines in culture media samples, we analyzed the median fluorescent intensity data using a 5-parameter logistic or spline curve-fitting method. Murine macrophage RAW 264.7 cells were grown in DMEM (ThermoFisher Scientific, Waltham, MA) supplemented with 10% FBS, 1% penicillin–streptomycin, 0.5 mM 2-mercaptoethanol, and 40 mM HEPES buffer (all from Sigma, St. Louis, MO) at 37°C in a 5% CO₂ incubator. Cells, maintained for E. coli O55:B5 (Sigma, St. Louis, MO) in Opti-MEM (Invitrogen Life Technologies, Waltham, MA) after which IFN-γ and LPS containing media were replaced with DMEM as previously. Activated RAW cells displayed morphological changes (Zhang et al., 2007). Prior to treating these cells with 20 μM PS or 1 μM paclitaxel or 20 μM PS plus 1 μM paclitaxel for 24 h, their culture medium was changed to EMEM:DMEM, 1:1 supplemented with 10% FBS. A software license for Prism 10.0.2 was purchased from Graphpad Software, LLC (Boston, MA, USA) to analyze dose–response data and to calculate the EC₅₀ for phosphosulindac efficacy in restoring mechanical allodynia scores back to normal control levels prior to the administration of paclitaxel. The data were analyzed using the software’s Nonlinear fit – [Agonist] vs. response – Variable Slop (four parameter) routine. The data are fit to the equation where: The EC₅₀ is the dose of agonist that gives a response half way between Bottom and Top. The Hill slope describes the steepness of the family of curves. A Hill slope of 1.0 is standard; a Hillslope >1.0 is steeper and a Hill slope of Y axis. Results are expressed as mean ± SEM. All values obtained have been included in the analysis. Analysis of variance (ANOVA) tests were conducted and followed by the Bonferroni post hoc test. Differences were determined to be significant at p < 0.05/*. We assessed the effect of PS in mice with established CIPN, reflecting the clinical situation in which patients present with CIPN after their chemotherapy is completed. As shown in Figures 1A–C, each of the three anticancer drugs that we studied induced significant CIPN, evidenced by allodynia. Topical treatment with PS 8% gel 3x/day was started after the neuropathy was established. This dose was chosen from previous studies in our laboratory investigating the efficacy of PS as an anti-cancer and anti-arthritis agent (Mackenzie et al., 2010; Huang et al.,...

Purpose: To evaluate the hypothesis that 3 novel compounds, OXT-328, Q-922, and CL-717 show efficacy in the treatment of oxygen-induced retinopathy (OIR) and whether or not their route of administration is intravitreal, topical, or systemic. Methods: The OIR mouse model, characterized by an avascular area (AVA) and a neovascular area (NVA) of the retina, was used to study retinopathy of prematurity and other retinal diseases characterized by abnormal vessel growth. We measured the effect of our compounds on both the AVA and NVA in whole mounts of mouse retinal tissue. We also evaluated their ability to prevent new vessel formation in chicken chorioallantoic membranes (CAMs). Finally, we measured the in vitro uptake and biodistribution of topically applied CL-717 in human eye explants. Results: In mice with OIR, compared to controls, a single intravitreal administration of Q-922 or OXT-328 significantly reduced both AVA and NVA. CL-717 administered as eye drops over 5 days also reduced AVA and NVA, whereas OXT-328 eye drops had no effect. Q-922 given intraperitoneal (150 mg/kg/day × 5 days) reduced AVA and NVA. Remarkably, explanted human eyes bathed in CL-717 show rapid uptake and biodistribution in ocular tissues. In the chicken CAM model, all 3 compounds reduced the formation of new blood vessels by about one-third. No side effect in mice was observed, except for mild ocular surface irritation with Q-922. Conclusions: Systemic administration of Q-922 or topical administration of CL-717 holds particular promise for a simplified treatment of proliferative retinopathies without the necessity of intravitreal injections.

Purpose: Dry eye disease (DED) is classified as aqueous deficient, evaporative, or mixed. We investigated the therapeutic effect of the novel anti-inflammatory drug phosphosulindac (PS) in rabbit models of DED encompassing its pathogenesis, and its transition to chronicity. Methods: We treated three rabbit models of DED with PS (hydrogel formulation) or vehicle topically applied 1 × /day. We induced aqueous-deficient DED (acute and chronic) by injecting Concanavalin A into lacrimal glands; evaporative DED by injecting into the upper eyelid inactivated Mycobacterium tuberculosis in complete Freund's adjuvant; and mixed DED through desiccative stress, induced by holding open the eye for 3 h. We determined corneal sensitivity, tear break-up time (TBUT), Schirmer's tear test (STT), tear osmolality, and fluorescein staining of the ocular surface. Results: PS reversed all abnormal DED parameters. In acute DED, PS dose dependently normalized corneal sensitivity and tear osmolality; and improved TBUT, STT, and fluorescein staining. PS normalized corneal sensitivity and improved all other parameters in chronic aqueous-deficient DED. In evaporative DED, PS normalized corneal sensitivity and improved TBUT and fluorescein staining (osmolality and STT were not significantly changed in this model). In the desiccative stress model, PS improved TBUT and fluorescein staining but had no effect on STT or tear osmolality. Conclusions: PS rapidly reversed almost all DED parameters in its three subtypes. The normalization of the suppressed corneal sensitivity suggests the possibility of marked symptomatic relief by PS. The hydrogel formulation allows once-daily dosing. PS merits further development as a potential treatment for DED.

To develop a more efficient impression cytology (IC) method for the transfer of ocular surface cells onto glass microscope slides for cytochemical, immunocytochemical, and immunofluorescence studie...