Tear film osmolarity and ocular surface disease in two rabbit models for keratoconjunctivitis sicca

We report the natural history of keratoconjunctivitis sicca (KCS) in two rabbit models. The first one (full KCS model) was created by closing the lacrimal gland excretory duct, and removing the nictitating membrane and harderian gland. We created the second one (lacrimal gland duct only [LGDO]-KCS model) by closing the lacrimal gland excretory duct. Although tear film osmolarity was abnormally high in both models, it was higher in the full KCS model. Decreases in corneal epithelial glycogen and in conjunctival goblet cell density, and morphological abnormalities of the conjunctiva correlated with increases in tear film osmolarity and duration of disease.     

Tear film and ocular surface changes after closure of the meibomian gland orifices in the rabbit

To determine whether meibomian gland dysfunction can increase tear film osmolarity and produce ocular surface changes analogous to those seen with lacrimal gland disease (keratoconjunctivitis sicca [KCS]), the authors closed the meibomian gland orifices in the right eyes of 11 rabbits by light cautery and studied the changes for 20 weeks. Tear film osmolarity was increased throughout the observation period. Conjunctival goblet cell density and corneal epithelial glycogen levels declined progressively. Closure of the meibomian gland orifices thus increased tear film osmolarity in the presence of normal lacrimal gland function and caused ocular surface abnormalities similar to KCS.     

Tear diluents in the treatment of keratoconjunctivitis sicca

To determine the optimum solution concentration for lowering elevated tear film osmolarity in keratoconjunctivitis sicca (KCS), tear osmolarity was measured in four KCS patients before and after instillation of either an isotonic saline solution or one of four hypotonic saline solutions (range, 75-225 mOsm/L). Average tear osmolarity one minute after instillation was significantly lower with the hypotonic solutions than with the isotonic saline (mean +/- SEM, 290 +/- 3 mOsm/L vs. 317 +/- 1 mOsm/L, P less than 0.0005). Solutions 150 mOsm/L or less were most effective in lowering osmolarity; the 75 mOsm/L solution was occasionally associated with irritation. In 16 KCS patients, we then compared the therapeutic efficacy of the 150 mOsm/L solution with that of an otherwise identical isotonic solution in a two-week, double-masked, crossover study. The 150 mOsm/L solution was superior for symptom relief by nearly 2:1 (P = 0.01).     

Open-label crossover study of vitamin A ointment as a treatment for keratoconjunctivitis sicca

The authors evaluated the efficacy of all-trans retinoic acid (vitamin A) ointment as a treatment for keratoconjunctivitis sicca (KCS) in a group of 11 patients selected on the basis of clinical history, slit-lamp examination results, rose Bengal staining, and tear film osmolarity. In this open-label crossover study, vitamin A ointment was no more effective than placebo in increasing tear secretion, as indicated by Schirmer test with proparacaine or tear film osmolarity, or in decreasing ocular surface disease, as indicated by rose Bengal staining. Seven patients stated some preference for the placebo ointment, two patients for the vitamin A ointment, and two patients had no preference.     

Keratoconjunctivitis sicca in male patients infected with human immunodeficiency virus type 1

Keratoconjunctivitis sicca (KCS) has not been reported as occurring as a single entity in the acquired immune deficiency syndrome (AIDS) population. In a survey of human immunodeficiency virus type 1 (HIV-1) infected male patients, the authors found that 21% (9/42) had signs and symptoms compatible with KCS with positive Schirmer test results. Tear osmolarity determinations were obtained from this group and from an age- and sex-matched group of HIV-infected patients without symptoms of KCS and with negative Schirmer test results. Eighty-nine percent of the suspect group had increased tear osmolarity, whereas none of the control patients had a hyperosmolar tear film (P less than 0.0001). Results strongly suggest that KCS occurs at a significantly greater rate in male individuals infected with HIV-1 than in the general population.     

An electrolyte-based solution that increases corneal glycogen and conjunctival goblet-cell density in a rabbit model for keratoconjunctivitis sicca.

Thirty-two rabbits with monocular surgically induced keratoconjunctivitis sicca (KCS) underwent masked treatment for 12 weeks with 1 of 4 artificial tear solutions. Disease in each group of treated rabbits was compared with disease in untreated KCS controls. One of the solutions tested was a unique electrolyte-based formulation shown previously to preserve normal goblet-cell density after extended exposure in normal rabbits. Only the electrolyte-based solution decreased elevated tear osmolarity and sodium after 9 weeks of treatment (P less than 0.05). At 20 weeks, mean corneal glycogen and conjunctival goblet-cell density in eyes treated with the electrolyte-based solution increased significantly relative to untreated KCS controls (P less than 0.01). With the other three solutions, mean glycogen levels and goblet-cell densities were either decreased relative to untreated KCS controls (P less than 0.05) or were unchanged. The electrolyte-based solution is the first treatment to increase corneal glycogen and conjunctival goblet cells in a rabbit model of KCS.     

P2Y(2) receptor stimulation increases tear fluid secretion in rabbits

PURPOSE: The purinergic P2Y(2) receptor agonists stimulate active Cl-transport across the excised rabbit conjunctival tissue in vitro. We determined whether UTP or ATP could increase the tear volume and change tear fluid composition in normal rabbits in vivo. METHODS: Fifty mL was applied to rabbit eyes of UTP, ATP at concentrations of 0.1, 0.3, 1, 3, 8.5% (1.8-154 mM) or saline. A modified Schirmer test with topical anesthesia was performed 5, 15, 30 and 60 min after the instillation. In studies lasting 30 days, 50 microL of 0.5% UTP was applied 6 times a day for 4 weeks. Tear samples were collected from the conjunctival sac with a glass microcapillary. The protein profile of the tear fluid was analyzed by SDS-polyacrylamide gel electrophoresis and total protein was measured with the Bradford assay. The Easy-Titer rabbit IgG assay kit was used for the determination of immunoglobulin G (IgG). RESULTS: UTP had dose-dependent stimulatory effects on tear secretion. It maximally increased tear secretion about 4-fold 15 min after its application. Similar effects were obtained with ATP. Repeated treatment with UTP reproducibly increased tear volume. Furthermore, UTP did not decrease total protein and IgG concentration in tear fluid and it had no effect on the protein profile. CONCLUSION: These data indicate that activation of P2Y(2) receptor increases tear fluid secretion accompanied with some proteins in normal rabbits. The purinergic agonists, UTP and ATP, have potential therapeutic value in the treatment of dry eye.     

Basal and Reflex Human Tear Analysis: I. Physical Measurements: Osmolarity,Basal Volumes,and Reflex Flow Rate

Minimally stimulated, retained “basal” tears and stimulated reflex tears were collected from normal controls, keratoconjunctivitis sicca (KCS) patients, and contact lens (CL) wearers. Basal tear samples were collected on small filter paper strips (Periopaper®) over a five-second period, and volume was measured by means of an electronic device (Periotron®). Collected basal tear volumes for KCS patients (0.84 ± 0.42 μl) were significantly lower (P <; 0.01) than normal controls (1.18 ± 0.36) and CL wearers (1.24 ± 0.27). Reflex tear flow rates were measured over a five-minute period on Schirmer strips. Volume was calculated by comparison of wet length with known volumes of 1% egg white lysozyme solution. The reflex tear flow rates in KCS patients (3.29 ± 3.57 μl/minute) were significantly lower than normal controls (5.71 ± 5.86) and CL wearers (6.96 ± 6.07). The elevation in CL wearers was not statistically significant when compared to normals. KCS patients are deficient in both basal and reflex tears compared to normals but have a more significant deficiency of basal tears. Female normals and CL wearers over 40 years of age have a higher tear osmolarity than those under 41 years of age. Female KCS patients over 40 years of age have a tear osmolarity that is not significantly different from female KCS patients under 41 years of age.     

Potentiation of sympathetic neurosecretion by forskolin and cyclic AMP in the rabbit iris-ciliary body

Forskolin has been reported to stimulate cAMP formation and reduce intraocular pressure in rabbit and primate eyes. In view of recent evidence for the involvement of cAMP in modulation of transmitter release at adrenergic synapses, we have investigated the presynaptic effects of forskolin and other cAMP activators on field-stimulated secretion of 3H-norepinephrine (3H-NE) in the isolated, perfused rabbit iris-ciliary body. Forskolin (10(-7)-10(-5) M) was found to markedly enhance stimulation-evoked 3H-NE release without affecting basal (spontaneous) release. The response to forskolin was potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 0.5 mM) and was mimicked by the cell-permeant cyclic nucleotide analog 8-bromo-cAMP. 8-bromo-cGMP also produce a small enhancement of stimulus-evoked 3H-NE secretion, whereas IBMX alone had little effect on either stimulated or basal secretion. These results suggest that cAMP may play an important neuromodulatory role in regulation of norepinephrine release at intraocular synapses, and raise the possibility that the ocular hypotensive response to forskolin in rabbit eyes may be mediated, in part, by enhanced adrenergic neurosecretion.     

Comparison of tear secretion and tear film instability after photorefractive keratectomy and laser in situ keratomileusis

PURPOSE: To evaluate and compare tear secretion and tear film instability following photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, Yonsei University School of Medicine, Seoul, Korea. METHODS: In a prospective study, 36 eyes (21 patients) had PRK and 39 eyes (25 patients) had LASIK to correct myopia. Tear secretion and tear film instability were tested preoperatively and 3 and 6 months postoperatively using Schirmer test values, tear breakup time (BUT) scores, and tear osmolarity. RESULTS: Six months after surgery, the change in Schirmer test values from preoperative levels was -14.57% +/- 6.39% (SD) in the PRK eyes and -23.40% +/- 5.94% in the LASIK eyes and the change in BUT scores, -12.54% +/- 8.28% and -18.79% +/- 13.01%, respectively. The change in tear osmolarity was 14.95% +/- 6.46% and 35.63% +/- 8.51%, respectively. CONCLUSIONS: The decrease in tear secretion was greater after LASIK than after PRK at 6 months. Proper treatment of dry eye is required after LASIK and PRK, particularly in the LASIK postoperative period.     

人工高眼压家兔血液,虹膜及房水cAMP,cGMP含量的关系

目的研究家兔血液、虹膜及房水中环腺苷酸（ｃｙｃｌｉｃａｄｅｎｏｓｉｎｅｍｏｎｏｐｈｏｓｐｈａｔｅ，ｃＡＭＰ）和环鸟苷酸（ｃｙｃｌｉｃｇｕａｎｏｓｉｎｅｍｏｎｏｐｈｏｓｐｈａｔｅ，ｃＧＭＰ）含量与高眼压之间的关系。方法用放射免疫方法测定家兔血浆、虹膜和房水中ｃＡＭＰ、ｃＧＭＰ含量。结果ｃＡＭＰ含量（ｎｍｏｌ／Ｌ）：血浆为７２．８１±７．６７，虹膜为１４１．０８±１６．７０，房水中为２８．４０±０．４９；ｃＧＭＰ含量（ｎｍｏｌ／Ｌ）：血浆为１０．３７±６．１５，虹膜为３５．６４±７．０２，房水中为１２．９０±０．６１。ｃＡＭＰ含量高于对照组（Ｐ＜０．０１），ｃＧＭＰ低于对照组（Ｐ＜０．０１）。结论高眼压与ｃＡＭＰ、ｃＧＭＰ含量关系显著。     

Cyclic nucleotide distribution in identified layers of suprafused rabbit retinas

To assess the effects of pharmacologic agents on metabolite levels in identified retinal layers we have devised an apparatus that allows suprafusion of everted eye cups (vitreal surface exposed) or isolated retinas (photoreceptor surface exposed), recording of electroretinograms (ERG), and rapid freezing in a configuration which permits obtaining frozen tangential sections of the retina. Samples from identified layers of the freeze-dried sections can be subsequently weighed and processed for biochemistry by Lowry methods (Lowry and Passonneau , 1972), or cyclic nucleotides can be extracted from weighed samples and assayed. To test the usefulness of the apparatus, we have compared levels of guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP), measured in defined layers of suprafused rabbit retinas, to concentrations reported for retinal layers from eyes removed from light- and dark-adapted rabbits and quickly frozen. For most layers the concentrations of cyclic nucleotides were similar in both the suprafused and whole eye preparations. In experimental applications we suprafused dark-adapted eye cups and retinas with a physiological saline whose free calcium level was sharply reduced by chelation, and observed that cGMP levels were elevated only in those retinal layers containing photoreceptor portions. Ethyleneglycol bis-(beta-amino-ethyl ether)N,N'-tetraacetic acid (EGTA) (3 mM) yielded a threefold increase in cGMP in 10 min when applied to the photoreceptor surface of isolated retinas. However, when the vitreal surfaces of everted eye cups were suprafused with calcium-chelated medium for 10 min, a threefold increase in cGMP required 20 mM EGTA, 3 mM EGTA being ineffective. In another study, vitreal surfaces of eye cups or photoreceptor surfaces of retinas were suprafused with physiological saline containing 3 mM 3-isobutyl-1-methylxanthine (IBMX) in order to alter cAMP retinal levels. In both cases, eightfold increases in cAMP were measured in the inner nuclear and inner plexiform layers, while three- to fourfold increases occurred in the outer nuclear and outer plexiform layers. This agent also caused a large increase in cAMP in the pigment epithelium.     

Development of a rabbit model of tear film instability and evaluation of viscosity of artificial tear preparations

OBJECTIVE: The purposes of this study were to establish a quantitative method for evaluating rabbit tear film status and investigate the efficacy of artificial tear preparations through ocular surface bathing or eye drop application. METHODS: The rabbit tear film was evaluated using a noninvasive specular reflection video recording system. The appearance of a tear break area (TBA) on the tear film images (7.4 mm2/mm) after 30 seconds of eye opening was quantified by image analysis. To induce disruption of the rabbit tear film, the ocular surface was challenged for 60 minutes with 1 ppm hypochloric acid (HOCl). Immediately after irrigation, artificial tear preparations composed of viscosity agents sodium hyaluronate (SH), hydroxypropylmethycellulose (HPMC), hydroxyethylcellulose (HEC), or chondroitin sulfate (CS) were applied to the rabbit eye through ocular surface bathing or eye drop application, and the recovery of the disrupted tear film was compared for each preparation. RESULTS: A dramatic increase in TBA was observed immediately after the ocular surface was challenged with HOCl, and it returned to the initial level after 6 hours. Immediately after ocular surface bathing and eye drop application, a dramatic recovery of TBA was observed in all the test solution-treated eyes. One hour after treatments, prolonged amelioration of the tear film instability was observed after ocular surface bathing, but not by eye drop application, with the artificial tear preparations composed of HPMC or SH. CONCLUSION: Ocular surface bathing with artificial tear preparations composed of a suitable viscosity agents could be useful in managing tear film instability.     

局部抗青光眼药物中苯扎氯胺导致干眼症的研究进展

A large part of glaucoma patients who have been treated with topical anti-glaucoma drugs for a period have experienced dry eye of varying degrees. Preservatives in anti-glaucoma drugs, particularly benzalkonium chloride (BAK）, are believed to play a major role in the development of dry eye in patients. Based on existing research, this paper summarizes several main mechanisms of benzalamine causing dry eye. BAK can directly acts on tear film and destroy tear film stability, resulting in tear evaporation increasing and tear film osmolarity elevating. BAK has cytotoxic effects on corneal epithelial cells, conjunctival goblet cells and meibomian gland cells, thus can affect the secretion of mucus and lipid in tear film. BAK can also cause chronic inflammation of the eye surface, leading to discomfort in patients. By exploring the mechanism of BAK causing dry eye, we hope to provide ideas for guiding the use of glaucoma and developing a new antiseptic system for eyedrops.(Int Rev Ophthalmol, 2021, 45：420-425)     

The ocular penetration of topical forskolin and its effects on intraocular pressure, aqueous flow rate and cyclic AMP level in the rabbit eye

The ocular penetration of 14C-forskolin in suspension was studied using albino rabbits. The effects of topical forskolin suspension on cyclic AMP (cAMP) synthesis, aqueous flow and intraocular pressure (IOP) were also studied. It was shown that only 0.03% of the instilled forskolin penetrated the ocular tissue. The calculated kep value for forskolin was 0.2 X 10(-4) cm/hr. The peak concentrations were calculated to be 4 X 10(-7) mole/liter, 4.6 X 10(-7) and 2.7 X 10(-7) mole/1,000 g tissue in aqueous, iris and ciliary body, respectively, after instillation of 1% forskolin suspension. Topical 1% forskolin suspension caused cAMP increase in the aqueous humor 30 minutes after instillation, but cAMP returned to baseline level 60 minutes after instillation. The cAMP level in the ciliary body was not increased by forskolin. Aqueous flow did not change, and the IOP was slightly decreased 45 and 60 minutes after instillation of forskolin suspension. The in vivo least-effective concentration of forskolin in the ciliary epithelium was considered to be about 2.7 X 10(-7) mole/1,000 g tissue. The weak IOP lowering effect of topical forskolin suspension was considered to be due to its poor ocular penetration. However, slight modification of molecular structure might increase ocular penetration. Present results suggest only a slight increase in penetrative potential would be needed to make forskolin effective in antiglaucoma therapy.     

Investigation of tear film change after recovery from acute conjunctivitis

PURPOSE: To study the changes of tear film after recovery from acute conjunctivitis. METHODS: This study involved 73 eyes of 56 consecutive patients who complained of dry eye after recovery from acute conjunctivitis at the Zhongshan Ophthalmic Center. Excluded were other factors that could affect the stability of the tear film. Tear film breakup time (BUT), Schirmer 1 test (S1T), tear meniscus height (TMH), and fluorescein staining (FL) were performed on both recovered and healthy eyes. The scores were measured at 3, 7, 14, 21, and 30 days after recovery. RESULTS: Compared with the results of the healthy eyes, most scores of BUT, S1T, TMH, and FL were all abnormal until day 30 after recovery from acute conjunctivitis. BUT decreased at 3, 7, 14, and 21 days (P < 0.05). S1T decreased at 3, 7, and 21 days (P < 0.05). TMH values became less than normal at 3, 7, and 14 days (P < 0.05). FL increased significantly at 7, 14, and 21 days (P < 0.05). At 30 days after recovery, all of the test scores returned to normal (P > 0.05). CONCLUSIONS: During acute conjunctivitis, inflammation and topical therapeutic agents can alter the tear film secretion, resulting in dry eye for nearly 1 month in recovered eyes. To minimize the effect of topical agents to the tear film, individualized treatment instead of frequent instillation of topical agents is recommended for acute conjunctivitis.     

Keratoconjunctivitis sicca in juvenile rheumatoid arthritis

PURPOSE: To compare the symptoms, signs, and results of objective tests for keratoconjunctivitis sicca (KCS) in patients with juvenile rheumatoid arthritis (JRA) and controls. METHODS: Sixty-four patients with JRA and 64 age- and sex-matched controls were compared in terms of symptoms, signs, and results of objective tests for KCS. Relation between tear film breakup time (TBUT), Schirmer test results, and JRA-related variables such as age of onset, duration, and type of JRA; presence of antinuclear antibodies (ANAs); and history of uveitis were evaluated. Analysis of variance, multivariate regression analysis, Kruskall-Wallis, Student t tests, and chi tests were used for statistical analysis. RESULTS: Twelve and a half percent of patients with JRA complained of dry eye symptoms compared with 1.5% of the controls (P = 0.031). Dry eye signs were detected in 10.9% of patients with JRA compared with 1.5% of controls (P = 0.038). TBUT and Schirmer test results were lower in the JRA group than in controls (P = 0.032 and P = 0.029, respectively). Seven patients (10.9%) had definite and 1 (1.5%) had probable diagnosis of KCS in the JRA group compared with no children in the control group (P = 0.034). Within the JRA group, Schirmer test and TBUT results were significantly lower in male patients and ones with longer duration of disease. CONCLUSIONS: The prevalence of symptoms, signs, and definite diagnosis of KCS is higher and basal tear secretion and tear film stability are lower in children with JRA than in controls. Among children with JRA, male sex and longer duration of disease are independent risk factors for having decreased basal tear secretion and tear film stability.     

Effects of carbamylcholine on nuclear cyclic nucleotide-dependent protein kinase activity in cultured corneal epithelial cells of the rabbit

Cyclic nucleotide-dependent protein kinase activity and cyclic nucleotide binding were assayed in cell homogenates and subcellular fractions of cultured rabbit corneal epithelium, and effects of carbamylcholine (1 mM) on activity in each fraction were evaluated. In cell homogenates and in nuclei, carbamylcholine significantly elevated cGMP-dependent kinase activity and [3H]cGMP binding, and reduced cAMP-dependent kinase activity and [3H]cAMP binding. In the cytosol, the drug significantly reduced cAMP kinase and cAMP binding but did not alter cGMP binding or kinase activity. In both mitochondria/lysosomes and microsomes, cGMP binding was significantly enhanced and cAMP binding reduced, but differences in protein kinase activity were not significant. The drug did not alter cyclic nucleotide-independent protein kinase activity. All observed effects were blocked by atropine. Kinase activity in the purified nuclear fraction also was assayed over a range of carbamylcholine, substrate, and cyclic nucleotide concentrations. Vmax for cGMP kinase in control nuclei was 295 +/- 18 pmol/mg protein/min (KM = 367 +/- 70 micrograms/ml histone) vs. Vmax = 846 +/- 6 pmol/mg/min (KM = 131 +/- 13 mu/ml histone) in carbamylcholine-treated nuclei. Vmax for cAMP kinase in control nuclei was 282 +/- 12 pmol/mg/min (KM = 172 +/- 7 micrograms/ml histone); 1 mM carbamylcholine abolished nuclear cAMP-dependent kinase activity. Cyclic nucleotide concentrations at half-maximal binding were 5.4 +/- 0.9 nM cGMP and 9.3 +/- 0.4 nM cAMP, as compared to 15.4 +/- 5.0 nM cGMP and 36.2 +/- 6.2 nM cAMP for half-maximal protein kinase activity. Regression analysis of Hill plots for variation of nuclear cyclic nucleotide binding and kinase activity as a function of carbamylcholine concentration indicated half-maximal drug effects on activity of both enzymes at approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tear lipocalin and lysozyme in Sj?gren and non-Sj?gren dry eye

PURPOSE: To evaluate the concentration of tear lipocalin, lysozyme, and total protein in Sj?gren's Syndrome (SS), non-Sj?gren's keratoconjunctivitis sicca (KCS), and non-dry-eyed (NDE) individuals. METHODS: Seventy-six subjects were recruited for this study: 25 SS subjects; 25 KCS subjects, and 26 NDE individuals. Symptoms were measured with a visual analogue scale. Tear flow was measured by the Schirmer I test without anesthesia. Tears were collected using an eye wash technique. Total tear protein was quantified using the DC Protein Assay Kit. Tear lipocalin and lysozyme were quantified via Western blotting performed on a Phast System. RESULTS: By definition, the SS and KCS groups both had significantly lower mean Schirmer scores (5.12 +/- 5.96 mm and 7.84 +/- 7.35 mm) compared with the NDE group (23.83 +/- 7.85 mm; p < 0.0001). There was no difference in mean Schirmer scores between SS and KCS groups (p = 0.19). The tear film of the SS group was characterized by significantly reduced (p < 0.0001) total protein and lipocalin concentrations compared with both KCS and NDE groups. No difference between the KCS and NDE groups was found in total protein (p = 0.92) or lipocalin (p = 0.19) concentration. In contrast, the concentration of tear film lysozyme was found to be statistically similar in all three groups examined. No statistically significant correlation was found in any group between mean Schirmer values compared with total protein, lipocalin or lysozyme concentration. CONCLUSION: Our data demonstrate a biochemical distinction between the Sj?gren's group compared with both KCS and control groups, in that both tear lipocalin and total tear protein were significantly reduced. Although correlations were not found between protein measurements and tear flow, a combination of tests including Schirmer I and quantitation of tear film biomarkers may allow for the identification of SS patients without the need for invasive testing.