Stimulated Tear Secretion Research Articles

Novel CFTR Activator Cact-3 Ameliorates Ocular Surface Dysfunctions in Scopolamine-Induced Dry Eye Mice.

Cystic fibrosis transmembrane conductance regulator (CFTR) is highly expressed on the ocular epithelium and plays a pivotal role in the fluid secretion driven by chloride transport. Dry eye disease is one of the most common diseases with limited therapeutic options. In this study, a high-throughput screening was performed to identify novel CFTR activators capable of inducing chloride secretion on the ocular surface. The screening of 50,000 small molecules revealed three novel CFTR activators. Among them, the most potent CFTR activator, Cact-3 (7-(3,4-dimethoxyphenyl)-N-(4-ethoxyphenyl)pyrazolo [1,5-α]pyrimidine-2-carboxamide), produced large and sustained Cl− currents in WT-CFTR-expressing FRT cells with no alterations of ANO1 and hERG channel activity. The application of Cact-3 strongly activated CFTR in the ocular epithelia of mice and it also significantly increased CFTR-mediated Cl− transport in a primary cultured human conjunctival epithelium. Cact-3 strongly stimulated tear secretion in normal mice. In addition, Cact-3 significantly reduced ocular surface damage and the expression of proinflammatory factors, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in an experimental mouse model of dry eye disease. These results suggest that Cact-3, a novel CFTR activator, may be a potential development candidate for the treatment of dry eye disease.

Reply re: “Loss of Stimulated Tear Secretion After Orbital Reconstruction”

Dr. Cho is a consultant for Horizon Therapeutics. Dr. Madhan has no financial or conflicts of interest to disclose. Correspondence: Raymond I. Cho, M.D., F.A.C.S., Department of Ophthalmology and Visual Sciences, The Ohio State University Wexner Medical Center and The Ohio State University Comprehensive Cancer Center, James Cancer Hospital and Solove Research Institute, Columbus, OH ([email protected])

Re: "Loss of Stimulated Tear Secretion After Orbital Reconstruction".

Re: "Loss of Stimulated Tear Secretion After Orbital Reconstruction".

Inhibition of Cathepsin S Reduces Lacrimal Gland Inflammation and Increases Tear Flow in a Mouse Model of Sj\xf6gren\u2019s Syndrome

Cathepsin S (CTSS) is highly increased in Sjögren’s syndrome (SS) patients tears and in tears and lacrimal glands (LG) of male non-obese diabetic (NOD) mice, a murine model of SS. To explore CTSS’s utility as a therapeutic target for mitigating ocular manifestations of SS in sites where CTSS is increased in disease, the tears and the LG (systemically), the peptide-based inhibitor, Z-FL-COCHO (Z-FL), was administered to 14–15 week male NOD mice. Systemic intraperitoneal (i.p.) injection for 2 weeks significantly reduced CTSS activity in tears, LG and spleen, significantly reduced total lymphocytic infiltration into LG, reduced CD3+ and CD68+ cell abundance within lymphocytic infiltrates, and significantly increased stimulated tear secretion. Topical administration of Z-FL to a different cohort of 14–15 week male NOD mice for 6 weeks significantly reduced only tear CTSS while not affecting LG and spleen CTSS and attenuated the disease-progression related reduction of basal tear secretion, while not significantly impacting lymphocytic infiltration of the LG. These findings suggest that CTSS inhibitors administered either topically or systemically can mitigate aspects of the ocular manifestations of SS.

Preganglionic Parasympathetic Denervation Rabbit Model for Innervation Studies.

Tear secretion from the main lacrimal gland (LG) is mainly regulated by parasympathetic nerves. We performed several innervation studies to investigate lacrimation. In male rabbits, we performed a retrograde dye-tracing study of LG innervation, evaluated preganglionic parasympathetic denervation, and administered glial cell-derived neurotrophic factor (GDNF) in the surgical area after parasympathetic denervation. Accumulation of fluorescent dye was observed in the pterygopalatine ganglion cells on the same side as the dye injection into the main LG. Fewer stained cells were observed in the cervical and trigeminal ganglia. After parasympathetic denervation surgery, tear secretion was decreased, and fluorescein and rose bengal staining scores were increased at day 1 after surgery and remained increased for 3 months on the denervated side only. Most of the effects in rabbits with parasympathetic denervation were not recovered by administration of GDNF. The main LG is primarily innervated by parasympathetic nerves to stimulate tear secretion. After preganglionic parasympathetic denervation, lacrimation was decreased, resulting in dry eyes, and this was maintained for at least 3 months. Administration of GDNF only minimally altered the effects of denervation.

NOD and NOR mice exhibit comparable development of lacrimal gland secretory dysfunction but NOD mice have more severe autoimmune dacryoadenitis

The male Non-Obese Diabetic (NOD) mouse is an established model of autoimmune dacryoadenitis characteristic of Sjögren's Syndrome (SS), but development of diabetes may complicate studies. The Non-Obese Diabetes Resistant (NOR) mouse is a MHC-II matched diabetes-resistant alternative, but development of autoimmune dacryoadenitis is not well-characterized. We compare features of SS in male NOD and NOR mice at 12 and 20 weeks. Stimulated tear secretion was decreased in 12 week NOD relative to BALB/c mice (p < 0.05), while by 20 weeks both NOD and NOR showed decreased stimulated tear secretion relative to BALB/c mice (p < 0.001). Tear CTSS activity was elevated in NOD and NOR relative to BALB/c mice (p < 0.05) at 12 and 20 weeks. While NOD and NOR lacrimal glands (LG) showed increased LG lymphocytic infiltration at 12 and 20 weeks relative to BALB/c mouse LG (p < 0.05), the percentage in NOD was higher relative to NOR at each age (p < 0.05). Gene expression of CTSS, MHC II and IFN-γ in LG were significantly increased in NOD but not NOR relative to BALB/c at 12 and 20 weeks. Redistribution of the secretory effector, Rab3D in acinar cells was observed at both time points in NOD and NOR, but thinning of myoepithelial cells at 12 weeks in NOD and NOR mice was restored by 20 weeks in NOR mice. NOD and NOR mice share features of SS-like autoimmune dacryoadenitis, suggesting common disease etiology. Other findings suggest more pronounced lymphocytic infiltration in NOD mouse LG including increased pro-inflammatory factors that may be unique to this model.

Novel tear secretion system - the effect and the mechanism of PACAP on tear secretion

Dry eye syndrome is defined as a disorder of the tear film caused by either a decreased production in tears or a disruption to the stability of the complex tear film, which causes damage to the ocular surface. It has been developed the medicine for dry eye syndrome focusing anti-inflammation or mucin secretion, however, no treatment has been developed focusing on the effect of elevation of the lacrimal secretion. We recently identified that pituitary adenylate cyclase-activating polypeptide (PACAP)-null mice develop dry eye-like symptoms such as corneal keratinization and tear reduction. PACAP receptor (PAC1-R) immunoreactivity was observed in the acinar cells of the mouse lacrimal gland. PACAP eye drop significantly stimulated tear secretion level, and the effect was suppressed by pretreatment with PAC1-R antagonist or adenylate cyclase inhibitor. PACAP eye drop on the PACAP KO mouse significantly increased the tear secretion, and continuous eye drop suppressed progression of the corneal keratinization. PACAP eye drops increase aquaporin 5 (AQP5) levels in the membrane of acinar cells in lacrimal glands. AQP5 siRNA treatment significantly attenuates PACAP-induced tear secretion. Based on these results, PACAP might be clinically useful to treat dry eye disorder.

The role of dinucleoside polyphosphates on the ocular surface and other eye structures.

Dinucleoside polyphosphates comprises a group of dinucleotides formed by two nucleosides linked by a variable number of phosphates, abbreviated NpnN (where n represents the number of phosphates). These compounds are naturally occurring substances present in tears, aqueous humour and in the retina. As the consequence of their presence, these dinucleotides contribute to many ocular physiological processes. On the ocular surface, dinucleoside polyphosphates can stimulate tear secretion, mucin release from goblet cells and they help epithelial wound healing by accelerating cell migration rate. These dinucleotides can also stimulate the presence of proteins known to protect the ocular surface against microorganisms, such as lysozyme and lactoferrin. One of the latest discoveries is the ability of some dinucleotides to facilitate the paracellular way on the cornea, therefore allowing the delivery of compounds, such as antiglaucomatous ones, more easily within the eye. The compound Ap4A has been described being abnormally elevated in patient's tears suffering of dry eye, Sjogren syndrome, congenital aniridia, or after refractive surgery, suggesting this molecule as biomarker for dry eye condition. At the intraocular level, some diadenosine polyphosphates are abnormally elevated in glaucoma patients, and this can be related to the stimulation of a P2Y2 receptor that increases the chloride efflux and water movement in the ciliary epithelium. In the retina, the dinucleotide dCp4U, has been proven to be useful to help in the recovery of retinal detachments. Altogether, dinucleoside polyphosphates are a group of compounds which present relevant physiological actions but which also can perform promising therapeutic benefits.

PACAP suppresses dry eye signs by stimulating tear secretion.

Dry eye syndrome is caused by a reduction in the volume or quality of tears. Here, we show that pituitary adenylate cyclase-activating polypeptide (PACAP)-null mice develop dry eye-like symptoms such as corneal keratinization and tear reduction. PACAP immunoreactivity is co-localized with a neuronal marker, and PACAP receptor (PAC1-R) immunoreactivity is observed in mouse infraorbital lacrimal gland acinar cells. PACAP eye drops stimulate tear secretion and increase cAMP and phosphorylated (p)-protein kinase A levels in the infraorbital lacrimal glands that could be inhibited by pre-treatment with a PAC1-R antagonist or an adenylate cyclase inhibitor. Moreover, these eye drops suppress corneal keratinization in PACAP-null mice. PACAP eye drops increase aquaporin 5 (AQP5) levels in the membrane and pAQP5 levels in the infraorbital lacrimal glands. AQP5 siRNA treatment of the infraorbital lacrimal gland attenuates PACAP-induced tear secretion. Based on these results, PACAP might be clinically useful to treat dry eye disorder.

The Effect of Caffeine on Tear Secretion

Caffeine, probably the most widely consumed psychoactive substance, is claimed to have conflicting effects on some tear film dynamics. This study sought to investigate the effect of orally ingested caffeine on tear secretion. In an examiner-masked, placebo-controlled, crossover experimental model, the effect of caffeine intake on tear secretion was studied in 41 healthy volunteers aged 20 to 26 years (mean, 23.0 ± 2.1 years). Participants were randomly assigned into two groups, A and B, to receive two different treatments in two sessions. Subjects in group A were exposed to 5.0 mg/kg body weight of caffeine dissolved in 200 mL of water on their first visit, whereas those in group B were exposed to 200 mL of water. On the second visit, however, the order of treatment was reversed. Schirmer 1 scores were measured repeatedly at 45, 90, 135, and 180 minutes after treatment. The baseline Schirmer 1 scores were compared with posttreatment scores. Schirmer 1 scores increased after caffeine intake. The increase was statistically significant at 45 and 90 minutes (p < 0.05) after caffeine intake. Age, body mass, and blood pressure had no correlation with Schirmer 1 scores (Spearman correlation test, p > 0.05). There was no influence of gender in caffeine's effect on tear secretion (F = 0.994, p = 0.399). From our study, orally ingested caffeine appears to stimulate tear secretion in healthy non-dry eye subjects.

Effect of lacritin, a novel glycoprotein, on ocular surface and tear film integrity

AbstractPurpose Dry eye is extremely common and debilitating. Treatment options are palliative at best and do not address the underlying pathology. Lacritin, a newly discovered glycoprotein, is secreted naturally in human tears by the lacrimal gland. Human recombinant lacritin stimulated tear secretion in normal rabbits and is well tolerated, leading us to hypothesize that topical application of lacritin will not adversely affect ocular surface and tear film integrity.Methods We compared full length lacritin construct with cyclosporin (Restasis®), in terms of tear break up time (TBUT) and goblet cell density using impression cytology. Lacritin (10, 50 μg/ml), or cyclosporin (0.05%), were administered bilaterally to New Zealand white rabbits three times daily for 14d (n=4/group).Results TBUT after lacritin (10 or 50 μg/ml) treatment was 12±2sec, which was similar to vehicle alone, but significantly decreased after cyclosporine treatment for 14d (6±3sec, p&lt;0.001). Preliminary results indicate that goblet cell density decreased by 7±8% after cyclosporine but was not decreased after lacritin. No local irritant effects were noted by slit lamp examination after lacritin treatment, but redness and mild congestion were present in the cyclosporin group.Conclusion Thus, treatment with lacritin was better tolerated than cyclosporin. This data suggests that lacritin does not adversely ocular surface and tear film integrity when compared to cyclosporine. Evaluation with inflammatory markers, such as CD‐11c, is currently ongoing. Glycoproteins, such as, lacritin, represent a new, unique therapeutic approach that may more closely address the pathology of dry eye. Commercial interest

UTP and diadenosine tetraphosphate accelerate wound healing in the rabbit cornea.

Nucleotides are naturally occurring substances present in tear film that can stimulate tear secretion in animals and humans. We investigated whether certain nucleotides can affect the rate of wound healing in the cornea of white rabbits. In the absence of any added compound, the rate of healing was 72.4 +/- 2.2 microm h(-1). Of all the tested nucleotides, UTP and Ap(4)A were the most active ones, maximally increasing the rate of healing to 121.6 +/- 3.7 and 93.7 +/- 3.2 microm h(-1), respectively. Responses to UTP were dose dependent. UTP had a pD(2) value of 8.9 +/- 0.1 (EC(50): 1.25 nM). P2 purinoceptor antagonists such as suramin and reactive blue-2, inhibited the effect of UTP indicating the involvement of P2Y receptors. Mitogen-activated protein kinase (MAPK) cascade inhibitors also abolished the effects of UTP, suggesting that P2Y receptors are coupled to the MAPK cascade, and that this is involved in controlling the rate of epithelial cell migration.

Effects of diadenosine polyphosphates on tear secretion in New Zealand white rabbits.

Extracellular diadenosine polyphosphates play important signaling functions in a number of physiological responses. Here we show that diadenosine polyphosphates are normal constituents of tear fluid and are potent stimulators of tear secretion through their interaction with P2Y receptors. Diadenosine tetraphosphate (Ap(4)A) and Ap(5)A were found in rabbit tears under basal conditions at concentrations of 2.92 and 0.58 microM, respectively. Single applications of UTP, ATP, and Ap(4)A increased tear secretion to 160 +/- 8% (n = 16) (P < 0.001), 131 +/- 6% (P < 0.05), and 162 +/- 11% (P < 0.05) of placebo values, respectively. Ap(4)A, Ap(5)A, and Ap(6)A, but not Ap(2)A and Ap(3)A, were able to stimulate tear secretion in a dose-dependent manner. Concentration-response studies produced pD(2) values of 5.56 +/- 0.03, 5.75 +/- 0.12, and 5.50 +/- 0.09 for Ap(4)A, Ap(5)A, and Ap(6)A, respectively, with Ap(4)A showing the greatest efficacy. Diadenosine polyphosphates also stimulated P2Y(1) and P2Y(2) receptors expressed in 1321N1 cells with no apparent effect on the other P2Y receptors tested. Nonselective P2 antagonists did not modify the tear secretion induced by UTP or Ap(4)A in rabbit eyes in vivo or in cloned receptors, except for a weak but significant reduction in stimulated tear secretion by reactive blue 2. These results suggest that diadenosine polyphosphates stimulate tear secretion via a P2Y receptor-mediated mechanism. Comparing the effects of diadenosine polyphosphates applied to the rabbit eye and to cloned P2Y receptors, it appears that the P2Y(2) receptor subtype is responsible for the prosecretory effects of these compounds.

Stimulation of Tear Secretion and Treatment of Dry-Eye Disease With 3-Isobutyl-1-methylxanthine

We examined the effect of topically applied 3-isobutyl-1-methylxanthine (IBMX), a known secretagogue, on tear secretion and dry-eye disease in a clinical study. We found that IBMX produced a dose-dependent decrease in tear film osmolarity that was significant at 3.0 mmol/L (P less than .0005) in patients with dry-eye disease. This effect was not blocked by prior administration of proparacaine hydrochloride (P less than .05). Throughout a 4-week, open-label, vehicle-controlled study, IBMX decreased tear film osmolarity significantly, whereas vehicle alone did not. After 4 weeks, mean (+/- SEM) osmolarity in IBMX-treated eyes decreased from 325 +/- 3.2 mOsm/L to 312 +/- 1.8 mOsm/L but remained unchanged in vehicle-treated eyes (323 +/- 4.4 mOsm/L vs 320 +/- 4.2 mOsm/L). In our study, IBMX was significantly more effective than vehicle alone in decreasing rose bengal staining (P less than .02). Hence, topical IBMX stimulated tear secretion and decreased ocular surface disease in patients with dry-eye disease.

Symptoms of Canalicular Obstruction

The need for surgical repair of monocanalicular lacerations is controversial. The authors used temporary punctal plugs of hydroxypropyl cellulose to assess the symptoms associated with upper and lower monocanalicular obstruction in 18 normal eyes. Fifty percent of those experimental obstructions resulted in mild intermittent symptoms of epiphora, increased tear film, discomfort, or blurred vision. Constant epiphora was not reported. The results suggest that a single upper or lower canaliculus is sufficient for drainage of minimally stimulated tear secretion but may not completely drain reflex tear secretion. This information may help patients and ophthalmologists reach an informed decision as to the need for surgical repair of a severed canaliculus.

Examination of Mouse Exorbital Lacrimal Gland after Exposure to 2-Chloroacetophenone Vapor

The compound 2-chloroacetophenone has been used to stimulate tear secretion from human subjects, yet the morphological response of the lacrimal gland to this agent has not been experimentally determined. This study used light and electron microscopic techniques to examine the effect of this agent with time on the morphology of the mouse exorbital lacrimal gland. In brief, exposure to 2-chloroacetophenone vapor causes rapid exocytosis of acinar cell granules as well as vacuolation of the secretory and ductal epithelial cells. Concomitantly, intralobular ductal epithelial cells which are normally cuboidal in shape, enlarge in size and release electron-dense granules into the duct lumen. Within 15 min after exposure, acinar cells show a preponderance of cells containing only pale granules and the ductal epithelium returns to a more cuboidal shape. By 60 min after exposure, the gland is not readily distinguishable from the unexposed, control gland.