Multiple cedar pollen challenge diminishes involvement of histamine in allergic conjunctivitis of Guinea pigs.

It has been reported that antihistamines do not fully modify symptoms of allergic conjunctivitis in clinical settings, suggesting that histamine is not the only contributor to symptom generation in the disease. However, in the majority of experimental allergic conjunctivitis models, antihistamines are very effective in the reduction of symptoms. In the present study, we used our recently developed guinea pig model of allergic conjunctivitis and evaluated whether involvement of histamine in the induction of symptoms of allergic conjunctivitis is altered by multiple antigen challenges. Guinea pigs were sensitized by intraperitoneal injection of Japanese cedar pollen extracts adsorbed on aluminum hydroxide gel, and then challenged by dropping a pollen suspension without the adjuvant on each eye once a week until the 15th challenge. The magnitude of the conjunctivitis intensity score (CIS), itch-associated scratching response and albumin leakage were found to increase with repeated challenges. At the 1st-3rd challenges, histamine H(1) receptor antagonist, mepyramine (10 mg/kg, p.o.), strongly reduced all these symptoms. However, symptoms at the 5th-15th challenges were not inhibited by mepyramine. On the other hand, a nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (10 mg/kg, i.v.), potently inhibited the increase of CIS and albumin leakage at the 15th challenge. In conclusion, histamine involvement in the induction of conjunctivitis symptoms in our model was diminished by multiple antigen challenges. The allergic conjunctivitis at the chronic stage is partly mediated by nitric oxide (NO) derived from NOSs that may be activated by mediators other than histamine. The histamine-independent allergic conjunctivitis may be useful for analyzing mechanisms underlying chronic conjunctivitis.

P2Y(2) receptor elicits PAS-positive glycoprotein secretion from rabbit conjunctival goblet cells in vivo.

We investigated in vivo whether UTP and ATP increased periodic acid and Schiff's reagent (PAS)-positive glycoprotein release from rabbit conjunctival goblet cells. Fifty microL of UTP or ATP at the concentrations of 0.003, 0.03, 0.3, 3.0, 8.5% (54 microM-154 mM) or saline were applied to rabbit eyes. Impression cytology was performed on the upper nasal bulbar conjunctiva and the cells were stained with PAS. To clarify purinergic receptor-mediated involvement in this response, suramin (1%; 7 mM), P2Y(2) antagonist and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 0.01%; 167 microM), P2Y(1) antagonist were applied in the rabbit conjunctival sac for 10 min. before UTP or ATP application. Images of the specimens were taken with a digital camera mounted on a microscope and the PAS staining area was measured using an image analyzing system. UTP or ATP eye drop instillation transiently decreased the PAS staining area in a dose-dependent manner, but it gradually recovered after another 30 min. Saline instillation had no effect until 60 min. later. All of the agonists-induced declines were inhibited by pretreatment with 1% (7 mM) suramin but not 0.01% (167 microM) PPADS. UTP and ATP stimulate PAS-positive glycoprotein secretion via P2Y(2) receptor on goblet cells in the rabbit bulbar conjunctiva in vivo.

Effects of substance P and IGF-1 in corneal epithelial barrier function and wound healing in a rat model of neurotrophic keratopathy.

PURPOSE: To establish a rat model of neurotrophic keratopathy and to examine the effects of the combination of substance P (SP) and insulin-like growth factor (IGF)-1 on corneal epithelial barrier function and wound healing in this model. METHODS: Corneal denervation was achieved by thermocoagulation of the ophthalmic branch of the trigeminal nerve. A modified Schirmer test was performed without topical anesthesia. Corneal epithelial barrier function was assessed by measurement of fluorescein permeability with an anterior fluorophotometer. Epithelial wound healing was evaluated by measurement of the area of the defect at various times after removal of the entire epithelium. Eye drops containing both 1 mM SP and IGF-1 (1 micro g/mL) were administered six times daily. RESULTS: The Schirmer test result in eyes subjected to trigeminal denervation was lower than that in control eyes. The fluorescein permeability of the corneal epithelium of denervated eyes was increased relative to that of control eyes. Furthermore, trigeminal denervation induced a delay in corneal epithelial wound healing. Application of eye drops containing SP and IGF-1 to denervated corneas restored the fluorescein permeability of the corneal epithelium to control levels and abolished the delay in epithelial wound healing. CONCLUSIONS: A rat model of neurotrophic keratopathy, characterized by reduced tear secretion, loss of corneal sensation, impaired epithelial barrier function, and delayed epithelial wound healing, was established by trigeminal denervation. Treatment with both SP and IGF-1 improved corneal epithelial barrier function and stimulated corneal epithelial wound healing in this model.

Abnormal protein profiles in tears with dry eye syndrome.

PURPOSE: To verify the hypothesis that protein concentrations, such as lactoferrin, epidermal growth factor (EGF), and aquaporin 5 (AQP5), in tears are abnormal in patients with dry eye. DESIGN: Prospective case-control study. METHODS: One hundred three dry eye patients were divided into three groups: dry eye not associated with the Sjögren syndrome (non-SS; n = 71), Sjögren syndrome (SS; n = 23), and Stevens-Johnson syndrome (SJS; n = 9). Sixteen normal control subjects were also checked. The concentrations of lactoerrin, EGF, and AQP5 were measured by enzyme-linked immunosorbent assay. RESULTS: The concentration of lactoferrin was significantly decreased in tears of non-SS (P =.0001), SS (P =.00005), and SJS (P =.0006) patients compared with control subjects. The concentration of EGF was significantly decreased in non-SS (P =.0005), SS (P =.00002), and SJS (P =.0001) patients compared with control subjects. The concentration of AQP5 was significantly increased in tears of only SS patients (P =.01) compared with control subjects and increased in tears of only SS patients compared with non-SS patients (P =.007). CONCLUSIONS: The decrease in both lactoferrin and EGF was found not only in SS patients but also in non-SS patients, indicating that tear components in dry eyes differ in their quantity and quality. Quantification of AQP5 increased only in SS patients, suggesting that AQP5 protein leaks into the tears when acinar cells of the lacrimal gland are damaged by lymphocytic infiltration.

Restoration of corneal epithelial barrier function and wound healing by substance P and IGF-1 in rats with capsaicin-induced neurotrophic keratopathy.

PURPOSE: To investigate the effects of topical application of the combination of substance P (SP) and insulin-like growth factor (IGF)-1 on corneal epithelial barrier function and epithelial wound closure in rats with capsaicin-induced neurotrophic keratopathy. METHODS: Neonatal rats were injected subcutaneously with a single dose of capsaicin to induce neurotrophic keratopathy. Corneal epithelial barrier function was evaluated with an anterior fluorophotometer. Tear fluid secretion was measured by the Schirmer test. Corneal epithelial wound healing was determined by measurement of the size of the epithelial defect after debridement of the entire epithelium. The combination of SP (1 mM) and IGF-1 (1 micro g/mL) in phosphate-buffered saline was administered in eye drops six times daily. RESULTS: Corneal epithelial barrier function was impaired and corneal epithelial wound healing was delayed in rats injected with capsaicin. The application of eye drops containing the combination of SP and IGF-1 to capsaicin-injected rats resulted in a significant improvement in corneal epithelial barrier function compared with that apparent in capsaicin-injected animals that received eye drops containing vehicle alone. Such treatment with SP and IGF-1 also significantly increased the rate of corneal epithelial wound closure in capsaicin-injected animals. CONCLUSIONS: Topical application of the combination of SP and IGF-1 improved both corneal epithelial barrier function and epithelial wound healing in an animal model of neurotrophic keratopathy.

Novel noninvasive sensitive determination of tear volume changes in normal cats.

We describe a novel, high-resolution and noninvasive method for measuring tear volume changes in cats. The method entails photographing at the lid margin the tear meniscus area defined by instillation of 0.1% fluorescein solution into the cul-de-sac. The inferior tear meniscus area was obtained from the digitized images with computer-assisted software. The tear meniscus area increased in proportion to the saline volume applied into the conjunctival sac, which validates the technique. Furthermore, this technique detected with high sensitivity previously described increases in tear fluid secretion induced by the P2Y(2) agonist. We demonstrate in cats that changes in conjunctival sac tear volume can be evaluated by measurement of its inferior tear meniscus area.

Artificial human corneas: scaffolds for transplantation and host regeneration.

PURPOSE: To review the development of artificial corneas (prostheses and tissue equivalents) for transplantation, and to provide recent updates on our tissue-engineered replacement corneas. METHODS: Modified natural polymers and synthetic polymers were screened for their potential to replace damaged portions of the human cornea or the entire corneal thickness. These polymers, combined with cells derived from each of the three main corneal layers or stem cells, were used to develop artificial corneas. Functional testing was performed in vitro. Trials of biocompatibility and immune and inflammatory reactions were performed by implanting the most promising polymers into rabbit corneas. RESULTS: Collagen-based biopolymers, combined with synthetic crosslinkers or copolymers, formed effective scaffolds for developing prototype artificial corneas that could be used as tissue replacements in the future. We have previously developed an artificial cornea that mimicked key morphologic and functional properties of the human cornea. The addition of synthetic polymers increased its toughness as it retained transparency and low light scattering, making the matrix scaffold more suitable for transplantation. These new composites were implanted into rabbits without causing any acute inflammation or immune response. We have also fabricated full-thickness composites that can be fully sutured. However, the long-term effects of these artificial corneas need to be evaluated. CONCLUSIONS: Novel tissue-engineered corneas that comprise composites of natural and synthetic biopolymers together with corneal cell lines or stem cells will, in the future, replace portions of the cornea that are damaged. Our results provide a basis for the development of both implantable temporary and permanent corneal replacements.

INS365 suppresses loss of corneal epithelial integrity by secretion of mucin-like glycoprotein in a rabbit short-term dry eye model.

P2Y2 receptor agonists, like UTP and ATP, stimulate mucin secretion from goblet cells in vitro. Therefore, mucin stimulants could be good candidates for the treatment of dry eye syndrome because mucin increases the tear film stability and protects against desiccation of ocular surface. INS365 is a more stable P2Y2 receptor agonist than UTP. In the present study, we evaluated, in normal rabbit eyes, its effectiveness to release mucin from goblet cells and to protect the corneal damage induced by desiccation. For mucin secretion, impression cytology was performed following the instillation of INS365 solution or saline into the conjunctival sac. The specimens were stained with periodic acid and Schiff (PAS) reagent, and then the staining area was calculated using computer software. INS365 dose-dependently decreased the PAS staining area of conjunctival goblet cells from 2 to 15 min post-application. Furthermore, we utilized the rabbit short-term dry eye model to evaluate if INS365 eyedrops could protect against any of the damage produced by blockage of blinking with ocular speculum. INS365 significantly suppressed corneal damage at concentrations of more than 0.1% w/v. These results suggest that this P2Y2 agonist is a good candidate for the treatment of dry eye disease.

Effect of gefarnate on the ocular surface in squirrel monkeys.

PURPOSE: To investigate the ability of gefarnate (geranyl farnesylacetate) to stimulate goblet cell function in the primate eye after a mild alkali injury of the tarsal conjunctiva. METHODS: A bilateral injury was created on the conjunctival surface of the lower eye lid of squirrel monkeys by means of a 30-second application of a 4-mm diameter piece of filter paper wetted with 0.5% NaOH. Gefarnate drops (1%) were administered to one eye of each monkey and vehicle alone in the contralateral eye six times a day, 5 days a week for 4 weeks. Slit-lamp biomicroscopy, impression cytology staining of the ocular surface, fluorescein and rose bengal staining, and Western blot for mucin were performed before injury and weekly thereafter. Light microscopy was used to evaluate the lower conjunctiva. RESULTS: Topical application of gefarnate was not associated with any adverse ocular surface effects. Goblet cell repopulation after injury was significantly greater in the gefarnate-treated eyes compared with the vehicle-treated eyes. In the gefarnate-treated eyes, tear mucin content was significantly greater at 1 week after injury. Fluorescein staining was significantly reduced at 3 weeks after injury, and rose bengal staining was significantly reduced in the area of the wound at 2 weeks in the gefarnate-treated eyes compared with the vehicle-treated eyes; at other times, conjunctival staining in the two groups of eyes was not significantly different. CONCLUSIONS: Gefarnate promotes goblet cell repopulation and increases mucin production after a conjunctival injury. No adverse affects of the treatment were found. Thus, this agent may be useful in conditions that diminish goblet cell function.