The effects of proinflammatory cytokines on the apoptosis of corneal endothelial cells following argon laser iridotomy.

The aim of this study was to evaluate the relationship between the expression of proinflammatory cytokines and the apoptosis of corneal endothelial cells after argon laser iridotomy (ALI). ALI was performed on each quadrant of the iris in the right eye of mice (ALI1 group). Left eyes were used as control group. The levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß, and interferon (IFN)-γ in mice eyes were measured, and TUNEL staining was performed 12 h after ALI. Mice in the ALI-Dexa group were pretreated daily with an intraperitoneal injection of dexamethasone for 4 days before undergoing ALI and compared with mice without dexamethasone pretreatment (ALI2 group). Twelve corneas from six rabbits were incubated ex vivo with (n = 6) or without (n = 6) IL-1ß. TUNEL staining was performed 24 h after ex vivo incubation. In the mice experiment, the levels of IL-1ß, TNF-α, TGF-ß, and IFN-γ were increased in the ALI1 group compared to the control group. Although many TUNEL-positive cells were observed in the ALI1 group, those were not detected in the control group. Dexamethasone pretreatment inhibited the increase in the levels of all four proinflammatory cytokines and reduced TUNEL-positive cells. In the rabbit experiment, TUNEL-positive cells were increased in the incubated corneas with IL-1ß compared to those without IL-1ß. Expression of proinflammatory cytokines following ALI seems to play a role in the apoptosis of corneal endothelial cells after ALI. Dexamethasone pretreatment inhibited increases in proinflammatory cytokines and reduced the apoptosis of corneal endothelial cells.

mRNA capping enzyme activity is coupled to an early transcription elongation.

One of the temperature-sensitive alleles of CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongation defect. This temperature-sensitive allele, ceg1-63, has an impaired ability to induce PUR5 in response to 6AU treatment and diminished enzyme-GMP formation activity. However, this cellular and molecular defect is not primarily due to the preferential degradation of the transcript attributed to a lack of cap structure. Our data suggest that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation as well as cap formation. First, in addition to the 6AU sensitivity, ceg1-63 is synthetically lethal with elongation-defective mutations in RNA polymerase II. Secondly, it produces a prolonged steady-state level of GAL1 mRNA after glucose shutoff. Third, it decreases the transcription read through a tandem array of promoter-proximal pause sites in an orientation-dependent manner. Taken together, we present direct evidence that suggests a role of capping enzyme in an early transcription. Capping enzyme ensures the early transcription checkpoint by capping of the nascent transcript in time and allowing it to extend further.

Changes in P2Y4 receptor expression in rat cochlear outer sulcus cells during development.

Extracellular adenosine triphosphate (ATP) released from cellular sources plays an important role in variety of the cochlear physiologic processes. The primary purinergic receptor subtype in the cochlea is the P2X2 receptor, which is a subtype of P2X receptor. This receptor appears to mediate a protective decrease in the electrical driving force in response to acoustic overstimulation. Outer sulcus cells (OSCs) in the cochlear lateral wall appear to maintain an adequate K+ concentration in the cochlear endolymph in response to varying intensities of auditory stimulation. However, little is known about developing OSCs. The purpose of this study was to investigate subtypes of purinergic receptors in developing rat OSCs using a voltage-sensitive vibrating probe. Results showed that only two P2 receptors (P2Y4 and P2X2) contributed to the regulation of short circuit currents in neonatal OSCs. ATP increased cation absorption via apical nonselective cation channels after activating P2Y4 receptors in early neonatal OSCs. P2Y4 expression rapidly declined postnatally and reached near adult levels on postnatal day 14. P2X2 was co-expressed with P2Y4 in early neonatal OSCs. Temporal changes in P2Y4 during OSC development might be involved in the establishment of the endolymphatic ion composition needed for normal auditory transduction and/or specific cellular differentiation.

Effects of purinergic stimulation on ciliary beat frequency and chloride secretion in sinusitis.

OBJECTIVES: We aimed to identify the functional abnormality of sinusitis-affected mucosa by observing the responsiveness of the mucosa to purinergic stimulation after the onset of sinusitis and during the recovery period. We also aimed to identify possible beneficial effects of purinergic agonists on sinusitis. METHODS: A rabbit sinusitis model was developed by blocking maxillary ostia. Sinus mucosae were harvested immediately and 1 and 4 weeks after reopening the ostia. We measured chloride secretion and ciliary beat frequencies responding to purinergic stimulation. RESULTS: The increases of ciliary beat frequency by adenosine triphosphate (100 micromol/L) were 3.2%+/-8.5%, 7.9%+/-2.3%, and 12.2%+/-1.9% immediately after establishment of sinusitis and 1 week and 4 weeks after reopening of ostia, respectively. Chloride secretion stimulated by adenosine triphosphate also showed gradual increase during the recovery period. Grossly, the mucosae appeared to have normalized in 80% (4 of 5) after 4 weeks; however, functional and microscopic improvements were still incomplete. CONCLUSIONS: Mucosal functions, assessed by increase of ciliary activity and ion secretion by purinergic stimulation, and microscopic findings showed gradual but incomplete recovery after 4 weeks of recovery, in contrast to the gross normalization. Purinergic agonists may have beneficial effects on sinusitis by stimulating decreased ciliary motility and chloride secretion in sinusitis.

Reactive blue 2, an antagonist of rat P2Y4, increases K+ secretion in rat cochlea strial marginal cells.

Extracellular ATP decreases K+ secretion in strial marginal cells via apical P2Y4 receptors. We investigated the effect of reactive blue 2 (RB-2), an antagonist of rat P2Y4, on rat strial marginal cells using a voltage-sensitive vibrating probe. The application of RB-2 increased K+ secretion in a dose-dependent manner, and this increase was characterized as a peak followed by a partial relaxation to a steady-state. Moreover, this response was similar to that caused by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Suramin had no similar effect, except at high concentration. Thus, we tested the effects of these chemicals on P2Y4 receptors in strial marginal cells. Both RB-2 and DIDS had antagonistic activities at P2Y4, and the antagonist potency at P2Y4 paralleled the potency of K+ secretion. Interestingly, 2'- and 3'-O-(4-benzoyl-benzoyl)adenosine 5'-triphosphate (BzATP) exhibited an agonistic effect at P2Y4 receptor, which was blocked by RB-2, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Based on these results, we speculate that direct and/or indirect inhibitory mechanisms between P2Y4 and KENQ1/KCNE1 K+ channels exist in strial marginal cell.

Effect of Titanium Dioxide Nanoparticle Exposure on the Ocular Surface: An Animal Study.

PURPOSE: To evaluate the effect of titanium dioxide (TiO2) nanoparticle exposure on the ocular surface. METHODS: Eighty eyes of 40 rabbits were used. The TiO2-1D group (n = 20) received a single instillation of TiO2 in the right eye. The TiO2-4D group (n = 20) received a TiO2 instillation in the right eye once a day for four days. The 40 untreated left eyes were used as controls. Ocular surface staining (n = 5 for each group) was performed with rose bengal dye, tear secretion (n = 5) was measured using the phenol red thread test, lactic dehydrogenase (LDH) activity (n = 5) and MUC5AC levels (n = 5) were measured in tears, and the area of the conjunctival goblet cells (n = 5) was measured through impression cytology and scanning electron microscopy 24 hours after the last TiO2 instillation. RESULTS: Ocular surface staining was increased but the tear secretion was not changed after TiO2 exposure. The TiO2-1D (1.39 OD) and TiO2-4D groups (0.58 OD) had higher median tear LDH activity than the control groups (0.57 OD and 0.29 OD, respectively). Although the median tear MUC5AC level in the TiO2-1D group (92.7 ng/ml) was higher than that of control 1 group (37.4 ng/ml), there was no significant difference in MUC5AC levels between the TiO2-4D and control 2 groups. Conjunctival goblet cell area decreased after TiO2 exposure. CONCLUSIONS: Exposure to TiO2 nanoparticles induced ocular surface damage. Although the tear MUC5AC level increased after a single exposure, it decreased to normal levels after repeated exposures. The area of conjunctival goblet cells decreased after TiO2 exposure.

Comparison of Cytotoxic Effects on Rabbit Corneal Endothelium between Preservative-free and Preservative-containing Dorzolamide/timolol.

PURPOSE: To evaluate and compare the toxic effects of eyedrops containing a fixed combination of 2.0% dorzolamide and 0.5% maleate timolol with or without preservatives on rabbit corneal endothelium. METHODS: This study was performed with 22 eyes of New Zealand white rabbits. Dorzolamide/timolol eyedrops with preservative (Cosopt group) or without preservative (Cosopt-S group) were diluted with a balanced salt solution at a 1 : 1 ratio. We injected 0.1 mL of diluted Cosopt into the anterior chamber of left eyes and an equal volume of diluted Cosopt-S into the anterior chamber of right eyes. Corneal thickness, corneal haze, and conjunctival injection were measured before and 24 hours after treatment. Endothelial damage was compared between both eyes by vital staining (alizarin red/trypan blue staining), live/dead cell assay, TUNEL assay, and scanning electron microscopy. RESULTS: Corneal endothelial damage was severe in the Cosopt group. Cosopt-treated eyes exhibited remarkable corneal edema and prominent apoptosis of endothelial cells. In addition, the live/dead cell assay revealed many dead cells in the endothelium, and scanning electron microscopy analysis showed that corneal endothelial cells exhibited a partial loss of microvilli on the surface as well as extensive destruction of intercellular junctions. However, in the Cosopt-S group, corneal edema was mild and the damage to the corneal endothelium was minimal. CONCLUSIONS: The main cause of corneal endothelial toxicity was due to the preservative in the dorzolamide/timolol fixed combination eyedrops, and not the active ingredient. Thus, it appears to be safer to use preservative-free eyedrops during the early postoperative period.

Effects of argon laser iridotomy on the corneal endothelium of pigmented rabbit eyes.

PURPOSE: In Asian countries, laser iridotomy for the treatment of angle-closure glaucoma is a common cause of bullous keratopathy, which may be associated with a shallow anterior chamber and dark iris pigmentation in Asians. Several cases of corneal decompensation after argon laser iridotomy have been reported. In the present study, we evaluated the harmful effects of argon laser iridotomy on the corneal endothelium. METHODS: Argon laser iridotomy was performed on the right eyes of pigmented rabbits. Changes in corneal thickness and endothelial cell density after laser iridotomy were evaluated. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed for assessment of corneal endothelial cell apoptosis. Combined staining with alizarin red and trypan blue, as well as a live/dead cell assay, were performed for evaluation of damage to the corneal endothelium induced by laser iridotomy. RESULTS: Corneal thickness did not change immediately after laser iridotomy; however, a significant increase was observed 24 hours after iridotomy (p = 0.001). The endothelial cell density of laser-treated eyes four days after laser iridotomy was significantly decreased compared with control eyes (p < 0.001). TUNEL staining showed many TUNEL-positive cells in the corneal endothelium and corneal stroma. No endothelial trypan blue-stained cell nuclei were observed after laser iridotomy; however, several large endothelial cells with damaged membrane integrity were observed. The live/dead cell assay clearly showed a large number of dead cells stained red in several areas throughout the entire corneal button 24 hours after iridotomy. CONCLUSIONS: Argon laser iridotomy induces corneal endothelial cell apoptosis in pigmented rabbit eyes, resulting in decreased endothelial cell density.

Comparison of aqueous levels of inflammatory mediators between toxic anterior segment syndrome and endotoxin-induced uveitis animal models.

PURPOSE: To compare clinical findings and the aqueous levels of inflammatory mediators between toxic anterior segment syndrome (TASS) and endotoxin-induced uveitis (EIU) animal models and to evaluate the efficacy of systemic steroid pretreatment in both animal models. METHODS: Rats were used in this study. Ortho-phthalaldehyde solution was injected into the anterior chamber to produce TASS (n=30), and lipopolysaccharide was injected into one hind footpad to produce EIU (n=30). Clinical findings were evaluated under slit-lamp examination, and the aqueous levels of prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α) were measured 24 hours after these procedures. Twelve of the rats in each animal model were pretreated with intraperitoneal injection of dexamethasone for 4 days before the development of TASS and EIU. RESULTS: Corneal haze scores were significantly higher for TASS than EIU, but clinical scores for anterior uveitis were not different between the two animal models. Although aqueous levels of PGE2 were markedly increased in both animal models, PGE2 levels were significantly higher for TASS than for EIU. However, an increase in aqueous levels of TNF-α was observed only in EIU. Dexamethasone pretreatment reduced the corneal haze score and clinical score for anterior uveitis in both animal models and inhibited the increase in aqueous levels of PGE2 and TNF-α. CONCLUSIONS: Prostaglandin E2 and TNF-α in aqueous humor seem to be regulated differently in animal models of TASS and EIU. However, dexamethasone pretreatment improved the clinical findings and inhibited the increases in PGE2 and TNF-α in both animal models.

Protective effects of dispersive viscoelastics on corneal endothelial damage in a toxic anterior segment syndrome animal model.

PURPOSE: We evaluated whether viscoelastics have protective effects on the corneal endothelial cell damage in a toxic anterior segment syndrome (TASS) animal model depending on the types of viscoelastics. METHODS: A TASS animal model was established with an injection of 0.1 mL o-phthaldehyde solution (0.14%) into the anterior chamber of New Zealand white rabbits. One of two different viscoelastics, 1% sodium hyaluronate (cohesive group) or a 1:3 mixture of 4% chondroitin sulfate and 3% sodium hyaluronate (dispersive group), was injected into the anterior chamber. After five minutes, it was removed using a manual I/A instrument, and then 0.1 mL of o-phthaldehyde solution (0.14%) was injected into the anterior chamber. Damage to corneal endothelial cells was compared between the two groups. RESULTS: The corneal thickness increased quickly in both groups after the disinfectant injection. However, the dispersive group showed relatively mild corneal edema compared to the cohesive group. The mean corneal haze score in the dispersive group also was lower than that of the cohesive group. These partial protective effects of the dispersive viscoelastic were demonstrated by the different findings of a live/dead cell assay, TUNEL staining, and scanning electron microscopy between the two groups. CONCLUSIONS: The TASS animal model seems to be a useful means to evaluate corneal endothelial cell damage caused by toxic substances to find ways to protect or reduce endothelial cell damage. Dispersive viscoelastics were shown to have partial protective effects against corneal endothelial cell damage caused by a toxic disinfectant.

Differential regulation of gene expression by RNA polymerase II in response to DNA damage.

Cells change their gene expression profile dynamically in various conditions. By taking the advantage of ChIP, we examined the transcription profile of Saccharomyces cerevisiae genes in response to DNA damaging agents such as MMS or 4NQO. Gene expression profiles of different groups of genes roughly correlated with that revealed by Northern blot assay or microarray method. Damage-inducible genes showed increased cross-linking signals of RNA polymerase II, TFIIH, and TFIIF, meanwhile damage repressible genes decreased them, which means that gene expression is mainly regulated at the level of transcription. Interestingly, the characteristic occupancy pattern of TFIIH and polymerase with phosphorylated carboxy-terminal domain (CTD) in promoter or in coding regions was not changed by the presence of DNA damaging agents in both non-inducible and inducible genes. ChIP data showed that the extent of phosphorylation of CTD per elongating polymerase complex was still maintained. These findings suggest that overall increase in CTD phosphorylation in response to DNA damage is attributed to the global shift of gene expression profile rather than modification of specific polymerase function.