Abstract: PURPOSE: To compare the effects of four commercially available multipurpose solutions (MPSs) on the structure and barrier function of corneal epithelial tight junctions. METHODS: Human corneal epithelial cells were cultured on collagen-coated slides and then exposed to MPS A (polyhexamethylene biguanide, macrogolglycerol hydroxystearate), MPS B (polyhexamethylene biguanide, poloxamine), MPS C (Alexidine, poloxamine), and MPS D (POLYQUAD, poloxamine) for 60 minutes. Tight junction integrity of the corneal epithelial cells was evaluated with ZO-1 (tight junction-related protein) labeling under laser confocal microscopy. To investigate the changes of ultrastructure in tight junctions of human corneal epithelial cells, an ultrathin cross-section of the cell on collagen membrane was also observed by transmission electron microscopy. For quantitative evaluation of barrier functions, transepithelial electrical resistance of the epithelial cell was measured 30, 60, and 120 minutes after MPS exposure by using a volt ohmmeter. RESULTS: The control (i.e., without MPS treatment) and MPS A-treated epithelial cells showed a normal, continuous linear pattern in ZO-1 staining along with cell-cell junctions. However, epithelial cells treated with MPS B, MPS C, and MPS D showed discontinuous, disrupted line structures at cell-cell borders. This may correspond to a partial breakdown of epithelial tight junctions. Treatment of epithelial monolayers with MPS B, MPS C, and MPS D caused a time-dependent decrease in transepithelial electrical resistance, whereas there was no significant difference between the MPS A-treated group and the control group. CONCLUSIONS: These results suggest the possibility that frequent use of a MPS with high cytotoxicity may lead to the breakdown of epithelial barrier functions and increase the risk of associated microbial infections in hydrogel lens wearers.
Abstract: PURPOSE: To investigate the role of mitogen-activated protein kinase (MAPK), such as p44/42 MAPK, p38 MAPK and stress-activated protein kinase (SAPK), in corneal epithelial cells during the wound healing process. METHODS: A single non-penetrating incision was produced on rat cornea. Then the corneal wound healing process was observed with an immunocytochemical technique using specific antibodies reacting only with phosphorylated p44/42 MAPK, p38 MAPK or SAPK. Cell lysates of corneal epithelial cells in rabbits stimulated with keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were processed for Western blot using antibodies to phosphorylated p44/42 MAPK. RESULTS: Maximum activation of p44/42 MAPK was observed in wing and basal cells at wounded regions in rat cornea at 1 hour after the incision. Activation of p44/42 MAPK was still detected in all basal and wing cells at wounded regions at up to 24 hours when the incisions were completely closed, and then receded to normal intensity after 7 days. Neither p38 MAPK nor SAPK were activated during the wound healing process. Western blot analysis of cultured corneal epithelial cells in rabbits showed phosphorylation of p44/42 MAPK after 30 minutes in response to KGF and HGF, whereas non-activated p44/42 MAPK was ordinarily detected even at the absence of KGF or HGF. CONCLUSIONS: These results demonstrate that p44/42 MAPK is activated during the corneal wound healing process and suggest that KGF and HGF play an important role in initiation of cell migration and proliferation in the initial wound healing process by activating p44/42 MAPK.
Abstract: With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.
Abstract: PURPOSE: To assess the chronic effects of rigid gas-permeable (RGP) contact lenses on corneal swelling and glucose-lactate metabolism in the rabbit cornea during 1 month of continuous extended wear and to establish the relationship between these effects and the oxygen transmissibility (Dk/L) of the test lens polymer. METHODS: Four RGP lenses of varying Dk/L were tested in 8 rabbits per test group (left eyes served as controls). After 7 days and 1 month extended wear, the concentrations of lactate and glucose in the corneal epithelium, stroma and aqueous humor were determined by enzyme assay; and epithelial and stromal ATP concentrations were separately measured by bioluminescence techniques. Corneal thickness was measured at a standard morning time by ultrasonic pachymetry before and after 1, 7, 15 days and 1 month extended wear. RESULTS: After 7 days and 1 month extended wear, generalized decreases were found in aqueous humor lactate levels for all test lenses, while concomitant increased aqueous glucose concentrations were observed. Total epithelial lactate levels correlated inversely with decreasing Dk/L levels for lower oxygen transmissible lenses (R = 0.951, P = 0.0051); and remained unchanged after extended wear of the hyper-oxygen transmissible Dk/L 125 test lens. By contrast, stromal lactate levels consistently decreased at all time points measured forextended wear of all test lenses. As expected, both epithelial and stromal ATP concentrations simultaneously decreased in extended wear. Overnight corneal swelling values after 24 hours wear of Dk/L = 27, 43, 70 and 125 test lenses were increased by 9.8, 7.1, 5.5, and 5.2% while persistent (residual) stromal swelling after one month extended wear was 16.8, 10.1, 8.6, and 5.6% respectively, in excess of baseline values. CONCLUSIONS: Chronic RGP contact-lens induced hypoxia is associated with altered glucose-lactate metabolism in the cornea and aqueous humor with excess production of increased levels of lactate in the epithelium for lower Dk/L test lenses, but decreased lactate concentration in the stroma and aqueous humor. Extended wear of the hyper-oxygen transmissible test lens (Dk/L = 125) however, produced no increase in epithelial lactate levels. Expected lens-induced decreases in epithelial and stromal ATP were not dependent on lens-oxygen transmissibility. Despite the persistence of lower than normal stromal levels of lactate during 1 month of extended wear for all test lenses, residual corneal swelling values remained consistently elevated above baseline values. Taken together, these data establish that increased stromal lactate accumulation cannot account for persistent stromal edema in chronic extended wear of RGP lenses; and that this effect appears to be independent of lens-oxygen transmissibility and may thus represent the prolonged mechanical effect of lens wear itself.
Abstract: PURPOSE: To assess adverse effects of contact lens-induced hypoxia on the rabbit cornea in vivo and determine the relation between binding of Pseudomonas aeruginosa and oxygen transmissibility for rigid and hydrogel lenses. METHODS: Six rigid lenses with Dk/Ltotal values between 0 and 97 x 10(-9) (cm/second) (ml O2/ml mmHg) and four hydrogel lenses (Dk/Ltotal 9, 20, 39, 51) were tested. All lenses had 14.0-mm diameters and a thickness (parallel) of 0.12 or 0.15 mm. Tear lactate dehydrogenase activity and tandem scanning confocal microscopy determinations were performed after the lens was worn for 24 hours. Binding of P. aeruginosa then was separately determined by the colony-forming unit method. Scanning electron microscopy was used to confirm in vivo tandem scanning confocal microscopy findings. RESULTS: Lens oxygen transmissibility determines binding of P. aeruginosa to the cornea after the lens is worn for 24 hours; epithelial damage produced by lenses of lower Dk/Ltotal appears to be the dominant biologic factor for P. aeruginosa binding and not lens rigidity. CONCLUSIONS: These results suggest that the risk of P. aeruginosa keratitis developing with overnight wear will be enhanced significantly for contact lenses with Dk/Ltotal values less than 50 x 10(-9) (cm/second) (ml O2/ml mmHg) (human equivalent oxygen percentage < or = 15%), and this risk will increase with further decreases in oxygen transmissibility. Because no hydrogel lenses approved by the Food and Drug Administration are available with oxygen transmission at this level, patients should be made aware of the increased risk of infectious keratitis associated with the overnight wear of current extended wear hydrogel lenses. Results of this study also demonstrate that quantitative clinical tandem scanning confocal microscopy imaging and tear lactate dehydrogenase activity measurements can provide prospective, noninvasive methods for assessing the ongoing interaction between contact lens and cornea in vivo.
Abstract: We evaluated the effects on the rabbit cornea of daily wear of rigid gas permeable (RGP) contact lenses treated with preserved care solutions by measuring concomitant tear lactate dehydrogenase (LDH) activity followed by in vivo tandem scanning confocal microscopy (TSCM). In vivo morphologic changes were confirmed by in vitro scanning electron microscopy (SEM). Two standard commercial RGP lens wetting and soaking solutions from the same manufacturer were tested: solution A with 0.004% benzalkonium chloride (BAK) and solution B with 0.003% chlorhexidine digluconate (CHX) and 0.002% thimerosal. Two experimental PBS-based wetting and soaking solutions were also tested: solution C with 0.005% BAK and 2% hydroxypropylmethylcellulose (HPMC) and solution D with 0.005% BAK without HPMC. Instillation of solution A without contact lens wear caused significant (P < 0.01) increases in desquamation of the superficial corneal epithelium and tear LDH activity compared with control eyes. After 3 weeks of RGP contact lens daily wear (8 hours/day), modified Draize scores of ocular surface lesions on the eyes wearing RGP lenses treated with solution A increased according to the duration of lens wear. Solution B did not produce significant change. With daily wear for 4 days (8 hours/day), RGP lenses treated with solution C and solution D produced increased corneal epithelium desquamation and an increase of LDH activity in tears. These effects were greater with HPMC (solution C) than without HPMC (solution D).(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: We used noninvasive biochemical techniques to study the effects on rabbit corneas of 7-day extended wear of rigid gas permeable (RGP) contact lenses of varying oxygen transmissibilities. Corneal effects were assessed through measurement of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities and albumin levels in tears. The RGP contact lenses used had Dk/Ltotal values ranging from 33 to 64 x 10(-9) (cm/sec) (mL O2/mL mmHg) and were of uniform 0.15 mm center thickness. Extended wear of high Dk (Dk/Ltotal = 34) and super high Dk (Dk/Ltotal = 56) lenses caused an increase in tear LDH activity from 1,190 U/L (before lens wear) to over 18,000 U/L during 7 days of continuous wear. These contact lenses also caused gradual increases in tear MDH activity from 431 U/L (before lens wear) to over 750 U/L after 7 days of continuous wear. Extended wear of the ultra high Dk lens (Dk/Ltotal = 64), however, caused no significant increase in LDH or MDH activity in tears. Tear albumin levels in all contact lens wearing eyes increased after 1 day of lens wear, then gradually recovered to normal values after 2 days of continuous wear. The changes in albumin levels did not correlate with Dk/Ltotal values of lenses worn.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Lactate dehydrogenase (LDH) activity in tears was measured in 202 myopic rigid gas permeable (RGP) and 79 hydrogel human contact lens wearers and 48 normal controls by noninvasive microcapillary sampling. The oxygen permeabilities (Dk) of five selected RGP contact lenses ranged between 0 and 230 x 10(-11) (cm2/s) ml O2/ml mm Hg), and the water content (WC) of the hydrogel lenses was 38, 72, and 80%. When normal diurnal variation of tear LDH activity and tear sample volume (0.3-0.6 microliters) were carefully controlled, the tear LDH activity of RGP lens wearers in daily and extended wear correlated as an inverse function of Dk/L, with the highest enzyme activity observed in wearers of polymethylmethacrylate daily wear lenses (347.4 U/L). The tear LDH activity in Menicon EX (Dk 108) lens wearers was higher in the extended wear (192.7 U/L) than in the daily wear group (161.9 U/L). There was no significant difference in tear LDH activity between the Menicon SF-P (Dk 230) extended wear group (132.0 U/L) and controls (133.5 U/L). In the hydrogel lens daily wear group, tear LDH activity of Experimental 72 (WC 72%) and Experimental 80 (WC 80%) was higher than that of hydroxyethylmethacrylate (HEMA) despite having high Dk value. Experimental 80 extended wearers showed lower LDH activity in tears sampled between 0.3 and 0.6 microliters than that of HEMA wearers. These results suggest that sequential measurement of LDH levels in tears may offer a new and unique method for the assessment of the physiologic effects of contact lens wear on the ocular surface, and provide a new clinical paradigm for the interaction of the contact lens with the cornea.
Abstract: We have established a quantitative method for evaluation of ocular surface lesions on the basis of lactate dehydrogenase (LDH) activity, malate dehydrogenase (MDH) activity, and albumin levels in rabbit tears. Lesions were produced with solutions of 0.005-0.02% benzalkonium chloride (BAK), 0.01-0.03% chlorhexidine digluconate (CHX), and 0.01-0.03% polyhexamethylene biguanide hydrochloride (PHMB), all of which are widely used in topical ophthalmic preparations. Two drops of test solution were instilled 15 times into rabbit eyes at 5 minute intervals. Sixty minutes after 0.02% BAK instillation, tear LDH activity increased from 1,840 U/L (without instillation) to 26,100 U/L, and concomitantly tear albumin levels rose from 0.11 mg/mL (without instillation) to 9.48 mg/mL. Instillation of 0.03% CHX and 0.03% PHMB caused smaller increases in LDH and MDH activity and albumin tear levels. LDH activity and albumin levels in tears were significantly correlated with the degree of total ocular surface lesions in both cornea and conjunctiva as observed by slit lamp biomicroscopy quantified using a modified Draize score. Based on the results of this study, we believe that LDH activity and albumin level in tears can be used as objective indicators for the quantitative evaluation of ocular surface lesions on both cornea and conjunctiva following application of topical ophthalmic preparations containing cytotoxic preservatives in animals and man.
