Artificial Aqueous Humor Research Articles

Electrochemical behavior of surgical 316L stainless steel eye glaucoma shunt (Ex-PRESS) in artificial aqueous humor.

A novel system of electrodeposited gold nanoparticles on a carbon paste electrode was utilized as an electrochemical sensor to monitor the corrosion performance of 316L stainless steel alloy in aqueous humor containing moxifloxacin hydrochloride (MFH) drug. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and potentiodynamic polarization measurements were used to estimate the corrosion performance of 316L stainless steel alloy in aqueous humor with immersion time. The experimental data was confirmed by scanning electron microscopy (SEM), Energy dispersive X-ray analysis (EDX) and antibacterial activity. All techniques conform well to each other and confirmed that the tested alloy corrosion decreases with increasing immersion time in aqueous humor. Corrosion is more inhibited after the addition of MFH drug. In addition, as the concentration of the drug increases, the protection efficiency of the tested alloy increases. This behavior was confirmed by sensing the drug concentration with time using the modified carbon paste electrode.

Active Maintenance of the Gradient of Refractive Index Is Required to Sustain the Optical Properties of the Lens.

To determine whether the cellular physiology of the lens actively maintains the optical properties of the lens and whether inhibition of lens transport affects overall visual quality. One lens from a pair of bovine lenses was cultured in artificial aqueous humor (AAH), while the other was cultured in either AAH-High-K+ or AAH + 0.1 mM ouabain for 4 hours. Lens pairs or whole enucleated eyes were then imaged in 4.7 Tesla (T) high-field small animal magnet. Lens surface curvatures, T1 measurements of water content, and T2 measurements of water/protein ratios were extracted from cultured lenses, while the geometrical parameters that define the optical pathway were obtained from whole eyes. Gradients of refractive index (GRIN), calculated from T2 measurements, and the extracted geometric parameters were inputted into optical models of the isolated lens and the whole bovine eye. Inhibiting circulating fluxes by inhibiting the Na/K-ATPase with ouabain or depolarization of the lens potential by High K+ caused changes to lens water content, the water/protein ratio (GRIN) and surface geometry that manifested as an increase in optical power and a decrease in negative spherical aberration in cultured lenses. Changes to optical properties of the lens resulted in a myopic shift that impaired vision quality in the optical model of the bovine eye. The cellular physiology of the lens actively maintains its optical properties and inhibiting the Na/K/ATPase induces a myopic shift in vision similar to that observed clinically in patients who go on to develop cataract.

Identification and Functional Characterization of a GSH Conjugate Efflux Pathway in the Rat Lens

To identify and functionally characterize transporters involved in the release of glutathione (GSH) conjugates from the rat lens. Polymerase chain reaction and Western blotting were used to screen for the presence of multidrug resistance-associated protein (Mrp) and organic anion transporting polypeptide (Oatp) isoforms, and immunohistochemistry used to localize Mrp isoforms. To test for Mrp function, lenses were loaded with 5-chloromethylfluorescein diacetate and monochlorobimane to form the fluorescent GSH conjugates glutathione methylfluorescein (GS-MF) and glutathione bimane (GS-B), respectively, and cultured in artificial aqueous humour (AAH) in the presence or absence of MK571, an Mrp-specific inhibitor, or benzbromarone, a nonspecific organic anion transporter inhibitor. Glutathione-MF and GS-B fluorescence were measured in the AAH media and lenses. Multidrug resistance-associated proteins 1, 4, 5, and Oatp1a4 were present at the transcript level, but only Mrp1, 4, and 5 were detected at the protein level. Multidrug resistance-associated proteins 1 and 5 localized to the epithelium and peripheral fiber cells, whereas Mrp4 strongly labeled the nuclei. Glutathione-MF and GS-B efflux was significantly decreased and accumulation in the lens significantly increased in the presence of MK571, indicating that the Mrps are the predominant transporters involved in GSH conjugate release from the lens. Glutathione-B conjugate efflux was further inhibited in the presence of benzbromarone, suggesting that alternative organic anion pathways were involved in mediating GS-B efflux. Multidrug resistance-associated proteins are present in the lens and may be used to remove endogenous and exogenous compounds from the lens via GSH conjugation. This may represent an important pathway of detoxification required to minimize oxidative stress and maintain lens homeostasis.

Evaluation of the effect of <i>Spathodea campanulata</i> flower bud exudate on cataractogenesis in rat lenses

Background: The flower bud exudates of Spathodea campanulata is commonly used as a local eye drop in Nigeria and is anecdotally claimed to improve vision. This study evaluated the anti-cataract activity of Spathodea campanulata (SPCM) flower bud exudates against cataractogenesis using rat lenses. This was done with a view to providing empirical scientific support for the use of the exudates from SPCM in the treatment and management of certain eye disorders.Materials and Methods: Fifty (50) rat lenses were used in this study, i.e. five groups of ten (10) lenses per group. Lenses were incubated in artificial aqueous humor (Normal control) with simultaneous incubation in 30 mM galactose (Untreated cataract). Co-incubation with captopril (Captopril treated cataract); 0.1 mg/ml of the exudate (Exudate treated cataract - Low dose) and 0.2 mg/ml of exudate (Exudate treated cataract- High dose) constituted the study groups. After 72 hr of incubation, lenses were observed for cataract and the preventive potential of the exudate against cataractogenesis was evaluated through the determination of the levels of anti-oxidant parameters such as total protein (TP), glutathione (GSH), malondialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT) were evaluated in the lens homogenates.Results: Photographic evaluation of the lenses showed the development of cataract in the untreated cataract group while opacification was retarded in higher dose exudate-treated and captopril treated lenses. Significant decrease in the levels of GSH and total protein, reduction in SOD and CAT activities as well as increase in MDA content were observed in cataractous lenses when compared with the normal control. SPCM displayed a doserelated anti-cataract activity. Captopril showed comparable anti-cataract potential to (0.2 mg/ml) SPCM. GSH, SOD and CAT activity in captopril treated cataract lenses increased to 75.55%, 64.08% and 72.45% of the normal control values respectively. In (0.2 mg/ml) exudate treated cataract lenses; GSH, SOD and CAT activity increased to 57.7%, 76.69% and 80.61% respectively.Conclusion: The study concluded that exudate of SPCM elicited its anti-cataract potential through its anti-oxidant activities.Key words: Eye, Cataract, Spathodea campanulata

Investigation of Carvedilol as an Antidenaturing and Anticataract Agent

Cataract is an opacification of the lens of eye. Causes of cataract formation are aging, generation of free radicals, diabetes, protein denaturation by oxidative stress or any inducer etc. Antioxidants and antioxidant enzymes, antidenaturing agent protect the eyes by reducing free radical damage and eye lenses by inhibiting protein denaturation. The present study evaluated the in vitro anticataract and antidenaturing activities of carvedilol against glucose-induced cataractogenesis using goat lenses and heat induced egg albumin denaturation. Transparent isolated goat lenses are incubated in artificial aqueous humor and divided into five experimental groups. The carvedilol at a dose of 20μg/ml is incubated simultaneously with glucose (55 mM) and glucose (5.5 mM) for a period of 72 h. ascorbic acid (20 μg/ml) is used as the standard drug. At the end of the incubation lense opacity is measured by photographic evaluation. Antidenaturing activity is evaluated by taking different concentration of carvedilol (10 p.p.m – 500 p.p.m) with egg albumin and subjected for protein denaturation by using heat for 15 min. Standard drug is used as Diclofenac sodium, at the end protein denaturation is analyse by taking absorbance at 440 nm and 660 nmrsp. The carvedilol shows significant inhibition of cataractogenesis of eye lenses by carvedilol at conc. 20 p.p.m. and also thermally induced protein denaturation inhibited by carvedilol at conc. 500 p.p.m. The present supports carvedilol as an antidenaturing and anticataract agent.

Evaluation of Echitamine, Ditamine and Echitenine for Anticataract Activity using in vitro cataract Model

The alkaloids such as echitamine, ditamine and echitenine were subjected for anti cataract activity using in vitro cataract model. Goat lenses were incubated in artificial aqueous humor containing 55 mM glucose with test alkaloids in different concentrations at room temperature for 72 h. Studied Biochemical parameters were sodium ion, potassium ion level and Na+-K+-ATPase activity. Glucose induced opacification of goat lens began 8-10 hrs after incubation and was complete in 72-80 hrs. Cataractous lenses treated with echitamine shown lower sodium ion, higher the potassium ion and higher Na+-K+-ATPase activity. Echitamine treated lens prevented formation and progress of cataract as evidenced by biochemical parameters and visual observation. Ditamine and echitenine were devoid of anti - cataract activity.

EVALUATION OF THE EFFECT OF SPATHODEA CAMPANULATA FLOWER BUD EXUDATE ON CATARACTOGENESIS IN RAT LENSES

Background: The flower bud exudates of Spathodea campanulata is commonly used as a local eye drop in Nigeria and is anecdotally claimed to improve vision. This study evaluated the anti-cataract activity of Spathodea campanulata (SPCM) flower bud exudates against cataractogenesis using rat lenses. This was done with a view to providing empirical scientific support for the use of the exudates from SPCM in the treatment and management of certain eye disorders.
 Materials and Methods: Fifty (50) rat lenses were used in this study, i.e. five groups of ten (10) lenses per group. Lenses were incubated in artificial aqueous humor (Normal control) with simultaneous incubation in 30 mM galactose (Untreated cataract). Co-incubation with captopril (Captopril treated cataract); 0.1 mg/ml of the exudate (Exudate treated cataract - Low dose) and 0.2 mg/ml of exudate (Exudate treated cataract- High dose) constituted the study groups. After 72 hr of incubation, lenses were observed for cataract and the preventive potential of the exudate against cataractogenesis was evaluated through the determination of the levels of anti-oxidant parameters such as total protein (TP), glutathione (GSH), malondialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT) were evaluated in the lens homogenates.
 Results: Photographic evaluation of the lenses showed the development of cataract in the untreated cataract group while opacification was retarded in higher dose exudate-treated and captopril treated lenses. Significant decrease in the levels of GSH and total protein, reduction in SOD and CAT activities as well as increase in MDA content were observed in cataractous lenses when compared with the normal control. SPCM displayed a dose-related anti-cataract activity. Captopril showed comparable anti-cataract potential to (0.2 mg/ml) SPCM. GSH, SOD and CAT activity in captopril treated cataract lenses increased to 75.55%, 64.08% and 72.45% of the normal control values respectively. In (0.2 mg/ml) exudate treated cataract lenses; GSH, SOD and CAT activity increased to 57.7%, 76.69% and 80.61% respectively.
 Conclusion: The study concluded that exudate of SPCM elicited its anti-cataract potential through its anti-oxidant activities.

Preventive effect of Zea mays L. (purple waxy corn) on experimental diabetic cataract.

Recently, substances possessing antioxidant can prevent cataractogenesis of diabetic cataract. Therefore, this study was carried out to determine the anticataract effect of Zea mays L. (purple waxy corn), a flavonoids rich plant, in experimental diabetic cataract. Enucleated rat lenses were incubated in artificial aqueous humor containing 55 mM glucose with various concentrations of Zea mays L. (purple waxy corn) ranging between 2, 10, and 50 mg/mL at room temperature for 72 h. At the end of the incubation period, the evaluation of lens opacification, MDA level, and the activities of SOD, CAT, GPx, and AR in lens were performed. The results showed that both medium and high doses of extract decreased lens opacity together with the decreased MDA level. In addition, medium dose of extract increased GPx activity while the high dose decreased AR activity. No other significant changes were observed. The purple waxy corn seeds extract is the potential candidate to protect against diabetic cataract. The mechanism of action may occur via the decreased oxidative stress and the suppression of AR. However, further research in vivo is still essential.

Ethanol extract of Moringa oliefera prevents in vitro glucose induced cataract on isolated goat eye lens

Aim of Study:The aim of current work was to evaluate in vitro anticataract potential of Moringa oliefera extract.Materials and Methods:Goat eye lenses were divided into 4 groups; Group served as control, Group II as toxic control, Group III and Group IV were incubated in extract (250 μg/ml and 500 μg/ml of extract of M. oliefera) Group II, III and IV were incubated in 55 mM glucose in artificial aqueous humor to induce lens opacification. Estimation of total, water soluble protein, catalase, glutathione and malondialdehyde along with photographic evaluation of lens was done.Results:Group II (toxic control) lenses showed high amount of MDA (Malondialdehyde), soluble, insoluble protein, decreased catalase and glutathione levels, while lenses treated with Moringa oliefera extract (Group III and Group IV) showed significant (* P < 0.05) reduction in MDA and increased level of catalase, glutathione, total and soluble protein.Conclusion:Results of present findings suggest protective effect of Moringa oliefera in prevention of in vitro glucose induced cataract.

Magnetic resonance and confocal imaging of solute penetration into the lens reveals a zone of restricted extracellular space diffusion

It has been proposed that in the absence of blood supply, the ocular lens operates an internal microcirculation system that delivers nutrients to internalized fiber cells faster and more efficiently than would occur by passive diffusion alone. To visualize the extracellular space solute fluxes potentially generated by this system, bovine lenses were organ cultured in artificial aqueous humor (AAH) for 4 h in the presence or absence of two gadolinium-based contrast agents, ionic Gd(3+), or a chelated form of Gd(3+), Gd-diethylenetriamine penta-acetic acid (Gd-DTPA; mol mass = 590 Da). Contrast reagent penetration into the lens core was monitored in real time using inversion recovery-spin echo (IR-SE) magnetic resonance imaging (MRI), while steady-state accumulation of [Gd-DTPA](-2) was also determined by calculating T1 values. After incubation, lenses were fixed and cryosectioned, and sections were labeled with the membrane marker wheat germ agglutinin (WGA). Sections were imaged by confocal microscopy using standard and reflectance imaging modalities to visualize the fluorescent WGA label and gadolinium reagents, respectively. Real-time IR-SE MRI showed rapid penetration of Gd(3+) into the outer cortex of the lens and a subsequent bloom of signal in the core. These two areas of signal were separated by an area in the inner cortex that limited entry of Gd(3+). Similar results were obtained for Gd-DTPA, but the penetration of the larger negatively charged molecule into the core could only be detected by calculating T1 values. The presence of Gd-DTPA in the extracellular space of the outer cortex and core, but its apparent absence from the inner cortex was confirmed using reflectance imaging of equatorial sections. In axial sections, Gd-DTPA was associated with the sutures, suggesting these structures provide a pathway from the surface, across the inner cortex barrier to the lens core. Our studies have revealed inner and outer boundaries of a zone within which a narrowing of the extracellular space restricts solute diffusion and acts to direct fluxes into the lens core via the sutures.

Visualizing ocular lens fluid dynamics using MRI: manipulation of steady state water content and water fluxes

Studies using various MRI techniques have shown that a water-protein concentration gradient exists in the ocular lens. Because this concentration is higher in the core relative to the lens periphery, a gradient in refractive index is established in the lens. To investigate how the water-protein concentration profile is maintained, bovine lenses were incubated in different solutions, and changes in water-protein concentration ratio monitored using proton density weighted (PD-weighted) imaging in the absence and presence of heavy water (D(2)O). Lenses incubated in artificial aqueous humor (AAH) maintained the steady state water-protein concentration gradient, but incubating lenses in high extracellular potassium (KCl-AAH) or low temperature (Low T-AAH) caused a collapse of the gradient due to a rise in water content in the core of the lens. To visualize water fluxes, lenses were incubated in D(2)O, which acts as a contrast agent. Incubation in KCl-AAH and low T-AAH dramatically slowed the movement of D(2)O into the core but did not affect the movement of D(2)O into the outer cortex. D(2)O seemed to preferentially enter the lens cortex at the anterior and posterior poles before moving circumferentially toward the equatorial regions. This directionality of D(2)O influx into the lens cortex was abolished by incubating lenses in high KCl-AAH or low T-AAH, and resulted in homogenous influx of D(2)O into the outer cortex. Taken together, our results show that the water-protein concentration ratio is actively maintained in the core of the lens and that water fluxes preferentially enter the lens at the poles.

In vitro prevention of cataract by Oyster Mushroom Pleurotus florida extract on isolated goat eye lens

Objectives:The aim of the present work was to evaluate the in vitro effect of Pleurotus florida extract cataract induced by glucose.Materials and Methods:Goat eye lenses were divided into four groups. Group I lenses were incubated in artificial aqueous humor with glucose concentration 5.5 mM (normal control). Group II lenses were incubated with glucose concentration 55 mM (toxic control). Group III and IV lenses incubated with glucose concentration 55 mM were incubated along with hydroethanolic extract of P. florida 250 μg/ml and 500 μg/ml and subjected to morphological and biochemical evaluation.Results:Group II lenses showed high amount of malondialdehyde (MDA) soluble and insoluble protein and decreased catalase and glutathione levels, while lenses treated with P. florida extract showed significant (P < 0.05) reduction in MDA, increased level of catalase (P < 0.001), glutathione (P < 0.005) and total and soluble protein.Conclusions:Hydroethanolic extract of P. florida showed prevention of in vitro glucose induced cataract. Thus, the goat lens model could be used for testing of various anticataract agents.

Adherence and kinetics of biofilm formation of Staphylococcus epidermidis to different types of intraocular lenses under dynamic flow conditions

To compare the adherence and biofilm formation of Staphylococcus epidermidis under in vitro flow conditions on intraocular lenses (IOLs) made of 4 biomaterials: poly(methyl methacrylate) (PMMA), silicone, hydrophilic acrylic, and hydrophobic acrylic. Department of Ophthalmology, Croix-Rousse University Hospital and University Research Laboratory, Lyon, France. Intraocular lenses were placed in a bioreactor designed to replicate intraocular conditions. The model consisted of Tygon tubing connected to a vial. Three septa allowed the entry and elimination of the artificial aqueous humor and inoculation of the bacterial suspension. The first of 2 pumps moved the aqueous humor along the circuit; the second pump regulated the flow at which the nutritive environment was regenerated. At various times (12, 16, 24, 40, 48, 60, and 72 hours), IOLs were taken from this environment and the bound bacteria were removed and counted. The distribution of bacterial adhesion on the IOLs was modeled using polynomial Poisson regression. To test the effect of the IOL biomaterial on bacterial adhesion, likelihood ratio tests were performed. The model provided the kinetics of S epidermidis biofilm growth on IOLs. The biofilm growth on each of the 4 biomaterials occurred in 3 phases: latent, dynamic or accelerated growth, and linear growth. The extent of bacterial binding to IOLs increased from hydrophilic acrylic polymer to PMMA, hydrophobic acrylic, and silicone. The differences were statistically significant. Bacterial adhesion to and biofilm development on the IOL surface depended on the characteristics of the biomaterial.

A Novel In Vitro Model to Study Staphylococcal Biofilm Formation on Intraocular Lenses under Hydrodynamic Conditions

To develop a novel in vitro model to study the formation of Staphylococcus epidermidis biofilm on intraocular lenses (IOLs) from the primary-attachment phase to the biofilm-accumulation phase. The model was designed to replicate intraocular conditions especially by taking into account intraocular hydrodynamics. The model consisted of Tygon tubing connected to a vial containing acrylic hydrophobic IOLs. Three septa, placed along the tubing, allowed, respectively, the artificial aqueous humor's arrival and its elimination and the bacterial suspension's inoculation. A first pump allowed the aqueous humor's movement along the circuit, whereas a second one regulated the flow at which the nutritive environment was regenerated. The whole circuit was placed in a 34 degrees C water bath. Every 2 to 4 hours, lenses were taken from this environment. Bound bacteria were removed by scraping of optical faces and counted. All data are presented as the mean, SD, and coefficient of variation (CV). Comparisons among experiments were performed by one-way analysis of variance (ANOVA). Calculated CVs were close to 30, showing that biofilm formation was homogeneous. Differences between experiments were nonsignificant for each removal time. The model provided the full kinetics of S. epidermidis biofilm growth on acrylic hydrophobic IOLs, with a stationary phase reached after 28 hours of incubation. Biofilm development is modulated by many variables, including environmental factors. The findings in the present study of bacterial colonization of IOLs under intraocular physiological conditions allow understanding and more accurate targeting of biomedical device-related infections such as endophthalmitis.

A Novel Approach to Suprachoroidal Drainage for the Surgical Treatment of Intractable Glaucoma

In glaucoma surgery, scarring of the artificial fistula is the limiting factor for long-term control of intraocular pressure (IOP). Several devices and surgical techniques have been developed for artificial aqueous humor drainage in intractable glaucoma. The authors describe a novel surgical technique that uses a silicone tube as a shunt for aqueous flow from the anterior chamber to the suprachoroidal space. Thirty-one eyes of 31 patients with uncontrollable refractory glaucoma were included in this prospective consecutive case-control study. Each eye had undergone an average of 3.5+/-1.9 previous interventions for glaucoma. The baseline IOP was 44.25+/-8.7 mm Hg despite maximum therapy. As in trabeculectomy, a limbus-based scleral flap was prepared. The suprachoroidal space was accessed via a deep posterior scleral flap. The silicone tube was inserted as an intrascleral connection from the anterior chamber to the suprachoroidal space. Cyclodialysis was avoided by this surgical approach. Success was defined as a lowering of IOP to below 21 mm Hg without the need for further medication or intervention. The mean functional shunt survival was 55.9+/-45.6 weeks. IOP was reduced to 12.9+/-5.2 mm Hg in 70% of all eyes after 30 weeks postoperatively. After 52 weeks, 60% of the eyes could be classified as representing success, and 76 weeks after surgery, 40% of the eyes still showed controlled IOP. In none of the eyes were severe postoperative hypotony or suprachoroidal bleeding observed. No localized or general inflammation or infection was seen in connection with the silicon tube. Two patients needed anterior chamber lavage because of bleeding. In 2 patients the tube had to be removed because of corneal endothelial contact. Shunt failure of the tube was caused in some cases by connective tissue formation at the posterior lumen of the tube. This novel surgical approach and the placement of the silicone tube described here have several advantages. Its intrascleral course minimizes the risk of conjunctival erosion and associated infections. No cyclodialysis is performed. Connection to the suprachoroidal space exploits the resorptive capability of the choroid. It guarantees drainage but also provides a natural counterpressure, avoiding severe postoperative hypotony. The suprachoroidal shunt presented here achieves good follow-up results in terms of IOP control. No serious complications have been observed. This new method promises to be an effective surgical technique and presents a new therapeutic option in intractable glaucoma. Fibroblast reaction obstructing the posterior lumen, seemed to be the only factor limiting drainage. Further studies and experiments will be needed to elucidate the exact physiologic mechanisms underlying the draining, the capacity and duration of the draining effect, and the histologic background of suprachoroidal scarring.

Roles for KCC Transporters in the Maintenance of Lens Transparency

To determine whether the potassium chloride cotransporter (KCC) family is expressed in the rat lens and to ascertain whether the transporters are involved in the regulation of lens volume and transparency. RT-PCR was performed on RNA extracted from fiber cells to identify members of the KCC family expressed in the lens. Western blot analysis and immunocytochemistry, using KCC isoform-specific antibodies, were used to verify expression at the protein level and to localize KCC isoform expression. Organ-cultured rat lenses were incubated in isotonic artificial aqueous humor (AAH) that contained either the KCC-specific inhibitor [(dihydronindenyl)oxy] alkanoic acid (DIOA), the KCC activator N-ethylmaleimide (NEM), or the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) for up to 18 hours. Lens wet weight was monitored, and lens transparency and tissue morphology were recorded with dark-field and confocal microscopy, respectively. Molecular experiments to characterize KCC isoform expression showed that KCC1, -3, and -4 were all expressed in the lens at both the transcript and protein levels and that KCC2 was not. Immunocytochemistry indicated that the three KCC isoforms exhibited distinct differentiation-dependent expression patterns, with KCC1 and -3 being restricted to the lens cortex, whereas KCC4 was found throughout the entire lens, including the lens core. In the lens cortex, most of the labeling for all KCC isoforms was cytoplasmic, whereas in the lens core, KCC4 labeling was associated with the membrane. Incubation of lenses in 100 microM DIOA for 18 hours caused lenses to increase their wet weight and induced a cortical opacity that was caused by extensive damage to peripheral fiber cells located up to 150 microm in from the lens capsule, whereas deeper fiber cells appeared unaffected by DIOA exposure. Lower concentrations of DIOA (10 microM) revealed that this damage was initiated primarily by the swelling of peripheral fiber cells. In contrast, NPPB-treated lenses exhibited a deeper zone (>100 microm) of cell damage that was initiated by the dilation of the extracellular space between fiber cells. Exposure of lenses to the KCC activator NEM caused cell shrinkage in peripheral fiber cells but extensive cell swelling in deeper fiber cells. Peripheral cell swelling caused a differential recruitment of KCC isoforms from a cytoplasmic pool to the plasma membrane. DIOA-induced cell swelling increased the association of KCC4 with membrane, whereas hypotonic cell swelling dramatically increased the association of KCC1 with the membrane. The rat lens expresses three KCC transporter isoforms (KCC1, -3, and -4) in a differentiation-dependent manner. Modulation of transporter activity and subcellular localization suggests that multiple KCC transporters mediate KCl efflux in peripheral fiber cells in a dynamic fashion. These results indicate that, in addition to Cl- channels, KCC transporters play a role in mediating a circulating flux of Cl- ions, which contributes to the maintenance of lens transparency through controlling the steady state volume of lens fiber cells.

In vitroprevention by ACE inhibitors of cataract induced by glucose

Objectives: To study, the anticataract activity of lisinopril and enalapril on cataract induced by glucose, in goat lenses. Materials and Methods: Goat lenses were incubated in artificial aqueous humor containing 55 mM glucose (cataractogenesis) with lisinopril or enalapril in different concentrations at room temperature for 72 h. Biochemical parameters studied in the lens were electrolytes (Na+, K+), Na+-K+-ATPase activity, malondialdehyde (MDA) and proteins. Results: Glucose induced opacification of goat lens began 8-10 hrs after incubation and was complete in 72-80 hrs. Cataractous lenses showed higher Na+, MDA (P Conclusion: The anticataract activity of lisinopril and enalapril may be because of the antioxidant and free radical scavenging activity, as evidenced by a decrease in MDA in treated lenses. Further in-vitro and in-vivo studies in various experimental models and long term clinical trials are required to validate the anticataract activity of ACE-inhibitors.

Determination of Glucose in Human Aqueous Humor Using Raman Spectroscopy and Designed-Solution Calibration

Glucose concentrations of in vitro human aqueous humor (HAH) samples from cataract patients were determined using 785 nm Raman spectra and partial least squares (PLS) calibration. PLS models were created from spectra of prepared calibration solutions rather than aqueous humor samples. Spectra were obtained with an excitation energy (100 mW for 150 s), which was higher than can be applied in vivo, to decrease the models' contribution to prediction uncertainty. The solutions contained experimentally designed levels of glucose, bicarbonate, lactate, urea, and ascorbate. Multiplicative signal correction of spectra helped compensate for the +/-20% drift in laser power observed at the sample over six noncontiguous days of data collection. Seventeen HAH samples containing 38-775 mg/dL of glucose exhibited a root-mean-square error (RMSEP) of 22 mg/dL, coefficient of determination (r(2)) of 0.989, and bias of 6 mg/dL when predicted from lower energy (30 s) spectra collected contemporaneously with fifty calibration spectra. Similar results were obtained even when spectral data were gathered separately for human aqueous humor samples and calibration samples: 10 HAH samples, calibrated on 25 solutions measured 3.6 weeks earlier, exhibited an RMSEP of 23 mg/dL, r(2) of 0.992, and bias of 9 mg/dL. The results demonstrate progress toward the determination of glucose levels in patient-derived aqueous humor using laboratory-derived "artificial aqueous humor" calibration solutions.

Phototransformations of Advanced Glycation End Products in the Human Eye Lens due to Ultraviolet A Light Irradiation

Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP-lysine and argpyrimidine are two UVA-absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation. The changes of OP-lysine, OP-phenethylamine (a phenethylamine analogue of OP-lysine), and argpyrimidine due to irradiation with UVA light in the presence or absence of air and ascorbic acid were followed by different spectral methods. Aged human lenses were similarly irradiated in artificial aqueous humor. The amounts of OP-lysine in the irradiated lenses and in the corresponding dark controls were determined by HPLC. Both OP-lysine and argpyrimidine decreased 20% when irradiated with UVA light in the absence of ascorbic acid. Under the same conditions, OP-lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UVA light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP-phenethylamine after UVA irradiation in the presence of ascorbic acid was phenethylamine, which indicates that the entire heterocycle of this AGE was cleaved and the initial amino group was restored. Some AGEs in the human eye lens can be transformed by UVA light.

Glucose determination in human aqueous humor with Raman spectroscopy

It has been suggested that spectroscopic analysis of the aqueous humor of the eye could be used to indirectly predict blood glucose levels in diabetics noninvasively. We have been investigating this potential using Raman spectroscopy in combination with partial least squares (PLS) analysis. We have determined that glucose at clinically relevant concentrations can be accurately predicted in human aqueous humor in vitro using a PLS model based on artificial aqueous humor. We have further determined that with proper instrument design, the light energy necessary to achieve clinically acceptable prediction of glucose does not damage the retinas of rabbits and can be delivered at powers below internationally acceptable safety limits. Herein we summarize our current results and address our strategies to improve instrument design.