Effect of UVA and UVB irradiation on the metabolic profile of rabbit cornea and lens analysed by HR-MAS 1H NMR spectroscopy

PURPOSE: The aim of the study was to investigate the metabolic profiles of intact rabbit corneas and lenses exposed to UVA and UVB radiation by using high-resolution (HR) magic angle spinning (MAS) (1)H nuclear magnetic resonance (NMR) spectroscopy and pattern recognition methods. METHODS: Adult albino rabbits were exposed to UVA (366 nm, 0.589 J/cm(2)) or UVB (312 nm, 1.667 J/cm(2)) radiation for 8 min, once a day for 5 days. Three days after the last irradiation day, samples of corneas and lenses were dissected. HR-MAS (1)H NMR spectroscopy combined with pattern recognition methods (principal component analysis and soft independent modelling of class analogy) and one-way ANOVA were applied to obtain metabolic information from intact corneal and lens tissue. RESULTS: UVB irradiation caused statistically significant metabolic changes in the rabbit corneas. A decrease in metabolites as ascorbate (84%), myo-inositol (59%), hypotaurine (91%) and choline (76%) was observed. Exposure to UVA radiation caused no significant metabolic alteration in this tissue. The metabolic profile of the rabbit lenses showed no detectable changes after UVA or UVB exposure. CONCLUSIONS: The combination of HR-MAS (1)H NMR spectroscopy and multivariate methods proved effective to analyse intact corneal and lens tissue after exposure to UV radiation of different wavelengths. By avoiding extraction methods and obtaining complete metabolic profiles from one sample, HR-MAS (1)H NMR spectroscopy provided important information about metabolic alteration occurring in rabbit corneal and lens tissue after UV exposure.     

High-resolution 1H NMR spectroscopy of aqueous humour from rabbits

Background: Detection of different substances in aqueous humour is important for evaluation of the disorders affecting the eye. The purpose of the present study was to apply highresolution proton (¹H) nuclear magnetic resonance (NMR) spectroscopy for extensive characterisation of the metabolites in the aqueous humour from rabbits. Methods: High-resolution ¹H NMR spectroscopy, including two-dimensional shift correlated (COSY) technique, was performed on aqueous humour from rabbits. Results: More than 20 metabolites were simultaneously detected and identified in high-resolution ¹H NMR spectra of aqueous humour from rabbits. Some of these were also quantified. Conclusion: High-resolution ¹H NMR spectroscopy is a valuable method for simultaneous detection of many different metabolites in aqueous humour.     

Effect of oxygen on aqueous humor dynamics in rabbits

A study was made in albino rabbits of the effect on aqueous humor dynamics of 100% oxygen, administered by face mask. A mean decrease in intraocular pressure of 4.9 mm Hg was found. This was accompanied by a decrease in episcleral venous pressure of 4.5 mm Hg. Anterior chamber aqueous humor flow decreased transiently after oxygen administration but returned to pre-oxygen levels after about 60 min. It was concluded that the sustained decrease in intraocular pressure which was caused by oxygen was secondary to the decrease in episcleral venous pressure and not to a decrease in the production of aqueous humor.     

Analysis of human aqueous humour by high resolution 1H NMR spectroscopy

High-resolution proton NMR spectra from nine samples of human aqueous humour extracted from the anterior chamber of the eye are reported. They were obtained within the order of 45 min from small volumes (ca 0.3 ml) of sample with minimal pretreatment and the method was non-destructive. Metabolites detected included acetate, acetoacetate, alanine, ascorbate, citrate, creatine, formate, glucose, glutamine-glutamate, beta-hydroxybutyrate, lactate, threonine and valine. Concentrations of lactate, valine, alanine and acetate were determined. These novel metabolite profiles may be of value in the study of clinical disorders.     

Time dependency of metabolic changes in rat lens after in vivo UVB irradiation analysed by HR-MAS 1H NMR spectroscopy

The lens ability to protect against, and repair ultraviolet radiation (UVR) induced damages, is of crucial importance to avoid cataract development. The influence of UVR-induced damage and repair processes on the lens metabolites are not fully understood. Observation of short- and long-term changes in light scattering and the metabolic profile of pigmented rat lenses after threshold UVR exposure might serve to better understand the protective mechanisms in the lens. By using high resolution magic angle spinning (HR-MAS) 1H NMR spectroscopy it was possible to investigate the metabolites of intact rat lenses. Brown-Norway rats were exposed to 15 kJm(-2) UVB irradiation. One eye was exposed and the contralateral served as control. The rats were sacrificed 5, 25, 125, and 625 hr post-exposure and the lenses were removed. The degree of cataract was quantified by measurement of lens forward light scattering. Thereafter, proton NMR spectra from intact lenses were obtained and relative changes in metabolite concentrations were determined. The light scattering in the lens peaked at 25 hr post-exposure and decreased thereafter. The lowest level of light scattering was measured 625 hr after exposure. No significant changes in concentration were observed for the metabolites 5 and 25 hr post-exposure except the total amount of adenosine tri- and diphosphate (ATP/ADP) that showed a significant decrease already 5 hr after exposure. At 125 hr the lens concentrations of lactate, succinate, phospho-choline, taurine, betaine, myo-inositol, and ATP/ADP showed a significant decrease (p<0.05). Phenylalanine was the only metabolite that revealed a significant increase 125 hr post-exposure. At 625 hr most of the metabolic changes seemed to normalise back to control levels. However, the concentration of betaine and phospho-choline were still showing a significant decrease 625 hr after UVB irradiation. The impact of UVB irradiation on the metabolic profile did not follow the same time dependency as the development of cataract. While the light scattering peaked at 25 hr post-exposure, significant changes in the endogenous metabolites were observed after 125 hr. Both the metabolic changes and the light scattering seemed to average back to normal within a month after exposure. Significant decrease in osmolytes like taurine, myo-inositol and betaine indicated osmotic stress and loss of homeostasis. This study also demonstrated that HR-MAS 1H NMR spectroscopy provides high quality spectra of intact lenses. These spectra contain a variety of information that might contribute to a better understanding of the metabolic response to drugs or endogenous stimuli like UVB irradiation.     

Effect of topical prostaglandin D2 on the aqueous humor dynamics in rabbits

Effects of topically applied prostaglandin (PG) D2 on the aqueous outflow facility, uveoscleral flow, and aqueous flow rate were studied in rabbits to reveal its intraocular pressure (IOP)-reducing mechanism. The outflow facility after PGD2 application (50 micrograms) as measured by 2-min tonography was 0.19 microliters/min/mm Hg and did not differ from that before the application (0.22) or from that of the control contralateral eye (0.21). Direct measurement of the uveoscleral flow by perfusion of the anterior chamber for 30 min with fluorescein isothiocyanate-labeled dextran solution showed the flow rate to be about 0.15 microliters/min in indomethacin-treated (10 mg/kg, i.p.) rabbits. In these animals, PGD2 (50 micrograms) was ineffective in changing the flow rate, while PGF2 alpha (50 micrograms) significantly increased the flow by about 35%. Indirect assessment of the effects of PGs on this flow system was made using the uveoscleral flow-antagonizing ability of topical pilocarpine (1.5 mg). IOP reduction by PGD2 (50 micrograms) was only slightly inhibited by pilocarpine, while that by PGF2 alpha (10 micrograms) was markedly reduced. Aqueous flow rate measured fluorophotometrically was about 3.5 microliters/min in normal eyes. After PGD2 (50 micrograms) the flow rate was significantly reduced to 3.0 microliters/min. The magnitude of this reduction was estimated to be enough to account for the IOP reduction after application of 50 micrograms of PGD2. These results indicate that IOP reduction caused by topical PGD2 application is due mostly to the inhibition of aqueous flow rate.     

Triamterene measurements in the aqueous humor of rabbits.

The kinetics of topical triamterene penetration were estimated from the time-course measurements of triamterene (in Dyazide) concentrations in the anterior chamber of six rabbits (n=12, left and right eyes). The two-compartment model of Jones and Maurice (1) was fitted to the measurements. We found the apparent elimination rate constant oftriamterene A = 0.33 +/- 0.12 hr(-1), the apparent absorption rate constant of triamterene B = 2.68 +/- 0.55 hr(-1), the cornea-aqueous transfer coefficient in reference to the corneal volume of triamterene kc.ca = 0.28 +/- 0.05 hr(-1), the loss coefficient of triamterene from the anterior chamber ko = 0.43 +/- 0.16 hr(-1) and the amount of triamterene in the cornea at time zero Mo = 483 +/- 125 ng/ml. The mean of ko = 0.43 hr(-1) is significantly lower (p = 0.04% using ZTEST) than the lower limit of aqueous loss coefficient = 0.58 hr(-1) usually found in rabbits (2). We conclude that Dyazide lowers the aqueous flow rate in the positive direction, considering glaucoma treatment. Peak triamterene concentration in the anterior chamber was P = 120 +/- 32 ng/ml. Half-life for elimination from the aqueous humor was T1/2 = 1.84 +/- 0.65 hr (Mean +/- SD).     

Fibrinogen concentration in the aqueous humor of buphthalmic rabbits

Fibrinogen concentration in the aqueous humor of buphthalmic rabbits, AXBU/J, and those of the normal parent strain AX/J are 0.36 +/- 0.14 and 0.08 +/- 0.01 mg/ml, respectively. There was no detectable fibrinogen in the aqueous humor of normal inbred strain III/J rabbits. Among the buphthalmic rabbits, elevated fibrinogen concentration in the aqueous humor was observed in two month old prodromal rabbits, indicating a small leakage of fibrinogen into the anterior chamber before the development of buphthalmia. Protein concentration in the aqueous humor was close to normal, in spite of an elevated level of fibrinogen in the young buphthalmic rabbits. Severe protein leakage was seen in only some of the old buphthalmic rabbits.     

Effect of ifenprodil on aqueous humor dynamics and optic nerve head circulation in rabbits.

The purpose of this study was to study the effects of ifenprodil, a cerebral vasodilator with alpha and N-methyl-D-aspartate (NMDA) receptor antagonistic activities, on aqueous humor dynamics and optic nerve head (ONH) circulation in rabbits. Experiments were performed during the dark phase in rabbits conditioned to a schedule of alternating 12-hr periods of light and dark. Effects on blood-aqueous barrier permeability (K(d)), aqueous flow rate (F), outflow facility to general blood circulation (C(gen)), and uveoscleral outflow (F(u) were determined fluorophotometrically. Effects on ONH tissue circulation were estimated using the laser speckle method. Unilateral topical administration of 0.5% ifenprodil solution decreased intraocular pressure (IOP) with a maximum reduction of 3.4 mmHg and an effect duration of 3 hr without effects on the contralateral eye. A single instillation of 0.5% ifenprodil had no significant effect on K(d), F, or C(gen), whereas it substantially increased F(u). Twenty-day, twice-daily unilateral 0.5% ifenprodil instillation significantly increased tissue blood velocity in the ONH only in the treated eye.     

Interleukin-1 beta in the aqueous humor in aphakic and pseudophakic eyes of rabbits]

The amounts of interleukin-1 beta (IL-1 beta) (by enzyme linked immunosorbent assay (ELISA) using anti-rabbit recombinant IL-1 beta (rIL-1 beta) antibody) and total protein content (by Bradford's method) were assayed in the aphakic and pseudophakic aqueous humor in 24 eyes of 16 rabbits. Posterior chamber intraocular lenses were examined scanning electron microscopically and immunohistochemically. The amount of detected IL-1 beta was 10.79 +/- 4.67 ng/ml (mean +/- SD) in the pseudophakic eyes 3 days postoperatively, 10.12 +/- 3.96 ng/ml in the pseudophakic eyes and 3.94 +/- 0.86 ng/ml in the aphakic eyes 7 days postoperatively. A significantly higher amounts (p less than 0.001) of IL-1 beta were detected in the pseudophakic eyes compared with the aphakic eyes at 7 days postoperatively. The amount of IL-1 beta varied in each rabbit 1 month postoperatively. In total protein content in the aqueous humor, pseudophakic eyes showed significantly higher compared with the aphakic eyes at 7 days (pseudophakic eyes: 29.7 +/- 11.7 mg/ml, aphakic eyes: 8.58 +/- 6.90 mg/ml, p less than 0.05) and 1 month (pseudophakic eyes: 8.30 +/- 1.21 mg/ml, aphakic eyes: 2.10 +/- 0.309 mg/ml, p less than 0.001) postoperative. Scanning electron microscopic study showed that small round cells, spindle-shaped cells and giant cells could be observed on the surface of intraocular lenses. All of these types of cells showed positive staining of IL-1 beta immunohistochemically. These results suggest that IL-1 beta might be an inflammatory mediator in eyes with lens extraction, especially pseudophakic eyes.     

Effects of systemic desmopressin on aqueous humor dynamics in rabbits

Intravenous desmopressin, a synthetic antidiuretic hormone, resulted in a dose-dependent increase in intraocular pressure (IOP) in rabbits. IOP was increased 3.6 +/- 0.8 mm Hg 6 hr following injection of desmopressin 200 mUnits/kg with the increase lasting over 10 hr. IOP returned to baseline 24 hr after the injection. Systemic blood pressure, plasma osmolarity and arterial blood gases were not altered by desmopressin. The increased IOP was not associated with alterations in measured outflow facility or episcleral venous pressure. Five hours after desmopressin injection, calculated aqueous humor flow was increased approximately 57%. Aqueous humor ascorbate measurements for calculation of flow to diffusion ratios and anterior chamber fluorophotometry also were consistent with an increased rate of aqueous humor formation as the mechanism for the IOP elevation. Desmopressin administration did not increase aqueous humor protein or aqueous humor cyclic AMP concentration. Systemic pretreatment with indomethacin only partially blocked the IOP increase. Systemic pretreatment with demeclocycline completely blocked the desmopressin-induced increase in IOP.     

哌仑西平滴眼剂在兔房水中的药代动力学研究

目的：研究哌仑西平滴眼剂在兔眼房水中的药代动力学，为其临床开发提供依据。方法：将40只兔随机分为8组，每组5只兔。配制不同pH值和不同粘度的哌仑西平滴眼剂，其中哌仑西平浓度均为5％。单眼单次滴入兔眼结膜囊后，分别于8个时间点抽取房水，应用高效液相色谱法(HPLC)测定房水哌仑西平浓度随时间的变化情况，并每隔两周更换处方给药：结果：哌仑西平难以透过角膜到达眼内。随着pH值的降低，哌仑西平角膜透过率逐渐提高。滴眼剂的粘度对角膜透过率无明显作用。结论：哌仑西平在临床开发中需要改变剂型，以进一步提高眼内浓度。     

Biological response in various compartments of the rat lens after in vivo exposure to UVR-B analyzed by HR-MAS 1H NMR spectroscopy

PURPOSE: The purpose of the present study was to investigate metabolic changes in different compartments of the rat lens (anterior, nuclear, posterior, and equatorial) after exposure to an acute double threshold dose of ultraviolet-B radiation (UVR-B) by using high-resolution magic angle spinning (HR-MAS) (1)H nuclear magnetic resonance (NMR) spectroscopy and pattern recognition (PR) METHODS: methods. One eye in each of 28 6-week-old female albino Sprague-Dawley rats was exposed to in vivo 7.5 kJ/m2 UVR-B for 15 minutes. The contralateral eye was left unexposed. One week after irradiation, all rats were killed, and both lenses were isolated. Each lens was cored by a trephine, and the cylinder was sliced into three portions (anterior, nuclear, and posterior). The lens material that remained after the coring process was analyzed as the equatorial region. Analysis of lens metabolism was performed by HR-MAS 1H NMR spectroscopy (14.1 T; Avance DRX600; Bruker BioSpin GmbH, Rheinstetten, Germany), and the metabolic profiles were statistically analyzed by the PR method of principal component analysis (PCA). RESULTS: Metabolic differences were detected among the compartments in the lens, both in samples from the contralateral nonexposed lenses and in samples from lenses exposed to in vivo UVR-B. In the rat lens, exposure to UVR-B caused changes in GSH, phosphocholine, myo-inositol, succinate, formate, and adenosine triphosphate (ATP)/adenosine diphosphate (ADP) and in levels of the amino acids phenylalanine, taurine, hypo-taurine, tyrosine, alanine, valine, isoleucine, and glutamate, that varied among lens compartments. CONCLUSIONS: HR-MAS 1H NMR spectroscopy, combined with PR methods (PCA), is effective for analysis of separate parts of the intact rat lens. To understand the biochemistry of the lens, it is important to divide the lens into sections, representing functionally and anatomically distinct compartments.     

Effects of brinzolamide on aqueous humor dynamics in monkeys and rabbits.

This study examines the mechanisms by which brinzolamide reduces intraocular pressure (IOP) in healthy rabbits and in monkeys with unilateral ocular hypertension. Intraocular pressures were measured by pneumatonometry and aqueous flow was determined by fluorophotometry before and after three twice-daily drops of 1% brinzolamide to both eyes per monkey and after similar treatment to one eye per rabbit. In monkeys, outflow facility was determined by fluorophotometry and uveoscleral outflow was calculated. In rabbits, outflow facility was determined by two-level constant pressure infusion and uveoscleral outflow was measured by an intracameral tracer technique. Compared with contralateral vehicle-treated rabbit eyes, IOP was reduced in brinzolamide-treated eyes by 2.5 +/- 1.9 mmHg (mean +/- standard deviation; p =.006) at four hours after the second dose. Aqueous flow was reduced by 0.50 +/- 0.65 microl/min (p =.02). This effect was found in rabbits previously treated with brinzolamide but not in naive rabbits. Treated hypertensive eyes of monkeys had a reduction in IOP of 7.3 +/- 8.8 mmHg (p = 0.01) and aqueous flow of 0.69 +/- 1.10 microL/min (p = 0.05) when compared with baseline. Brinzolamide did not affect outflow facility or uveoscleral outflow in either rabbits or monkeys. It is concluded that, in normotensive eyes of rabbits and hypertensive eyes of monkeys, brinzolamide reduces IOP by reducing aqueous flow and not by affecting aqueous humor drainage.     

The effects of body immersion on aqueous humor dynamics in rabbits

Rabbits subjected to free-floating body immersion demonstrated decreases in intraocular pressure followed by later increases. These intraocular pressure alterations were associated with parallel changes in aqueous humor production. Episcleral venous pressure was elevated during immersion but outflow facility was unaltered. The decrease in intraocular pressure could be partially blocked by pretreatment with vasopressin or desoxycorticosterone acetate. In cross-circulation studies between an immersed and non-immersed rabbit, a decrease in intraocular pressure was observed in both animals. This observation further supported the involvement of humoral factors in the body-immersion induced intraocular pressure response.