Effect of UV-A and UV-B irradiation on the metabolic profile of aqueous humor in rabbits analyzed by 1H NMR spectroscopy

PURPOSE: This study was conducted to investigate metabolic changes in aqueous humor from rabbit eyes exposed to either UV-A or -B radiation, by using (1)H nuclear magnetic resonance (NMR) spectroscopy and unsupervised pattern recognition METHODS: methods. Both eyes of adult albino rabbits were irradiated with UV-A (366 nm, 0.589 J/cm(2)) or UV-B (312 nm, 1.667 J/cm(2)) radiation for 8 minutes, once a day for 5 days. Three days after the last irradiation, samples of aqueous humor were aspirated, and the metabolic profiles analyzed with (1)H NMR spectroscopy. The metabolic concentrations in the exposed and control materials were statistically analyzed and compared, with multivariate methods and one-way ANOVA. RESULTS: UV-B radiation caused statistically significant alterations of betaine, glucose, ascorbate, valine, isoleucine, and formate in the rabbit aqueous humor. By using principal component analysis, the UV-B-irradiated samples were clearly separated from the UV-A-irradiated samples and the control group. No significant metabolic changes were detected in UV-A-irradiated samples. CONCLUSIONS: This study demonstrates the potential of using unsupervised pattern recognition methods to extract valuable metabolic information from complex (1)H NMR spectra. UV-B irradiation of rabbit eyes led to significant metabolic changes in the aqueous humor detected 3 days after the last exposure.     

LMWH inhibits anterior chamber inflammation after extra capsular lens extraction through down regulation of bFGF content in aqueous humor

AIM: To observe the changes of basic fibroblast growth factor (bFGF) content in anterior chamber before and after extra capsular lens extraction for investigating the mechanism of low molecular weight heparin (LMWH) inhibiting anterior chamber inflammation. METHODS: Eighty-four rabbits were randomly divided into control and experimental group, 42 rabbits in each group. Extra capsular lens extraction was done on unilateral eye in each rabbit. LMWH was perfused into anterior chamber by the concentration of 50U/mL at the end of operation in experimental group. The degrees of corneal edema, aqueous flare and fibrin were evaluated with slit lamp microscope on postoperative day 1, 3, 6, 15, 30, 45 and 60, respectively. Six eyes of each group were at each time point. Contents of bFGF in aqueous humor were determined by ELISA after animals were killed. Another six eyes were used for determining the base line level of bFGF in aqueous humor. RESULTS: The degrees of corneal edema, aqueous flare and fibrin in experimental group were significantly lighter than those in control group (P<0.01) on postoperative day 1, 3 and 6, respectively. No difference was showed between the two groups at other point time. Contents of bFGF in aqueous humor increased at the same time. bFGF content reached peak on postoperative day 1 in experimental group, while on postoperative day 6 in control group. Contents of bFGF in the two groups declined slowly after reaching peak. The bFGF content in control group were significantly higher than that in experimental group 1-30 days after surgery (P<0.05). No significant differences were shown between the two groups on postoperative day 45 and 60, respectively. CONCLUSION: Perfusion with LMWH by the concentration of 50U/mL can significantly reduce anterior chamber inflammation after extra capsular lens extraction in rabbits, which may be related to down regulation of bFGF content in aqueous humor.     

Metabolic changes in rat lens after in vivo exposure to ultraviolet irradiation: measurements by high resolution MAS 1H NMR spectroscopy

PURPOSE: In the present study, high-resolution magic angle spinning proton nuclear magnetic resonance (HR-MAS (1)H NMR) spectroscopy was used to investigate changes in the metabolic profile of intact rat lenses after UVB irradiation of the eyes. METHODS: Three groups of Sprague-Dawley rats were exposed to UVB radiation at 2.5, 5.0, and 7.5 kJ/m(2). One eye was exposed, and the contralateral eye served as the control. One week after exposure, the lenses were removed and forward light-scattering was quantified. Thereafter, proton NMR spectra from the intact lenses were obtained. Relative changes in metabolite concentrations were determined. RESULTS: The lenses in all three groups showed significant increases in light-scattering after UVB irradiation. The high-quality HR-MAS (1)H NMR spectra permitted more than 30 different metabolites to be identified. UVB irradiation caused a significant decrease (P < 0.05) in concentrations of taurine, hypotaurine, tyrosine, phenylalanine, valine, myo-inositol, phosphocholine, betaine, succinate, and glutathione at all three UV doses. For glycine, glutamate, and lactate, significant decreases in concentration were observed at the two lowest UVR-B doses. The total amount of adenosine tri- and diphosphate and (ATP, ADP) decreased significantly and that of adenosine monophosphate AMP increased significantly at the two highest doses. Alanine was the only amino acid that increased after UVB irradiation. None of these metabolites exhibited a significant UVB dose-dependent relationship. CONCLUSIONS: This study demonstrates for the first time the potential of HR-MAS (1)H NMR spectroscopy as an analytical tool for use on intact lenses. Near-threshold UVR-B doses led to a generally significant decrease in water-soluble metabolites 1 week after exposure. The lack of dose-dependent changes in the metabolites indicates that repair processes during the first week after UVB irradiation overcome the immediate metabolic disturbances.     

Disposable 1-day Acuvue contact lenses for the delivery of lomefloxacin to rabbits' eyes.

PURPOSE: To investigate the ability of a disposable soft contact lens (1-Day Acuvue) to deliver lomefloxacin, a fluoroquinolones antibiotic, into the cornea and aqueous humor of rabbits. METHODS: Experiments were conducted on adult albino rabbits. 1-Day Acuvue lenses were immersed for 1 hour in commercially-available lomefloxacin eye solution and then placed on the cornea. After 0.5, 2, 4,6, and 8 hours, the animals were killed and the lenses were removed and placed into a 20 mL saline bath. Corneal tissue and aqueous humor samples were also obtained at these times. The release of lomefloxacin from the lenses was calculated by measuring the amount of drug remaining in the lenses after removal from the rabbit eyes. The concentration of lomefloxacin in the cornea and anterior chamber following the wearing of lomefloxacin-loaded lenses was compared with the concentrations following frequent-drop therapy (one drop of lomefloxacin hourly for 8 hours). RESULTS: In vivo, lomefloxacin was released from 1-Day Acuvue lenses gradually over 8 hours. The cornea and aqueous humor levels in the eyes following the wear of lomefloxacin-loaded lenses were significantly higher than those achieved by frequent-drop therapy. The drug levels in the cornea and aqueous humor generally remained above the 90% minimal inhibitory concentration for 8 hours in the 1-Day Acuvue lens group. CONCLUSIONS: 1-Day Acuvue contact lenses soaked in 0.3% lomefloxacin can release sufficient amounts of lomefloxacin and in this study produced higher levels in both the cornea and aqueous humor than that achieved by frequent-drop therapy for up to 8 hours. We conclude that 1-Day Acuvue contact lens can be used as a drug delivery system for lomefloxacin.     

家兔晶状体机械性损伤后虹膜和房水中组织金属蛋白酶抑制物活性的改变

目的　探讨家兔晶状体机械性损伤后 ,基质金属蛋白酶抑制物 (TIMPs)在虹膜组织和房水中的动态变化及其意义。方法　建立单眼晶状体机械性损伤的家兔模型 ,应用反向酶谱法检测并定量分析损伤后 1、3、7和 15d眼内虹膜组织和房水中两种主要的TIMPs (TIMP 1、2 )的活性 ,并应用蛋白酶谱法检测TIMPs的作用底物基质金属蛋白酶 (MMPs)的活性。结果　健康家兔眼和实验家兔未损伤眼的虹膜组织和房水中 ,均未检测到TIMP 1、2的相应蛋白活性条带。伤后 1d家兔损伤眼的虹膜组织中的TIMP 1、2活性显著增加 (P <0 0 5 ) ,而MMP 2的活性受抑制 ;伤后 1d损伤眼的房水中 ,TIMP 1、2的活性均显著增加 (P <0 0 5 ) ;此后逐渐减少 ,至伤后 7d与健康家兔比较无差异 (P值分别为 0 0 97和 0 777) ;酶谱分析结果显示MMP 2活性的改变与此对应 ,即在损伤后 1d受到抑制 ,此后逐渐恢复。结论　TIMPs参与家兔晶状体机械性损伤后的眼内急性反应 ,对抑制炎性反应、促进创伤修复可能具有重要意义。     

Increase of aqueous humor proteins with aging

Quantitative analyses by crossed immunoelectrophoresis were carried out on 31 eyes of 26 cataract patients, age 4-80 years, to detect age changes in the human aqueous humor especially in the following aqueous humor proteins: prealbumin, albumin, alpha 1-acid glycoprotein and transferrin. There was a significant correlation between age and concentration of each of the 4 proteins. The results reported in this paper may be due to senile alteration of the pathway that serum proteins follow from the leaky vessels of the ciliary body to the anterior chamber and/or to a decrease of the aqueous flow rate with advancing age. The relationship between the ages and the aqueous humor protein levels established in this study can be utilized as controls in studying the aqueous humor proteins in uveitis and other ocular diseases.     

Intracameral and lenticular penetration of locally applied stable isotope-labeled vitamin E

PURPOSE: To confirm the aqueous humor and lens dynamics of 1% deuterium-labeled alpha-tocopherol acetate (D(3)-VEA) solution. METHODS: The concentrations of D(3)-VEA and D(3)-alpha-tocopherol (D(3)-VE), derived from D(3)-VEA, in the aqueous humor and lens were measured after continuous instillation of 1% D(3)-VEA into the cul-de-sac of rat eyes for 1 or 3 weeks. D(3)-VEA and D(3)-VE concentrations were determined by gas chromatography/mass selective detector. RESULTS: In the aqueous humor, the concentrations of D(3)-VEA and D(3)-VE were, respectively, 93.1 and 9.4 ng/mL after 1 week, and, respectively, 498.9 and 21.5 ng/mL after 3 weeks. In the lens, they were, respectively, 15.0 and 9.8 ng/g after 1 week, and 6.1 and 4.8 ng/g after 3 weeks. CONCLUSION: The penetration levels of alpha-tocopherol acetate by eyedrop application were confirmed in the aqueous humor and lens.     

The alpha and gamma crystallin content in aqueous humor of eyes with clear lenses and with cataracts.

Specific radioimmunoassays were used to measure the concentration of α- and γ-crystallins in human aqueous humor. It was demonstrated that these crystallins are normally present in aqueous humor from healthy eyes. The crystallin concentration did not seem to increase with age. The normal upper limit for the α-crystallin concentration was found to be 10 ng/ml and for the γ-crystallin concentration 60 ng/ml. In the aqueous humor of eyes with cortical cataract the concentration of both crystallins was increased. With nuclear cataracts the α-crystallin concentration was increased while the γ-crystallin concentration was decreased. Experiments in rabbits showed that the crystallins in the aqueous humor left the anterior chamber at the same rate as the aqueous bulk flow. The demonstration of lens crystallins in the aqueous humor is compatible with the hypothesis that they leak from the lens.     

人工晶体植入后房水中透明质酸钠残留量的测定

目的测定人工晶体植入后房水中透明质酸钠的残留量。方法用岛津ＵＶ３１０１型紫外分光光度仪测量２３例（２５只眼）人工晶体植入后,注吸前及注吸２ｍｌ、５ｍｌ、８ｍｌ时房水中透明质酸钠（ｓｏｄｉｕｍｈｙａｌｕｒｏｎａｔｅ）的百分比浓度。结果注吸前、后不同的相对百分比浓度分别为６５５０％±１８１２％、１０２０％±４８０％、２１９％±１８３％及０７０％±０９１％。结论房水置换２ｍｌ可清除房水中大部分透明质酸钠,而置换８ｍｌ可使透明质酸钠残留减至微量,保持术后眼压正常。     

柔红霉素脂质体的制备及兔房水中柔红霉素及其脂质体浓度的测定

目的:探讨药物柔红霉素(daunorubicine,DNR)及其脂质体在房水中维持的浓度及时间。方法:20只兔眼于晶体囊外摘除术中分别注入0.1ml之1mg/ml DNR及其脂质体(LDNR),术后12、24、48小时和1周时间采集房水,应用高效液相色谱法测定房水中DNR的浓度。结果:术后12、24、48小时注入DNR和LDNR眼均可测出DNR,且LDNR眼浓度明显高于DNR眼,术后1周LDNR眼仍可测出DNR,但注入DNR眼无法测出。结论:应用高效液相色谱法可测定兔房水中的DNR浓度,且LDNR眼浓度明显高于DNR眼,有明显的缓释延时效应。眼科学报1998;14:232～235。     

低分子肝素下调房水bFGF含量抑制晶状体囊外摘除术后前房炎症

目的:观察房水中碱性成纤维细胞生长因子(basic fi-bro blast growth factor,bFGF)含量的变化,探讨低分子肝素(low molecular weight heparin,LMWH)抑制晶状体摘除术后前房炎症的机制。方法:家兔84只随机分为对照组和实验组,每组42只,行单眼透明晶状体囊外摘除术。实验组手术结束时用浓度为50kU/L的LMWH进行前房灌注。分别于术后1,3,6,15,30,45,60d每组各取6只家兔,裂隙灯显微镜下对角膜水肿、房水混浊及前房纤维蛋白渗出的程度进行观察和分级,然后处死动物,抽取房水用Elisa法测定bFGF的含量。另选6只健康家兔测定前房bFGF含量作为基线值。结果:术后1～6d,实验组角膜水肿、房水混浊及前房纤维蛋白渗出的程度明显轻于对照组(P<0.01),15d后两组比较无差异。术后实验组和对照组房水bFGF含量同时升高,实验组1d达峰值,而对照组6d达峰值;达峰值后两组bFGF含量均缓慢下降。术后1～30d对照组bFGF含量明显高于实验组(P<0.05),45d后两组比较无显著性差异。结论:应用浓度为50kU/L的LMWH前房灌注能显著降低兔眼晶状体囊外摘除术后前房炎症反应,其机制可能与房水中bFGF含量下调有关。     

Dimethylthiourea inhibits the inflammatory response to intravitreally-injected endotoxin

Dimethylthiourea, a potent scavenger of toxic oxygen metabolites such as the hydroxyl radical, hypochlorous acid, and hydrogen peroxide, was tested for its ability to inhibit an experimentally induced inflammatory response. Inflammation was induced in one eye of male New Zealand white rabbits by intravitreal injection of 10 ng Escherichia coli endotoxin; the contralateral eye received an equal volume of pyrogen-free saline vehicle. Dimethylthiourea was administered intraperitoneally to these animals at 0, 300, 450 and 600 mg kg-1. At 24 h post-endotoxin injection, all vehicle-injected eyes appeared normal with the exception of a small, but significant increase in aqueous humor protein concentration in the 600 mg kg-1 dimethylthiourea group. In endotoxin-injected eyes, treatment with dimethylthiourea, especially at the highest dose, significantly reduced iridal hyperemia, aqueous humor cell number and protein and prostaglandin-E concentrations, and the ex vivo release of prostaglandin-E from the lens. The ability of dimethylthiourea to significantly inhibit the inflammatory response to intravitreally-injected endotoxin suggests that toxic oxygen metabolites may play an important role in the initiation and/or propagation of this form of acute anterior uveitis. Furthermore, the data are consistent with an important interaction between toxic oxygen and arachidonic acid metabolites.     

Aqueous humor composition in Fuchs' dystrophy

Several studies have suggested differences in aqueous humor composition in Fuchs' dystrophy, including elevations in fibrinogen-related factors, compared with controls. In the current study, aqueous humor was obtained at surgery from 10 uninflamed eyes with advanced Fuchs' dystrophy and cataracts and 11 control eyes with cataracts alone. Total fibrinogen-related antigen was measured in a masked manner using an enzyme-linked immunosorbant assay (ELISA). There was no statistically significant difference between the means of the two groups (0.281 +/- 0.292 (SD) and 0.176 +/- 0.090 mg/ml, respectively). Similarly, there was no difference in ELISA-determined aqueous humor small molecular weight fibrinogen-derived metabolites between the two groups, 623 +/- 141 and 550 +/- 55 fibrinogen equivalents, respectively. Also, no statistically significant difference was detected between Fuchs' dystrophy and control eyes in aqueous humor ascorbate, glucose, carbon dioxide, bicarbonate, and pH. Therefore, this study found no evidence of alterations in aqueous humor composition in Fuchs' dystrophy and supports the hypothesis that the disease is a primary disorder of the corneal endothelium.     

Proteolytic enzymes and antiproteases in eye tissue and blood serum. and the protein composition of aqueous humor in experimental corneal burns

After deep termic and alkaline burns of rabbit's cornea the increasing of activity of neutral trypsinlike proteases, elastase and simultaneous increasing of antitryptic activity are observed in burned part of cornea, part of cornea which is surrounding the burn, and in ciliary body, iris and aqueous humor. One week after termic burn and two weeks after alkaline burn the means of these activities began decrease. The time, when ulceration and perforation of cornea are observed, is corresponded to the period of maximal activity of proteolytic enzymes in the eye tissues. At the peripherical blood there are significant changes of level of main inhibitors of proteolysis (reactants of acute phase of inflammation -alpha 1 antitrypsin and alpha 2 macroglobulin). There are qualitative and quantitative changes of proteins content of aqueous humour in the burned eyes, which have phase characteristics and depend upon the stage of inflammation process.     

High-resolution magic angle spinning <Superscript>1</Superscript>H NMR spectroscopy of metabolic changes in rabbit lens after treatment with dexamethasone combined with UVB exposure

BACKGROUND: Long-term steroid treatment and UVB exposure are well-known cataractogenic factors. The purpose of this study was to investigate metabolic changes in the rabbit lens after long-term dexamethasone treatment in combination with UVB exposure, using high-resolution magic angle spinning proton nuclear magnetic resonance (HR-MAS (1)H NMR) spectroscopy to analyse intact lens tissues. METHODS: Rabbits received topical doses of 0.1% dexamethasone or 0.9% saline (50 microl) four times daily for 36 days. On day 37, the eyes were exposed to UVB radiation (2.05 J/cm(2)). Twenty-four hours later the animals were killed, and HR-MAS (1)H NMR spectra of lens tissues were obtained. RESULTS: More than 15 major metabolites were assigned in NMR spectra of rabbit lenses. The combined treatment with dexamethasone and UVB induced large reductions in the concentration of reduced glutathione, inositols, taurine and lactate compared with normal lenses. Concurrently, the levels of glucose, sorbitol and sorbitol-3-phosphate were increased. After exposure to UVB radiation only, the most significant finding was a decrease in the concentration of lactate. No lens opacities were detected. CONCLUSIONS: HR-MAS (1)H NMR spectroscopy was found to be an efficient tool for analysis of intact lens tissues. High-resolution NMR spectra of intact lens tissue enabled metabolic changes to be quantified. Long-term treatment with dexamethasone combined with UVB exposure induced substantial metabolic changes, dominated by osmolytic regulation processes and loss of glutathione.     

Influence of cataract and posterior chamber lens implantation on the dynamics of the aqueous humor. Prospective study in fluorophotometry

The influence of posterior chamber lens implantation on the aqueous humor dynamics was investigated using anterior chamber fluorophotometry before and 9 weeks after cataract extraction and IOL implantation. 34 Patients finished the protocol. Patients with preexisting glaucoma or those having a history of ocular or systemic inflammation were excluded from the study, also those taking topical or systemic drugs with potential influence on the aqueous humor dynamics. 9 weeks after IOL implantation, a mean increase in aqueous humor flow was notified. This increase was highly significant (p less than 0.01, Students paired t-test). There were no significant changes in aqueous humor dynamics in the unoperated, fellow eyes which served as controls. The significant difference of aqueous humor flow 9 weeks after cataract extraction and IOL implantation indicates that the liberation of prostaglandins may cause an increase of outflow facility as well as pseudo-facility.     

Regional distribution of calcium in alloxan diabetic rabbit lens

A diabetic rabbit model was developed for investigation of cataractogenesis and other changes in the anterior segment. Rabbits were fasted, injected with 0.7 mg/kg alloxan, fed 1% glucose solution for 24 hrs and returned to a normal diet. Animals showing and maintaining blood glucose of greater than 300 mg% within two days were used in this study. Concomitant with increase in blood glucose was a rise in aqueous humor glucose and osmolality, together with a decrease in ascorbate concentration. Vacuoles and small discrete opacities developed, and in some cases, at longer time periods complete opacity of anterior or posterior aspects was found. Total calcium content of the whole lens increased up to 2-fold, especially after 60 days, and was correlated with a decrease in lens transmittance of a He/Ne laser beam and also with high osmolality of the aqueous humor. Free calcium was six-fold higher in opaque areas than clear areas, and was 100-fold higher in vacuoles. It is suggested that, in addition to the recognized role in sugar cataractogenesis of osmotic stress due to sorbitol accumulation in the lens, changes of intracellular calcium in localized areas of the lens and stresses imposed by changes in aqueous humor osmolality may also be important.     

晚期青光眼的房水动力学变化

目的：观察晚期青光眼高眼压对睫状体功能的损害作用及对角膜透明性的影响。方法：应用扫描荧光光度计及Ｓｃｈｉｏｔｚ电子眼压计分别测定１５只正常眼和１５只晚期原发性青光眼眼压、房水排出率和房水流畅系数,并观察角膜的透明性。结果：晚期青光眼眼房水排出率显著下降,下降程度与眼压水平及病程成正比。房水排出率降低至０．８μｌ／ｍｉｎ。角膜出现水肿混浊,房水排出率下降愈甚,角膜透明性改变愈明显。结论：持续高眼压将     

缝线固定人工晶状体植入术后房水细胞学实验研究

目的研究兔眼缝线固定式后房型人工晶状体植入术后早期炎症反应中房水的细胞学动态变化。方法分为缝线固定人工晶状体植入术组；晶状体囊外摘出组；正常对照组。术后1、3、7和14天抽取房水计数白细胞总数及分类。结果缝线固定人工晶状体植入术组术后房水炎症细胞数明显高于单纯晶状体囊外摘出组。结论术后早期房水白细胞总数和嗜中性粒细胞增加可能与手术所致的机械性创伤及血房水屏障破坏有关；术后房水巨噬细胞增多可能是对人工晶状体材料的一种免疫反应。     

Age-related changes in the vitreus and lens of rhesus monkeys (Macaca mulatta)

Biomicroscopic observations of the lens and vitreus, and biochemical analyses of vitreus and aqueous humor, were carried out on free-ranging rhesus monkeys (Macaca mulatta) ranging in age from 1 to 19 years. No senile cataracts were observed. After an initial decrease, the thickness of the lens increased in animals above 4 years of age, concomitant with a decrease in the depth of the anterior chamber. Three patterns of branching of the hyaloid arteriole and the capsulo-pupillary vascular system on the posterior surface of the lens were found. Six stages of vitreus patterns are described showing development of vitreous tracts and liquid pockets and corresponding closely to similar stages observed in human vitreus. The concentration and the molecular size of Na-hyaluronate and the protein concentration in the vitreus showed no significant change with age. The amount of liquid vitreus which could be collected showed a significant increase with age. Biochemical analysis of the aqueous humor revealed no significant differences in Na-hyaluronate or protein concentration in young and old adults.