Two-Photon Excited Fluorescence Lifetime Imaging of Tetracycline-Labeled Retinal Calcification.

Deposition of calcium-containing minerals such as hydroxyapatite and whitlockite in the subretinal pigment epithelial (sub-RPE) space of the retina is linked to the development of and progression to the end-stage of age-related macular degeneration (AMD). AMD is the most common eye disease causing blindness amongst the elderly in developed countries; early diagnosis is desirable, particularly to begin treatment where available. Calcification in the sub-RPE space is also directly linked to other diseases such as Pseudoxanthoma elasticum (PXE). We found that these mineral deposits could be imaged by fluorescence using tetracycline antibiotics as specific stains. Binding of tetracyclines to the minerals was accompanied by increases in fluorescence intensity and fluorescence lifetime. The lifetimes for tetracyclines differed substantially from the known background lifetime of the existing natural retinal fluorophores, suggesting that calcification could be visualized by lifetime imaging. However, the excitation wavelengths used to excite these lifetime changes were generally shorter than those approved for retinal imaging. Here, we show that tetracycline-stained drusen in post mortem human retinas may be imaged by fluorescence lifetime contrast using multiphoton (infrared) excitation. For this pilot study, ten eyes from six anonymous deceased donors (3 female, 3 male, mean age 83.7 years, range 79-97 years) were obtained with informed consent from the Maryland State Anatomy Board with ethical oversight and approval by the Institutional Review Board.

Fluorescence Lifetime Imaging of Human Sub-RPE Calcification In Vitro Following Chlortetracycline Infusion.

We have shown that all sub-retinal pigment epithelial (sub-RPE) deposits examined contain calcium phosphate minerals: hydroxyapatite (HAP), whitlockite (Wht), or both. These typically take the form of ca. 1 µm diameter spherules or >10 µm nodules and appear to be involved in the development and progression of age-related macular degeneration (AMD). Thus, these minerals may serve as useful biomarkers the for early detection and monitoring of sub-RPE changes in AMD. We demonstrated that HAP deposits could be imaged in vitro by fluorescence lifetime imaging microscopy (FLIM) in flat-mounted retinas using legacy tetracycline antibiotics as selective sensors for HAP. As the contrast on a FLIM image is based on the difference in fluorescence lifetime and not intensity of the tetracycline-stained HAP, distinguishing tissue autofluorescence from the background is significantly improved. The focus of the present pilot study was to assess whether vascular perfusion of the well tolerated and characterized chlortetracycline (widely used as an orally bioavailable antibiotic) can fluorescently label retinal HAP using human cadavers. We found that the tetracycline delivered through the peripheral circulation can indeed selectively label sub-RPE deposits opening the possibility for its use for ophthalmic monitoring of a range of diseases in which deposit formation is reported, such as AMD and Alzheimer disease (AD).

Role of ultraviolet irradiation and oxidative stress in cataract formation-medical prevention by nutritional antioxidants and metabolic agonists.

PURPOSE: Cataract is a significant cause of visual disability with relatively high incidence. It has been proposed that such high incidence is related to oxidative stress induced by continued intraocular penetration of light and consequent photochemical generation of reactive oxygen species, such as superoxide and singlet oxygen and their derivatization to other oxidants, such as hydrogen peroxide and hydroxyl radical. The latter two can also interact to generate singlet oxygen by Haber-Weiss reaction. It has been proposed that in addition to the endogenous enzymatic antioxidant enzymes, the process can be inhibited by many nutritional and metabolic oxyradical scavengers, such as ascorbate, vitamin E, pyruvate, and xanthine alkaloids, such as caffeine. METHODS: Initial verification of the hypothesis has been done primarily by rat and mouse lens organ culture studies under ambient as well as ultraviolet (UV) light irradiation and determining the effect of such irradiation on its physiology in terms of its efficiency of active membrane transport activity and the levels of certain metabolites such as glutathione and adenosine triphosphate as well as in terms of apoptotic cell death. In vivo studies on the possible prevention of oxidative stress and cataract formation have been conducted by administering pyruvate and caffeine orally in drinking water and by their topical application using diabetic and galactosemic animal models. RESULTS: Photosensitized damage to lens caused by exposure to visible light and UVA has been found to be significantly prevented by ascorbate and pyruvate. Caffeine has been found be effective against UVA and UVB. Oral or topical application of pyruvate has been found to inhibit the formation of cataracts induced by diabetes and galactosemia. Caffeine has also been found to inhibit cataract induced by sodium selenite and high levels of galactose. Studies with diabetes are in progress. CONCLUSIONS: Various in vitro and in vivo studies summarized in this review strongly support the hypothesis that light penetration into the eye is a significant contributory factor in the genesis of cataracts. The major effect is through photochemical generation of reactive oxygen species and consequent oxidative stress to the tissue. The results demonstrate that this can be averted by the use of various antioxidants administered preferably by topical route. That they will be so effective is strongly suggested by the effectiveness of pyruvate and caffeine administered topically to diabetic and galactosemic animals.

Inhibition of selenite-induced cataract by caffeine.

PURPOSE: The objective of the investigation was to study possible inhibition of oxidative stress and cataract formation by caffeine in vivo. METHODS: Oxidative stress and consequent cataract formation was induced by intraperitoneal administration of a single dose of sodium selenite (1.16 µmol) to Sprague-Dawley rat pups on day 9 postnatally. In experiments designed to inhibit such cataract formation, the pups were pretreated intraperitoneally with caffeine (5.15 µmol), starting 2 days prior to the administration of selenite and continuing such treatment till day 21, when the experiments were terminated. The extent of tissue damage caused by the selenite was assessed biochemically by measurements of the levels of GSH and ATP in the isolated lenses. Cataract formation and its prevention were monitored by examining the eye with pen light illumination and subsequent photography of the isolated lenses. RESULTS: Injection of selenite led to a significant loss of lens clarity because of cataract formation. In the group treated with caffeine, the formation of cataract was significantly prevented. In the caffeine-untreated group, the levels of lens GSH and ATP were substantially lower than in the caffeine-treated group. The levels of GSH decreased from a value of ∼8.2 µmol to ∼2 µmol/g wet weight of the lens. The content of ATP decreased from ∼2.5 µmol to about ∼ 1 µmol. In the case of caffeine-treated group, these decreases were significantly prevented from taking place, the corresponding values of GSH and ATP being ∼5.8 and ∼1.6 µmol/g, respectively. CONCLUSION: Over all, the results suggest that caffeine can exert a significant preventive effect against cataract formation induced by agents generating reactive oxygen species such as sodium selenite.

Kynurenine-induced photo oxidative damage to lens in vitro: protective effect of caffeine.

Photochemical generation of reactive species of oxygen in the lens and aqueous and consequent physiological damage to the tissue has been implicated in the genesis of human cataracts. The present studies were undertaken to examine the feasibility of possible prevention of such damage to the lens initiated by UV activation of kynurenine, a well-known photosensitizer in the human lens. The studies were done by organ culturing intact mouse lenses in medium containing kynurenine and exposed to UVA. Tissue damage was assessed by the inhibition of its ability to carry active transport of rubidium ions and the associated decrements in the levels of GSH and ATP. These deleterious effects were significantly prevented by caffeine, an alkaloid present in many common beverages and known to chemically deactivate the said oxygen derivatives. Further studies on the pharmacological significance of the findings are hence in progress.

Prevention of oxidative damage to lens by caffeine.

PURPOSE: The primary objective of this study was to examine the possibility of inhibiting oxidative damage to the lens in vitro by caffeine. METHODS: Oxidative damage was inflicted by incubating mouse lenses in Tyrode medium containing 0.1 mM Fe(8)Br(8), an iron complex soluble in aqueous medium. Parallel incubations were conducted in the presence of caffeine (5 mM). RESULTS: Lenses incubated in the medium containing Fe(8)Br(8) undergo oxidative stress, as evidenced by the inhibition of Na(+)-K(+) ATPase-driven rubidium transport and the loss of tissue glutathione and ATP. These effects were prevented in presence of caffeine. That the effects are due to the oxyradicals produced was ascertained further by parallel studies with Tempol (5 mM), a well-known scavenger of reactive oxygen species (ROS) with its activity being more pronounced with hydroxyl radicals as compared to other ROS. CONCLUSIONS: Caffeine was found to be effective in preventing oxidative stress to the lens induced by iron under ambient conditions. The protective effect is attributable to its ability to scavenge ROS, particularly the hydroxyl radical.

UV-B-induced damage to the lens in vitro: prevention by caffeine.

Ultraviolet (UV) irradiation is one of the significant risk factors in the genesis of cataracts. Pathogenetically, the process can be triggered by the intraocular generation of various reactive species of oxygen that are well known to be initiated by the penetration of light, especially of the UV frequencies. The contribution of UV exposure in the etiology of this disease is likely to increase further due to ozone depletion in the upper atmosphere. The present studies were undertaken to examine if the UV effects can be attenuated with the xanthine-based alkaloids primarily present in tea and coffee. We have examined this possibility by in vitro lens culture studies with caffeine. As expected, mice lenses incubated in Tyrode solution exposed to UV at 302 nm are physiologically damaged, as evidenced by the inhibition of the active transport of (86)Rb(+), an ion acting as a surrogate of the K(+). There was a simultaneous decrease in the levels of adenosine triphosphate and glutathione. The addition of caffeine to the medium prevented such deleterious effects. That caffeine and perhaps other xanthinoids have a protective effect against cataract formation induced by UV has hence been demonstrated for the first time.

Prevention of oxidative stress to the retina by pyruvate. A preliminary report.

AIM: Oxidative stress is considered to be a significant risk factor in the pathogenesis of many eye diseases associated with aging. The purpose of this study was to investigate the susceptibility of retinal tissue to reactive oxygen species (ROS)-induced damage, and to examine if this can be prevented by compounds having the ability to scavenge ROS as well as support the tissue bioenergetically via normal as well as anaplerotic metabolic pathways. METHODS: Experiments were conducted on retinas isolated from CD-1 mice. The isolated retinas were incubated in Medium 199, generating ROS by the action of xanthine oxidase and uricase on xanthine. Parallel experiments were conducted in the presence of pyruvate (10 mM). The extent of oxidative damage to the tissue was assessed by determining the levels of glutathione. Malonaldehyde was used as an index of the peroxidative degradation of lipids. RESULTS: Exposure of the tissue to ROS resulted in a significant decrease in its glutathione content. There was a simultaneous increase in the level of malonaldehyde. These effects were substantially attenuated by pyruvate. CONCLUSIONS: While ROS has been previously implicated in the genesis of many aging diseases of the retina, methods to prevent or treat them are currently in their infancy. The present investigations suggest that this can be accomplished by the use of certain compounds of endogenous origin, such as pyruvate, which have the dual properties of acting as ROS scavengers while also acting as metabolic agonists. By virtue of its oxyradical scavenging property, it is expected to inhibit the oxidative degradation of polyunsaturated fatty acids required to maintain the structural and functional integrity of the tissue.

Induction of ultraviolet cataracts in vitro: prevention by pyruvate.

Ultraviolet (UV) radiation is one of the important cataract risk factors. The present studies examined the hypothesis that this effect is due to the UV penetration through the cornea and subsequent induction of a photochemical generation of reactive species of oxygen (ROS) in the aqueous and lens. The hypothesis was ascertained by rat lens organ culture studies conducted under UV (365 nm), with media containing micromolar levels of riboflavin, with and without pyruvate, the latter acting as an ROS scavenger. The implication of ROS in the UV-induced damage was confirmed by measurements of peroxide generation. Damage to the lens was assessed physiologically by measuring the decrease in its active transport of rubidium ions. Biochemically, it was assessed by measuring the lowering of adenosine triphosphate and glutathione. The incorporation of pyruvate in the medium protected the lens against these deleterious effects. That the beneficial effect of pyruvate is attributable to its ROS-scavenging property was proven by the peroxide depletion in its presence, commensurate with its own utilization in parallel. A protective effect of this keto acid against UV-induced tissue damage has been shown for the first time, suggesting its clinical usefulness against UV irradiation-induced pathologies. Hence, further studies on the possible protective effects of such alpha-keto acids against UV damage are in progress.

Susceptibility of the ocular lens to nitric oxide: implications in cataractogenesis.

Oxides of nitrogen, such as nitric oxide (NO), are now biologically referred to as reactive nitrogen species. The generation of NO gives rise to several other reactive species, such as NO+, NO-, NO2, N2O3, and ONOO- and so forth, which are all capable of inflicting tissue damage. Indeed, NO generation is known to be associated with retinal degeneration and glaucoma. Its level has also been found to increase in the aqueous and vitreous humors in diabetes. We hypothesize that such an increase would have a detrimental effect on the biochemistry and metabolism of tissues, including the lens, bathed by the aqueous containing elevated levels of NO. The primary aim of our investigations was, therefore, to examine the susceptibility of the lens to damage by NO in vitro in the presence of nitroaspirin, a novel NO donating agent. The extent of physiologic damage to the lens was initially assessed by determining the integrity of its active transport mechanism. The overall status of tissue metabolism was determined by measuring the adenosine triphosphate (ATP) levels. The levels of glutathione (GSH) and glutathione disulfide, reflecting the status of its antioxidant reserve, were also determined. That NO is indeed deleterious to the lens was apparent by the inhibition of the active transport of Rb(+). This was associated with a substantial decrease in the contents of ATP and GSH, the decrease in the latter directly suggesting that the NO effects are caused by oxidative stress. That the effects are caused by NO generated from nitroaspirin was proven by a substantial increase in NO level in the medium during incubation of the lenses with nitroaspirin, as compared to the controls. The results, therefore, were highly suggestive of a contribution of the oxides of nitrogen in cataract formation associated with diabetes and other aging diseases.

Lens thiol depletion by peroxynitrite. Protective effect of pyruvate.

Pyruvate (PY) is known to be a potent scavenger of H(2)O(2 )by undergoing its peroxidative decarboxylation. While doing so, it also inhibits .OH generation, in addition to its direct .OH scavenging effect. We now hypothesize that PY would also be decarboxylated by cleaving the -O-O- bond in peroxynitrite (ONOO(-)) (PN), with the effect of protecting tissues against NO(x )induced damage. We have verified this by measuring (14)CO(2) formation on incubation of 1-(14)C-PY with 3-morpholinosydnonimine (SIN-1). Its protective effect against PN induced thiol depletion was initially assessed by determining its ability to inhibit oxidation of pure GSH. This was further evaluated by incubating lens homogenate with SIN-1 with or without PY. As conceived, PY did inhibit PN induced loss of protein as well as non-protein -SH. The findings therefore appear potentially useful to protect against nitrite induced damage to the lens and other tissues known to occur with aging and certain diseases such as diabetes.