Longitudinal Study of Age-Related Cataract Using Dynamic Light Scattering: Loss of α-Crystallin Leads to Nuclear Cataract Development.

PURPOSE: To conduct a longitudinal study on age-related nuclear cataracts using dynamic light scattering (DLS) to determine if cataract progression is associated with loss of the unbound form of the lens molecular chaperone protein, α-crystallin. DESIGN: Natural history and cohort study. PARTICIPANTS: Patients 30 years of age or older of either gender seeking treatment at the Wilmer Eye Institute Cornea-Cataract Department. METHODS: All patients underwent a comprehensive dilated eye examination every 6 months, including slit-lamp grading of their lenses using the Age-Related Eye Disease Study (AREDS) clinical lens grading system and obtaining an estimate of unbound α-crystallin level in the nucleus, the α-crystallin index (ACI), using the National Aeronautics and Space Administration-National Eye Institute DLS device. We used a random effects statistical model to examine the relationship of lens opacity changes over time with ACI changes. MAIN OUTCOME MEASURES: α-Crystallin Index (ACI) and AREDS nuclear cataract grade. RESULTS: Forty-five patients (66 eyes) 34 to 79 years of age with AREDS nuclear lens grades of 0 to 3.0 were followed up every 6 months for a mean of 19 months (range, 6-36 months). We found that lenses with the lowest baseline levels of ACI had the most rapid progression of cataracts, whereas lenses with higher ACI at baseline had no or slower cataract progression. Lenses that lost α-crystallin at the highest rates during the study also had faster progression of nuclear cataracts than lenses with a slower rate of ACI loss. Kaplan-Meier survival curves showed that lenses with the lowest initial ACI had the highest risk of undergoing cataract surgery. CONCLUSIONS: This longitudinal study corroborates our previous cross-sectional study finding that higher levels of unbound α-crystallin as assessed by ACI are associated with lower risk of cataract formation and that loss of ACI over time is associated with cataract formation and progression. This study suggested that assessment of ACI with the DLS device could be used as a surrogate for lens opacity risk in clinical studies, and for assessing nuclear cataract events in studies where cataract development may be a side effect of a drug or device.

Isoelectric focusing in a drop.

A novel approach to molecular separations is investigated using a technique termed droplet-based isoelectric focusing. Drops are manipulated discretely on a superhydrophobic surface, subjected to low voltages for isoelectric focusing, and split-resulting in a preparative separation. A universal indicator dye demonstrates the generation of stable, reversible pH gradients (3-10) in ampholyte buffers, and these gradients lead to protein focusing within the drop length. Focusing was visually characterized, spectroscopically verified, and assessed quantitatively by noninvasive light scattering measurements. It was found to correlate with a quantitative model based on 1D steady-state theory. This work illustrates that molecular separations can be deployed within a single open drop, and the differential fractions can be separated into new discrete liquid elements.

Measurement of lens protein aggregation in vivo using dynamic light scattering in a guinea pig/UVA model for nuclear cataract.

The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model. DLS size distribution analysis conducted at the same location in the lens nucleus of control and UVA-irradiated animals showed a 28% reduction in intensity of small diameter proteins in experimental lenses compared with controls (P < 0.05). In addition, large diameter proteins in UVA-exposed lens nuclei increased five-fold in intensity compared to controls (P < 0.05). The UVA-induced increase in apparent size of lens nuclear small diameter proteins was three-fold (P < 0.01), and the size of large diameter aggregates was more than four-fold in experimental lenses compared with controls. The diameter of crystallin aggregates in the UVA-irradiated lens nucleus was estimated to be 350 nm, a size able to scatter light. No significant changes in protein size were detected in the anterior cortex of UVA-irradiated lenses. It is presumed that the presence of a UVA chromophore in the guinea pig lens (NADPH bound to zeta crystallin), as well as traces of oxygen, contributed to UVA-induced crystallin aggregation. The results indicate a potentially harmful role for UVA light in the lens nucleus. A similar process of UVA-irradiated protein aggregation may take place in the older human lens nucleus, accelerating the formation of human nuclear cataract.

Non-invasive Detection of Unique Molecular Signatures in Laser-Induced Retinal Injuries.

Unintentional laser exposure is an increasing concern in many operational environments. Determining whether a laser exposure event caused a retinal injury currently requires medical expertise and specialized equipment that are not always readily available. The purpose of this study is to test the feasibility of using dynamic light scattering (DLS) to non-invasively detect laser retinal injuries through interrogation of the vitreous humor (VH). Three grades of retinal laser lesions were studied: mild (minimally visible lesions), moderate (Grade II), and severe (Grade III). A pre-post-treatment design was used to collect DLS measurements in vivo at various time points, using a customized instrument. VH samples were analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) and relative protein abundances were determined by spectral counting. DLS signal analysis revealed significant changes in particle diameter and intensity in laser-treated groups as compared with control. Differences in protein profile in the VH of the laser-treated eyes were noted when compared with control. These results suggest that laser injury to the retina induces upregulation of proteins that diffuse into the VH from the damaged tissue, which can be detected non-invasively using DLS.

Aggregation of lens crystallins in an in vivo hyperbaric oxygen guinea pig model of nuclear cataract: dynamic light-scattering and HPLC analysis.

PURPOSE: The role of oxygen in the formation of lens high-molecular-weight (HMW) protein aggregates during the development of human nuclear cataract is not well understood. The purpose of this study was to investigate lens crystallin aggregate formation in hyperbaric oxygen (HBO)-treated guinea pigs by using in vivo and in vitro METHODS: methods. Guinea pigs were treated three times weekly for 7 months with HBO, and lens crystallin aggregation was investigated in vivo with the use of dynamic light-scattering (DLS) and in vitro by HPLC analysis of water-insoluble (WI) proteins. DLS measurements were made every 0.1 mm across the 4.5- to 5.0-mm optical axis of the guinea pig lens. RESULTS: The average apparent diameter of proteins in the nucleus (the central region) of lenses of HBO-treated animals was nearly twice that of the control animals (P < 0.001). Size distribution analysis conducted at one selected point in the nucleus and cortex (the outer periphery of the lens) after dividing the proteins into small-diameter and large-diameter groups, showed in the O2-treated nucleus a threefold increase in intensity (P < 0.001) and a doubling in apparent size (P = 0.03) of large-diameter aggregate proteins, compared with the same control group. No significant changes in apparent protein diameter were detected in the O2-treated cortex, compared with the control. The average diameter of protein aggregates at the single selected location in the O2-treated nucleus was estimated to be 150 nm, a size capable of scattering light and similar to the size of aggregates found in human nuclear cataracts. HPLC analysis indicated that one half of the experimental nuclear WI protein fraction (that had been dissolved in guanidine) consisted of disulfide cross-linked 150- to 1000-kDa aggregates, not present in the control. HPLC-isolated aggregates contained alphaA-, beta-, gamma-, and zeta-crystallins, but not alphaB-crystallin, which is devoid of -SH groups and thus does not participate in disulfide cross-linking. All zeta-crystallin present in the nuclear WI fraction appeared to be there as a result of disulfide cross-linking. CONCLUSIONS: The results indicate that molecular oxygen in vivo can induce the cross-linking of guinea pig lens nuclear crystallins into large disulfide-bonded aggregates capable of scattering light. A similar process may be involved in the formation of human nuclear cataract.

Ocular static and dynamic light scattering: a noninvasive diagnostic tool for eye research and clinical practice.

The noninvasive techniques of static and dynamic light scattering are emerging as valuable diagnostic tools for the early detection of ocular and systemic diseases. These include corneal abnormalities, pigmentary dispersion syndrome, glaucoma, cataract, diabetic vitreopathy, and possibly macular degeneration. Systemic conditions such as diabetes and possibly Alzheimer's disease can potentially be detected early via ocular tissues. The current state of development of these techniques for application to ophthalmic research and ultimately clinical practice is reviewed.

New optical scheme for a polarimetric-based glucose sensor.

A new optical scheme to detect glucose concentration in the aqueous humor of the eye is presented. The ultimate aim is to apply this technique in designing a new instrument for, routinely and frequently, noninvasively monitoring blood glucose levels in diabetic patients without contact (no index matching) between the eye and the instrument. The optical scheme exploits the Brewster reflection of circularly polarized light off of the lens of the eye. Theoretically, this reflected linearly polarized light on its way to the detector is expected to rotate its state of polarization, owing to the presence of glucose molecules in the aqueous humor of a patient's eye. An experimental laboratory setup based on this scheme was designed and tested by measuring a range of known concentrations of glucose solutions dissolved in water. (c) 2004 Society of Photo-Optical Instrumentation Engineers.

In vivo assessment of diabetic lenses using dynamic light scattering.

One of the most threatening aspects of diabetes mellitus is the development of visual impairment. For example, cataracts are 1.6 times more common in people with diabetes than in those without diabetes. Cataract extraction is the only treatment. In many cases, diabetes-related ocular pathologies go undiagnosed until visual function is compromised. This paper compares and contrasts the ocular changes observed using dynamic light scattering (DLS) and conventional ophthalmic techniques during long-term maintenance of the sand rat (Psammomys obesus) on a high caloric diet. P. obesus is a wild rodent in the subfamily Gerbillinae that inhabits the desert areas of the Middle East and Africa. This animal is unique in that it develops mild to moderate obesity, hyperglycemia, pancreatic atrophy, impaired renal function, ketoacidosis, vision loss, and other diabetic complications when it consumes a high caloric diet. In this study, five animals were fed Purina sand rat chow and thus served as normal control animals. Five animals were fed a commercially prepared rodent diet (Purina 5002) consisting of only 4-5% fiber and a grain-based rabbit supplement (BioServe Rabbit Stix Appetite Stimulant) consisting of 50% carbohydrate to provide a high caloric (diabetogenic) diet and thus induce diabetes. Blood samples for the biochemical analyses, DLS, and other optical examinations were obtained on alternate weeks. Our preliminary results have demonstrated subtle changes in the lens of the diabetic sand rats as early as 2 months on a diabetogenic diet. This is an ongoing joint project with Food and Drug Administration and the National Aeronautics and Space Administration. This technique is proving to be a practical, sensitive, noninvasive diagnostic tool useful for the early detection of ocular pathologies and understanding the mechanism of cataract formation.

A hypothesis on biological protection from space radiation through the use of new therapeutic gases as medical counter measures.

Radiation exposure to astronauts could be a significant obstacle for long duration manned space exploration because of current uncertainties regarding the extent of biological effects. Furthermore, concepts for protective shielding also pose a technically challenging issue due to the nature of cosmic radiation and current mass and power constraints with modern exploration technology. The concern regarding exposure to cosmic radiation is biological damage that is associated with increased oxidative stress. It is therefore important and would be enabling to mitigate and/or prevent oxidative stress prior to the development of clinical symptoms and disease. This paper hypothesizes a "systems biology" approach in which a combination of chemical and biological mitigation techniques are used conjunctively. It proposes using new, therapeutic, medical gases as chemical radioprotectors for radical scavenging and as biological signaling molecules for management of the body's response to exposure. From reviewing radiochemistry of water, biological effects of CO, H2, NO, and H2S gas, and mechanisms of radiation biology, it can be concluded that this approach may have therapeutic potential for radiation exposure. Furthermore, it also appears to have similar potential for curtailing the pathogenesis of other diseases in which oxidative stress has been implicated including cardiovascular disease, cancer, chronic inflammatory disease, hypertension, ischemia/reperfusion (IR) injury, acute respiratory distress syndrome, Parkinson's and Alzheimer's disease, cataracts, and aging. We envision applying these therapies through inhalation of gas mixtures or ingestion of water with dissolved gases.

Hydrogen therapy may reduce the risks related to radiation-induced oxidative stress in space flight.

Cosmic radiation is known to induce DNA and lipid damage associated with increased oxidative stress and remains a major concern in space travel. Hydrogen, recently discovered as a novel therapeutic medical gas in a variety of biomedical fields, has potent antioxidant and anti-inflammatory activities. It is expected that space mission activities will increase in coming years both in numbers and duration. It is therefore important to estimate and prevent the risks encountered by astronauts due to oxidative stress prior to developing clinical symptoms of disease. We hypothesize that hydrogen administration to the astronauts by either inhalation or drinking hydrogen-rich water may potentially yield a novel and feasible preventative/therapeutic strategy to prevent radiation-induced adverse events.

Clinical detection of precataractous lens protein changes using dynamic light scattering.

OBJECTIVE: To use dynamic light scattering to clinically assess early precataractous lens protein changes. METHODS: We performed a cross-sectional study in 380 eyes of 235 patients aged 7 to 86 years with Age-Related Eye Disease Study clinical nuclear lens opacity grades 0 to 3.8. A dynamic light-scattering device was used to assess alpha-crystallin, a molecular chaperone protein shown to bind other damaged lens proteins, preventing their aggregation. The outcome measure was the alpha-crystallin index, a measure of unbound alpha-crystallin in each lens. The association of the alpha-crystallin index with increasing nuclear opacity and aging was determined. RESULTS: There was a significant decrease in the alpha-crystallin index associated with increasing nuclear lens opacity grades (P < .001). There were significant losses of alpha-crystallin even in clinically clear lenses associated with aging (P < .001). The standard error of measurement was 3%. CONCLUSIONS: Dynamic light scattering clinically detects alpha-crystallin protein loss even in clinically clear lenses. alpha-Crystallin index measurements may be useful in identifying patients at high risk for cataracts and as an outcome variable in clinical lens studies. CLINICAL RELEVANCE: The alpha-crystallin index may be a useful measure of the protective alpha-crystallin molecular chaperone reserve present in a lens, analogous to creatinine clearance in estimating renal function reserve.

Pharmacologic vitreolysis with microplasmin increases vitreous diffusion coefficients.

BACKGROUND: Pharmacologic vitreolysis is a new approach to improve vitreo-retinal surgery and ultimately to liquefy and detach the vitreous from the retina to eliminate the contribution of the vitreous to retinopathy. The mechanism of action of the agents being developed for pharmacologic vitreolysis remains unclear. The effect of microplasmin on vitreous diffusion coefficients was investigated using the non-invasive technique of dynamic light scattering (DLS). METHODS: Vitreous diffusion coefficients in 18 intact porcine eyes were measured in vitro with dynamic light scattering (DLS). DLS was performed on all specimens at 37 degrees C 30 min after injections of human recombinant microplasmin at doses ranging from 0.125 to 0.8 mg, with 20-nm tracer nanospheres. RESULTS: DLS findings in untreated porcine vitreous were similar to the previously described findings in bovine and human vitreous. Microplasmin increased porcine vitreous diffusion coefficients in a dose-dependent manner (correlation coefficient, r=0.93), with an 85% increase after a 30-min exposure to the maximum dose. CONCLUSIONS: Pharmacologic vitreolysis with human recombinant microplasmin increases vitreous diffusion coefficients in vitro. The results of these studies have implications for the dosing, route of administration, duration of action and methods of determining efficacy in future studies of pharmacologic vitreolysis to enhance vitreo-retinal surgery, as well as the design of clinical trials to induce prophylactic posterior vitreous detachment.

Particle-size and velocity measurements in flowing conditions using dynamic light scattering.

The noninvasive optical technique of dynamic light scattering (DLS) is routinely used to characterize dilute and transparent submicrometer particle dispersions in laboratory environments. A variety of industrial and biological applications would, however, greatly benefit from on-line monitoring of dispersions under flowing conditions. We present a model experiment to study flowing dispersions of polystyrene latex particles of varying sizes under varying flow conditions by using a newly developed fiber-optic DLS probe. A modified correlation function proposed in an earlier study is applied to the analysis of extracting the size and velocity of laminar flowing particulate dispersions. The complementary technique of laser Doppler velocimetry is also used to measure the speed of moving particles to confirm the DLS findings.

A clinical study of the human lens with a dynamic light scattering device.

A study was conducted to determine the potential usefulness and repeatability of a new dynamic light scattering (DLS) device for clinical studies of the human lens and early cataract. Studies using the cold cataract model showed this new device to be more sensitive than the Scheimpflug cataract imaging system in detecting the earliest cataractous changes. A miniaturized clinical DLS device developed by NASA using fiber optic probes was mounted on a Keratoscope (Optikon 2000), which has a 3-dimensional aiming system for accurate repeated sampling of the same area of the lens. A test/retest study was then conducted on the nuclear region of the lenses of 12 normal eyes. After a full, dilated eye examination, DLS data were obtained using the new device on the same eyes twice, 30-60 min apart. Particle size distributions and mean log particle size data were obtained. The mean percent differences between the larger and smaller of the test--retest pairs was 6.4% (range 0.05--10.8%); the between-test S.D. was 0.116. Actual numerical margin of error was +/- 0.023. In addition, the mean coefficient of variation was 4.2% (range 0.3--7.3%). A useful clinical end point obtained from data produced by the device was the mean log particle size. These results suggest that the DLS will be useful in the detection and study of the beginning and earliest stages of cataract formation in humans.