Cold Cataract Research Articles

Fast and Mobile Cataract Detection by Applying Line Laser Eye Illumination

Cataract is observed when the eye lens becomes opaque. This condition causes blurred vision and is the main cause of blindness worldwide. Cataract diagnosis is usually performed during ophthalmologist examination using a slit lamp, which requires expertise, is expensive and bulky. In this study, we present a small handheld illumination setup for cataract detection. Ex-vivo porcine eyes are investigated to determine whether colored line lasers, 450 nm (blue), 520 nm (green) and 650 nm (red), which shine obliquely into the eye, are principally suited for detection of the Y shaped suture cataract and of cold cataract, respecting exposure limits of EU guideline 2006/25/EC. Camera images of the cataract exhibited good results under illumination with all line lasers. Observations with the physician’s eye led to an even better diagnosis of cataract. Generally, green laser light illumination was the best choice for cataract detection. With red laser light illumination it was also possible, but least suitable for this purpose. With this method, line lasers are a good choice for cataract identification, as cataract can be detected quickly and without much effort. This type of line laser illumination of the eye is safe and both types of cataract are detectable with all wavelengths. For the human eye, a further development of this system is conceivable.

Phase-Decorrelation Optical Coherence Tomography Measurement of Cold-Induced Nuclear Cataract.

The purpose of this work is to determine the sensitivity of phase-decorrelation optical coherence tomography (OCT) to protein aggregation associated with cataracts in the ocular lens, as compared to OCT signal intensity. Six fresh porcine globes were held at 4°C until cold cataracts developed. As the globes were re-warmed to ambient temperature, reversing the cold cataract, each lens was imaged repeatedly using a conventional OCT system. Throughout each experiment, the internal temperature of the globe was recorded using a needle-mounted thermocouple. OCT scans were acquired, their temporal fluctuations were analyzed, and the rates of decorrelation were spatially mapped. Both decorrelation and intensity were evaluated as a function of recorded temperature. Both signal decorrelation and intensity were found to change with lens temperature, a surrogate of protein aggregation. However, the relationship between signal intensity and temperature was not consistent across different samples. In contrast, the relationship between decorrelation and temperature was found to be consistent across samples. In this study, signal decorrelation was shown to be a more repeatable metric for quantification of crystallin protein aggregation in the ocular lens than OCT intensity-based metrics. Thus, OCT signal decorrelation measurements could enable more detailed and sensitive study of methods to prevent cataract formation. This dynamic light scattering-based approach to early cataract assessment can be implemented on existing clinical OCT systems without hardware additions, so it could quickly become part of a clinical study workflow or an indication for use for a pharmaceutical cataract intervention.

Protein and Water Distribution Across Visual Axis in Mouse Lens: A Confocal Raman MicroSpectroscopic Study for Cold Cataract.

Purpose: The aims of the study were to investigate cellular mechanisms of cold cataract in young lenses of wild-type C57BL/6J (B6WT) mice treated at different temperatures and to test a hypothesis that cold cataract formation is associated with the changes in lens protein and water distribution at different regions across lens fiber cells by Raman spectroscopy (RS). Methods: RS was utilized to scan the mouse lens at different regions with/without cold cataract. Three regions with various opacification along the equatorial axis in the anterior–posterior lens section were scanned. The intensity ratio of Raman bands at 2,935 and 3,390 cm−1 (Ip/Iw) were used to evaluate lens protein and water distribution. We further determined water molecular changes through Gaussian profiles of water Raman spectra. Results: Three specific regions 1, 2, and 3, located at 790–809, 515–534, and 415–434 μm away from the lens center, of postnatal day 14 B6WT lenses, were subjected to RS analysis. At 37°C, all three regions were transparent. At 25°C, only region 3 became opaque, while at 4°C, both regions 2 and 3 showed opacity. The sum of the difference between Ip/Iw and the value of linear fitting line from scattered-line at each scanning point was considered as fluctuation degree (FD) in each region. Among different temperatures, opaque regions showed relatively higher FD values (0.63 and 0.79 for regions 2 and 3, respectively, at 4°C, and 0.53 for region 3 at 25°C), while transparent regions provided lower FD values (less than 0.27). In addition, the decrease in Gaussian peak II and the rising of Gaussian peak III and IV from water Raman spectra indicated the instability of water molecule structure in the regions with cold cataract. Conclusion: Fluctuation degrees of RS data reveal new mechanistic information about cold cataract formation, which is associated with uneven distribution of lens proteins and water across lens fiber cells. It is possible that RS data partly reveals cold temperature-induced redistribution of lens proteins such as intermediate filaments in inner fiber cells. This lens protein redistribution might be related to unstable structure of water molecules according to Gaussian profiles of water RS.

Integrated Transcriptomic and Proteomic Analysis Reveals Up-Regulation of Apoptosis and Small Heat Shock Proteins in Lens of Rats Under Low Temperature

Cold cataract is the reversible opacification of the lens when the temperature decreases. However, we observed that when temperature of the rats’ lens was maintained at a lower temperature for a prolonged time, the opacification of lens was only partly reversible. To review the potential molecular mechanism of the irreversible part of opacification under cold stimulation, we applied comparative transcriptomic and proteomic analysis to systematically investigate the molecular changes that occurred in the lens capsules of rats under low temperature treatments. The RNA sequencing based transcriptomic analysis showed a significant up-regulation of genes related to the lens structure and development in the Hypothermia Group. Hub genes were small heat shock proteins (sHSPs). Besides the same findings as the transcriptomic results, the liquid chromatography-tandem mass spectrometry based proteomic analysis also revealed the up-regulation of the apoptotic process. To further analyze the regulatory mechanism in this process, we subsequently performed integrated analysis and identified the down-regulation of Notch3/Hes1 and PI3K/Akt/Xiap signaling axis. Our research revealed the activation of the apoptotic process in rats’ lens under cold stimulation, and the sHSP related heat shock response as a potential protective factor through our transcriptomic and proteomic data.

Simultaneously imaging and quantifying in vivo mechanical properties of crystalline lens and cornea using optical coherence elastography with acoustic radiation force excitation.

The crystalline lens and cornea comprise the eye’s optical system for focusing light in human vision. The changes in biomechanical properties of the lens and cornea are closely associated with common diseases, including presbyopia and cataract. Currently, most in vivo elasticity studies of the anterior eye focus on the measurement of the cornea, while lens measurement remains challenging. To better understand the anterior segment of the eye, we developed an optical coherence elastography system utilizing acoustic radiation force excitation to simultaneously assess the elasticities of the crystalline lens and the cornea in vivo. A swept light source was integrated into the system to provide an enhanced imaging range that covers both the lens and the cornea. Additionally, the oblique imaging approach combined with orthogonal excitation also improved the image quality. The system was tested through first ex vivo and then in vivo experiments using a rabbit model. The elasticities of corneal and lens tissue in an excised normal whole-globe and a cold cataract model were measured to reveal that cataractous lenses have a higher Young’s modulus. Simultaneous in vivo elasticity measurements of the lens and cornea were performed in a rabbit model to demonstrate the correlations between elasticity and intraocular pressure and between elasticity and age. To the best of our knowledge, we demonstrated the first in vivo elasticity of imaging of both the lens and cornea using acoustic radiation force-optical coherence elastography, thereby providing a potential powerful clinical tool to advance ophthalmic research in disorders affecting the lens and the cornea.

Inducible brown/beige adipocytes in retro-orbital adipose tissues

Beige adipocytes and brown adipocytes can generate heat by using mitochondrial uncoupling protein 1 (Ucp1), a thermogenic protein. Browning/beiging is the emergence of beige adipocytes in white adipose tissues (WAT) for cold acclimatization. Here we show the existence of brown/beige adipocytes in retro-orbital WAT in mice. Histologically, Ucp1-positive cells with multilocular lipid droplets were abundant in retro-orbital WAT of immature mice; those cells decreased in number with age. However, Ucp1-positive adipocytes with multilocular lipid droplets emerged in retro-orbital WAT in adult mice, due to cold exposure as short as 3 h. Consistent with this observation, the expression level of Ucp1 mRNA was enhanced in tissues upon cold exposure. Furthermore, eye surface temperature remained within a physiological range during cold challenge. RT-qPCR suggested a mixed phenotype of brown and beige adipocytes in retro-orbital WAT. Transmission electron microscopic observation showed multiple lipid droplets and numerous mitochondria with high cristae density in retro-orbital WAT cells from both control and cold-exposed mice. Our results suggest that warming of the orbital cavity by browning/beiging in retro-orbital WAT is a protective mechanism against cold cataract caused by lowered lens temperature.

Optical coherence elastography of cold cataract in porcine lens.

.Cataract is one of the most prevalent causes of blindness around the world. Understanding the mechanisms of cataract development and progression is important for clinical diagnosis and treatment. Cold cataract has proven to be a robust model for cataract formation that can be easily controlled in the laboratory. There is evidence that the biomechanical properties of the lens can be significantly changed by cataract. Therefore, early detection of cataract, as well as evaluation of therapies, could be guided by characterization of lenticular biomechanical properties. In this work, we utilized optical coherence elastography (OCE) to monitor the changes in biomechanical properties of ex vivo porcine lenses during formation of cold cataract. Elastic waves were induced in the porcine lenses by a focused micro air-pulse while the lenses were cooled, and the elastic wave velocity was translated to Young’s modulus of the lens. The results show an increase in the stiffness of the lens due to formation of the cold cataract (from to ). These results show a relation between lens opacity and stiffness and demonstrate that OCE can assess lenticular biomechanical properties and may be useful for detecting and potentially characterizing cataracts.

Uncovering the Role of Surface Residues and Buffer Composition in Liquid-Liquid Phase Separation of Eye Lens Crystallins from an Antarctic Toothfish

The Antarctic toothfish, Dissostichus mawsoni, lives in the Southern Ocean around Antarctica, requiring adaptations to deal with the challenges of living in a subfreezing environment. One such adaptation is that the D. mawsoni eye lens is completely resistant to liquid-liquid phase separation (LLPS) of the eye lens proteins, also known as cold cataract. Despite this observation, a sub-fraction of the D. mawsoni eye lens proteins was found to undergo LLPS above the body temperature of the toothfish. Six γ-crystallins were identified from this fraction and characterized for their LLPS behavior and general features of stability. No obvious relationship could be discerned between the critical temperatures at which these γ-crystallins undergo LLPS and the various measures of stability. Additionally, mutagenesis of predicted surface exposed residues was performed to study their role in the LLPS behavior. Mutagenesis revealed that replacing exposed arginine for lysine results in a decrease of the phase transition temperature, while replacing lysine with arginine had the opposite effect, but similar magnitude. These finding highlights the importance of arginine in increasing attractive protein-protein interactions even when the site specific charge is largely unchanged. Furthermore, we have shown that the components of the buffer solution can further modulate the phase transition temperature of these crystallins.

Controlling Liquid–Liquid Phase Separation of Cold-Adapted Crystallin Proteins from the Antarctic Toothfish

Liquid–liquid phase separation (LLPS) of proteins is important to a variety of biological processes both functional and deleterious, including the formation of membraneless organelles, molecular condensations that sequester or release molecules in response to stimuli, and the early stages of disease-related protein aggregation. In the protein-rich, crowded environment of the eye lens, LLPS manifests as cold cataract. We characterize the LLPS behavior of six structural γ-crystallins from the eye lens of the Antarctic toothfish Dissostichus mawsoni, whose intact lenses resist cold cataract in subzero waters. Phase separation of these proteins is not strongly correlated with thermal stability, aggregation propensity, or cross-species chaperone protection from heat denaturation. Instead, LLPS is driven by protein–protein interactions involving charged residues. The critical temperature of the phase transition can be tuned over a wide temperature range by selective substitution of surface residues, suggesting general principles for controlling this phenomenon, even in compactly folded proteins.

Understanding the dynamics of biological colloids to elucidate cataract formation and develop a methodology for its early diagnosis

The eye lens is the most notable example of mammalian tissues exhibiting complex colloidal behavior. In this paper, we briefly describe how dynamics in colloidal suspensions can address selected aspects of cataracts, which is ultimately related to protein self-assembly under pathological conditions. Results from dynamic light-scattering of eye lens homogenates over a wide protein concentration were analyzed and the various relaxation modes were identified in terms of collective and self-diffusion processes. Using this information as an input, the complex relaxation pattern of the intact lens nucleus was rationalized. The model of cold cataract–a phase separation effect of the lens cytoplasm with cooling–was used to simulate a lens cataract under in vitro conditions to determine the correlation function parameters, which could be used as reliable indicators of cataract onset. The applicability of dynamic light scattering as a non-invasive, early diagnostic tool for ocular diseases is also demonstrated in light of these findings.

Dynamic light scattering study on phase separation of a protein-water mixture: Application on cold cataract development in the ocular lens

We present a detailed dynamic light scattering study of the phase separation in the ocular lens emerging during cold cataract development. Cold cataract is a phase separation effect that proceeds via spinodal decomposition of the lens cytoplasm with cooling. The intensity autocorrelation functions of the lens protein content are analyzed with the aid of two methods, providing information on the populations and dynamics of the scattering elements associated with cold cataract. It is found that the temperature dependence of many measurable parameters changes appreciably at the characteristic temperature approximately 16+/-1 degrees C which is associated with the onset of cold cataract. By extending the temperature range of this work to previously inaccessible regimes, i.e., well below the phase separation or coexistence curve at Tcc, we have been able to accurately determine the temperature dependence of the collective and self-diffusion coefficients of proteins near the spinodal. The analysis showed that the dynamics of proteins bears some resemblance to the dynamics of structural glasses, where the apparent activation energy for particle diffusion increases below Tcc, indicating a highly cooperative motion. Application of ideas developed for studying the critical dynamics of binary protein-solvent mixtures, as well as the use of a modified Arrhenius equation, enabled us to estimate the spinodal temperature Tsp of the lens nucleus. The applicability of dynamic light scattering as a noninvasive, early-diagnostic tool for ocular diseases is also demonstrated in light of the findings of the present paper.

γD-Crystallin–Associated Protein Aggregation and Lens Fiber Cell Denucleation

To understand the underlying molecular mechanism for a dominant cataract caused by a point mutation in the gammaD-crystallin gene. A dominant cataractous mouse line was identified from chemically induced mouse mutations by phenotypic screening with slit lamp examination. Genomewide linkage analysis and DNA sequencing were used to determine the causative gene mutation. Histology, immunohistochemistry, Western blotting, and in vitro transfection studies were used to characterize mutant lenses. Cataracts in mutant mice were caused by a point mutation in the gammaD-crystallin gene (gammaD-V76D). Intranuclear gamma-crystallin aggregates, incomplete denucleation, and decreased connexins were observed in mutant lens fiber cells. Mutant gammaD-V76D proteins became less soluble in the lens, and structural modeling suggested that the substituted aspartic acid residue (D) altered hydrogen bond formation and surface electrostatic potential of the protein. Unexpectedly, the formation of cold cataracts, which occurred in wild-type lenses at low temperature, was abolished in gammaD-V76D mutant lenses. In vitro transfection studies revealed that wild-type gammaD proteins were uniformly distributed in the cytosol and nucleus of transfected cells, whereas gammaD-V76D proteins formed cytosolic and nuclear aggregates. Mutant gammaD-V76D reduces protein solubility in the lens and forms substantial intranuclear aggregates that disrupt the denucleation process of inner lens fiber cells. Sustained fiber cell nuclei and nuclear remnants scatter light, whereas other downstream events, such as decreased connexins, presumably disrupt gap junction communication and lens homeostasis, further contributing to the cataract phenotype in mutant lenses. This work also suggests that gammaD-crystallin is one of the crucial components for the formation of cold cataracts in vivo.

The lens of the eye as a focusing device and its response to stress

The continued peripheral growth of the lens, resulting in the concentration of older tissue toward the center, has the important optical consequence of producing a lens of variable refractive index. An approach consisting of the projection of fine laser beams through excised lenses in physiological solution has been used for in vitro study of lens optical quality. By varying the separation of the incident beams and/or the wavelength characteristics of the laser used, lens refractive properties and relative transparency may be examined. In the review provided, these optical properties are correlated to lens suture anatomy, lens mitochondrial morphology and function and the function of lens heat shock proteins. In addition, lens spherical aberration is evaluated as a function of accommodation. This work can be highlighted as follows: Mammalian lens suture morphology has a direct impact on lens optical function and, while suture structure of mammalian and avian lenses are very different, they both show an age-related deterioration in morphology and focusing ability. The distribution and appearance of mitochondria of the lens epithelium and superficial fiber cells are similar in all vertebrates. Lens mitochondrial integrity is correlated to lens focusing ability, suggesting a correlation between lens optical properties and lens metabolic function. The induction of cold cataract measured optically in cultured mammalian lenses is enhanced by thermal (heat) shock and this effect is prevented by inhibiting heat shock protein production. Finally, lens accommodative function can be studied by measuring lens refractive change using a physiological model involving an intact accommodative apparatus.

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, 30–60min 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.

Lens crystallins and oxidation: the special case of γS

Among lens crystallins, γ-crystallins are particularly sensitive to oxidation, because of their high amount of Cys and Met residues. They have the reputation to induce, upon ageing, lens structural modifications leading to opacities. A combination of small angle X-ray scattering and chromatography was used to study the oxidation of γ-crystallins. At pH 7.0, all the γ-crystallins under study were checked to have the same structure in solution. Under gentle oxidation conditions at pH 8.0, human γS (hγS) and bovine γS (bγS) formed disulfide-linked dimers, whereas the other bγ-crystallins did not. Cys20 was shown to be responsible for dimer formation since the C20S mutant only formed monomers. The hγS dimers were stable for weeks and did not form higher oligomers. In contrast, monomeric γS-crystallins freshly prepared at pH 8.0, and submitted to more drastic oxidation by X-ray induced free radicals, were rapidly transformed into higher oligomers. So, only extensive oxidation causing partial unfolding could be detrimental to the lens and linked to cataract formation. The γS-crystallins lack the temperature-induced opacification observed with the other γ-crystallins and known as cold cataract. The oxidation-induced associative behaviour and cold cataract are therefore demonstrated to be uncoupled.

Cold cataracts: a naturally occurring aqueous two-phase system

The cytoplasm of the eye lenses shows a liquid–liquid phase transition similar to the one observed in aqueous two-phase systems. This phenomenon is known as cold cataracts. We have studied the solution behavior of the main protein fractions that constitute the lenses’ cytoplasm using small-angle neutron scattering and dynamic light scattering. Our results provide evidence that an intricate balance of forces underlines the physical phenomena responsible for the optical properties of the lenses and for the phase transition that is observed as the temperature is lowered below some critical value. These forces include solvent-mediated forces besides the more conventional Coulombic and dispersion forces. This study suggests that solvent mediated forces must be included to successfully model liquid–liquid phase transitions like the ones observed in cold cataracts or in aqueous two-phase systems.

Eye lens transparency or cold cataract: a question of protein interaction potentials

Eye lens transparency or cold cataract: a question of protein interaction potentials

Precataractous changes affect lens transparency in the selenite cataract.

Selenite treatment of the preweanling rat stabilized the transparency of the lens nucleus to decreasing temperature. Hence, we compared properties of the cortex and nucleus from lenses of selenite-treated and age-matched control rats. A subcutaneous dose of 30 nmol Na2SeO3/g body weight was administered to 10- to 13-day-old Sprague-Dawley rats. Uninjected, age-matched littermates served as controls. As required, lenses were frozen in liquid N2 and separated into nuclear and cortical-epithelial fractions. Transparency of solutions of lens proteins (90-100 mg per ml) was monitored from 30 to 2 degrees C as percent transmittance (%T) at 490 nm. The critical phase separation temperature, Tc, was the temperature at 80%T. Protein associations were monitored with gel filtration chromatography. The nuclear 'cold cataract', in intact lenses, formed at similar temperatures at 14 and 15 days of age, but at a significantly lower temperature when the lenses were from a selenite-treated rat. The Tc, however, was greater by 1.5-2 degrees C for solutions of proteins isolated from whole lenses or lens nuclei from rats 24 and 48 h after treatment with selenite. Further, less gamma-crystallin was associated with the alpha-crystallin fraction in extracts from the nucleus of lenses from treated rats. Altered phase separation properties occurred as an early event in the etiology of selenite cataract. The different in vivo and in vitro responses to temperature indicated that properties of lens crystallins do not solely establish transparency in the intact lens.

13C-NMR off-resonance rotating frame spin-lattice relaxation studies of bovine lens γ-crystallin self association: effect of ‘macromolecular crowding’

The NMR technique of 13C off-resonance rotating frame spin-lattice relaxation, which provides an accurate assessment of the effective rotational correlation time (τ 0,eff) for macromolecular rotational diffusion, was applied to the study of γ-crystallin association as a function of protein concentration and temperature. Values of the effective rotational correlation time for γ-crystallin rotational diffusion were obtained at moderate to high protein concentrations (80–350 mg/ml) and at temperatures above, and below, the cold cataract phase transition temperature. With increasing concentration γ-crystallin was observed to increasingly associate as reflected by larger values of τ 0,eff Decreasing temperature in the range of 35 to 22°C was found to result in no change in the temperature corrected value of τ 0,eff at a γ-crystallin concentration of 80 mg/ml, whereas at temperatures of 18°C or below, this parameter was approx. twofold larger, suggesting the occurrence of a well defined phase transition, which correlated well with the cold cataract phase transition temperature. At higher protein concentrations, by contrast, τ 0,eff (temperature corrected) was found to increase by approx. 1.6- to 2-times in the temperature interval 35°C to 22°C, a result consistent with the dependence of the cold cataract phase transition temperature on γ-crystallin concentration. Analysis of intensity ratio dispersion curves, using an assumed model of isodesmic association, permitted the estimation of the association constant characterizing the aggregation under particular conditions of concentration and temperature. The significant increase in the value of the association constant with moderate increases in protein concentration was rationalized by invoking the effect of ‘macromolecular crowding’. The results obtained in this study suggest that in the intact lens, where high protein concentrations prevail, γ-crystallin is unlikely to be found in the monomeric state, but more likely, as a significantly aggregated species, representing a broad molecular weight distribution.

Micrometer-Scale Resolution Imaging of the Anterior Eye In Vivo With Optical Coherence Tomography

To demonstrate a new diagnostic technique, optical coherence tomography, for high-resolution cross-sectional imaging of structures in the anterior segment of the human eye in vivo. Optical coherence tomography is a new, noninvasive, noncontact optical imaging modality that has spatial resolution superior to that of conventional clinical ultrasonography (< 20 microns) and high sensitivity (dynamic range, > 90 dB). Survey of intraocular structure and dimension measurements. Laboratory. Convenience sample. Correlation with range of accepted normal intraocular structure profiles and dimensions. Direct in vivo measurements with micrometer-scale resolution were performed of corneal thickness and surface profile (including visualization of the corneal epithelium), anterior chamber depth and angle, and iris thickness and surface profile. Dense nuclear cataracts were successfully imaged through their full thickness in a cold cataract model in calf eyes in vitro. Optical coherence tomography has potential as a diagnostic tool for applications in noncontact biometry, anterior chamber angle assessment, identification and monitoring of intraocular masses and tumors, and elucidation of abnormalities of the cornea, iris, and crystalline lens.