A comparative study on the viscoelastic properties of human and animal lenses

A new method of compression between two parallel plates is used to measure the viscoelastic properties of whole and decapsulated human lenses and compare them with other animal species. Compressive load relaxation was performed by deforming the lens by 10% and measuring the force relaxation response for 100 s to obtain thickness, stiffness and relaxation of the induced loading force and Maxwell parameters for human, monkey, porcine and leporine whole and decapsulated lenses. Thickness and percentage loading force relaxation increased linearly with lens age, whereas stiffness and induced loading force increased exponentially. Human and monkey lenses aged at different rates. Loading force relaxation in a generalized Maxwell model was described by three time constants ranging from 1 to 1000 s. Compressive load relaxation is a very versatile method to study the viscoelastic properties of whole and decapsulated lenses and potentially also artificial accommodating lenses. The data presented in the study will help researchers choose the most suitable animal lenses based on the desired properties and age to be mimicked from the human lenses.     

Mapping elasticity in human lenses using bubble-based acoustic radiation force

This study uses acoustic radiation pressure to displace a femtosecond laser-produced bubble in human lens tissue. Bubble displacement is monitored with low-amplitude, high-resolution ultrasound. Displacements are compensated by bubble size determined from ultrasonic backscatter. The Young's modulus is proportional to the inverse of the compensated displacement with the constant of proportionality determined from similar measurements in a controlled gelatin sample. Multiple measurements were obtained on 12 human lens specimens grouped into two age categories, middle-age (about 40 years old) and old-age (63-70 years old). There were 3 lenses from 2 donors in the middle-age group and 9 lenses from 5 donors in the old-age group. At each radial position, the median value was computed for all measurements within each group. For middle-age lenses, Young's modulus ranged from 5.2kPa in the center to 1.1kPa on the periphery. For old-age lenses, Young's modulus ranged from 10.6kPa in the center to 1.4kPa on the periphery. These values are the same order of magnitude as previous measurements using other techniques. The age related change in elasticity distribution is also similar to a previous study. Radially varying elasticity may provide insight into the mechanics of accommodation.     

Dynamic multi-arm radial lens stretcher: a robotic analog of the ciliary body

We developed a dynamic lens stretching device to quantitatively determine the relationships between force, equatorial displacement, and anterior curvature. A computer-controlled four-arm lens stretcher, equipped with real-time force transducers in each arm, was designed and constructed to obtain transient force measurements during lens stretching. The force-decay spectrum was fitted with a seven-parameter viscoelastic model characterized by three time constants. A corneal topography unit was used to measure the curvature of fresh porcine lenses ex vivo and in vitro in a four-arm lens stretcher at various equatorial displacements. The lens stretcher and corneal topography unit provided detailed information regarding the behavior of the porcine lens in vitro. For all lenses, the central portion of the anterior surface flattened as zonular force increased. Force increased nonlinearly with equatorial displacement. Relaxation time constants for accommodation were 34.5+/-12.2 ms, 310+/-122 ms, and 12,800+/-9490 ms. Time constants for disaccommodation were 34.9+/-4.7 ms, 291+/-79.1 ms, and 3400+/-775 ms, which were not statistically different from those measured for accommodation. The lens stretcher acts as a robotic analog of the ciliary body. This device allows direct, quantitative measurement of the forces and curvature changes relevant to accommodation. However, distortions were present due to the use of only four arms.     

Viscoelastic shear properties of the fresh porcine lens

AIM: To determine the viscoelastic properties of the porcine lens METHODS: Linear viscoelastic shear properties of the stroma of four porcine lenses were measured within 5 hours post-mortem, using sinusoidal oscillatory shear deformation. The elastic shear modulus, viscous shear modulus, dynamic viscosity, damping ratio, and phase shift of the lenses were quantified by a controlled-strain, linear simple-shear rheometer at frequencies of 10-50 Hz. RESULTS: The mean viscoelastic properties and their standard deviations across the frequencies examined were: the elastic shear modulus, G' = 6.2+/-4.0 Pa, the viscous shear modulus, G' = 19.2+/-2.5 Pa, the dynamic viscosity, eta' = 0.16+/-0.1 Pa x sec, the damping ratio zeta = 4.06+/-1.25, and the phase shift, delta = 76 degrees +/- 5.6 degrees. CONCLUSIONS: The measured viscoelastic shear properties of the porcine lens reflect a low dynamic viscosity with a high damping ratio. The porcine lens is viscoelastic and is more viscous than elastic. The magnitude of the complex shear modulus of the porcine lens, |G*|, is similar to the shear modulus of the young human lens. Understanding these viscoelastic properties of the natural lens may provide guidance in developing a lens substitute capable of accommodation in the post cataract patient.     

Stiffness gradient in the crystalline lens

Background While the overall stiffness of the lens has been measured in a number of studies, the knowledge about the stiffness distribution within the lens is still limited. The purpose of this study was to determine the stiffness gradient in the human crystalline lens. A secondary purpose was to determine whether the stiffness gradient depends on age. Methods The local dynamic stiffness was measured in 10 human crystalline lenses (age range: 19 to 78 years). The lenses were stored at −70°C before being measured. The influence of freezing on the mechanical properties has been determined in a previous study. A small oscillating probe was used to measure the local dynamic shear modulus as a measure of lens stiffness. The measurements were taken in the cross-sectional plane through the lens equator. Results The local dynamic shear modulus varied with location for all tested lenses. The central stiffness of the oldest lens (78 years) was 10⁴ times higher than the youngest (19 years) lens. The equatorial stiffness of the oldest lens was 10² times higher than the youngest lens. For the older lenses, the centre was 5.8–210 times stiffer than the periphery, as opposed to earlier results described by Fisher (1971), who found that the periphery was up to 3 times softer than the centre for lenses younger than 70-years-old. For the three youngest lenses (19 to 49 years), the periphery was 2.2–16.6 times stiffer than the centre. Conclusions The dynamic stiffness of the crystalline lens varies with location within the lens. The stiffness gradient depends on the age of the lens. The results of the 10 lenses indicate that the stiffness of both centre and periphery increase with age, but at a different rate.     

Dynamic mechanical properties of human lenses

The purpose of this study was to determine the shear compliance of human crystalline lenses as a function of age and frequency. Dynamic mechanical analysis was performed on 39 human lenses, ranging in age from 18 to 90 years, within the frequency range of 0.001-30 Hz. The lenses were stored at -70 degrees C before being measured. The influence of freezing on the mechanical properties was determined using pairs of porcine lenses, with one lens measured directly after enucleation and the other after freezing. The measurement method had a repeatability standard deviation of 4 and 6% for the storage and loss compliance, respectively. The reproducibility standard deviation was 31 and 33% for the storage and loss compliance respectively. On average, freezing increased the storage compliance by 8% and increased the loss compliance by 32%, both depending slightly on age and frequency. The human lenses exhibited a distinct viscoelastic behavior. The storage and loss compliance depended strongly on age and decreased a factor 1000 over a lifetime. Dynamic mechanical analysis has proven to be a successful technique for characterizing the mechanical properties of the human crystalline lens. The shear compliance decreases exponentially with age.     

Wettability of silicone-hydrogel contact lenses in the presence of tear-film components

INTRODUCTION: Modern application of soft contact lenses demands safe and comfortable wear over extended time periods up to one month. Lenses that exhibit and sustain complete water wetting allow thick tear-film deposition, minimize film rupture, and permit smooth tear recovery upon lid closure. Water contact angles determined using an air bubble captive on a lens best gauge the in-vivo wetting state. To achieve highly water wetting lenses demands that contact-angle hysteresis be eliminated and that the advancing and receding angles both approach zero. Since lens wear exposes the anterior surface to tear proteins, lens wettability should be measured in the presence of tear-film components. METHODS: A captive-bubble technique is applied to measure the advancing and receding contact angles of two commercial silicone-hydrogel lenses: PureVision (PV) and Focus Night & Day (CF) and a standard HEMA (hydroxethyl-methacrylate) hydrogel lens: Acuvue (AV). In the captive-bubble method, an air bubble immersed in aqueous solution is brought into contact with the contact lens. The contact angle through water during bubble expansion yields the receding angle. Bubble contraction gives the water advancing angle. Contact-angle hysteresis is the difference between the advancing and receding angles. RESULTS: In isotonic solution, all three lenses display considerable contact-angle hysteresis with advancing angles of almost 90 degrees. When lysozyme and/or mucin were added to the aqueous solution, hysteresis was eliminated, and equivalent and high water wetting was achieved for the three lenses. Only the advancing angle in isotonic solution provided discriminating evidence for differences in surface chemistry. Covalent attachment of polyethyleneglygol (PEG) to the PV lens surface achieved complete water wetting independent of the presence of tear protein in the solution. CONCLUSIONS: The captive-bubble technique provides contact angles that are relevant to on-eye lens wear. Both advancing and receding contact angles are important to lens wettability performance. When lysozyme and/or mucin are present in the solution, PV, CF, and AV lenses display low advancing and receding contact angles indicative of equivalent wettability performance. This result is due to molecular adsorption of the proteins onto the lens external surface. Covalently attached PEG on the PV lens not only provides complete water wetting but also minimizes or even eliminates protein adsorption.     

Mouse lens stiffness measurements

Presbyopia is a gradual loss of accommodation with age. Various studies have shown that an age-related increase in lens stiffness may be one factor involved. Lens stiffness has previously been measured using lens spinning experiments, resistance to conical probe penetration and dynamic mechanical analysis. In the current study, two different techniques have been used to evaluate the stiffness of isolated mouse lenses. In the first method, compressive forces were applied to mouse lenses using microscope cover-slips to exert incremental forces on the lens. Lens images were captured for analysis of change in diameter. In the second method, a fully automated squeezer system with an actuator, electronic scale and a CCD camera was used to apply incremental compressive forces to the lenses. The actuator exerted forces comparable to those exerted by cover-slips. Force and actuator displacement data together with images of the lenses as they were compressed were captured. Images were analyzed for change in lens diameter on application of force and also with actuator displacement. Lenses from 19 young male mice (4-weeks old) and 28 male retired breeders (7-9 months old) were tested. Lenses were used immediately after sacrificing the mice and extracting the lenses. The lenses from the older male mice were stiffer compared to the lenses from the younger male mice. This was determined by comparing the average change in lens diameter at various force values used. The two methods provide a good indication of the stiffness properties of mouse lenses.     

Primate lens capsule elasticity assessed using Atomic Force Microscopy

The purpose of this project is to measure the elasticity of the human and non-human primate lens capsule at the microscopic scale using Atomic Force Microscopy (AFM). Elasticity measurements were performed using AFM on the excised anterior lens capsule from 9 cynomolgus monkey (5.9–8.0 years), 8 hamadryas baboon (2.8–10.1 years), and 18 human lenses (33–79 years). Anterior capsule specimens were obtained by performing a 5 mm continuous curvilinear capsulorhexis and collecting the resulting disk of capsular tissue. To remove the lens epithelial cells the specimen was soaked in 0.1% trypsin and 0.02% EDTA for 5 min, washed, and placed on a Petri dish and immersed in DMEM. Elasticity measurements of the capsule were performed with a laboratory-built AFM system custom designed for force measurements of ophthalmic tissues. The capsular specimens were probed with an AFM cantilever tip to produce force–indentation curves for each specimen. Young’s modulus was calculated from the force–indentation curves using the model of Sneddon for a conical indenter. Young’s modulus of elasticity was 20.1–131 kPa for the human lens capsule, 9.19–117 kPa for the cynomolgus lens capsule, and 13.1–62.4 kPa for the baboon lens capsule. Young’s modulus increased significantly with age in humans (p = 0.03). The age range of the monkey and baboon samples was not sufficient to justify an analysis of age dependence. The capsule elasticity of young humans (<45 years) was not statistically different from that of the monkey and baboon. In humans, there is an increase in lens capsule stiffness at the microscale that could be responsible for an increase in lens capsule bulk stiffness.     

Biometric, optical and physical changes in the isolated human crystalline lens with age in relation to presbyopia.

The biometric, optical and physical properties of 19 pairs of isolated human eye-bank lenses ranging in age from 5 to 96 years were compared. Lens focal length and spherical aberration were measured using a scanning laser apparatus, lens thickness and the lens surface curvatures were measured by digitizing the lens profiles and equivalent refractive indices were calculated for each lens using this data. The second lens from each donor was used to measure resistance to physical deformation by providing a compressive force to the lens. The lens capsule was then removed from each lens and each measurement was repeated to ascertain what role the capsule plays in determining these optical and physical characteristics. Age dependent changes in lens focal length, lens surface curvatures and lens resistance to physical deformation are described. Isolated lens focal length was found to be significantly linearly correlated with both the anterior and posterior surface curvatures. No age dependent change in equivalent refractive index of the isolated lens was found. Although decapsulating human lenses causes similar changes in focal length to that which we have shown to occur when human lenses are mechanically stretched into an unaccommodated state, the effects are due to nonsystematic changes in lens curvatures. These studies reinforce the conclusion that lens hardening must be considered as an important factor in the development of presbyopia, that age changes in the human lens are not limited to the loss of accommodation that characterizes presbyopia but that the lens optical and physical properties change substantially with age in a complex manner.     

Shear modulus data for the human lens determined from a spinning lens test

The paper describes a program of mechanical testing on donated human eye bank lenses. The principal purpose of the tests was to obtain experimental data on the shear modulus of the lens for use in future computational models of the accommodation process. Testing was conducted using a procedure in which deformations are induced in the lens by spinning it about its polar axis. Shear modulus data were inferred from these observed deformations by means of a finite element inverse analysis procedure in which the spatial variation of the shear modulus within the lens is represented by an appropriate function (see Burd et al., 2011 for a detailed specification of the design of the spinning lens test rig, experimental protocols and associated data analysis procedures that were employed in the tests). Inferred data on lens shear modulus are presented for a set of twenty-nine lenses in the age range 12 years to 58 years. The lenses were tested between 47 h and 110 h from the time of death (average post-mortem time 74 h). Care was taken to exclude any lenses that had been affected by excessive post-mortem swelling, or any lenses that had suffered mechanical damage during storage, transit or the testing process. The experimental data on shear modulus indicate that, for young lenses, the cortex is stiffer than the nucleus. The shear modulus of the nucleus and cortex both increase with increasing age. The shear modulus of the nucleus increases more rapidly than the cortex with the consequence that from an age of about 45 years onwards the nucleus is stiffer than the cortex. The principal shear modulus data presented in the paper were obtained by testing at a rotational speed of 1,000 rpm. Supplementary tests were conducted at rotational speeds of 700 rpm and 1,400 rpm. The results from these supplementary tests are in good agreement with the data obtained from the principal 1,000 rpm tests. Studies on the possible effects of lens drying during the test suggested that this factor is unlikely to have led to significant errors in the experimental determination of the shear modulus. The shear modulus data presented in the paper are used to develop 'age-stiffness' models to represent the shear modulus of the lens as a function of age. These models are in a form that may be readily incorporated in a finite element model of the accommodation process. A comparison is attempted between the shear modulus data presented in the current paper and equivalent data published by previous authors. This comparison highlights various limitations and inconsistencies in the data sets.     

Refractive index distribution in the porcine eye lens for 532 nm and 633 nm light

AIMS: To measure the refractive index distribution in porcine eye lenses for two wavelengths from the visible spectrum: 532 and 633 nm, in order to determine whether there are any discernible wavelength dependent differences in the shape of the profile and in the magnitude of refractive index. METHODS: Rays were traced through 17 porcine lenses of the same age group and of similar size. Ray trace parameters were used to calculate the refractive index distributions for 633 nm light in all 17 lenses and for 532 nm light in 10 lenses. The effect of the refractive index at the edge of the lens, on the rest of the profile, was considered because the mismatch between refractive index at the lens edge and the refractive index of the surrounding gel necessitated a further step in calculations. RESULTS: The shape of the refractive index distributions is parabolic. There is a small wavelength dependent difference in the magnitude of the refractive index across the profile and this increases very slightly into the centre of the lens. The value of the refractive index at the edge of the lens does not appreciably affect the index profile. CONCLUSIONS: The wavelength dependent differences in refractive index between light of 633 and 532 nm are small but discernible.     

EphA2 and Ephrin-A5 Guide Eye Lens Suture Alignment and Influence Whole Lens Resilience

PurposeFine focusing of light by the eye lens onto the retina relies on the ability of the lens to change shape during the process of accommodation. Little is known about the cellular structures that regulate elasticity and resilience. We tested whether Eph–ephrin signaling is involved in lens biomechanical properties.MethodsWe used confocal microscopy and tissue mechanical testing to examine mouse lenses with genetic disruption of EphA2 or ephrin-A5.ResultsConfocal imaging revealed misalignment of the suture between each shell of newly added fiber cells in knockout lenses. Despite having disordered sutures, loss of EphA2 or ephrin-A5 did not affect lens stiffness. Surprisingly, knockout lenses were more resilient and recovered almost completely after load removal. Confocal microscopy and quantitative image analysis from live lenses before, during, and after compression revealed that knockout lenses had misaligned Y-sutures, leading to a change in force distribution during compression. Knockout lenses displayed decreased separation of fiber cell tips at the anterior suture at high loads and had more complete recovery after load removal, which leads to improved whole-lens resiliency.ConclusionsEphA2 and ephrin-A5 are needed for normal patterning of fiber cell tips and the formation of a well-aligned Y-suture with fiber tips stacked on top of previous generations of fiber cells. The misalignment of lens sutures leads to increased resilience after compression. The data suggest that alignment of the Y-suture may constrain the overall elasticity and resilience of the lens.     

Time-resolved studies of Nd:YAG laser-induced breakdown. Plasma formation, acoustic wave generation, and cavitation

The use of high intensity ultrashort pulsed laser radiation to produce optical breakdown is an important approach for the surgical treatment of intraocular structures. We have investigated the transient properties of Nd:YAG laser induced breakdown in a saline model using time-resolved spectroscopic techniques. Spatially resolved pump and probe techniques are applied to study the dynamic behavior of the plasma formation, acoustic wave generation, and cavitation processes which accompany the optical breakdown. Measurements of plasma shielding and luminescence indicate that the laser induced plasma forms on a subnanosecond time scale and has a lifetime of several nanoseconds. An acoustic transient is generated at the breakdown site and propagates spherically outward with an initial hypersonic velocity, then loses energy and propagates at sound velocity. Transient heating following the plasma formation produces a liquid-gas phase change and gives rise to cavitation or gas bubble formation. This gas bubble expands rapidly for several microseconds, then slows to reach its maximum size and finally collapses.     

Determination of the state and content of water in normal avian, fish, porcine, bovine, and human lenses as studied by differential scanning calorimetry

Differential scanning calorimetry was used to measure the relative amounts of 'bulk' and 'bound' water in normal avian, bovine, fish, human, and porcine lenses. The amounts of bound water (mg bound water/mg lens dry weight) found in avian and porcine lenses were statistically different from each other in addition to being statistically different from fish, human, and bovine lenses. There were no significant differences in the mean values between human, fish, and bovine lenses. Avian lenses had the highest amount of bound water, while fish lenses had the lowest bound water content. Significant differences in total water content (mg total water/mg lens dry weight) were observed between all of the lenses, with the exception of bovine and human lenses which were not statistically different. Fish lenses had the lowest amount of total water, and avian lenses had the highest total water content. There were significant differences in bulk water content (mg bulk water/mg lens dry weight) between all of the lenses. Avian lenses had the highest bulk water content, and fish lenses had the lowest bulk water content.     

Teaching continuous curvilinear capsulorhexis using a postmortem pig eye with simulated cataract(2)(2).

We modified an inexpensive, easily prepared model using a postmortem pig eye for training ophthalmology residents to perform continuous curvilinear capsulorhexis. To reduce the tension and elasticity of the porcine anterior lens capsule, 0.05 mL of formalin mixed with hydroxyethylcellulose or a viscoelastic material is injected into the anterior chamber via the corneal limbus to fix the surface of the central anterior lens capsule of the pig eye in situ. One or 2 minutes after the injection, only the anterior lens capsule is fixed and corneal transparency is maintained. The reduction in tension and elasticity of the anterior lens capsule caused by its fixation increases the resemblance of the simulated cataract to the human cataract.     

The stiffness of human cataract lenses is a function of both age and the type of cataract

Surgical evidence suggests that nuclear cataract lenses are generally harder than normal lenses. We examined this quantitatively using dynamic mechanical analysis of cataract lenses removed during surgery and compared the results with data from normal lenses. Stiffness of the lens centre was found to depend on the type of cataract and the age of the patient. Nuclear cataract lenses were generally stiffer than those extracted from patients with predominantly cortical cataract, with some in the latter group appearing not to differ significantly from age-matched normals. At age 40-50, the nuclear region of advanced nuclear cataract lenses was found to be approximately 46 times harder than that of normal lenses of the same age. By age 70-80 the stiffness of advanced nuclear cataract lenses had doubled, however, by this age, normal lenses had also increased significantly in stiffness so that the difference between cataract and normal lenses was much less pronounced, being a factor of approximately 2.5.     

Cytoplasmic filaments in the crystalline lens of various species: functional correlations.

The distribution of cytoplasmic filaments in lenses of five species was studied with the electron microscope. Two distinct patterns emerged. One pattern, in which filaments are grouped in characteristic bundles around the nucleus, in processes, and throughout the subcortical cytoplasm of epithelial cells, is typical of spherical, non-accommodating lenses of mice and rats. The second pattern is associated with anteriorly-flattened, accommodating lenses of infant human, squirrel and frog. In these, filaments are scattered in epithelial cells, but are accumulated on either side of the plasma membrane junction between epithelial cells and lens fibers. They are especially dense on the lens fiber side of the junction, and form a lattice associated with the lens fiber plasma membrane. The lattice is less extensive along the sides of lens fibers not in contact with epithelial cells. In spherical lenses the epithelial-fiber lattice is greatly reduced. Filaments in both types of lenses ranged in diameter between 5 and 11 nm. The filaments are thought to be a mixture of thin and intermediate filaments.It is hypothesized that the role of cytoplasmic filaments in lens, depending on the pattern present, is either to structurally support a spherical shape, or to provide a contractile force or elasticity to return the flattened anterior surface to the accommodated state in conjunction with the elasticity of the lens capsule.     

Comparison of the behavior of natural and refilled porcine lenses in a robotic lens stretcher

The mechanism by which the eye dynamically changes focal distance (accommodation), and the mechanism by which this ability is lost with age (presbyopia), are still contested. Due to inherent confounding factors in vivo, in vitro measurements have been undertaken using a robotic lens stretcher to examine these mechanisms as well as the efficacy of lens refilling - a proposed treatment for presbyopia. Dynamic forces, anterior and posterior curvatures, and lens thickness are all correlated for young natural and refilled porcine lenses. Comparisons are made to lenses refilled with a homogeneous polymer system. The amplitude of accommodation of the young porcine lens is very small such that it may be a suitable model for presbyopia. The behavior of refilled lenses was highly dependent on the refill volume. The volume could be tuned to maximize accommodative amplitude in the refilled lens.     

Porcine eye lens crystallins: antigenic similarity with human crystallins and tool for the detection of anti-crystallin antibodies

BACKGROUND: The usual sources of antigenic material for investigations on circulating immunoglobulins with anti-lens crystallins specificity are saline extracts of human cataract lenses. This practice has a number of drawbacks, especially the possible antigenic alterations that may have occurred in cataract lenses. The aim of this investigation was to compare the antigenic properties of porcine eye lens crystallins and human crystallins, with regard to the possibility for an alternative source of antigenic material for detection of anti-crystallin antibodies in human sera. METHODS: We produced rabbit antisera against saline extracts of human and porcine eye lenses. These sera were applied for the antigenic characterizations of the two extracts with indirect and absorption enzyme-linked immunosorbent assay. The two antigens were further compared by testing them against 30 human sera from cataract patients and 30 sera from blood donors. RESULTS: The antibodies raised against human eye lens cross-reacted with antigens of the porcine lens. This finding was supported by the absorption experiments - the antigens of the porcine eye lens strongly inhibit the reactivity of the rabbit serum raised against human eye lens and vice versa. We established a significant positive correlation (Spearman, r=0.89, P<0.0001) between the reactivity of the tested sera against human and porcine lens extracts. CONCLUSION: These data demonstrated a strong antigenic similarity between human and porcine lens crystallins, suggesting the appropriateness of the use of porcine lens extracts for the detection of humoral anti-lens autoimmune response in patients with eye diseases.