Lens Deformations and Ruptures in Human Lenses With Cortical Cataract Subjected to Ex Vivo Simulated Accommodation Ralph Michael,1–3 Justin Christopher D’Antin,1,2 Laura Pinilla Cortés,1,2 Harvey John Burd,4 Brian Sheil,4 and Rafael I. Barraquer1,2,5 1 Centro de Oftalmología Barraquer, Barcelona, Spain 2 Institut Universitari Barraquer, Universitat Autònoma de Barcelona, Barcelona, Spain 3 University Eye Clinic, Paracelsus Medical University, Salzburg, Austria 4 Department of Engineering Science, University of Oxford, Oxford, United Kingdom 5 Universitat Internacional de Catalunya, Barcelona, Spain Correspondence: Rafael I. Barraquer, PURPOSE. Human cortical opacities are most commonly accompanied by changes in lens Centro de Oftalmología Barraquer, fiber structure in the equatorial region at the lens nucleus–cortex interface. Cortex and Carrer de Muntaner, 314, 08021 nucleus have different elastic properties, which change with age. We therefore subjected Barcelona, Spain; ex vivo lenses to simulated accommodation and studied the internal deformations to ralphm@barraquer.com. better understand the mechanism of cortical cataract formation. Received: November 3, 2020 METHODS. Nine human donor lenses (33–88 years old) were tested using a bespoke Accepted: December 16, 2020 Published: January 11, 2021 radial stretching device for anterior eye segments. Seven of the lenses exhibited corti- cal cataracts. The other two lenses, without cataract, were used as controls. Frontal and Citation: Michael R, D’Antin JC, cross-sectional images of the lens obtained during stretching facilitated measurements Pinilla Cortés L, Burd HJ, Sheil B, on equatorial lens diameter and central lens thickness in the stretched and unstretched Barraquer RI. Deformations and ruptures in human lenses with states. cortical cataract subjected to ex vivo RESULTS. Stretching caused the lens equatorial diameter to increase in all cases. simulated accommodation. Invest Conversely, the lens central thickness showed no systematic variation during stretching. Ophthalmol Vis Sci. 2021;62(1):12. For four of the lenses with cortical cataract, ruptures were observed during stretching https://doi.org/10.1167/iovs.62.1.12 at the nucleus–cortex boundary adjacent to the cortical cataracts. Ruptures were not observed in the control lenses or in the three other lenses with cortical cataract. CONCLUSIONS. Internal ruptures can occur in aged ex vivo lenses subjected to simulated disaccommodation. These ruptures occur at the nucleus–cortex interface; at this location, a significant stiffness discontinuity is expected to develop with age. It is hypothesized that ruptures occur in in vivo lenses during accommodation—or attempted accommodation. Keywords: accommodation, presbyopia, cortical cataract C ataracts are generally considered a multifactorial disease that is mainly caused by lifelong accumulation of oxidative stress, influenced by environmental and genetic physiological milieu, allowing environmental factors such as ultraviolet radiation or other oxidative stress to alter the transparency in this area. Recently, we have shown that factors, life style, exposure to ultraviolet radiation, diet, and emmetropes and hyperopes (who are known to accommo- systemic diseases.1,2 Nuclear cataracts are generally believed date more than myopes) have a higher prevalence of corti- to be caused by the accumulation of high-molecular-weight cal cataract than myopes.8 The ex vivo simulated accom- aggregates and increasing protein insolubility, resulting in modation tests described in the current paper explore the lens opacity. Accumulation of fluorescent chromophores potential link between cortical cataract and accommodation- leads to a yellow to dark brunescent nuclear opacity.3 For induced internal lens deformations. cortical cataracts, the repeated internal deformations that The morphology of cortical cataracts is characterized by develop within the lens during accommodation may be a an irregular fiber architecture and undulated, folded, and contributing causal factor, in addition to changes in the lens fractured groups of fiber cells at the border zone between proteins. the cortex and nucleus (Fig. 1).9 These morphological obser- The suggestion that accommodation forces might vations indicate the presence of significant internal defor- contribute to the formation of cortical cataract was first mations within the lens. Previous finite element studies on proposed by Wilhelm Schoen4 in a lecture at Leipzig Univer- simulated disaccommodation reported by Belaidi and Pier- sity in 1896. This hypothesis has been discussed, more scionek10 and Wang et al.11 demonstrate that significant recently, by Fisher5 and later by Pau.6 Angra et al.7 suggested concentrations of stress appear to develop at locations in that shear stresses in the lens cortex could lead to a disturbed the lens where cortical cataracts typically occur. Copyright 2021 The Authors iovs.arvojournals.org | ISSN: 1552-5783 1 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Accommodation Forces IOVS | January 2021 | Vol. 62 | No. 1 | Article 12 | 2 Localized concentrations of stress could, initially, cause misalignment of intracellular lens protein, causing an increase in light scattering prior to gross lens fiber damage. Such an increase in light scattering at the border zone between the cortex and adult lens nucleus has been observed in Scheimpflug studies.12 First occurrence of corti- cal cataracts has been documented from around 40 years of age onward in human donor lenses.13 Data on lens shear modulus determined from inden- tation tests14,15 and spinning lens tests16 indicate that in young lenses the nucleus is less stiff (i.e., has a lower value of shear modulus) than the cortex. The nucleus and cortex both increase in stiffness with age, but the stiff- ness of the nucleus increases at a faster rate than that of the cortex, with the consequence that, from middle age onward, the nucleus becomes increasingly stiffer than the cortex. These age-related stiffness changes mean that a sharp stiffness gradient develops at the nucleus–cortex boundary in later life. It is considered that repeated radial forces due to attempted accommodation will continue to be applied to the lens via the zonules, even beyond the age when the ability of the lens to accommodate has been lost. The stiffness gradient at the nucleus–cortex bound- ary seems likely to exacerbate the internal strains that develop in the equatorial cortex during attempted accommo- dation,1,9,17,18 with consequential implications for the devel- opment of cortical cataract in late middle-aged and elderly subjects. The current paper describes a set of ex vivo simulated disaccommodation tests on aged human lenses. The purpose of the tests is to explore potential correlations between the observed mechanical behavior of the lenses and the pres- ence, or absence, of cortical cataract. A bespoke radial lens stretching device was used to conduct the tests.19,20 Fisher21 was probably the first to design a device for mounting anterior eye segments and imposing on them a prescribed radial stretch. Fisher’s design imposed radial stretching via eight arms. Similar devices have been developed by Pierscionek,22 Glasser and Campbell,23 and Koopmans et al.24 Enhanced forms of lens stretching apparatus incorpo- rate the measurement of the external force applied to the sample.25–28 In interpreting the lens stretching tests, we consider the adult lens nucleus to be as defined in the morphological studies by Taylor et al.29 Using this definition, the anterior and posterior cortex is approximately 650 μm thick and the equatorial cortex is approximately 750 μm. This cortex size corresponds to the location of a diffusion barrier found by Sweeney and Truscott30 and peeling or hydrodissection experiments by Garland et al.31 and Augusteyn.32 There are alternative definitions of the dimensions of the lens cortex and nucleus, such as the Oxford Classification System, based FIGURE 1. Human donor lens with cortical cataract. Frontal view on Scheimpflug images and which considers the lens at (top) and cut at the axial plane (center), both imaged with dark-field birth to be the lens nucleus.33 In the Oxford Classification illumination. Boxed area with groups of opaque fibers is shown below with scanning electron microscopy (bottom). Lens fibers at System, the anterior and posterior lens cortex is approxi- the border zone between the nuclear and cortical lens regions are mately 1200 μm thick, and the thickness of the equatorial broken (arrows), and the broken ends are directed against the cortex is approximately 2000 μm. nuclear fibers, which maintain a regular, uninterrupted organiza- In lenses with cortical cataracts, a clear morphological tion. Further, note the curled (asterisk) and folded (arrowheads) distinction between lens cortex and adult nucleus is typically fibers in the region adjoining the broken fibers. n, nuclear side; c, observed (Fig. 1). This border zone is usually found from cortical side. Reprinted with permission from Michael R, Barraquer RI, Willekens B, van Marle J, Vrensen GFJM. Morphology of age- 500 to 700 μm below the lens capsule.9,34 Such a border related cuneiform cortical cataracts: the case for mechanical stress. zone has also been observed in in vivo studies using optical Vision Res. 2008;48(4):626–634. Copyright 2007 Elsevier Ltd. coherence tomography 35,36 Accommodation Forces IOVS | January 2021 | Vol. 62 | No. 1 | Article 12 | 3 TABLE. Human Donor Lens Information and Descriptive Results of Lens Stretching Cortical Cortical Cataract Rupture Maximum Nuclear Cataract Lens Diameter (mm) Depth (mm) Depth Rupture Stretching Age Postmortem Cataract Circumference† Unstretched Circumference‡ Force ID (y) (h) Grade* (%) Unstretched Stretched Unstretched Stretched (mm) (%) (mN) M71 33 45 0 — 9.30 9.71 — — — — 75 M80 46 115 0 — 9.13 9.44 — — — — 54 M82 78 107 4 100 9.64 9.76 0.590 0.640 — — 53 M86 87 58 3 100 9.39 9.92 0.550 0.710 — — 67 M85 74 73 5 31 9.83 10.28 0.550 0.730 — — 69 M87 88 68 3 42 9.82 10.07 0.490 0.570 0.400 7 53 M83 85 97 4 39 9.74 10.08 0.710 0.810 0.690 10 68 M79 60 78 3 61 9.38 9.81 0.530 0.670 0.510 28 56 M69 86 40 3 44 9.97 10.38 0.540 0.710 0.480 44 75§ Mean — 76 — 60 — — 0.570 0.690 0.520 22 — * Nuclear cataract grade according to BCN 10 scale (0–10). † Cortical cataract along lens circumference as percentage of total lens equatorial perimeter. ‡ Rupture along lens circumference as percentage of total lens equatorial perimeter. § The force for specimen M69 was estimated, because the ciliary body was not cut as in all other samples due to an oversight. METHODS 29-year lens in vivo from a fully accommodated to a disac- commodated state is in the region 42 to 55 mN (depending We studied the lenses of nine human donors provided on assumptions on the material properties).14,15 Burd and by the Banco de Ojos para Tratamiento de la Ceguera Wilde39 also employed finite-element analysis and reported (Barcelona, Spain), which included (1) seven lenses with that a computed force of 49 mN was required to stretch a cortical cataracts (between 60 and 88 years of age), and (2) 45-year lens from a fully accommodated to a disaccommo- two, relatively young control lenses (33 and 46 years of age) dated state. Moreover, ex vivo lens stretching data in Pinilla without cataracts (see Table). The donor eyes were stored at et al.19 indicate that a radial force in the region of 50 mN 8°C with a mean postmortem time of 76 hours (range, 40– is needed to stretch the lens from an accommodated to a 115 hours). This research was conducted under the tenets disaccommodated state. Augusteyn et al.26 found a median of the Declaration of Helsinki and conformed to the Spanish force of 80 mN to stretch human lenses ex vivo from an regulations for the use of human tissues from organ donors. accommodated to a disaccommodated state; however, this Stretching tests of anterior segments of the donor eyes force was obtained with an intact ciliary body. The data in were conducted using a bespoke apparatus previously Panilla et al.19 suggest that, when the ciliary body is intact, described in Pinilla et al.19 and Michael et al.20 The lenses the force actually transmitted to the lens is about 50% of the were stretched using a stepper motor, and the result- externally applied force. Thus, the 80 mN from Augusteyn ing forces were measured using a 0.1-mN precision load et al.26 is also broadly consistent with the 50 to 60 mN cell from a commercial digital precision balance (Precisa applied in the current tests. BJ 210C; Precisa Gravimetrics AG, Dietikon, Switzerland). The stretching protocol consists of applying increments A digital camera (EOS 550D, 3456 × 2304 pixel resolu- of radial displacement (250 μm) to the loading hooks that tion; Canon, Tokyo, Japan) mounted on a surgical micro- connect with the ciliary body.19 The applied radial force is scope (Carl Zeiss Meditec AG, Jena, Germany) was used measured at the end of each displacement increment; this to take frontal images of the donor lens during stretching. system does not allow the application of preset values of Scheimpflug images of the lens cross-section were captured radial force. The measured radial forces increased as the at each loading stage using the charge-coupled device sensor displacement increments were applied; maximum values of from a digital camera (NEX-5, 2448 × 1624 pixel resolution; the force—corresponding to the end of increment 10—are Sony Corporation, Tokyo, Japan) and axial illumination from listed in the Table. We selected the increment (8, 9, or 10) a Zeiss slit lamp. Each lens test was comprised of three or where the measured force was in the range of 50 to 60 mN four measurement cycles, each involving 10 increments of (considered to be representative of physiological conditions) stretching followed by 10 increments of relaxing (total of to allow the results to be compared at approximately the 2500 μm stretching distance per cycle).20 same value of radial force. Frontal images from the second stretching cycle were Analysis of stretching-induced changes to lens shape was used for subsequent analysis of the lens equatorial diam- undertaken for both the equatorial lens diameter (frontal eter; cross-sectional Scheimpflug images were taken during images) and the lens central thickness (cross-sectional a third or fourth cycle to evaluate lens thickness changes. Scheimpflug images). The lens equator is not perfectly circu- The frontal images were also used to describe the cortical lar; it was generally found to deform somewhat asymmet- cataract and to detect ruptures inside the lens during stretch- rically during stretching. To obtain data on lens diame- ing, recording both their depth below the lens capsule and ter change during stretching, therefore, the projected area their extension along the lens perimeter. Nuclear cataract within the lens equatorial perimeter in frontal images was was graded on a scale of 0 to 10 according to the BCN 10 determined using image processing software; an equivalent chart.37 lens diameter for a perfect circle with the same area was The protocol adopted in the radial lens stretching tests19 used for the evaluation. involves making radial cuts in the ciliary muscle during the sample preparation process. This ensures that the radial force applied to the lens can be determined directly from RESULTS external measurements. Finite-element analyses described in The two control lenses (33 and 46 years) had unstretched Hermans et al.38 indicate that the force required to stretch a equatorial diameters of 9.30 and 9.13 mm, respectively, and Accommodation Forces IOVS | January 2021 | Vol. 62 | No. 1 | Article 12 | 4 FIGURE 2. Two sample lenses, one control lens (above; M80, 46 years) and one with cortical cataract and internal ruptures after stretching (below; M69, 86 years). Frontal image on the left shows the first image, before the stretching experiments in the unstretched state. Frontal image on the right shows the last image after two or three cycles of stretching in the stretched state. The lens outline (right) is given for frontal view and cross-sectional view in the unstretched state (black) and with a stretching force of approximately 54 mN applied (red) during the same stretching cycle. showed no indications of nuclear or cortical cataract. The out cortical cataract; rupture occurred at 520 μm (on aver- unstretched equatorial diameter of the aged lenses (60 to age) below the lens capsule. The ruptures extended on aver- 88 years) ranged between 9.4 and 10.0 mm. They showed age 22% around the lens circumference, measured after four nuclear cataract of between 3 and 5 on the BNC 10 scale, stretching cycles with a maximum force of 75 mN applied which is typical for this age range. The degree of corti- (see Table). cal cataract was variable, occupying either the entire lens To allow for comparison, quantitative data on the circumference or only part; on average, the cortical cataract stretched lens dimensions were determined at representa- was estimated to occupy about 60% of the lens circumfer- tive values of radial force in the range of 50 to 60 mN; these ence. Cortical cataracts were found on average at a depth of data are represented in Figure 3 as a percentage relative to 570 μm below the lens capsule (Table). the unstretched state. Total equatorial diameter increased in Stretching-induced changes to lens shape were deter- all lenses after stretching: by 2% to 6% in the aged lenses mined by comparing measurements taken at an average and by 3% to 4% in the control lenses. Total central thickness applied force of 54 mN (range, 50–60 mN). Both control decreased in two aged lenses by 2%, remained unchanged lenses increased in equatorial diameter during stretching (±1%) in another two aged lenses, and increased by 4% with a rather homogeneous extension around the lens equa- or 5% in a further two aged lenses. Total central thick- tor (Fig. 2, Supplementary Fig. S1, Supplementary Videos ness in the two control lenses decreased by 13% (M71) S1 and S2). Because of the transparent media, no distinc- and 5% (M80). The equatorial diameter of the lens nucleus tion between stretching of the cortex and nucleus could be increased in both control lenses by 5% and remained prac- observed. The lenses with cortical cataract also increased in tically unchanged (±1%) in the aged lenses. Consistent with equatorial diameter during stretching; however, some areas the total central thickness changes, the central thickness of around the lens circumference stretched unsymmetrically, the nucleus in the control lenses decreased by 12% (M71) more in some areas and less in others. The cortex exhib- and 5% (M80) and remained practically unchanged (±1%) ited larger deformation near regions of observed opacifi- in the aged lenses (Fig. 3). cations compared to regions where the cortex was clear (Fig. 2, Supplementary Fig. S1). Four out of the seven catarac- DISCUSSION tous lenses showed ruptures at the nucleus–cortex interface adjacent to the cortical cataracts (Supplementary Videos S3 This study was undertaken on human tissue from post- and S4). These ruptures typically occurred at regions with- mortem donors. Due to regulatory conditions and clinical Accommodation Forces IOVS | January 2021 | Vol. 62 | No. 1 | Article 12 | 5 Equatorial lens diameter Central lens thickness 110 110 105 100 100 Lens thickness (%) Lens diameter (%) 90 95 90 80 85 70 80 60 75 70 50 M71 M80 M79 M85 M82 M83 M69 M86 M87 M71 M80 M79 M85 M82 M83 M69 M86 M87 33y 46y 60y 74y 78y 85y 86y 87y 88y 33y 46y 60y 74y 78y 85y 86y 87y 88y Total lens unstretched Total lens stretched Total lens unstretched Total lens stretched Rupture Nucleus unstretched Nucleus stretched Nucleus unstretched Nucleus stretched FIGURE 3. Equatorial lens diameter (left) and central lens thickness (right) for the total lens (dark color) and subdivided for the lens nucleus (light color) for the unstretched state (green) and stretched state (orange). Results are given as a percentage of the dimension with the total lens unstretched state set to 100%. Below are the specimen identification numbers (M71–M87) and the ages of the donor in years. Ruptures are indicated with a small white rectangle. Lenses are stretched with an average force of 54 mN (10−3 Newton). The radial force applied to the lens for specimen M69 was estimated, because the ciliary body was not cut as in all other samples due to an oversight. schedules for corneal transplants, the time between death eter change of 0.33 mm.26 Another similar smaller study and our experiments varied between 40 and 115 hours with human donor lenses (mean age 40 years) showed a (see Table). With longer postmortem times, the lens surface lens diameter change of about 0.45 mm.41 In vivo estima- can become more opaque, and there is an increased risk of tions with optical coherence tomography and magnetic reso- lens capsule detachment during stretching, as was observed nance imaging in subjects about 40 years old and with 5 or in one sample (M80). 8 diopters accommodation stimulus resulted in a lens diam- In most of the lenses, the equatorial lens diameter eter change between 0.30 and 0.40 mm.42,43 changed unsymmetrically around the lens circumference Our data on lens deformations due to stretching in the during stretching; therefore, we estimated the lens diame- aged lenses were somewhat surprising. Aged human donor ter by calculating the projected lens surface, in both the lenses showed similar, or even greater, changes in lens unstretched and fully stretched state and converted these diameter (2%–6%) than the control lenses (3%−4%) during surfaces to a circle with the same area. Determinations of the stretching at the reference radial force of 54 mN. Lens thick- surface area were conducted using high-resolution images. ness remained unchanged in five aged lenses but increased The lens thickness was measured in Scheimpflug images. In by 4% or 5% in two aged lenses during stretching. lenses with pronounced cataract, the posterior lens capsule These results demonstrate that the lens cortex remains is difficult to visualize, so there is some uncertainty in the deformable in aged lenses. The lens nucleus in aged lenses, lens thickness data, particularly in the aged lenses. however, appeared to remain undeformed during stretching; On the basis of the procedures in Pinilla et al.,19 the ciliary this is in contrast to the control lenses. These observations muscle was cut radially in the sample preparation protocol suggest that, consistent with prior data on lens shear modu- to ensure that all of the externally applied radial force was lus, the nucleus has a relatively high stiffness in aged lenses. applied directly to the lens. Due to an oversight, the ciliary Remarkably, the lens thickness increased in some of the aged body in sample M69 was not radially cut. In this case, on lenses (Fig. 3). In Supplementary Video S4 (sample M69), the basis of data in Pinilla et al.,19 we estimated the radial compression of the cortex at the equator is apparent, and force applied to the lens as 50% of the measured force for cortical lens material is pushed to the lens pole, resulting in the case where the ciliary body is intact. an increase of lens thickness during stretching. Considering an aged human lens with standard dimen- The morphology, severity, size, and extension along the sions according to Al-Ghoul et al.40 (9.8-mm diameter and lens circumference of a cortical cataract is highly vari- 4.87-mm thickness), the percentage changes in lens diame- able.3,9,34 However, a common feature is a transparent zone ter due to stretching for the control lenses were 3% and 4% below the lens capsule slightly above the cortical cataract (0.30 and 0.40 mm, respectively). The percentage changes (Fig. 1). In our dataset, this depth ranged from 490 to in lens thickness in the control lenses were 5% and 13% 710 μm, below which the cortical cataract was located. These (0.24 and 0.63 mm, respectively). This is in good agreement measured depths are in good agreement with the range with results from other groups. For example, in a large study 350 to 700 μm based on 29 cortical cataract samples previ- from the United States and India, 40-year-old ex vivo human ously documented in the literature.9 donor lenses, with intact ciliary bodies, were stretched at During our lens stretching tests, ruptures caused non- 100 mN (approximately equivalent to 50 mN in our samples uniform deformations to occur around the lens circumfer- with the ciliary body cut) and showed an average lens diam- ence; ruptures did not occur in all lenses. The ruptures Accommodation Forces IOVS | January 2021 | Vol. 62 | No. 1 | Article 12 | 6 were typically located adjacent to the cortical cataracts 5. Fisher RF. Human lens fibre transparency and mechanical (Fig. 2, Supplementary Fig. S1). For example, the two lenses stress. Exp Eye Res. 1973;16(1):41–49. (samples M82 and M86) with cortical cataract present along 6. Pau H. Cortical and subcapsular cataracts: significance of the entire circumference did not exhibit any ruptures. It is physical forces. Ophthalmologica. 2006;220(1):1–5. possible that the cortical cataract may behave similarly to 7. Angra SK, Adhikari KP, Dada VK. Refractive error stress scar tissue, with higher connectivity in these areas leading in the aetiology of senile cataract. Indian J Ophthalmol. to a reduced probability of rupture. 1986;34(1):1–5. The incidence of a rupture in the lens cortex did not 8. Michael R, Pareja-Arico L, Rauscher FG, Barraquer RI. appear to be correlated with postmortem time (Table). Cortical cataract and refractive error. Ophthalmic Res. 2019;62(3):157–165. Lenses with and without rupture both had a median post- mortem time of 73 hours, and a non-parametric Mann– 9. Michael R, Barraquer RI, Willekens B, van Marle J, Vrensen GF. Morphology of age-related cuneiform cortical cataracts: Whitney U test comparing both groups was not significant the case for mechanical stress. Vision Res. 2008;48(4):626– (P = 0.624). We also investigated whether or not there was 634. an effect of postmortem time on lens shape changes during 10. Belaidi A, Pierscionek BK. Modeling internal stress distri- stretching (Fig. 3). Spearman’s rank correlation test indicated butions in the human lens: can opponent theories coexist? that lens diameter and thickness changes did not correlate J Vis. 2007;7(11):1–12. with postmortem time; the coefficient ρ was –0.460 (P = 11. Wang K, Venetsanos D, Wang J, Pierscionek BK. Gradient 0.213) considering diameter and –0.167 (P = 0.668) consid- moduli lens models: how material properties and applica- ering thickness. tion of forces can affect deformation and distributions of In all cases, ruptures occurred at a depth that was shal- stress. Sci Rep. 2016;6:31171. lower than the cortical cataracts. One lens (M79) exhib- 12. Fujisawa K, Sasaki K. Changes in light scattering intensity of ited minor initial capsule damage at the 1 o’clock posi- the transparent lenses of subjects selected from population- tion (Supplementary Fig. S1). This minor damage may allow based surveys depending on age: analysis through liquid to enter the lens and fill the cavity opened by the Scheimpflug images. Ophthalmic Res. 1995;27(2):89–101. rupture, thereby allowing the rupture to propagate. 13. Vrensen G, Willekens B. Classification and prevalence of early senile lens opacities in human donor eyes. Dev Ophthalmol. 1989;17:181–187. CONCLUSIONS 14. 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Protein profiles in cortical and nuclear regions of aged human donor lenses: a SUPPLEMENTARY MATERIAL confocal Raman microspectroscopic and imaging study. Exp Eye Res. 2016;145:100–109. SUPPLEMENTARY VIDEO S1. Lens M80 in frontal view, 35. Uhlhorn SR, Borja D, Manns F, Parel JM. Refractive index first stretching cycle. measurement of the isolated crystalline lens using optical coherence tomography. Vision Res. 2008;48(27):2732–2738. SUPPLEMENTARY VIDEO S2. Lens M80 in cross- 36. de Castro A, Benito A, Manzanera S, et al. Three-dimensional sectional view, first stretching cycle. cataract crystalline lens imaging with swept-source opti- SUPPLEMENTARY VIDEO S3. Lens M69 in frontal view, cal coherence tomography. Invest Ophthalmol Vis Sci. first, second and third stretching cycle. 2018;59(2):897–903. SUPPLEMENTARY VIDEO S4. Lens M69 in cross- 37. Barraquer RI, Pinilla CL, Allende MJ, et al. Validation of the nuclear cataract grading system BCN 10. Ophthalmic Res. sectional view, first stretching cycle. 2017;57(4):247–251.