Measuring the effects of postmortem time and age on mouse lens elasticity using atomic force microscopy.

The mouse lens is frequently used both in vivo and ex vivo in ophthalmic research to model conditions affecting the human lens, such as presbyopia. The mouse lens has a delicate structure which is prone to damage and biomechanical changes both before and after extraction from the whole globe. When not properly controlled for, these changes can confound the biomechanical analysis of mouse lenses. In this study, atomic force microscopy microindentation was used to assess changes in the Young's Modulus of Elasticity of the mouse lens as a function of mouse age and postmortem time. Old mouse lenses measured immediately postmortem were significantly stiffer than young mouse lenses (p = 0.028). However, after 18 h of incubation, there was no measurable difference in lens stiffness between old and young mouse lenses (p = 0.997). This demonstrates the need for careful experimental control in experiments using the mouse lens, especially regarding postmortem time.

Operational immune tolerance towards transplanted allogeneic pancreatic islets in mice and a non-human primate.

AIMS/HYPOTHESIS: Patients with autoimmune type 1 diabetes transplanted with pancreatic islets to their liver experience significant improvement in quality of life through better control of blood sugar and enhanced awareness of hypoglycaemia. However, long-term survival and efficacy of the intrahepatic islet transplant are limited owing to liver-specific complications, such as immediate blood-mediated immune reaction, hypoxia, a highly enzymatic and inflammatory environment and locally elevated levels of drugs including immunosuppressive agents, all of which are injurious to islets. This has spurred a search for new islet transplant sites and for innovative ways to achieve long-term graft survival and efficacy without life-long systemic immunosuppression and its complications. METHODS: We used our previously established approach of islet transplant in the anterior chamber of the eye in allogeneic recipient mouse models and a baboon model of diabetes, which were treated transiently with anti-CD154/CD40L blocking antibody in the peri-transplant period. Survival of the intraocular islet allografts was assessed by direct visualisation in the eye and metabolic variables (blood glucose and C-peptide measurements). We evaluated longitudinally the cytokine profile in the local microenvironment of the intraocular islet allografts, represented in aqueous humour, under conditions of immune rejection vs tolerance. We also evaluated the recall response in the periphery of the baboon recipient using delayed-type hypersensitivity (DTH) assay, and in mice after repeat transplant in the kidney following initial transplant with allogeneic islets in the eye or kidney. RESULTS: Results in mice showed >300 days immunosuppression-free survival of allogeneic islets transplanted in the eye or kidney. Notably, >70% of tolerant mice, initially transplanted in the eye, exhibited >400 days of graft survival after re-transplant in the kidney without immunosuppression compared with ~30% in mice that were initially transplanted in the kidney. Cytokine and DTH data provided evidence of T helper 2-driven local and peripheral immune regulatory mechanisms in support of operational immune tolerance towards the islet allografts in both models. CONCLUSIONS/INTERPRETATION: We are currently evaluating the safety and efficacy of intraocular islet transplantation in a phase 1 clinical trial. In this study, we demonstrate immunosuppression-free long-term survival of intraocular islet allografts in mice and in a baboon using transient peri-transplant immune intervention. These results highlight the potential for inducing islet transplant immune tolerance through the intraocular route. Therefore, the current findings are conceptually significant and may impact markedly on clinical islet transplantation in the treatment of diabetes.

Measurement of Crystalline Lens Volume During Accommodation in a Lens Stretcher.

PURPOSE: To determine if the lens volume changes during accommodation. METHODS: The study used data acquired on 36 cynomolgus monkey lenses that were stretched in a stepwise fashion to simulate disaccommodation. At each step, stretching force and dioptric power were measured and a cross-sectional image of the lens was acquired using an optical coherence tomography system. Images were corrected for refractive distortions and lens volume was calculated assuming rotational symmetry. The average change in lens volume was calculated and the relation between volume change and power change, and between volume change and stretching force, were quantified. Linear regressions of volume-power and volume-force plots were calculated. RESULTS: The mean (± SD) volume in the unstretched (accommodated) state was 97 ± 8 mm3. On average, there was a small but statistically significant (P = 0.002) increase in measured lens volume with stretching. The mean change in lens volume was +0.8 ± 1.3 mm3. The mean volume-power and volume-load slopes were -0.018 ± 0.058 mm3/D and +0.16 ± 0.40 mm3/g. CONCLUSIONS: Lens volume remains effectively constant during accommodation, with changes that are less than 1% on average. This result supports a hypothesis that the change in lens shape with accommodation is accompanied by a redistribution of tissue within the capsular bag without significant compression of the lens contents or fluid exchange through the capsule.

The zonules selectively alter the shape of the lens during accommodation based on the location of their anchorage points.

PURPOSE: To determine the role of anterior and posterior zonular tension on the optomechanical lens response during accommodation simulation. METHODS: Ten eyes from nine hamadryas baboons (4.9 ± 0.7 years) and 20 eyes from 18 cynomolgus monkeys (5.4 ± 0.3 years) were dissected, leaving the lens, zonules, ciliary body, hyaloid membrane, anterior vitreous, and a segmented scleral rim intact. The lens preparation was mounted in a lens stretcher, and the outer scleral shell was displaced radially in a stepwise fashion. The load, lens, and ciliary body diameters, lens power, lens thickness, and the anterior and posterior radius of curvature were measured during stretching. The zonular fibers attached to either the posterior or anterior lens surface were then carefully transected and the experiment was repeated. Zonular transection was confirmed in four eyes via laser scanning confocal microscopy after immunostaining. The effect of zonular transection on the tissue response to stretching was quantified. RESULTS: Without anterior zonules, 48% and 97% of the changes in anterior and posterior radii are retained. Without posterior zonules, 81% and 67% of the changes in anterior and posterior radii are retained. The changes in lens shape were reduced after transecting either the anterior or posterior zonules; however, both surfaces still changed shape. CONCLUSIONS: While either the anterior or posterior zonules alone are capable of changing the shape of both lens surfaces, the anterior zonules have a greater effect on the anterior lens surface, and the posterior zonules have a greater effect on the posterior lens surface.

Semiautomated analysis of optical coherence tomography crystalline lens images under simulated accommodation.

Presbyopia is an age related, gradual loss of accommodation, mainly due to changes in the crystalline lens. As part of research efforts to understand and cure this condition, ex vivo, cross-sectional optical coherence tomography images of crystalline lenses were obtained by using the Ex-Vivo Accommodation Simulator (EVAS II) instrument and analyzed to extract their physical and optical properties. Various filters and edge detection methods were applied to isolate the edge contour. An ellipse is fitted to the lens outline to obtain central reference point for transforming the pixel data into the analysis coordinate system. This allows for the fitting of a high order equation to obtain a mathematical description of the edge contour, which obeys constraints of continuity as well as zero to infinite surface slopes from apex to equator. Geometrical parameters of the lens were determined for the lens images captured at different accommodative states. Various curve fitting functions were developed to mathematically describe the anterior and posterior surfaces of the lens. Their differences were evaluated and their suitability for extracting optical performance of the lens was assessed. The robustness of these algorithms was tested by analyzing the same images repeated times.

Technique for air bubble management during endothelial keratoplasty in eyes after penetrating glaucoma surgery.

Our purpose was to develop a technique for maintaining air within the anterior chamber during endothelial keratoplasty in eyes that have previously undergone trabeculectomy or a glaucoma drainage implant. Whole human globes and rabbits underwent penetrating glaucoma surgery to develop the technique. Without the aid of any additional device or manipulation, continuing to inject air into the anterior chamber as it escapes through the sclerostomy or tube eventually fills the subconjunctival space and allows for back pressure. This allows for a full anterior chamber air fill and brief elevation of intraocular pressure. We employed this overfilling technique on 3 patients with previous incisional glaucoma surgery to perform successful Descemet stripping endothelial keratoplasty without complication. We recommend using the overfilling technique when performing Descemet stripping endothelial keratoplasty surgery in eyes with previous penetrating glaucoma surgery because it is a simple technique without the need for pre- or postoperative manipulation.

Laser trabeculoplasty induces changes in the trabecular meshwork glycoproteome: a pilot study.

Laser trabeculoplasty (LT) is a commonly used modality of treatment for glaucoma. The mechanism by which LT lowers the intraocular pressure (IOP) is unknown. With the use of cat eyes, selective laser trabeculoplasty (SLT) with a Q-switched frequency doubled Nd:YAG laser was used to treat the trabecular meshwork (TM). Laser treated TM was then subjected to proteomic analysis for detection of molecular changes and histological analysis for the detection of structural and protein expression patterns. In addition, the protein glycosylation patterns of laser treated and nontreated TM was assessed and differentially glycosylated proteins were proteomically identified. SLT laser treatment to the TM resulted in elevated glycosylation levels compared to nonlasered TM. TM laser treatment also resulted in protein expression levels changes of several proteins. Elevated levels of biglycan, keratocan and prolargin were detected in laser treated TM compared to nonlasered controls. Further investigation is anticipated to provide insight into how glycosylation changes affect TM proteins and TM regulation of aqueous outflow in response to laser trabeculoplasty.

Effect of anterior zonule transection on the change in lens diameter and power in cynomolgus monkeys during simulated accommodation.

PURPOSE: To quantify the role of anterior zonular tension on the optomechanical lens response during simulation of accommodation in primates. METHODS: Postmortem cynomolgus monkey eyes (n = 14; age range, 3.0-11.5 years) were dissected leaving intact the lens, zonules, ciliary body, hyaloid membrane, anterior vitreous, and a scleral rim. The lens was mounted in a lens-stretching system and stretched radially in step-wise fashion. The load, and the lens diameter and power were measured at each step and the diameter- and power-load relationships were quantified. The anterior zonular fibers were then transected, and the experiment was repeated. The equatorial lens diameter and lens optical power before and after zonular transection were compared. RESULTS: Stretching increased the lens diameter by 0.25 +/- 0.09 mm (median +/- interquartile range) before and 0.25 +/- 0.19 mm after zonular transection. Stretching decreased the lens power by 13.0 +/- 6.5 D before and 10.6 +/- 8.0 D after zonular transection. The load required to change the diameter of the lens by 1 mm decreased from 18.8 +/- 10.7 g before to 15.0 +/- 7.8 g after zonular transection. The absolute change in power per gram of loading decreased from 2.5 +/- 1.1 before to 2.0 +/- 1.2 D after zonular transection. CONCLUSIONS: The cynomolgus monkey lens retains a significant fraction of its accommodative ability after transection of the anterior zonules in simulated accommodation experiments.