Comparison of human amniotic membrane decellularisation approaches for hESC-derived RPE cells culture.

OBJECTIVE: Recent clinical studies have shown that the transplantation of functional retinal pigment epithelium (RPE) cells can prevent the onset of RPE degeneration in age-related macular degeneration. This study aimed to investigate the potential of human amniotic membrane (hAM) as a viable scaffold for the growth and proliferation of pluripotent-derived RPE cells. METHODS AND ANALYSIS: Three enzymatic hAM de-epithelialisation methods (thermolysin, trypsin-EDTA and dispase II) were assessed by histological analysis and optical coherence tomography (OCT). We generated RPE cells from a human embryonic stem cell (hESC) line subjected to spontaneous differentiation in feeder-free conditions. The hESC-derived RPE cells were seeded over denuded hAM at a density of 2.0×105 cells/cm2 and maintained in culture for up to 4 weeks. Immnofluorescence was carried out to evaluate the development of a confluent monolayer of RPE cells on the top of the hAM. Conditioned medium was collected to measure pigment epithelium-derived factor (PEDF) concentration by ELISA. RESULTS: Laminin α5 and collagen IV staining confirmed the efficiency of the de-epithelialisation process. In particular, thermolysin showed good retention of tissue integrity on OCT images and greater preservation of the hAM basement membrane. The hESC-derived RPE cells formed patches of pigmented cells interspersed along the denuded hAM, but failed to form a regular sheet of RPE cells. These cells expressed typical RPE markers, such as PMEL17 and RPE65, but they secreted low levels of PEDF. CONCLUSION: The biological variability of the hAM could influence the adhesion and the expansion of hESC-derived RPE cells. Further studies are required to verify whether a non-confluent monolayer might represent a limit to transplantation.

Study of the force dynamics at the capsular interface related to ciliary body stimulation in a primate model.

PURPOSE: To study the mechanical dynamics between the zonuli-capsular complex and an accommodating intraocular lens mechanics simulator using pharmacological stimulation of the ciliary body in a primate eye. METHODS: Following phacoemulsification removal of the lens, force/movement gauges simulating the accommodating intraocular lens mechanics were implanted in 8 primate eyes (4 positioned in-the-bag and 4 on-the-bag). The gauge readings were taken under full effect of pilocarpine 4% or atropine 1% starting 6 weeks after surgery and following 3-month intervals for 30 months. RESULTS: An average movement of 390 µm for on-the-bag gauges and 145 µm for in-the-bag gauges was documented at 3 months postoperatively. The mechanical compliance of in-the-bag gauges rapidly deteriorated with capsular fibrosis developing inside the gauge and the capsular bag shrinking around it. On-the-bag gauges remained functional but the initial compliance was compromised over time by the pressure build up caused by the fibrotic capsular bag. CONCLUSIONS: Following phacoemulsification, capsular fibrosis causes disappearance of the mechanical forces detected by an in-the-bag gauge. An on-the-bag gauge detects active forces at the sulcus lasting at least 5 years, although the contracting capsule pressure compromises its compliance. These findings have important implications for future accommodating intraocular lens designs.

Visual and accommodative outcomes 1 year after implantation of an accommodating intraocular lens based on a new concept.

PURPOSE: To evaluate visual outcomes and accommodative gains 1 year after implantation of the NuLens accommodating intraocular lens (IOL). SETTING: Department of Refractive Surgery, Vissum Corp., Alicante, Spain. METHODS: This study comprised patients with cataract and atrophic macular degeneration. In each patient, the accommodating IOL was implanted in the eye with the worse visual acuity. At the 12-month follow-up visit, visual acuity and accommodation were measured to determine the efficacy of the IOL. Ultrasound biomicroscopy was used to measure accommodative amplitude. RESULTS: Ten eyes of 10 patients were evaluated. The mean number of lines patients could read increased from 1.0 preoperatively to 3.8 lines 6 months postoperatively, indicating improvement in uncorrected near visual acuity after IOL implantation. The mean change in cross-section measurements of the IOL was 0.06 mm at 1 month; the value peaked at 3 months (0.21 mm), after which it decreased steadily, becoming stable at 9 months (0.09 mm, which is equivalent to 10.00 diopters [D] of accommodation). Corrected near visual acuity improved slightly (0.7 Jaeger lines) at 12 months, with the best reading distance at 10 cm. These results suggest that the near and distance visual acuities were approximately equal and, therefore, the IOL can produce accommodation of 10.00 D. CONCLUSIONS: The accommodation mechanism of the IOL can produce an ocular power variation of 10.00 D. Near visual acuity improved without compromising distance visual acuity.

The NuLens accommodating intraocular lens.

The NuLens intraocular lens (IOL) uses a new optomechanical concept to generate a wide range of dioptric power from minimal movements of the ciliary apparatus during accommodative effort. Basic study of the concept of optomechanics in the primate eye led to successful construction and implantation of functioning IOL prototypes.

Feasibility and development of a high-power real accommodating intraocular lens.

PURPOSE: To develop an accommodating intraocular lens (IOL) that changes power with ciliary body action and generates a minimum of 8.0 diopters (D) by manipulation of a flexible material between a sulcus-fixated rigid plate and a ciliary muscle-operated capsular diaphragm. SETTING: Vissum-Instituto Oftalmológico de Alicante and Miguel Hernández University, Alicante, Spain. METHODS: The feasibility of the concept was tested by a laboratory lens model. An implantable measuring device was constructed to simulate the lens action. The device was implanted in monkey eyes to measure the various parameters involved with such IOLs. Based on these measurements, an accommodating IOL prototype was built and implanted in monkeys' eyes. Pharmacologic agents were used to achieve ciliary relaxation and spasm. Ultrasound biomicroscopy (UBM) imaging was used to document the active changes of the IOL flexible lens curvature as related to the ciliary muscles status. RESULTS: The laboratory model produced more than 50.0 D of accommodation. The UBM demonstrated changes in lens curvature between cyclospasm to cycloplegia of calculated 9.0 to 53.0 D for flexible material with a refractive index of 1.41. CONCLUSIONS: Flexible material is capable of being manipulated when placed between a scleral-fixated rigid plane and the ciliary muscles-operated capsular diaphragm. Such manipulation as used by the IOL concept presented here has generated an active change of more than 40.0 D in the monkey eye.

Retinal microvascular patency in the diabetic rat.

PURPOSE: To study whether the patency to erythrocytes in retinal microvessels of diabetic rats is reduced or blocked before the vessels lose their patency to plasma flow. METHODS: We used recognized techniques to induce diabetic and galactose related microvascular retinal lesions in rats: (1) alloxan induction (2) streptozotocin induction (3) galactose-containing diet. The rats were followed up to 17 months. We used our vascular trichrome technique to observe the effects of the ongoing diabetes on the retinal microcirculation. RESULTS: A focal leakage of a plasma-borne fluorescent dye was noted around the junction of the deep retinal capillaries and the ascending venules to the superficial retinal circulation in the streptozotocin and alloxan diabetic rats by the 14th month, and, by the 16th month, retinal capillary non-perfusion and retinal vascular malformations were present. The affected vessels showed patency to microspheres (0.2 microm in diameter) but no perfusion of erythrocytes. No such changes were seen in the galactose-fed rats. CONCLUSIONS: (1) The location between the deep retinal capillary net and the ascending venules may be the site of early vascular leakage in the diabetic rat model, (2) the erythrocytes' passage in the affected retinal microcirculation was blocked before the development of complete blockage to plasma in diabetic rats. The logical assumption that during the development stage of retinal capillary occlusion there may be a transient stage of microvascular insufficiency was examined. The lathyrogen, imino-diproprionitrile (IDPN), had previously been effective for creating a fast-developing model of retinal vasculopathy. Using that model, we demonstrated a stage in which the retinal microvasculature was blocked to erythrocytes but not to plasma [1]. However, we questioned the applicability of our findings to more slowly developing microvasculopathies, such as diabetic retinopathy. We designed the current study to examine the presence of such stage in slowly developing microvasculopathy. Animal models that are known to induce "diabetic retinopathy-like" changes used [2-4]. The diabetic animals were followed for a period of 17 months. Starting at the 12th month, a few animals of each group were killed and the retains were examined with our trichrome method [1] for relative capillary patency to erythrocytes and plasma, for functionality of endothelial cells, and for disturbances in the blood-retinal barrier. The results of this study support the hypothesis that retinal microvascular insufficiency does exist as a temporary stage that precedes the development of complete capillary blockage in long-term developing rat models of diabetic retinopathy.

Nucleus cryoinversion for extraction of highly complicated cataracts.

We describe a technique for cataract extraction in vitrectomized eyes that uses secured removal of the nucleus with no manipulation of the capsular bag. After a large scleral tunnel is prepared and a capsulorhexis created, a 20-gauge retinal cryoprobe enveloped in a plastic or a silicone sleeve is introduced into the anterior chamber. The cryoprobe engages the nucleus by a "freeze-grip" and rotates it 180 degrees so that the nucleus is elevated into the anterior chamber with no force applied to the capsule and the cryoprobe supporting it from below. The cryoprobe is replaced by a vectis under the nucleus and with the help of another instrument from above, the nucleus is removed. Aspiration of cortical material under low-pressure fluid maintenance of the anterior chamber completes removal of the cataract. An intraocular lens is then implanted in the ciliary sulcus for maximum capsule support.

Endoscopic photocoagulation of the ciliary body after repeated failure of trans-scleral diode-laser cyclophotocoagulation.

PURPOSE: To report the successful control of intraocular pressure with endoscopic cyclophotocoagulation after repeated failure of trans-scleral diode-laser cyclophotocoagulation. DESIGN: Interventional case report. METHODS: A 3 1/2-year-old child had intraocular pressure around 30 mm Hg in one eye despite full medical treatment and three procedures of trans-scleral diode laser photocoagulation. RESULTS: Endoscopic view of the ciliary body revealed many misplaced laser burns in the pars plana region. Using direct endoscopic visualization of the ciliary body, precise, confluent burns were applied to the ciliary body. Six months after the procedure, intraocular pressure has remained under 20 mm Hg. CONCLUSION: Endoscopic cyclophotocoagulation can effectively lower intraocular pressure after recurrent external diode-laser photocoagulation has failed to do so. The direct visualization during the endoscopic procedure is particularly advantageous in cases with atypical ciliary body morphology, such as in pediatric glaucoma.