Intraocular pressure and injection forces during intravitreal injection into enucleated porcine eyes.

Injection of biological molecules into the intravitreous humor is of increasing interest for the treatment of posterior segment eye diseases such as age-related degenerative macular degeneration. The injection volume is limited by an increase in intraocular pressure (IOP) and 50-100 µL are typically used for most intravitreally (IVT) applied commercial products. Direct measurement of IOP is difficult and has not been studied dependent on solution properties and injection rates. We used an instrumental set-up to study IOP ex vivo using healthy enucleated porcine eyes. IOP was determined as a function of injection volume for viscosities between 1 and 100 mPas, injection rates of 0.1, 1, and 1.5 mL/min, and needle length and diameter (27/30G and 0.5/0.75″) using Dextran solutions. IOP increased exponentially for injection volumes larger than 100 µL. We did not observe differences in IOP dependent on viscosity, injection rate, and needle diameter. However, variability increased significantly for injection volumes larger than 100 µL and, unexpectedly, declined with higher viscosities. We demonstrate that the exponential increase in IOP is not reflected by injection force measurements for typical configurations that are used for IVT application. The present findings may guide injection volumes for intravitreal injection and inform injection force considerations during technical drug product development.

Multifunctional carboxymethyl chitosan derivatives-layered double hydroxide hybrid nanocomposites for efficient drug delivery to the posterior segment of the eye.

Efficient ocular drug delivery to the posterior segment of the eye by topical administration is a great challenge to pharmacologists. To explore drug delivery system of organic-inorganic hybrid nanocomposites for the efficient delivery of dexamethasone disodium phosphate (DEXP), a targeted hybrid nanocomposite based on layered double hydroxide (LDH) and functional carboxymethyl chitosan (CMCS) derivatives was designed. A special substrate of peptide transporter-1 (PepT-1) and glutathione was modified on CMCS. CMCS-glutathione-glycylsarcosine (CMCG-GS) and CMCS-glutathione-valyl-valine (CMCG-VV)-LDH hybrid nanocomposites were prepared and structurally confirmed. The in vitro experiments on human conjunctival epithelial cells showed noncytotoxicity (LDH concentration ≤100.0 µg/mL) and enhanced permeability for hybrid nanocomposites. Additionally, cellular uptake of the CMCG-GS-DEXP-LDH (10:1) nanocomposite eye drops involved clathrin-mediated endocytosis and PepT-1 mediated actively targeting transport. Results of the in vivo precorneal retention study showed an 8.35-fold, 2.87-fold and 2.58-fold increase of AUC0-6 h, Cmax and MRT for CMCG-GS-DEXP-LDH (10:1) hybrid nanocomposite eye drops, respectively, compared to that of the commercial product. Fluorescence imaging of fluorescein isothiocyanate isome (FITC)-loaded LDH hybrid nanocomposites demonstrated that FITC could diffuse into the choroid-retina with the shelter of LDH and CMCG-GS. The presence of a strong fluorescence signal of FITC-conjugated LDH hybrid nanocomposites in the sclera revealed that integral LDH nanocarrier reached the sclera. In the tissue distribution evaluation of rabbit's eyes, DEXP of CMCG-GS-DEXP-LDH (10:1) nanocomposites group retained in the target of the choroid-retina for 3 h with final concentration at 120.04 ng/g. Furthermore, the results of fluorescence imaging and tissue distribution suggested that the intraocular transport pathway for the hybrid nanocomposites is the conjunctival-scleral route. Consequently, the developed hybrid nanocomposites offer a simple and efficient strategy for topically administered drug delivery to the posterior segment of the eye. STATEMENT OF SIGNIFICANCE: Efficient ocular drug delivery to the posterior segment of the eye by topical administration is a great challenge to pharmacologists. In this manuscript, hybrid nanocomposite based on layered double hydroxide (LDH) and functional carboxymethyl chitosan (CMCS) derivatives were designed. The multifunctional properties of these hybrid nanocomposites were attributed to active targeting, bioadhesive capacity and penetration enhancement. Visualization of transport routes of fluorescein isothiocyanate-conjugated LDH hybrid nanocomposites demonstrated that the integral LDH nanocarrier reached the sclera through the conjunctival-scleral pathway, and the loaded drug could further diffuse to the retina. The multifunctional CMCS derivatives-LDH hybrid nanocomposites could be applied for the efficient drug delivery to the posterior segment of the eye through noninvasive topical instillation.

In Vivo Elasticity Mapping of Posterior Ocular Layers Using Acoustic Radiation Force Optical Coherence Elastography.

Purpose: We used acoustic radiation force optical coherence elastography (ARF-OCE) to map out the elasticity of retinal layers in healthy and diseased in vivo rabbit models for the first time. Methods: A healthy rabbit eye was proptosed and imaged using ARF-OCE, by measuring the tissue deformation after an acoustic force is applied. A diseased retinal inflammation model was used to observe the contrast before and after disease formation. Retinal histologic analysis was performed to identify layers of the retina corresponding with the optical images. Results: The general trend of the retinal layer elasticity is increasing stiffness from the ganglion side to the photoreceptor side, with the stiffest layer being the sclera. In a healthy rabbit model, the mechanical properties varied from 3 to 16 kPa for the five layers that were identified via optical imaging and histology (3.09 ± 0.46, 3.82 ± 0.88, 4.53 ± 0.74, 6.59 ± 2.27, 16.11 ± 5.13 kPa). In the diseased model, we have induced optical damage in a live rabbit and observed a change in the stiffness trend in its retina. Conclusions: High sensitivity elasticity maps can be obtained using the ARF-OCE system to differentiate different retinal layers. Subtle changes in the mechanical properties during the onset of diseases, such as retinal degeneration, can be measured and aid in early clinical diagnosis. This study validates our imaging system for the characterization of retinal elasticity for the detection of retinal diseases in vivo.

Location and pressure dependent transmission of human and porcine sclera: an anterior to posterior examination.

PURPOSE: For diaphanoscopy or transscleral laser applications, the transmission of the sclera is an essential property. The study aimed to determine the pressure dependent transmission of human sclera from anterior to posterior. METHODS: Pressure dependent transmission measurements were performed by a pressure inducing setup at the range of 60-2058 kPa. The transmissions were measured within spectral range of 350-1100 nm. Specimens of human sclera were taken from corneo-scleral transplants. Those compounds were obtained at pars plicata residual sclera tissue. For an anterior to posterior examination of transmission, samples were taken from halved eye globes, which were formerly fixed in formalin. RESULTS: The pressure dependent transmission increased with rising load at all measured wavelengths for human sclera samples. The highest increase was observed for short wavelengths. With rising pressure, the increase of transmission aimed for a steady state. This behavior was fitted by a limited growing function. With an inducing burden of 2058 kPa, the steady state was already reached and exhibited an increase in transmission factor of 4.1 at 400 nm and 1.8 at 1000 nm. The anterior to posterior measurements of human sclera fixed in formalin were not corresponding to the results of the other human samples. For the porcine samples, the transmission increased from anterior to the equator of the eye globe. Further posterior the transmission decreased and rose again to N. opticus. With rising pressure, the transmission increased at all wavelengths and all locations. Posterior from the equator, with higher pressure the transmission became superior compared to anterior. CONCLUSIONS: The results of human sclera fixed in formalin could be related to formalin-induced cross-linking between the collagen fibers. Because of doubt about the physiological behavior of formalin-fixed samples, formalin-free porcine postmortem eye globes were also probed having a very similar thickness and histological structure as human sclera, so the results could be set in relation to human probes. These results can now be used to create an eye-map to determine maximum possible retina irradiation or illumination durations for transscleral applications in eye surgery.

Anterior chamber perfusion versus posterior chamber perfusion does not influence measurement of aqueous outflow facility in living mice by constant flow infusion.

Mice are now routinely utilized in studies of aqueous humor outflow dynamics. In particular, conventional aqueous outflow facility (C) is routinely measured via perfusion of the aqueous chamber by a number of laboratories. However, in mouse eyes perfused ex-vivo, values for C are variable depending upon whether the perfusate is introduced into the posterior chamber (PC) versus the anterior chamber (AC). Perfusion via the AC leads to posterior bowing of the iris, and traction on the iris root/scleral spur, which may increase C. Perfusion via the PC does not yield this effect. But the equivalent situation in living mice has not been investigated. We sought to determine whether AC versus PC perfusion of the living mouse eye may lead to different values for C. All experiments were conducted in C57BL/6J mice (all ♀) between the ages of 20 and 30 weeks. Mice were divided into groups of 3-4 animals each. In all groups, both eyes were perfused. C was measured in groups 1 and 2 by constant flow infusion (from a 50 µL microsyringe) via needle placement in the AC, and in the PC, respectively. To investigate the effect of ciliary muscle (CM) tone on C, groups 3 and 4 were perfused live via the AC or PC with tropicamide (muscarinic receptor antagonist) added to the perfusate at a concentration of 100 µM. To investigate immediate effect of euthanasia, groups 5 and 6 were perfused 15-30 min after death via the AC or PC. To investigate the effect of CM tone on C immediately following euthanasia, groups 7 and 8 were perfused 15-30 min after death via the AC or PC with tropicamide added to the perfusate at a concentration of 100 µM. C in Groups 1 (AC perfusion) and 2 (PC perfusion) was computed to be 19.5 ± 0.8 versus 21.0 ± 2.1 nL/min/mmHg, respectively (mean ± SEM, p > 0.4, not significantly different). In live animals in which tropicamide was present in the perfusate, C in Group 3 (AC perfusion) was significantly greater than C in Group 4 (PC perfusion) (22.0 ± 4.0 versus 14.0 ± 2.0 nL/min/mmHg, respectively, p = 0.0021). In animals immediately following death, C in groups 5 (AC perfusion) and 6 (PC perfusion) was computed to be 21.2 ± 2.0 versus 22.8 ± 1.4 nL/min/mmHg, respectively (mean ± SEM, p = 0.1196, not significantly different). In dead animals in which tropicamide was present in the perfusate, C in group 7 (AC perfusion) was greater than C in group 8 (PC perfusion) (20.6 ± 1.4 versus 14.2 ± 2.6 nL/min/mmHg, respectively, p < 0.0001). C in eyes in situ in living mice or euthanized animals within 15-30 min post mortem is not significantly different when measured via AC perfusion or PC perfusion. In eyes of live or freshly euthanized mice, C is greater when measured via AC versus PC perfusion when tropicamide (a mydriatic and cycloplegic agent) is present in the perfusate.

Refractive Outcomes of Posterior Chamber Phakic Intraocular Lens Implantation for Correction of Myopia and Myopic Astigmatism.

OBJECTIVES: The aim was to examine efficacy and safety after Implantable Collamer Lens (ICL) implantation for correction of myopia et myopic astigmatism. METHODS: This prospective clinical study included 28 eyes of 16 patients which underwent implantation of ICL for correction of myopia up to -18,00 diopters (D) and myopic astigmatism up to -6,00 D in the Eye Clinic Svjetlost Sarajevo, from January 2013 to January 2016. Uncorrected distance visual acuity (UDVA), spherical equivalent (SE), corrected distance visual acuity (CDVA), intraocular pressure (IOP), endothelial cell (EC) density were evaluated at one, six and twelve months. For statistical analysis SPSS for Windows and Microsoft Excel were used. RESULTS: Out of 16 patients, with mean age of 28,21 ± 4,06 years, 12 of them had binocular and 4 of them had monocular procedure. After 12 months mean UDVA was 0,76 ± 0,16 compared to UDVA 0,04 ± 0,03 preoperatively. Mean SE preoperatively was -0,21 ± 0,27 D compared to -9,52 ± 3,69 D preoperatively. At 12 months one eye (3,57%) lost 2 Snellen lines. In this study 8 eyes (28,57%) gained 1 line, 5 eyes gained (17,56%) 2 lines, and 3 eyes (10,72%) gained 3 lines. EC loss was 5,50±4.71% after 12 months. There was no significant change of IOP by the end of 12 months follow up period. One haptic crack was reported as the only intraoperative complication. Three postoperative complications were: two lens rotations and one retinal detachment. CONCLUSION: Implantation of ICL is an effective and safe method for reducing or correcting myopia and myopic astigmatism.

Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery.

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.

L'objectif de la présente étude était de développer un système murin de culture d'explant de rétine/choroïde/sclérotique afin de faciliter la livraison intra-vitréenne de vecteurs. Des explants de segments postérieurs provenant de souris adultes de deux groupes d'âge différents (4 sem et 15 sem) furent cultivés dans un milieu sans sérum pour des périodes de temps variables. La viabilité tissulaire fut évaluée par morphologie macroscopique, quantification de la survie cellulaire, expression de la caspase-3 activée, et immunohistochimie. Afin d'imiter la thérapie génique oculaire, les explants furent exposés pendant 48 h à des unités de transduction variables d'un vecteur lentiviral exprimant le gène pour la protéine fluorescente verte. Les explants de cellules de la rétine sont demeurés viables pour environ 1 sem, bien que dans la couche de cellules ganglionnaires on nota le développement de l'apoptose entre 4 à 7 j. Suite à l'infusion de vecteur dans le segment postérieur, la transduction virale fut notée dans plusieurs couches rétiniennes des animaux des deux groupes d'âge. Une influence de l'âge de la souris donneuse fut notée et chez les souris plus âgées la transduction ne se faisait pas aussi bien que chez les jeunes souris. Ce modèle d'explant permet la gestion facile de culture de segment oculaire postérieur avec un minimum de dérangement du tissu, et pourrait être utile pour étudier des méthodes visant à augmenter la thérapie génique sous conditions contrôlées.(Traduit par Docteur Serge Messier).

Age- and race-related differences in human scleral material properties.

PURPOSE: We tested the hypothesis that there are age- and race-related differences in posterior scleral material properties, using eyes from human donors of European (20-90 years old, n = 40 eyes) and African (23-74 years old, n = 22 eyes) descent. METHODS: Inflation tests on posterior scleral shells were performed while full-field, three-dimensional displacements were recorded using laser speckle interferometry. Scleral material properties were fit to each eye using a microstructure-based constitutive formulation that incorporates the collagen fibril crimp and the local anisotropic collagen architecture. The effects of age and race were estimated using Generalized Estimating Equations, while accounting for intradonor correlations. RESULTS: The shear modulus significantly increased (P = 0.038) and collagen fibril crimp angle significantly decreased with age (P = 0.002). Donors of African descent exhibited a significantly higher shear modulus (P = 0.019) and showed evidence of a smaller collagen fibril crimp angle (P = 0.057) compared to donors of European descent. The in-plane strains in the peripapillary sclera were significantly lower with age (P < 0.015) and African ancestry (P < 0.015). CONCLUSIONS: The age- and race-related differences in scleral material properties result in a loss of scleral compliance due to a higher shear stiffness and a lower level of stretch at which the collagen fibrils uncrimp. The loss of compliance should lead to larger high frequency IOP fluctuations and changes in the optic nerve head (ONH) biomechanical response in the elderly and in persons of African ancestry, and may contribute to the higher susceptibility to glaucoma in these at-risk populations.

New side-view imaging technique for observing posterior chamber structures during cataract surgery in porcine eyes.

BACKGROUND: To develop a side-view imaging technique for observing the dynamic behavior of posterior chamber structures (PCSs) in porcine eyes which mimics closed-eye cataract surgery in humans. METHODS: Enucleated porcine eyes were placed into liquid nitrogen for 5 seconds and immediately bisected at about a 45-degree angle to the equatorial plane. The anterior portion was attached firmly to a glass slide with superglue and sprinkled with wheat flour. Phacoemulsification and aspiration (PEA) was performed as in humans on 10 consecutive porcine eyes. The movements of the PCSs were monitored through the glass slide with a high-resolution video camera set below the cut surface of the eye. The intraocular pressure (IOP) was monitored during the surgery. The highest IOP, operation time, and volume of irrigation fluid of 10 whole eyes were compared to that obtained from the bisected eyes glued to a glass slide. In a second set of experiments, the strength of the seal between the bisected eye and the glass slide was tested in three sets of eyes: 1) frozen eye fixed with superglue with wheat flour for 3 min; 2) frozen eye fixed with superglue for 3 min; and 3) non-frozen eye fixed with superglue for 30 min. The highest IOP that led to a disruption of the seal was compared among the three groups. RESULTS: PEA was successfully performed on 9 of 10 (90%) eyes with the movements of the PCSs clearly observed. The average maximum intraocular pressure of the 9 bisected eyes was 55.8 ± 4.7 mmHg and that for the 10 unbisected eyes was 55.3 ± 5.0 mmHg (P = 0.650). The frozen eye fixed with superglue in combination with wheat flour (Group 1) had the strongest sealing strength with an average IOP at the breaking point of 117.3 ± 36.2 mmHg. CONCLUSIONS: Our side-view imaging technique can be used to evaluate the changes of the PCSs during intraocular surgery and for surgical training of new residents.

Clinical evaluation of optical quality and intraocular scattering after posterior chamber phakic intraocular lens implantation.

PURPOSE: We assessed the optical quality and intraocular scattering after posterior chamber phakic intraocular lens implantation. METHODS: We examined prospectively 38 eyes of 19 consecutive patients undergoing implantable contact lens (ICL) implantation (mean age ± SD 36.3 ± 5.7 years), and 38 age-matched normal eyes of 19 healthy volunteers (mean age 36.4 ± 4.9 years). We assessed quantitatively the values of modulation transfer function (MTF) cutoff frequency, Strehl ratio, objective scattering index (OSI), and the Optical Quality Analysis System (OQAS) values (OVs). We compared these variables in eyes undergoing ICL implantation to those in healthy eyes. RESULTS: The mean MTF cutoff frequency, Strehl ratio, OSI, OV 100%, OV 20%, and OV 9% were 28.69 ± 8.59 cycles/degree, 0.17 ± 0.04, 1.06 ± 0.48, 0.96 ± 0.29, 0.83 ± 0.31, and 0.83 ± 0.32, respectively, 3 months after ICL implantation. We found no significant differences in the MTF cutoff frequency (Mann Whitney U test, P = 0.31), Strehl ratio (P = 0.46), OSI (P = 0.30), or OVs at contrasts of 100% (P = 0.51), 20% (P = 0.46), and 9% (P = 0.36), between the ICL and control groups. CONCLUSIONS: The optical quality parameters, such as the MTF cutoff frequency, Strehl ratio, OSI, or OVs in the ICL group, were not significantly different from those in the control group, suggesting that the optical quality and intraocular scattering of eyes undergoing ICL implantation essentially was equivalent to those of healthy eyes.

Biomechanics of the human posterior sclera: age- and glaucoma-related changes measured using inflation testing.

PURPOSE: The objective of this study was to measure the biomechanical response of the human posterior sclera in vitro and to estimate the effects of age and glaucoma. METHODS: Scleral specimens from 22 donors with no history of glaucoma and 11 donors with a history of glaucoma were excised 3 mm posterior to the equator and affixed to an inflation chamber. Optic nerve cross-sections were graded to determine the presence of axon loss. The time-dependent inflation response was measured in a series of pressure-controlled load-unload tests to 30 mm Hg and creep tests to 15 and 30 mm Hg. Circumferential and meridional strains were computed from the digital image correlation displacements, and midposterior stresses were determined from pressure and deformed geometry. RESULTS: Among normal specimens, older age was predictive of a stiffer response and a thinner sclera. In the age group 75 to 93, diagnosed glaucoma eyes with axon damage were thicker than normal eyes. Both damaged and undamaged glaucoma eyes had a different strain response in the peripapillary sclera characterized by a stiffer meridional response. Undamaged glaucoma eyes had slower circumferential creep rates in the peripapillary sclera than normal eyes. Glaucoma eyes were not different from normal eyes in stresses and strains in the midposterior sclera. CONCLUSIONS: The observed differences in the biomechanical response of normal and glaucoma sclera may represent baseline properties that contribute to axon damage, or may be characteristics that result from glaucomatous disease.

Barriers to transport induced by periodic oscillations in a physical model of the human vitreous chamber.

Understanding mixing processes that occur in the human vitreous chamber is of fundamental importance due to the relevant clinical implications in drug delivery treatments of several eye conditions. In this article we rely on experimental observations (which demonstrated that dispersion coefficients largely dominate diffusive coefficients) on a physical model of the human eye to perform an analysis based on Lagrangian trajectories. In particular, we study barriers to transport in a particularly significant two-dimensional section of the eye model by using nonlinear dynamical systems theoretical and numerical tools. Bifurcations in the system dynamics are investigated by varying the main physical parameters of the problem.

[Computer simulation of hydraulic flows in a human eye].

A two-dimensional computer model was developed to describe hydraulic flows inside the human eye. The flow field was described by coupled Navier-Stokes and Darcy equations. The velocity and pressure profiles in the chambers, the wall, and the vitreous body of the normal eye were obtained using the finite-element method. The model includes the filtration of fluid from the retinal capillary and its drainage through the choroid. The applications of this model include the investigation of the contribution of convection and diffusion to the transport of drugs and study of the kinetics of biodistribution in the eye.