Treatment of many posterior-segment ocular indications would benefit from improved targeting of drug delivery to the back of the eye. Here, we propose the use of iontophoresis to direct delivery of negatively charged nanoparticles through the suprachoroidal space (SCS) toward the posterior pole of the eye. Injection of nanoparticles into the SCS of the rabbit eye ex vivo without iontophoresis led to a nanoparticle distribution mostly localized at the site of injection near the limbus and <15% of nanoparticles delivered to the most posterior region of SCS (>9 mm from the limbus). Iontophoresis using a novel microneedle-based device increased posterior targeting with >30% of nanoparticles in the most posterior region of SCS. Posterior targeting increased with increasing iontophoresis current and increasing application time up to 3 min, but further increasing to 5 min was not better, probably due to the observed collapse of the SCS within 5 min after injection ex vivo. Reversing the direction of iontophoretic flow inhibited posterior targeting, with just ~5% of nanoparticles reaching the most posterior region of SCS. In the rabbit eye in vivo, iontophoresis at 0.14 mA for 3 min after injection of a 100 μL suspension of nanoparticles resulted in ~30% of nanoparticles delivered to the most posterior region of the SCS, which was consistent with ex vivo findings. The procedure was well tolerated, with only mild, transient tissue effects at the site of injection. We conclude that iontophoresis in the SCS using a microneedle has promise as a method to target ocular drug delivery within the eye, especially toward the posterior pole.
PURPOSE. Rodent and primate models are commonly used in glaucoma research; however, both have their limitations. The tree shrew (Tupaia belangeri) is an emerging animal model for glaucoma research owing in part to having a human-like optic nerve head anatomy, specifically a collagenous load-bearing lamina. However, the anterior segment anatomy and function have not been extensively studied in the tree shrew. Thus, the purpose of this study was to provide the first detailed examination of the anterior segment anatomy and aqueous outflow facility in the tree shrew. METHODS. Aqueous outflow dynamics were measured in five ostensibly normal eyes from three tree shrews using the iPerfusion system over a range of pressures. Gross histological assessment and immunohistochemistry were performed to characterize anterior segment anatomy and to localize several key molecules related to aqueous outflow. RESULTS. Anterior segment anatomy in tree shrews is similar to humans, demonstrating a scleral spur, a multilayered trabecular meshwork and a circular Schlemm's canal with a single lumen. Average outflow facility was 0.193 μL/min/mm Hg (95% confidence interval, 0.153-0.244), and was stable over time. Outflow facility was more similar between contralateral eyes (approximately 5% average difference) than between eyes of different animals. No significant dependence of outflow facility on time or pressure was detected (pressure-flow nonlinearity parameter of 0.01 (95% % confidence interval, -0.29 to 0.31 CI μL/min/mm Hg). CONCLUSIONS. These studies lend support to the usefulness of the tree shrew as a novel animal model in anterior segment glaucoma and pharmacology research. The tree shrew's cost, load-bearing collagenous lamina cribrosa, and lack of washout or anterior chamber deepening provides a distinct experimental and anatomic advantage over the current rodent and nonhuman primate models used for translational research.
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Baptiste Coudrillier;
Diogo M. Geraldes;
Nghia T. Vo;
Robert Atwood;
Christina Reinhard;
Ian C. Campbell;
Yazdan Raji;
Julie Albon;
Richard L. Abel;
Christopher Ethier
The lamina cribrosa (LC) is a complex mesh-like tissue in the posterior eye. Its biomechanical environment is thought to play a major role in glaucoma, the second most common cause of blindness. Due to its small size and relative inaccessibility, high-resolution measurements of LC deformation, important in characterizing LC biomechanics, are challenging. Here we present a novel noninvasive imaging method, which enables measurement of the three-dimensional deformation of the LC caused by acute elevation of intraocular pressure (IOP). Posterior segments of porcine eyes were imaged using synchrotron radiation phase contrast micro-computed tomography (PC μCT) at IOPs between 6 and 37 mmHg. The complex trabecular architecture of the LC was reconstructed with an isotropic spatial resolution of 3.2 μm. Scans acquired at different IOPs were analyzed with digital volume correlation (DVC) to compute full-field deformation within the LC. IOP elevation caused substantial tensile, shearing and compressive devformation within the LC, with maximum tensile strains at 30 mmHg averaging 5.5%, and compressive strains reaching 20%. We conclude that PC μCT provides a novel high-resolution method for imaging the LC, and when combined with DVC, allows for full-field 3D measurement of ex vivo LC biomechanics at high spatial resolution.
It has been suggested that common mechanisms may underlie the pathogenesis of primary open-angle glaucoma (POAG) and steroid-induced glaucoma (SIG). The biomechanical properties (stiffness) of the trabecular meshwork (TM) have been shown to differ between POAG patients and unaffected individuals. While features such as ocular hypertension and increased outflow resistance in POAG and SIG have been replicated in mouse models, whether changes of TM stiffness contributes to altered IOP homeostasis remains unknown. We found that outer TM was stiffer than the inner TM and, there was a significant positive correlation between outflow resistance and TM stiffness in mice where conditions are well controlled. This suggests that TM stiffness is intimately involved in establishing outflow resistance, motivating further studies to investigate factors underlying TM biomechanical property regulation. Such factors may play a role in the pathophysiology of ocular hypertension. Additionally, this finding may imply that manipulating TM may be a promising approach to restore normal outflow dynamics in glauco ma. Further, novel technologies are being developed to measure ocular tissue stiffness in situ. Thus, the changes of TM stiffness might be a surrogate marker to help in diagnosing altered conventional outflow pathway function if those technologies could be adapted to TM.
PURPOSE. The sclera is believed to biomechanically influence eye size, facilitating the excessive axial elongation that occurs during myopigenesis. Here, we test the hypothesis that the sclera will be remodeled and exhibit altered biomechanics in the mouse model of form-deprivation (FD) myopia, accompanied by altered retinoid concentrations, a potential signaling molecule involved in the process. METHODS. Male C57 Bl/6J mice were subjected to unilateral FD (n = 44 eyes), leaving the contralateral eye untreated (contra; n = 44). Refractive error and ocular biometry were measured in vivo prior to and after 1 or 3 weeks of FD. Ex vivo measurements were made of scleral biomechanical properties (unconfined compression: n = 24), scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 18, and immunohistochemistry: n = 22), and ocular all-trans retinoic acid (atRA) concentrations (retina and RPE + choroid + sclera, n = 24). Age-matched naïve controls were included for some outcomes (n = 32 eyes). RESULTS. Significant myopia developed after 1 (−2.4 ± 1.1 diopters [D], P < 0.001) and 3 weeks of FD (−4.1 ± 0.7 D, P = 0.025; mean ± standard deviation). Scleral tensile stiffness and permeability were significantly altered during myopigenesis (stiffness = −31.4 ± 12.7%, P < 0.001, and permeability = 224.4 ± 205.5%, P < 0.001). Total scleral sGAG content was not measurably altered; however, immunohistochemistry indicated a sustained decrease in chondroitin-4-sulfate and a slower decline in dermatan sulfate. The atRA increased in the retinas of eyes form-deprived for 1 week. CONCLUSIONS. We report that biomechanics and GAG content of the mouse sclera are altered during myopigenesis. All scleral outcomes generally follow the trends found in other species and support a retina-to-sclera signaling cascade underlying mouse myopigenesis.
Diffuse correlation spectroscopy (DCS) is an optical modality used to measure an index of blood flow in biological tissue. This blood flow index depends on both the red blood cell flow rate and density (i.e., hematocrit), although the functional form of hematocrit dependence is not well delineated. Herein, we develop and validate a novel tissue-simulating phantom containing hundreds of microchannels to investigate the influence of hematocrit on blood flow index. For a fixed flow rate, we demonstrate a significant inverse relationship between hematocrit and blood flow index that must be accounted for to accurately estimate blood flow under anemic conditions.
PURPOSE: This study tested the hypothesis that highly targeted intrastromal delivery of bevacizumab using coated microneedles allows dramatic dose sparing compared with subconjunctival and topical delivery for treatment of corneal neovascularization.
METHODS: Stainless steel microneedles 400 μm in length were coated with bevacizumab. A silk suture was placed in the cornea approximately 1 mm from the limbus to induce corneal neovascularization in the eyes of New Zealand white rabbits that were divided into different groups: untreated, microneedle delivery, topical eye drop, and subconjunctival injection of bevacizumab. All drug treatments were initiated 4 days after suture placement and area of neovascularization was measured daily by digital photography for 18 days.
RESULTS: Eyes treated once with 4.4 μg bevacizumab using microneedles reduced neovascularization compared with untreated eyes by 44% (day 18). Eyes treated once with 2500 μg bevacizumab using subconjunctival injection gave similar results to microneedle-treated eyes. Eyes treated once with 4.4 μg subconjunctival bevacizumab showed no significant effect compared with untreated eyes. Eyes treated with 52,500 μg bevacizumab by eye drops three times per day for 14 days reduced the neovascularization area compared with untreated eyes by 6% (day 18), which was significantly less effective than the single microneedle treatment. Visual exam and histological analysis showed no observable effect of microneedle treatment on corneal transparency or microanatomical structure.
CONCLUSIONS: This study shows that microneedles can target drug delivery to corneal stroma in a minimally invasive way and demonstrates effective suppression of corneal neovascularization after suture-induced injury using a much lower dose compared with conventional methods.
Although effective drugs that lower intraocular pressure (IOP) in the management of glaucoma exist, their efficacy is limited by poor patient adherence to the prescribed eye drop regimen. To replace the need for eye drops, in this study we tested the hypothesis that IOP can be reduced for one month after a single targeted injection using a microneedle for administration of a glaucoma medication (i.e., brimonidine) formulated for sustained release in the supraciliary space of the eye adjacent to the drug's site of action at the ciliary body. To test this hypothesis, brimonidine-loaded microspheres were formulated using poly(lactic acid) (PLA) to release brimonidine at a constant rate for 35 days and microneedles were designed to penetrate through the sclera, without penetrating into the choroid/retina, in order to target injection into the supraciliary space. A single administration of these microspheres using a hollow microneedle was performed in the eye of New Zealand White rabbits and was found to reduce IOP initially by 6 mm Hg and then by progressively smaller amounts for more than one month. All administrations were well tolerated without significant adverse events, although histological examination showed a foreign-body reaction to the microspheres. This study demonstrates, for the first time, that the highly-targeted delivery of brimonidine-loaded microspheres into the supraciliary space using a microneedle is able to reduce IOP for one month as an alternative to daily eye drops.
We introduce Airy-beam tomographic microscopy (ATM) for high-resolution, volumetric, inertia-free imaging of biological specimens. The work exploits the highly adjustable Airy trajectories in the 3D space, transforming the conventional telecentric wide-field imaging scheme that requires sample or focal-plane scanning to acquire 3D information. The results present a consistent near-diffraction-limited 3D resolution across a tenfold extended imaging depth compared to widefield microscopy. We anticipate the strategy to not only offer a promising paradigm for 3D optical microscopy, but also be translated to other non-optical waveforms.
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Darryl R. Overby;
Enhua H. Zhou;
Rocio Vargas-Pinto;
Ryan M. Pedrigi;
Rudolf Fuchshofer;
Sietse T. Braakman;
Ritika Gupta;
Kristin M. Perkumas;
Joseph M. Sherwood;
Amir Vahabikashi;
Dang Quynh;
Jae Hun Kim;
Ross Ethier;
W. Daniel Stamer;
Jeffrey J. Fredberg;
Mark Johnson
Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm's canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes. Here we establish the existence of a reduced tendency for pore formation in the glaucomatous SC cell-likely accounting for increased outflow resistance-that positively correlates with elevated subcortical cell stiffness, along with an enhanced sensitivity to the mechanical microenvironment including altered expression of several key genes, particularly connective tissue growth factor. Rather than being seen as a simple mechanical barrier to filtration, the endothelium of SC is seen instead as a dynamic material whose response to mechanical strain leads to pore formation and thereby modulates the resistance to aqueous humor outflow. In the glaucomatous eye, this process becomes impaired. Together, these observations support the idea of SC cell stiffness-and its biomechanical effects on pore formation-as a therapeutic target in glaucoma.