Drug-Free, Nonsurgical Reduction of Intraocular Pressure for Four Months after Suprachoroidal Injection of Hyaluronic Acid Hydrogel.

Glaucoma is the leading cause of irreversible blindness. Current treatments use drugs or surgery to reduce intraocular pressure (IOP). In this study, a drug-free, nonsurgical method is developed that lowers IOP for 4 months without requiring daily patient adherence. The approach involves expanding the suprachoroidal space (SCS) of the eye with an in situ-forming hydrogel injected using a microneedle. This study tests the hypothesis that SCS expansion increases the drainage of aqueous humor from the eye via the unconventional pathway, which thereby lowers IOP. SCS injection of a commercial hyaluronic acid (HA) hydrogel reduces the IOP of normotensive rabbits for more than 1 month and an optimized HA hydrogel formulation enables IOP reduction for 4 months. Safety assessment by clinical ophthalmic examinations indicate the treatment is well tolerated. Histopathology shows minor hemorrhage and fibrosis at the site of injection. Further analysis by ultrasound biomicroscopy demonstrates a strong correlation of IOP reduction with SCS expansion. Outflow facility measurements show no difference in pressure-dependent outflow by the conventional pathway between treated and untreated eyes, supporting the hypothesis. In conclusion, SCS expansion with an in situ-forming hydrogel can enable extended IOP reduction for treating ocular hypertension and glaucoma without drugs or surgery.

Effects of General Anesthesia on Intraocular Pressure in Rabbits.

Measurement of intraocular pressure (IOP) is a standard procedure in ophthalmic research in animals, specifically in glaucoma research, and the control of IOP is essential during certain veterinary ophthalmic surgeries. We evaluated the effect of isoflurane on IOP in the clinically healthy laboratory rabbits and tested a way to minimize the alteration of IOP during isoflurane anesthesia. After measurement of the baseline IOP in each eye of 9 awake New Zealand white rabbits, animals were anesthetized by using either: (1) isoflurane without premedication, (2) a combination of ketamine and xylazine, or (3) isoflurane inhalation after an injection of ketamine-xylazine premedication. Isoflurane led to a sustained increase in IOP of approximately 12 mm Hg. In contrast, ketamine and xylazine decreased IOP by nearly 5 mm Hg (all values compared with baseline measurements in awake, unrestrained animals). The observed decrease in IOP after ketamine-xylazine anesthesia is consistent with anesthetic effects generally seen during anesthesia in other studies. The increased IOP after isoflurane anesthesia in rabbits in this study was an unexpected result that appears to be specific to this combination of anesthetic and animal species. Premedication with ketamine-xylazine diminished the effect of isoflurane inhalation on IOP. These results should be considered in the design of ophthalmic research studies using rabbits and in intraocular surgery where IOP stability is desired.

Collagenase injection into the suprachoroidal space of the eye to expand drug delivery coverage and increase posterior drug targeting.

Injection into the suprachoroidal space (SCS) allows drug delivery targeted to sclera, choroid, and retina. Here, we studied SCS injection formulated with collagenase to expand drug delivery coverage and increase posterior drug targeting within SCS by breaking down collagen fibrils that link sclera and choroid in the SCS. When 1 µm latex microparticles were injected with a collagenase formulation using microneedles into the SCS of rabbit eyes ex vivo and incubated at 37 °C for 4 h, microparticle delivery coverage increased from 20% to 45% and enhanced posterior drug targeting. Collagenase concentration was optimized to 0.5 mg/mL to maximize expanded posterior delivery and minimize tissue damage. Effects of collagenase injection within SCS increased and then plateaued 4 h after injection. Simultaneous injection of collagenase and microparticles had a greater effect on expanded delivery in the SCS compared to sequential injection. Collagenase injection into the SCS of rabbit eyes in vivo was also effective to expand delivery and was generally well-tolerated, showing transiently lowered IOP, but no apparent lasting adverse effects on ocular tissues such as sclera, choroid, and retina, as determined by analyzing histology, sclera tensile strength, and fundus imaging. We conclude that addition of collagenase during SCS injection can expand drug delivery coverage and increase posterior drug targeting.

Targeting drug delivery within the suprachoroidal space.

The suprachoroidal space (SCS), a potential anatomical space between the sclera and choroid, is a novel route for drug delivery targeting the chorioretinal layers of the eye. The safety and efficacy of SCS drug delivery have been shown in multiple clinical trials. Recent studies have developed methods for more precise targeting within the SCS at sites of action at the posterior pole (e.g., macula), near the limbus (e.g., ciliary body), and throughout the SCS using iontophoresis, swollen hydrogels, high-density particle emulsions, highly viscous and non-Newtonian fluids, and microstents. Here, we review novel technologies targeting the posterior, anterior, or entire SCS.

Nictitating membrane fixation improves stability of the contact lens on the animal corneal surface.

We evaluated the feasibility and safety of nictitating membrane fixation to address reduced contact lens stability by the nictitating membrane in a rabbit model. Under general anesthesia, twelve animals received a horizontal mattress suture between the nictitating membrane and the upper eyelid of one eye. To assess the effects of this technique and secondary side effects, contact lens stability test, Schirmer tear test, tear break-up time measurement, eye tissue pathology and morphology were evaluated. Contact lens stability was increased after nictitating membrane fixation. The percentage of contact lens retention in the nictitating membrane fixed rabbit after 4 hours was 90% whereas that in the untreated rabbit was 42.5%. In addition, there were no significant differences in tear quantity and quality between the fixed and untreated eyes. Furthermore, no remarkable pathological lesions were found in gross observation during the 1-month time period or the following pathological examination. In this study, we demonstrated that nictitating membrane fixation increases contact lens stability without specific side effects using a rabbit model. This minimally invasive procedure could be useful when designing animal models for testing new contact lenses and has potential to apply to other biomaterial research on the ocular surface.

Local delivery of a carbohydrate analog for reducing arthritic inflammation and rebuilding cartilage.

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation. Because OA has a multifactorial nature and complex interrelationship of the individual elements of a whole joint, there is a need for comprehensive therapeutic approaches for cartilage tissue engineering, which simultaneously address multiple aspects of disease etiology. In this work, we investigated a multifunctional carbohydrate-based drug candidate, tri-butanoylated N-acetyl-D-galactosamine analog (3,4,6-O-Bu3GalNAc) that induced cartilage tissue production by human mesenchymal stem cells (hMSCs) and human OA chondrocytes by modulating Wnt/ß-catenin signaling activity. The dual effects promoted chondrogenesis of human MSC and reduced inflammation of human OA chondrocytes in in vitro cultures. Translating these findings in vivo, we evaluated therapeutic effect of 3,4,6-O-Bu3GalNAc on the rat model of posttraumatic OA when delivered via local intra-articular sustained-release delivery using microparticles and found this method to be efficacious in preventing OA progression. These results show that 3,4,6-O-Bu3GalNAc, a disease modifying OA drug candidate, has promising therapeutic potential for articular cartilage repair.

Evaluation of the biocompatibility of regenerated cellulose hydrogels with high strength and transparency for ocular applications.

Prompt emergency treatment for ocular injury, particularly in a battlefield setting, is essential to preserve vision, reduce pain, and prevent secondary infection. A bandage contact lens that could be applied in the field, at the time of injury, would protect the injured ocular surface until hospital treatment is available. Cellulose, a natural polymer, is widely used in biomedical applications including bandage materials. Hydrogels synthesized from different cellulose sources, such as plants, cotton, and bacteria, can have the optical transparency and mechanical strength of contact lenses, by tailoring synthesis parameters. Thus, we optimized the fabrication of cellulose-based hydrogels and evaluated their in vivo biocompatibility and related physical properties. Our data demonstrate that along with tailorable physical properties, our novel cellulose-based hydrogels could be made with contact lens geometry, exhibit no significant signs of material toxicity after 22 days of in vivo testing, and show significant promise for use as a corneal bandage immediately following ocular trauma.

Physical and Biological Characterization of the Gamma-Irradiated Human Cornea.

PURPOSE: To compare the physical and biological characteristics of commercial gamma-irradiated corneas with those of fresh human corneas and to determine suitability for transplantation. METHODS: The physical properties of gamma-irradiated and fresh corneas were evaluated with respect to light transmittance, hydration (swelling ratio), elastic modulus (compressive modulus by the indentation method), matrix organization (differential scanning calorimetry), and morphology (light and transmission electron microscopy). The biological properties of the gamma-irradiated cornea, including residual cell content and cellular biocompatibility, were evaluated by quantifying DNA content and measuring the proliferation rate of human corneal epithelial cells, respectively. RESULTS: The hydration, light transmittance, elastic modulus, and proliferation rate of human corneal epithelial cells were not significantly different between fresh and gamma-irradiated corneas. However, differences were observed in tissue morphology, DNA content, and thermal properties. The density of collagen fibrils of the gamma-irradiated corneal sample (160.6 ± 33.2 fibrils/µm) was significantly lower than that of the fresh corneal sample (310.0 ± 44.7 fibrils/µm). Additionally, in the gamma-irradiated corneas, cell fragments-but not viable cells-were observed, supported by lower DNA content of the gamma-irradiated cornea (1.0 ± 0.1 µg/mg) than in fresh corneas (1.9 µg/mg). Moreover, the denaturation temperature of gamma-irradiated corneas (61.8 ± 1.1 °C) was significantly lower than that of fresh corneas (66.1 ± 1.9 °C). CONCLUSIONS: Despite structural changes due to irradiation, the physical and biological properties of the gamma-irradiated cornea remain similar to the fresh cornea. These factors, combined with a decreased risk of rejection and longer shelf life, make the gamma-irradiated tissue a viable and clinically desired option in various ophthalmic procedures.

Regeneration of corneal epithelium utilizing a collagen vitrigel membrane in rabbit models for corneal stromal wound and limbal stem cell deficiency.

PURPOSE: This study was performed to evaluate the potential of a collagen-based membrane, collagen vitrigel (CV), for reconstructing corneal epithelium in the stromal wound and limbal stem cell deficiency (LSCD) models. METHODS: Three groups of rabbits were used in the stromal wound model: CV affixed using fibrin glue (CV + FG group, n = 9), fibrin glue only (FG group, n = 3) and an untreated control group (n = 3). In the LSCD model, one group received CV containing human limbal epithelial cells (CV + hLEC group, n = 2) and the other was an untreated control (n = 1). Gross observation, including fluorescent staining, pathological examination, immunohistochemistry and electron microscopy, was used to evaluate the effect of CV on the corneal epithelium. RESULTS: In the stromal wound model, fluorescent staining showed that epithelial reconstruction occurred as rapidly in the CV + FG group as it did in the control group. The pathological examination proved that the CV supported a healthy corneal epithelium in the CV + FG group, whereas FG led to hypertrophy and inappropriate differentiation of corneal epithelium in the FG group. In the LSCD model, the corneas in the CV + hLEC group showed sustained tissue transparency with good epithelialization, low inflammatory response and reduced neovascularization. However, the control cornea was translucent and showed high amounts of inflammation and neovascularization. CONCLUSION: We have demonstrated that CV supports corneal epithelial differentiation and prevents epithelial hypertrophy, in addition to serving as a scaffold for hLEC transplantation, without complications.

Future perspectives for regenerative medicine in ophthalmology.

Repair and reconstruction of the cornea has historically relied on synthetic materials or tissue transplantation. However, the future holds promise for treatments using smart biomaterials and stem cells that direct tissue repair and regeneration to ultimately create new ocular structures that are indistinguishable from the original native tissue. The cornea is a remarkable engineering structure. By understanding the physical structure of the tissue and the resulting impact of the structure on biological function, we can design novel materials for a number of ophthalmic clinical applications. Furthermore, by extending this structure-function approach to characterizing corneal disease processes, new therapies can be engineered.