Improvement in Accommodation and Dynamic Range of Focus After Laser Scleral Microporation: A Potential Treatment for Presbyopia.

Purpose: To examine the ocular changes in accommodation, wavefront aberrations, and dynamic range of focus (DROF) after laser scleral microporation (LSM) for treating presbyopia. Methods: Four presbyopic aged cynomolgus macaques (>13 years; n = 8 eyes) were included. All eyes received LSM with erbium: yttrium-aluminum-garnet laser. Spherical equivalent, true accommodation, pseudo-accommodation, wavefront aberrations, and extended range of focus (EROF), or collectively known as DROF, were evaluated using a ray tracing aberrometer. True accommodation referred to the difference in spherical equivalent between distance and near vision, whereas EROF (sum of true and pseudo-accommodation) was determined by measuring the difference in diopters (D) between near and distance through-focus curves, at 50% threshold of the visual Strehl ratio of optical transfer function. Results: From before to seven months after surgery, there was a significant increase in true accommodation from 0.6 ± 1.0 D before surgery to 5.9 ± 2.8 D at seven months after surgery (P < 0.001). EROF increased significantly from 3.4 ± 1.0 D before surgery to 11.1 ± 4.6 D at seven months after surgery (P < 0.001). Ocular aberrations did not vary significantly between preoperative and various postoperative timepoints in either disaccommodated or accommodated states (P > 0.05). No adverse event such as scleral perforation or hypotony was noted. Conclusions: This non-human primate study demonstrated that LSM serves as a novel therapy for improving accommodation and DROF function biomechanically, with a positive response observed throughout the seven-month postoperative period. Translational Relevance: This proof-of-concept study highlights the potential of LSM as a novel treatment for vision recovery in presbyopic eyes.

Safety profiles of terahertz scanning in ophthalmology.

Terahertz (THz) technology has emerged recently as a potential novel imaging modality in biomedical fields, including ophthalmology. However, the ocular biological responses after THz electromagnetic exposure have not been investigated. We conducted a rabbit study to evaluate the safety profiles of THz scanning on eyes, at a tissue, cellular, structural and functional level. Eight animals (16 eyes) were analysed after excessive THz exposure (control, 1 h, 4 h, and 1 week after continuous 4-h exposure; THz frequency = 0.3 THz with continuous pulse generated at 40 µW). We found that at all the time points, the corneas and lens remained clear with no corneal haze or lens opacity formation clinically and histopathologically. No thermal effect, assessed by thermographer, was observed. The rod and cone cell-mediated electroretinography responses were not significantly altered, and the corneal keratocytes activity as well as endothelial viability, assessed by in-vivo confocal microscopy, was not affected. Post-exposed corneas, lens and retinas exhibited no significant changes in the mRNA expression of heat shock protein (HSP)90AB1), DNA damage inducible transcript 3 (DDIT3), and early growth response (EGR)1. These tissues were also negative for the inflammatory (CD11b), fibrotic (fibronectin and α-smooth muscle actin), stress (HSP-47) and apoptotic (TUNEL assay) responses on the immunohistochemical analyses. The optical transmittance of corneas did not change significantly, and the inter-fibrillar distances of the corneal stroma evaluated with transmission electron microscopy were not significantly altered after THz exposure. These results provide the basis for future research work on the development of THz imaging system for its application in ophthalmology.

Tissue Responses and Wound Healing following Laser Scleral Microporation for Presbyopia Therapy.

Purpose: To investigate the postoperative inflammatory and wound-healing responses after laser scleral microporation for presbyopia. Methods: Thirty porcine eyes were used for the optimization of laser intensities first. Six monkeys (12 eyes) received scleral microporation with an erbium yttrium aluminum garnet (Er:YAG) laser, and half of the eyes received concurrent subconjunctival collagen gel to modulate wound-healing response. The intraocular pressure (IOP) and the laser ablation depth were evaluated. The animals were euthanized at 1, 6, and 9 months postoperatively. The limbal areas and scleras were harvested for histologic analysis and immunofluorescence of markers for inflammation (CD11b and CD45), wound healing (CD90, tenascin-C, fibronectin, and HSP47), wound contraction (α-smooth muscle actin [α-SMA]), vascular response (CD31), nerve injury (GAP43), and limbal stem cells (P63 and telomerase). Results: In the nonhuman primate study, there was a significant reduction in IOP after the procedure. Overall, the ablation depth was 76.6% to 81.2% at 1 month and slightly decreased to 71.5% to 72.7% at 9 months. Coagulative necrosis around the micropores, as well as expression of CD11b, CD45, tenascin, fibronectin, HSP47, and GAP43, was distinct at 1 month but subsided with time. Collagen gel treatment significantly suppressed the upregulation of CD11b, CD45, fibronectin, and tenascin-C. The expression of CD90, α-SMA, and CD31 was minimal in all eyes. Conclusions: The study demonstrated the course of inflammatory and wound-healing responses following laser scleral microporation. The tissue responses were small and self-limited, resolved with time, and were suppressed by concurrent collagen treatment. It provides a useful understanding of this new procedure. Translational Relevance: The results would be helpful in the laser parameter modification to improve the long-term treatment stability.

Safety and Feasibility of Intrastromal Injection of Cultivated Human Corneal Stromal Keratocytes as Cell-Based Therapy for Corneal Opacities.

Purpose: To evaluate the safety and feasibility of intrastromal injection of human corneal stromal keratocytes (CSKs) and its therapeutic effect on a rodent early corneal opacity model. Methods: Twelve research-grade donor corneas were used in primary culture to generate quiescent CSKs and activated stromal fibroblasts (SFs). Single and repeated intrastromal injections of 2 to 4 × 104 cells to rat normal corneas (n = 52) or corneas with early opacities induced by irregular phototherapeutic keratectomy (n = 16) were performed, followed by weekly examination of corneal response under slit-lamp biomicroscopy and in vivo confocal microscopy with evaluation of haze level and stromal reflectivity, and corneal thickness using anterior segment optical coherence tomography (AS-OCT). Time-lapse tracing of Molday ION-labelled cells was conducted using Spectralis OCT and label intensity was measured. Corneas were collected at time intervals for marker expression by immunofluorescence, cell viability, and apoptosis assays. Results: Injected CSKs showed proper marker expression with negligible SF-related features and inflammation, hence maintaining corneal clarity and stability. The time-dependent loss of injected cells was recovered by repeated injection, achieving an extended expression of human proteoglycans inside rat stroma. In the early corneal opacity model, intrastromal CSK injection reduced stromal reflectivity and thickness, resulting in recovery of corneal clarity, whereas noninjected corneas were thicker and had haze progression. Conclusions: We demonstrated the safety, feasibility, and therapeutic efficacy of intrastromal CSK injection. The cultivated CSKs can be a reliable cell source for potential cell-based therapy for corneal opacities.

Higher-Order-Aberrations Following Hyperopia Treatment: Small Incision Lenticule Extraction, Laser-Assisted In Situ Keratomileusis and Lenticule Implantation.

PURPOSE: To compare the postoperative higher-order-aberrations (HOAs) after hyperopic small incision lenticule extraction (SMILE), hyperopic laser-assisted in situ keratomileusis (LASIK), and lenticule implantation for correction of hyperopia. METHODS: Eighteen monkeys were divided to six groups: +2.00 D and +4.00 D hyperopic SMILE, +2.00 D and +4.00 D hyperopic LASIK (n = 6 eyes for each), and lenticule implantation with a -2.00 D and -4.00 D lenticule (n = 3 eyes for each). The corneal HOAs were evaluated preoperatively and 3-month postoperatively. RESULTS: At 3-month postoperatively, the spherical aberrations significantly increased toward negative direction in all +4.00 D groups (all P < 0.05). There was a significant change toward more negative values in the third-order vertical coma in the SMILE +4.00 D and LASIK +4.00 D groups (P = 0.026 and P = 0.036, respectively). There were also significant changes in the third-order horizontal trefoil (P = 0.034) and oblique secondary astigmatism (P = 0.012) in the LASIK +4.00 D group. In the eyes that underwent +4.00 D lenticule implantation, the fourth-order horizontal quatrefoil significantly increased (P = 0.029). In low hyperopia correction (+2.00 D), treatment with lenticule implantation tended to have less changes in HOAs, compared to the other two groups. CONCLUSIONS: In hyperopic SMILE, hyperopic LASIK or lenticule implantation surgery, significant induction of third- and fourth-order HOAs were seen in moderate hyperopia correction but not in low hyperopia correction. In low hyperopia treatment, lenticule implantation might offer a favorable trend in the aspect of HOAs. TRANSLATIONAL RELEVANCE: The results provided the knowledge of surgically induced HOAs and understanding of the effects of surgery in different types of hyperopic correction.

A Biodegradable, Sustained-Released, Tacrolimus Microfilm Drug Delivery System for the Management of Allergic Conjunctivitis in a Mouse Model.

Purpose: To investigate the drug release profiles of a tacrolimus-loaded poly(D,L-lactide-co-ε-caprolactone) (PLC) microfilm, and to evaluate its efficacy on the treatment of allergic conjunctivitis using a mouse model. Methods: The in vitro and in vivo drug release profiles were first characterized. Balb/c mice were immunized with short ragweed (SRW) injection followed by re-challenges with topical SRW solution. The mice were divided into six groups (n = 12 in each): negative control (NC); positive control (PC); tacrolimus eye drops (Te); subconjunctival tacrolimus microfilm (Tm); dexamethasone eye drops (De); and tacrolimus + dexamethasone eye drops (Te+De). The mice were evaluated for 28 days by a scoring system for allergic conjunctivitis. Histopathologic and immunohistochemical staining with CD11c, CD4, and IL-4 were performed. Results: The microfilms were biocompatible and delivered clinically sufficient dose in a sustained manner, with a steady rate of 0.212 to 0.243 µg/day in vivo. Compared to the PC groups, the Te, Tm, De, and Te+De groups significantly reduced the allergic clinical scores throughout the study period (all P < 0.01; 0.0 ± 0.0, 5.6 ± 0.9, 3.3 ± 0.9, 3.2 ± 0.9, 1.9 ± 0.4 and 1.7 ± 0.8 for the NC, PC, Tm, Te, De, and Te+De groups, respectively, at 4 weeks after treatment). The suppressed eosinophils, CD11c, CD4, and IL-4 expression were also observed in all treatment groups, with more reduction in the Te+De group. Conclusions: Tacrolimus-loaded microfilms display good biocompatibility and desirable sustained drug release. It was as effective as conventional tacrolimus eye drops on the treatment of allergic conjunctivitis, providing a promising clinically applicable alternative for controlling allergic disease activity, or other immune-mediated ocular diseases.

Biological corneal inlay for presbyopia derived from small incision lenticule extraction (SMILE).

Corneal inlays are a relatively new treatment option for presbyopia. Using biological inlays, derived from lenticules extracted from small incision lenticule extraction, may offer advantages over commercialized synthetic inlays in the aspect of biocompatibility. We conducted a non-human primate study to evaluate the safety, predictability, efficacy and tissue response after autogeneic, decellularized xenogeneic and xenogeneic lenticule implantation. The lenticule implantation effectively resulted in central corneal steepening (simulated keratometric values increased by 1.8-2.3 diopters), central hyper-prolate changes (asphericity Q values changed by -0.26 to -0.36), corneal anterior surface elevation (7.7-9.3 µm) and reasonable effective zone (1.5-1.8 times of the lenticule physical diameter), with no differences among the three groups. Slit lamp microscopy, transmission electron microscopy, confocal microscopy, histology and immunohistochemistry analyses confirmed the biocompatibility of the autogeneic and decellularized lenticules, whereas one eye in the xenogeneic group developed corneal stromal rejection during the study period. Our results showed that lenticule implantation has the potential for the management of presbyopia, and provide the basis for future clinical studies. The decellularization process may increase the potential utilization of lenticules without changing the efficacy.

Early wound healing and refractive response of different pocket configurations following presbyopic inlay implantation.

BACKGROUND: Presbyopic inlays have mostly been implanted under a corneal flap. Implantation in a pocket has advantages including less postoperative dry eye and neurotrophic effect, and better biomechanical corneal stability. This study investigated the effect of different pocket and flocket dimensions on corneal stability and refractive power after Raindrop™ implantation, and the associated wound healing response. METHODOLOGY: Ten New Zealand White rabbits had bilateral pocket Raindrop™ implantation. Eyes were allocated to 4 groups: pockets with 4mm, 6mm, and 8mm diameters, and 8mm flocket. They were examined pre-operatively, at day 1, weeks 1, 2, 3 and 4 post-surgery with anterior segment optical coherence tomography, corneal topography and in-vivo confocal microscopy. After euthanasia (week 4), CD11b, heat shock protein (HSP) 47 and fibronectin corneal immunohistochemistry was performed. RESULTS: Corneal thickness (mean±SD) increased from 360.0±16.2µm pre-operatively to 383.9±32.5, 409.4±79.3, 393.6±35.2, 396.4±50.7 and 405±20.3µm on day 1, weeks 1,2,3 and 4 respectively (p<0.008, all time-points). Corneal refractive power increased by 11.1±5.5, 7.5±2.5, 7.5±3.1, 7.0±3.6 and 6.3±2.9D (p<0.001). Corneal astigmatism increased from 1.1±0.3D to 2.3±1.6, 1.7±0.7, 1.8±1.0, 1.6±0.9 and 1.6±0.9D respectively (p = 0.033). CT, refractive power change and astigmatism were not different between groups. The 8mm pocket and 8mm flocket groups had the least stromal keratocyte reflectivity. CD11b, fibronectin or HSP47 weren't detected. CONCLUSIONS: Anatomical and refractive stability was achieved by 1 week; the outcomes were not affected by pocket or flocket configuration. No scarring or inflammation was identified. The 8mm pocket and flocket showed the least keratocyte activation, suggesting they might be the preferred configuration.

Altered Anterior Segment Biometric Parameters in Mice Deficient in SPARC.

Purpose: Secreted protein acidic and rich in cysteine (SPARC) and Hevin are structurally related matricellular proteins involved in extracellular matrix assembly. In this study, we compared the anterior chamber biometric parameters and iris collagen properties in SPARC-, Hevin- and SPARC-/Hevin-null with wild-type (WT) mice. Methods: The right eyes of 53 WT, 35 SPARC-, 56 Hevin-, and 63 SPARC-/Hevin-null mice were imaged using the RTVue-100 Fourier-domain optical coherence tomography system. The parameters measured were anterior chamber depth (ACD), trabecular-iris space area (TISA), angle opening distance (AOD), and pupil diameter. Biometric data were analyzed using analysis of covariance and adjusted for age, sex, and pupil diameter. Expression of Col1a1, Col8a1, and Col8a2 transcripts in the irises was measured by quantitative polymerase chain reaction. Collagen fibril thickness was evaluated by transmission electron microscopy. Results: Mice that were SPARC- and SPARC-/Hevin-null had 1.28- and 1.25-fold deeper ACD, 1.45- and 1.53-fold larger TISA, as well as 1.42- and 1.51-fold wider AOD than WT, respectively. These measurements were not significantly different between SPARC- and SPARC-/Hevin-null mice. The SPARC-null iris expressed lower Col1a1, but higher Col8a1 and Col8a2 transcripts compared with WT. Collagen fibrils in the SPARC- and SPARC-/Hevin-null irises were 1.5- and 1.7-fold thinner than WT, respectively. The Hevin-null iris did not differ from WT in these collagen properties. Conclusions: SPARC-null mice have deeper anterior chamber as well as wider drainage angles compared with WT. Therefore, SPARC plays a key role in influencing the spatial organization of the anterior segment, potentially via modulation of collagen properties, while Hevin is not likely to be involved.

Sequentially-crosslinked bioactive hydrogels as nano-patterned substrates with customizable stiffness and degradation for corneal tissue engineering applications.

Naturally-bioactive hydrogels like gelatin provide favorable properties for tissue-engineering but lack sufficient mechanical strength for use as implantable tissue engineering substrates. Complex fabrication or multi-component additives can improve material strength, but often compromises other properties. Studies have shown gelatin methacrylate (GelMA) as a bioactive hydrogel with diverse tissue growth applications. We hypothesize that, with suitable material modifications, GelMA could be employed for growth and implantation of tissue-engineered human corneal endothelial cell (HCEC) monolayer. Tissue-engineered HCEC monolayer could potentially be used to treat corneal blindness due to corneal endothelium dysfunction. Here, we exploited a sequential hybrid (physical followed by UV) crosslinking to create an improved material, named as GelMA+, with over 8-fold increase in mechanical strength as compared to regular GelMA. The presence of physical associations increased the subsequent UV-crosslinking efficiency resulting in robust materials able to withstand standard endothelium insertion surgical device loading. Favorable biodegradation kinetics were also measured in vitro and in vivo. We achieved hydrogels patterning with nano-scale resolution by use of oxygen impermeable stamps that overcome the limitations of PDMS based molding processes. Primary HCEC monolayers grown on GelMA+ carrier patterned with pillars of optimal dimension demonstrated improved zona-occludin-1 expression, higher cell density and cell size homogeneity, which are indications of functionally-superior transplantable monolayers. The hybrid crosslinking and fabrication approach offers potential utility for development of implantable tissue-engineered cell-carrier constructs with enhanced bio-functional properties.

Wound healing profiles of hyperopic-small incision lenticule extraction (SMILE).

Refractive surgical treatment of hyperopia still remains a challenge for refractive surgeons. A new nomogram of small incision lenticule extraction (SMILE) procedure has recently been developed for the treatment of hyperopia. In the present study, we aimed to evaluate the wound healing and inflammatory responses of this new nomogram (hyperopic-SMILE), and compared them to those of hyperopic-laser-assisted in situ keratomileusis (LASIK), using a rabbit model. A total of 26 rabbits were used, and slit lamp biomicroscopy, autorefractor/keratometer, intraocular pressure measurement, anterior segment optical coherence tomography, corneal topography, and in vivo confocal microscopy examinations were performed during the study period of 4 weeks. The corneas were then harvested and subject to immunofluorescence of markers for inflammation (CD11b), wound healing (fibronectin) and keratocyte response (HSP47). The lenticule ultrastructual changes were also analyzed by transmission electron microscopy. Out results showed that hyperopic-SMILE effectively steepened the cornea. Compared to hyperopic-LASIK, hyperopic-SMILE had less postoperative wound healing response and stromal interface reaction, especially in higher refractive correction. However, compared to myopic-SMILE, hyperopic-SMILE resulted in more central deranged collagen fibrils. These results provide more perspective into this new treatment option for hyperopia, and evidence for future laser nomogram modification.

Evaluation of a Micro-Optical Coherence Tomography for the Corneal Endothelium in an Animal Model.

Recent developments in optical coherence tomography (OCT) systems for the cornea have limited resolution or acquisition speed. In this study we aim to evaluate the use of a 'micro-OCT' (µOCT ~1 µm axial resolution) compared to existing imaging modalities using animal models of corneal endothelial disease. We used established cryoinjury and bullous keratopathy models in Sprague Dawley rats comparing ex vivo µOCT imaging in normal and diseased eyes to (1) histology; (2) in vivo confocal microscopy (IVCM); and (3) scanning electron microscopy (SEM). Qualitative and quantitative comparisons amongst imaging modalities were performed using mean endothelial cell circularity [(4π × Area)/Perimeter(2)] with coefficient of variation (COV). We found that µOCT imaging was able to delineate endothelial cells (with nuclei), detect inflammatory cells, and corneal layers with histology-like resolution, comparable to existing imaging modalities. The mean endothelial cell circularity score was 0.88 ± 0.03, 0.87 ± 0.04 and 0.88 ± 0.05 (P = 0.216) for the SEM, IVCM and µOCT respectively, with SEM producing homogenous endothelial cell images (COV = 0.028) compared to the IVCM (0.051) and µOCT (0.062). In summary, our preliminary study suggests that the µOCT may be useful for achieving non-contact, histology-like images of the cornea for endothelial cell evaluation, which requires further development for in vivo imaging.

Early Corneal Wound Healing and Inflammatory Responses After SMILE: Comparison of the Effects of Different Refractive Corrections and Surgical Experiences.

PURPOSE: To investigate the early corneal wound healing and inflammatory responses after small incision lenticule extraction (SMILE) with different power of corrections and surgical experiences using a rabbit model. METHODS: Twenty-four rabbits underwent SMILE with -2.00, -4.00, and -8.00 diopters (D) correction. One eye of each rabbit was operated on by surgeon 1 (experienced) and the contralateral eye was operated on by surgeon 2 (novice). Slit-lamp examination, anterior segment optical coherence tomography (AS-OCT), and in vivo confocal microscopy were performed at 1 day and 1 week postoperatively. The corneas were harvested for immunofluorescence of markers for inflammation (CD11b), wound healing (fibronectin), and keratocyte response (HSP47). RESULTS: All corneas appeared clear throughout the follow-up period. In vivo confocal microscopy showed a greater reflectivity after -8.00 D than -2.00 D correction at day 1 at the lenticule anterior, posterior, and extracted lenticule planes (surgeon 1: P = .004, .041, and .038; surgeon 2: P = .012, .045, and .031). Different refractive corrections did not significantly affect the expression of CD11b, fibronectin, and HSP47. In the -2.00 D group, eyes operated on by surgeon 2 had thicker central corneal thickness evaluated by AS-OCT (P = .049) and exhibited more CD11b- and HSP47-positive cells at day 1 at the small vertical incision (P = .039 and .042). CONCLUSIONS: The early inflammatory and wound healing responses after SMILE were minimal. In the early postoperative period, less surgical experience resulted in an increased inflammatory response in low myopic corrections. Greater keratocyte response was seen in high myopic corrections irrespective of surgeon experience. [J Refract Surg. 2016;32(5):346-353.].

Biofilms of Pathogenic Nontuberculous Mycobacteria Targeted by New Therapeutic Approaches.

Microbial infections of the cornea are potentially devastating and can result in permanent visual loss or require vision-rescuing surgery. In recent years, there has been an increasing number of reports on nontuberculous mycobacterial infections of the cornea. Challenges to the management of nontuberculous mycobacterial keratitis include delayed laboratory detection, low index of clinical suspicion, poor drug penetration, slow response to therapy, and prolonged use of antibiotic combinations. The ability of nontuberculous mycobacteria to evade the host immune response and the ability to adhere and to form biofilms on biological and synthetic substrates contribute to the issue. Therefore, there is an urgent need for new antimicrobial compounds that can overcome these problems. In this study, we evaluated the biofilm architectures for Mycobacterium chelonae and Mycobacterium fortuitum in dynamic flow cell chamber and 8-well chamber slide models. Our results showed that mycobacterial biofilms were quite resistant to conventional antibiotics. However, DNase treatment could be used to overcome biofilm resistance. Moreover, we successfully evaluated a new antimicrobial compound (AM-228) that was effective not only for planktonic mycobacterial cells but also for biofilm treatment and was compared favorably with the most successful "fourth-generation" fluoroquinolone, gatifloxacin. Finally, a new treatment strategy emerged: a combination of DNase with an antibiotic was more effective than an antibiotic alone.

Effect of Intraoperative Corneal Stromal Pocket Irrigation in Small Incision Lenticule Extraction.

This study aimed at evaluating the effect of intraoperative corneal pocket irrigation in small incision lenticule extraction (SMILE) and compares it to that in femtosecond laser-assisted in situ keratomileusis (FS-LASIK). Sixteen rabbit eyes underwent a SMILE procedure, with 8 eyes having corneal pocket irrigation, while the other 8 eyes were without irrigation. Another 16 eyes underwent a FS-LASIK procedure for comparison, with 8 eyes having flap irrigation, while the other 8 eyes were without irrigation. The results showed that the changes in the total corneal thickness, anterior and posterior lamellar thickness, measured by the anterior segment optical coherence tomography, were comparable between the SMILE with and without irrigation groups, suggesting that the irrigation did not lead to significant changes in the corneal thickness. However, at postoperative 8 hours, in vivo confocal microscopy showed that the interface reflectivity in the SMILE with irrigation group was significantly higher than that in other three groups. The presence of interface fluid was further confirmed by the identification of fluid pockets with undulated collagen shown on histological section in the post-SMILE with irrigation eyes. Our findings might contribute to the occurrence of post-SMILE delayed immediate visual quality recovery and further clinical study is required.

A biodegradable ocular implant for long-term suppression of intraocular pressure.

Timolol maleate (TM) has been used for many years for the reduction of intraocular pressure (IOP) in glaucoma patients. However, the topical mode of administration (eyedrops) is far from optimal because of the issues of low bioavailability, high drug wastage, and lack of patient compliance. Suboptimal control of the IOP leads to disease progression and eventually to blindness. Ideally, TM is delivered to the patient so that its action is both localized and sustained for 3 months or more. In this work, we developed a subconjunctival TM microfilm for sustained, long-term delivery of TM to the eyes, using the biodegradable elastomer poly(lactide-co-caprolactone) (PLC). The copolymer is biocompatible and has flexibility and mechanical characteristics suitable for a patient-acceptable implant. Controlling the release of TM for 3 months is challenging, and this work describes how, by using a combination of multilayering and blending with poly(ethylene glycol) (PEG) copolymers, we were able to develop a TM-incorporated biodegradable film that can deliver TM at a therapeutic dose for 90 days in vitro. The data was further confirmed in a diseased primate model, with sustained IOP-lowering effects for 5 months with a single implant, with acceptable biocompatibility and partial degradation.

Use of anterior segment optical coherence tomography to predict corneal graft rejection in small animal models.

PURPOSE: To correlate the degree of anterior chamber (AC) inflammation and corneal thickness evaluated by anterior segment optical coherence tomography (ASOCT) with corneal graft rejection status and to explore the value of ASOCT in assisting the diagnosis or prediction of graft rejection using a rat penetrating keratoplasty (PK) model. METHODS: A total of 40 PKs were performed using Fisher rats (allogeneic groups) and Lewis rats (syngeneic group) as donors and Lewis rats as recipients: isograft control group (n=10), allograft untreated group (n=10), and allograft with 1% prednisolone acetate treatment group (n=20). All the grafts were evaluated for 28 days by a scoring rejection index (RI) to assess the graft opacity, edema, and neovascularization using slit lamp biomicroscopy. The AC inflammation and corneal graft thickness were assessed using ASOCT. RESULTS: All the allogeneic control grafts and four of the 20 allogeneic steroid-treated grafts developed rejection episodes. In the allogeneic treated group, the rejected grafts had a significantly higher mean AC inflammation grade at 1 week (grade 3.25±0.49 vs. 1.83±0.36, P<0.001), significantly thicker central graft thickness at 2 weeks (455.25±42.42 µm vs. 381.247±12.51 µm, P=0.047), and a significantly higher RI at 4 weeks (7.75±0.63 vs. 4.60±0.13, P<0.001) compared to the nonrejected grafts. Eyes with ≥grade 3 AC inflammation at 1 week, or with ≥400 µm central graft thickness at 2 weeks, were significantly associated with graft rejection (odds ratio [OR], 15.15, P=0.009, and OR, 9.75, P=0.014, respectively). CONCLUSIONS: The use of ASOCT to evaluate AC inflammation and corneal thickness aids in the early evaluation and diagnosis of graft rejection in animal models. Early increased AC inflammation was an early predictor of graft failure prior to definitive clinical evaluation.

A biodegradable, sustained-released, prednisolone acetate microfilm drug delivery system effectively prolongs corneal allograft survival in the rat keratoplasty model.

Frequent and long-term use of topical corticosteroids after corneal transplantation is necessary to prevent graft rejection. However, it relies heavily on patient compliance, and sustained therapeutic drug levels are often not achieved with administration of topical eye drops. A biodegradable drug delivery system with a controlled and sustained drug release may circumvent these limitations. In this study, we investigated the efficacy of a prednisolone acetate (PA)-loaded poly (d,l-lactide-co-ε-caprolactone) (PLC) microfilm drug delivery system on promoting the survival of allogeneic grafts after penetrating keratoplasty (PK) using a rat model. The drug release profiles of the microfilms were characterized (group 1). Subsequently, forty-eight PK were performed in four experimental groups: syngeneic control grafts (group 2), allogeneic control grafts (group 3), allogeneic grafts with subconjunctivally-implanted PA microfilm (group 4), and allogeneic grafts with PA eye drops (group 5; n = 12 in each). PA-loaded microfilm achieved a sustained and steady release at a rate of 0.006-0.009 mg/day, with a consistent aqueous drug concentration of 207-209 ng/ml. The mean survival days was >28 days in group 2, 9.9±0.8 days in group 3, 26.8±2.7 days in group 4, and 26.4±3.4 days in group 5 (P = 0.023 and P = 0.027 compared with group 3). Statistically significant decrease in CD4+, CD163+, CD 25+, and CD54+ cell infiltration was observed in group 4 and group 5 compared with group 3 (P<0.001). There was no significant difference in the mean survival and immunohistochemical analysis between group 4 and group 5. These results showed that sustained PA-loaded microfilm effectively prolongs corneal allograft survival. It is as effective as conventional PA eye drops, providing a promising clinically applicable alternative for patients undergoing corneal transplantation.

Optimization of subconjunctival biodegradable microfilms for sustained drug delivery to the anterior segment in a small animal model.

PURPOSE: We evaluated a biodegradable, sustained-release, prednisolone acetate (PA)-loaded poly[d,l-lactide-co-ε-caprolactone] (PLC) drug delivery system on its biocompatibility, feasibility and release characteristics in vitro and in vivo. METHODS: Blank and 40% PA-loaded PLC microfilms with a diameter of 2 mm were fabricated, and the degradation and drug release profiles of the microfilms were characterized in vitro and in vivo. The microfilms were implanted into the subconjunctival space of Lewis rats (n = 48). All eyes were assessed clinically using slit-lamp biomicroscopy, and graded with Hackett-McDonald ocular scoring system and anterior segment optical coherence tomography. Histologic and immunohistochemical analyses were performed comparing blank and PA-loaded microfilm groups. PA concentrations in the aqueous humor were determined by HPLC. RESULTS: Subconjunctivally-implanted PLC microfilms were able to deliver PA in a sustained manner over 3 months, with a steady rate of 0.002 mg/d in vivo. Eyes with either blank or PA-loaded implanted microfilms showed a very minimal inflammatory response at the insertion sites and mild degree of collagen encapsulation around the microfilms, with significantly less CD11c cells at 2 and 4 weeks (P = 0.001 and P = 0.002), and collagen formation at 2 weeks (P = 0.001) in the PA-loaded microfilm group. Anterior chamber PA levels were achieved, with concentrations at 76.7 ± 5.9, 70.3 ± 2.3, and 42.7 ± 4.1 ng/mL at 2, 4, and 12 weeks, respectively. CONCLUSIONS: PA-loaded PLC microfilms display good biocompatibility, feasibility, and desirable sustained drug release profiles, and have the potential to exhibit antifibrotic and anti-inflammatory effects. This device is applicable to use in small animal models of anterior segment inflammation.