Posterior Segment Tissues Research Articles

TLR4 deficiency does not alter glaucomatous progression in a mouse model of chronic glaucoma.

Glaucoma is a leading cause of irreversible blindness worldwide. Toll-like receptor 4 (TLR4) is a pattern-recognition transmembrane receptor that induces neuroinflammatory processes in response to injury. Tlr4 is highly expressed in ocular tissues and is known to modulate inflammatory processes in both anterior and posterior segment tissues. TLR4 activation can lead to mitochondrial dysfunction and metabolic deficits in inflammatory disorders. Due to its effects on inflammation and metabolism, TLR4 is a candidate to participate in glaucoma pathogenesis. It has been suggested as a therapeutic target based on studies using acute models, such as experimentally raising IOP to ischemia-inducing levels. Nevertheless, its role in chronic glaucoma needs further evaluation. In the current study, we investigated the role of TLR4 in an inherited mouse model of chronic glaucoma, DBA/2J. To do this, we analyzed the effect of Tlr4 knockout (Tlr4 -/-) on glaucoma-associated phenotypes in DBA/2J mice. Our studies found no significant differences in intraocular pressure, iris disease, or glaucomatous progression in Tlr4 -/- compared to Tlr4 +/+ DBA/2J mice. These data do not identify a role for TLR4 in this chronic glaucoma, but further research is warranted to understand its role in other glaucoma models and different genetic contexts.

May a post‐radiation ocular surface disorder compromise penetrating keratoplasty surgery? A case report

Aims/Purpose: Ocular surface disorder after ocular radiation therapy, even commonly reported, remains overlooked. Any delay in diagnosis may lead to vision threatening complications. The presented case highlights the clinical outcome of a severe post‐radiation ocular surface disorder, the importance of intense therapy and the limitations of further surgical interventions.Methods: Case report.Results: A 34‐year‐old woman was referred with complains of painful and reddish right eye since years. Her medical history revealed an iris melanoma excision at the age of 29 years. Due to recurrence, a proton beam treatment has been performed thereafter. Consequently, the patient developed post‐radiation retinopathy with macula edema, secondary glaucoma, cataract, as well as a severe ocular surface disorder with corneal decompensation and band keratopathy. Several surgical treatments have been attempted among which: phacoemulsification with IOL implantation, trabeculectomy with mitomycin C. Due to refractory glaucoma a Baerveldt glaucoma drainage was necessitated, thereafter. Despite these various therapeutic approaches, there was no improvement of the ocular surface problem. With increasing deterioration of the corneal situation, a penetrating keratoplasty, has been discussed.Conclusions: The continuous worsening of clinical symptomatology of the proton beam radiotherapy ocular surface disorder can be the result of a post‐radiation syndrome. An ischaemic retinopathy and anterior segment disorder lead to engorgement of posterior and anterior segment tissues. Gradual expansion of ischemia, vasculitis and inflammatory mediators compresses the retinal tissue leading to recurrent macular oedema; as well as to secondary glaucoma and corneal decompensation. Due to the worsening of the clinical presentation of ocular surface disorder a penetrated keratoplastic surgery has been discussed. It remains however still questionable whether such patients would make profit from penetrated keratoplastic surgery, as the resulting corneal stromal lymphangiogenesis and hemangiogenesis, in the presence of post‐radiation keratopathy, are essential conditions for allograft rejection.

Visualization of preretinal membranes using trypan blue in patients with traction retinal detachments

ObjectifLa réparation chirurgicale du décollement de rétine tractionnel (DRT) complexe peut présenter un défi en raison de la difficulté à distinguer la membrane fibrogliale de la surface rétinienne sous-jacente. Nous décrivons ici le taux de rattachement de la rétine et les résultats visuels lors de l'utilisation du bleu de trypan (BT) pour visualiser la membrane fibreuse proliférative pendant la réparation chirurgicale du DRT. NatureÉtude rétrospective. ParticipantsCohorte de 17 patients qui ont subi une vitrectomie par la pars plana en vue de la réparation chirurgicale d'un DRT s'accompagnant du BT entre le 1er janvier 2005 et le 31 décembre 2020. MéthodesLes dossiers des patients ont fait l'objet d'un examen rétrospectif pour en extraire différentes données, soit les paramètres chirurgicaux, la meilleure acuité visuelle corrigée (MAVC) en logMAR avant et après l'intervention et la qualité du rattachement de la rétine aux jours 30 et 90 de même qu'au moment de la visite de suivi la plus récente. RésultatsLes causes du DRT au sein de cette cohorte de 17 patients étaient les suivantes : rétinopathie diabétique proliférante, complications à la suite d'une endophtalmie, lésion à globe ouvert et néovascularisation secondaire à une occlusion veineuse rétinienne. La MAVC préopératoire moyenne ± é.-t. se chiffrait à 1,7 ± 1,7 logMAR (20/1000 sur l’échelle de Snellen), tandis que la MAVC postopératoire lors de la visite de suivi la plus récente s’élevait à 1,4 ± 1,2 logMAR (20/500 sur l’échelle de Snellen). L'utilisation du BT a permis de délinéer avec succès la membrane prérétinienne proliférative chez 100 % des patients, sans coloration résiduelle des tissus du segment postérieur ni réaction indésirable au colorant notée lors des visites postopératoires. Enfin, 88 % (15 rétines sur 17) et 76 % (13 rétines sur 17) des rétines étaient toujours rattachées 3 et 6 mois après l'intervention, respectivement. ConclusionLe BT peut être un outil utile pour visualiser la membrane épirétinienne fibreuse proliférative responsable de l'effet de traction afin de la retirer chez des patients présentant un DRT complexe, ce qui entraîne peut-être de meilleurs résultats chirurgicaux et un taux plus élevé de rattachement durable de la rétine.

Safety and Efficacy of Suprachorodial Injection of Triamcinolone Acetonide: Review of a Novel Treatment

Suprachoroidal injection is a unique, minimally invasive procedure for drug delivery to the posterior segment of the eye. Drugs can be delivered easily via microneedles that are slightly longer than the scleral and conjunctival thickness at pars plana. Suprachoroidal delivery of corticosteroids allows higher steroid concentration in the posterior segment tissues with minimal drug concentration in the anterior segment, which decreases the risk of other adverse ocular events like steroid-induced elevation of intraocular pressure and cataract progression. Several clinical trials have evaluated the safety and efficacy of suprachoroidal injection of triamcinolone acetonide in various ocular conditions. This article reviews the novel treatment as a promising route for drug delivery.

Progress in Ocular Drug Delivery: Challenges and Constraints.

The eye has several dynamic and static barriers in place to limit the entry of foreign substances including therapeutics. As such, efficient drug delivery, especially to posterior segment tissues, has been challenging. This chapter describes the anatomical and physiological challenges associated with ocular drug delivery before discussing constraints with regard to formulation parameters. Finally, it gives an overview of advanced drug delivery technologies with a specific focus on recently marketed and late-stage clinical trial products.

СРАВНИТЕЛЬНАЯ ХАРАКТЕРИСТИКА ВЛИЯНИЯ АЦЕТИЛЦИСТЕИНА И ДЕКСАМЕТАЗОНА НА ПОКАЗАТЕЛИ ОКИСЛИТЕЛЬНОГО СТРЕССА И АНТИОКСИДАНТНОЙ ЗАЩИТЫ В ТКАНЯХ ГЛАЗА ПРИ УВЕИТЕ

Objective. To study the effects of N-acetylcysteine (NAC), dexamethasone and their combination on the parameters of lipid peroxidation (LPO) and antioxidant defense (AOD) in the posterior segment tissues of rabbit eye under experimental immunogenic uveitis (EIU). Material and methods. The experimental study was carried out on 45 rabbits (90 eyes), 5 of which were intact. The animals with EIU were divided into 8 groups (5 animals each) in which daily intramuscular injections of placebo, NAC, dexamethasone or a combination of NAC and dexamethasone were used for 3 or 7 days. Subsequently the content of LPO products, concentration of reduced glutathione, oxidized glutathione and their ratios, activity of superoxide dismutase and catalase enzymes as well as the total antioxidant activity were determined in the posterior segment tissues of the rabbit eyes. Results. Application of NAC led to a significant decrease in the concentration of LPO products, as well as a significant elevation of AOD parameters. Dexamethasone had a negative effect on the glutathione system in the tissues of the eye. The combined use of NAC and dexamethasone revealed the synergism of their pharmacological action and made it possible to increase the overall antioxidant potential of the therapy. Conclusion. NAC parenteral administration showed a pronounced antioxidant effect. Combination therapy with NAC and dexamethasone significantly increased effectiveness of the treatment. The data obtained allows recommending NAC administration as part of the complex therapy of uveitis.

Mechanistic Model for the Prediction of Small-Molecule Vitreal Clearance Combining Diffusion-Limited and Permeability-Limited Clearance.

The discovery of new small-molecule drugs for intravitreal administration would benefit from simple models to predict vitreal clearance (CL). The current models available have limitations in their applicability to small-molecule drugs and translatability to humans. We developed a mechanistic model combining the diffusion rate of the molecule in the vitreous and permeability across posterior segment tissues and applied it to 30 small molecules with observed CL available mostly from literature. We used Caco-2 permeability as a surrogate for ocular tissue permeability. The model predicted rabbit vitreal CL well, with 80% of the predictions being within a 2-fold range of the observed CL. For an accurate prediction, it was crucial to consider the anterior diffusion CL from the vitreous to the aqueous and a limiting diffusion CL for the whole eye. We observed no major differences in model accuracy when using literature permeability values from retinal pigment epithelial cell models. Importantly, by adopting the specific dimensions of the human eye, the model was able to accurately predict vitreal CL of four compounds for which human vitreal CL data are available. In summary, this mechanistic model enables a simple, accurate, and translatable estimation of small-molecule vitreal CL.

Gelatin-Based Photocurable Hydrogels for Corneal Wound Repair.

In this study, an injectable, photocurable gelatin system, consisting of acrylated gelatin and thiolated gelatin, with tunable mechanical, biodegradation, and biological properties was used as a potential cell-supportive scaffold for the repair of focal corneal wounds. The mechanical property of hydrogels can be readily modified (postcure shear modulus of between 0.3 and 22 kPa) by varying the ratio of acrylate to thiol groups, photointensity, and solid content, and the biodegradation times also varied with the change of solid content. More importantly, the generated hydrogels exhibited excellent cell viability in both cell seeding and cell encapsulation experiments. Furthermore, the hydrogels were found to be biocompatible with rabbit cornea and aided the regeneration of a new tissue under a focal corneal wound (exhibiting epithelial wound coverage in <3d), and ultraviolet irradiation did not have any obvious harmful effect on the cornea and posterior eye segment tissues. Along with their injectability and tunable mechanical properties, the photocurable thiol-acrylate hydrogels showed promise as corneal substitutes or substrates to construct a new corneal tissue.

Ocular Distribution and Pharmacokinetics of Lifitegrast in Pigmented Rabbits and Mass Balance in Beagle Dogs.

Purpose: Lifitegrast is approved in the United States for the treatment of dry eye disease (DED). We assessed lifitegrast's ocular distribution/pharmacokinetic profile in rabbits, and 14C-lifitegrast mass balance/excretion in dogs.Methods: Female pigmented rabbits received a single topical ocular dose of lifitegrast (Formulation No. 1, n = 25; No. 2, n = 25) per eye twice daily (target, 1.75 mg/eye/dose). Blood/ocular tissues were collected on day 5. Beagle dogs received single intravenous (n = 10; target, 3 mg, 262 μCi/animal) and ocular (n = 8, target, 3 mg, 30 μCi/eye) doses of 14C-lifitegrast (∼8 weeks between doses). Blood, excreta, and cage rinse/wipes were collected. Concentrations were measured by mass spectrometry/liquid scintillation counting. Pharmacokinetic analyses (noncompartmental) included maximum concentration (Cmax), time to Cmax (tmax), and area under the concentration-time curve from 0 to 8 h (AUC0–8).Results: In rabbits, lifitegrast Cmax and AUC0–8 were similar between formulations. Cmax was highest in ocular anterior segment tissues: 5,190–14,200 ng/g [conjunctiva (palpebral/bulbar), cornea, anterior sclera]. Posterior segment tissues had lower concentrations (0–826 ng/g). AUC0–8 followed a similar trend. Plasma concentrations were low (Cmax <18 ng/mL). Tissue/plasma tmax was ∼0.25–1 h. In dogs, after intravenous/ocular doses, 14C-lifitegrast was eliminated primarily through feces. Excreted radioactivity was mainly unchanged lifitegrast.Conclusions: High exposure of lifitegrast in rabbit ocular anterior segment tissues and low exposure in posterior segment tissues/plasma suggests that lifitegrast reaches target tissues for DED treatment, with low potential for off-target systemic/ocular effects. Excretion of unchanged 14C-lifitegrast suggests minimal drug metabolism in vivo. This is consistent with lifitegrast clinical trial efficacy/safety data.

Racial Differences in the Extracellular Matrix and Histone Acetylation of the Lamina Cribrosa and Peripapillary Sclera.

We investigated the extracellular matrix (ECM) of the lamina cribrosa (LC) and peripapillary sclera (PPS) and compared histone acetylation and related enzymes to identify racial differences between Korean and Caucasian donor eyes. Posterior segment tissues were obtained from 30 Caucasian donors and 42 age and axial length-matched Korean donors. Histone modification was assessed for histone deacetylase (HDAC) 2, HDAC3, and acetylated histone H3. The promoter regions of the major ECM in the LC and PPS including collagen type I and III, and elastic fiber components (elastin and fibrillin-1) and lysyl oxidase enzymes including lysyl oxidase-like 1 and 2 (LOXL2) were evaluated by chromatin immunoprecipitation (ChIP) assay. Protein and mRNA expression of major ECM components were assessed using real-time polymerase chain reaction analysis, western blot analysis, and immunohistochemical staining. HDAC2 and HDAC3 expression levels were decreased and acetylated histone H3 was increased in the LC and PPS of Korean eyes than Caucasian eyes. The promoter regions of LOXL2, elastin, and fribrillin-1 genes were highly acetylated in Korean LC. Expression of LOXL2 and elastic fiber components (elastin and fibrillin-1) were significantly increased in Korean LC and PPS than Caucasians according to the real-time polymerase chain reaction, western blot analyses, and quantification of elastic fiber staining. Histone acetylation status differed in the promoter regions of the elastic fiber components and LOXL2 in the LC and PPS according to race. Further study to reveal the association with these findings to the pathogenesis of glaucoma in Korean eyes is needed.

Penetration Route of the Selective Glucocorticoid Receptor Agonist SA22465 and Betamethasone into Rabbit Meibomian Gland Based on Pharmacokinetics and Autoradiography

Meibomian glands are modified sebaceous glands embedded within specific types of dense connective tissues. This study investigated drug penetration into meibomian glands following a single topical administration in rabbits. We measured time course concentrations of the selective glucocorticoid receptor agonist (SEGRA) SA22465 and betamethasone in lid margins, palpebral conjunctival epithelium, and meibomian glands following a single instillation using a newly established collection method. We also visualized the distribution of 14C-SA22465 in eyelid tissue sections using microautoradiography. Concentrations of SA22465 and its major metabolite SA22313 were highest in lid margins, followed by palpebral conjunctival epithelium and meibomian glands in a 100-fold descending order. Betamethasone exhibited similar distribution profiles with smaller concentration differences. The distribution of silver grains as a quantitative index of radioactivity in eyes and eyelids was determined in a subjective manner using microautoradiographs, which revealed that the highest distribution of silver grains was associated with the cornea, followed by posterior segment tissues, such as the sclera, choroid, and retina. Low levels were associated with more internal ocular tissues and a greater number of compartments. Moderate levels of radioactivity were associated with meibomian glands and connective tissues, including the nictitating membrane. In contrast, meibomian ducts contained only background levels of radioactivity. Our findings indicate that the transconjunctiva is the most likely route of drug entry into meibomian glands following ocular administration rather than the central meibomian duct; however, this distribution is limited. A physiologic barrier may restrict drug penetration across the tarsal plate.

Nanoparticle-loaded biodegradable light-responsive in situ forming injectable implants for effective peptide delivery to the posterior segment of the eye

Diseases affecting the posterior segment the eye, such as age-related macular degeneration (AMD), are the leading cause of blindness worldwide. Conventional dosage forms, such as eye drops, have to surmount several elimination mechanisms and complex barriers to achieve therapeutic concentrations at the target site often resulting in low anterior segment bioavailability (ca. 2–5%) with generally none of the drug reaching posterior segment tissues. Thus, frequent intravitreal injections are currently required to treat retinal conditions which have been associated with poor patient compliance due to pain, risk of infection, hemorrhages, retinal detachment and high treatment related costs. To partially overcome these issues, ocular implants have been developed for some posterior segment indications; however, the majority require surgical implantation and removal at the end of the intended treatment period. The transparent nature of the cornea and lens render light-responsive systems an attractive strategy for the management of diseases affecting the back of the eye. Light-responsive in situ forming injectable implants (ISFIs) offer various benefits such as ease of application in a minimally invasive manner and more site specific control over drug release. Moreover, the biodegradable nature of such implants avoids the need for surgical removal after release of the payload. Incorporating drug-loaded polymeric nanoparticles (NPs) into these implants may reduce the high initial burst release from the polymeric matrix and further sustain drug release thus avoiding the need for frequent injections as well as minimizing associated side effects. However, light-responsive systems for ophthalmic application are still in their early stages of development with limited reports on their safety and effectiveness. We hypothesize that the innovative design and properties of NP-containing light-responsive ISFIs can serve as a platform for effective management of ocular diseases requiring long term treatment.

Ocular disposition of diazepam in rabbits: understanding its level in vitreous humour for forensic applications

The disadvantages associated with routine toxicological samples, such as blood samples, have encouraged the forensic toxicologist to use vitreous humour as an alternative matrix. However, its use is still limited. The reason for this is the lack of a systematic study in this domain. Moreover, the phenomena of drug disposition in ocular tissues are mostly unexplored. This paper presents a controlled study to investigate the distribution of diazepam in post-mortem blood, vitreous humour and ocular tissues using rabbit as an animal model. This study reveals that diazepam levels in vitreous and aqueous humour increase with their decrease in whole blood at post-mortem. A further study on the distribution of diazepam in ocular tissues reveals that the anterior and posterior segment tissues might be responsible for the increase in diazepam levels in aqueous humour and vitreous humour respectively. The present study strengthens the possibility of using vitreous humour as a complementary sample to blood in forensic toxicological investigations.

Infection of Human Retinal Pigment Epithelium with Chlamydia trachomatis.

PurposeLittle is known about the susceptibility of posterior segment tissues, particularly the human retinal pigment epithelium (hRPE), to Chlamydia trachomatis. The purpose of the study was to investigate the possibility of infecting the hRPE with Chlamydia trachomatis, and to examine the infectivity of different Chlamydia trachomatis clinical isolates for hRPE cells and the hRPE cell response to the infection.MethodsCultured hRPE and McCoy cells were inoculated with eight Chlamydia trachomatis (serovar E) clinical isolates at multiplicity of infection (MOI) of 2.0 or 0.3. To detect Chlamydia trachomatis, samples were stained immunohistochemically with anti-major outer membrane protein antibodies at 24h, 48h, and 72h postinoculation (PI). The changes in the expression of signaling molecules and proteins of cytoskeleton and extracellular matrix in hRPE cells were examined immunohistochemically.ResultsAll eight clinical isolates demonstrated ability to infect hRPE cells. At equal MOI of 0.3, the infectivity of Chlamydia trachomatis clinical isolates for RPE culture was found to be at least as high as that for McCoy cell culture. At 24h PI, the percentage of inclusion-containing cells varied from 1.5 ± 0.52 to 14.6 ± 3.3% in hRPE cell culture infected at MOI of 2.0 against 0.37 ± 0.34 to 8.9 ± 0.2% in McCoy cell culture infected at MOI of 0.3. Collagen type I, collagen type IV, basic fibroblast growth factor, transforming growth factor-beta and interleukin–8 expression at 48h PI were maximally increased, by 2.1-, 1.3-, 1.5-, 1.5- and 1.6-fold, respectively, in the Chlamydia trachomatis-infected compared with control hRPE cell culture specimens (P < 0.05).ConclusionsThis study, for the first time, proved the possibility of infecting hRPE cultured cells with Chlamydia trachomatis, which leads to proproliferative and proinflammatory changes in the expression of signaling molecules and extracellular matrix components.

Distribution of topical ocular nepafenac and its active metabolite amfenac to the posterior segment of the eye

Nepafenac ophthalmic suspensions, 0.1% (NEVANAC®) and 0.3% (ILEVRO™), are topical nonsteroidal anti-inflammatory drug (NSAID) products approved in the United States, Europe and various other countries to treat pain and inflammation associated with cataract surgery. NEVANAC is also approved in Europe for the reduction in the risk of postoperative macular edema (ME) associated with cataract surgery in diabetic patients. The efficacy against ME suggests that topical administration leads to distribution of nepafenac or its active metabolite amfenac to the posterior segment of the eye. This article evaluates the ocular distribution of nepafenac and amfenac and the extent of local delivery to the posterior segment of the eye, following topical ocular instillation in animal models. Nepafenac ophthalmic suspension was instilled unilaterally in New Zealand White rabbits as either a single dose (0.1%; one drop) or as multiple doses (0.3%, one drop, once-daily for 4 days, or 0.1% one drop, three-times daily for 3 days and one morning dose on day 4). Nepafenac (0.3%) was also instilled unilaterally in cynomolgus monkeys as multiple doses (one drop, three-times daily for 7 days). Nepafenac and amfenac concentrations in harvested ocular tissues were measured using high-performance liquid chromatography/mass spectrometry. Locally-distributed compound concentrations were determined as the difference in levels between dosed and undosed eyes. In single-dosed rabbit eyes, peak concentrations of locally-distributed nepafenac and amfenac showed a trend of sclera > choroid > retina. Nepafenac peak levels in sub-samples posterior to the eye equator and inclusive of the posterior pole (E-PP) were 55.1, 4.03 and 2.72 nM, respectively, at 0.25 or 0.50 h, with corresponding amfenac peak levels of 41.9, 3.10 and 0.705 nM at 1 or 4 h. By comparison, peak levels in sclera, choroid and retina sub-samples in a band between the ora serrata and the equator (OS-E) were 13- to 40-fold (nepafenac) or 11- to 23-fold (amfenac) higher, indicating an anterior-to-posterior directional concentration gradient. In multiple-dosed rabbit eyes, with 0.3% nepafenac instilled once-daily or 0.1% nepafenac instilled three-times daily, cumulative 24-h locally-distributed levels of nepafenac in E-PP retina were similar between these groups, whereas exposure to amfenac once-daily dosing nepafenac 0.3% was 51% of that achieved with three-times daily dosing of 0.1%. In single-dosed monkey eyes, concentration gradients showed similar directionality as observed in rabbit eyes. Peak concentrations of locally-distributed nepafenac were 1580, 386, 292 and 13.8 nM in E-PP sclera, choroid and retina, vitreous humor, respectively, at 1 or 2 h after drug instillation. Corresponding amfenac concentrations were 21.3, 11.8, 2.58 and 2.82 nM, observed 1 or 2 h post-instillation. The data indicate that topically administered nepafenac and its metabolite amfenac reach pharmacologically relevant concentrations in the posterior eye segment (choroid and retina) via local distribution, following an anterior-to-posterior concentration gradient. The proposed pathway involves a choroidal/suprachoroidal or periocular route, along with an inward movement of drug through the sclera, choroid and retina, with negligible vitreal compartment involvement. Sustained high nepafenac concentrations in posterior segment tissues may be a reservoir for hydrolysis to amfenac.

Hydrogel Ring for Topical Drug Delivery to the Ocular Posterior Segment

Purpose: To investigate the efficacy of a topical hydrogel ring for drug delivery to the posterior segment of the rabbit eye.Materials and methods: Novel hydrogel corneal lenses (CL), scleral/corneal lenses (S/CL), and rings were prepared using poly(hydroxyethyl methacrylate). The devices were immersed in 0.3% ofloxacin ophthalmic solution (OOS) to homogeneously distribute the drug throughout the hydrogel. The medicated CL, S/CL, Ring 1 (standard ring), or Ring 2 (shape-optimized ring) was applied to the surface of the cornea, cornea/bulbar conjunctiva, or bulbar conjunctiva of albino rabbits, respectively. Medicated rings did not touch the corneal surface. In another group, one OOS drop was administered to the eye. After 0.25–8 hours, the hydrogel devices were removed and ocular tissues were harvested. High-performance liquid chromatography (HPLC) was used to measure the ofloxacin concentration in the devices and tissues. The drug concentrations in the posterior segment tissues were compared among ofloxacin delivery methods.Results: One hour after placement, eyes treated with Ring 1 or S/CL had markedly higher ofloxacin levels in the posterior segment tissues (conjunctiva, sclera, and retina/choroid) than eyes treated with topical OOS or a CL. Lower levels of ofloxacin were found in anterior segment tissues (cornea and aqueous humor) in eyes treated with Ring 1 compared to those treated with S/CL. Ring 2 most effectively delivered ofloxacin to the retina/choroid. The tissue ofloxacin concentration in the fellow eye was markedly lower than the eye treated with Ring 2.Conclusions: Our results suggest that hydrogel rings are effective in delivering topical ophthalmic drugs to the posterior segment. The drugs are most likely delivered via the transconjunctival/scleral route by lateral diffusion across the bulbar conjunctiva and through the sclera. Systemic drug delivery to the posterior segment is minimal.

Topical drug delivery to retinal pigment epithelium with microfluidizer produced small liposomes

Drug delivery from topically instilled eye drops to the posterior segment of the eye has long been one of the greatest challenges of ocular drug development. We developed methods of liposome preparation utilizing a microfluidizer to achieve adjustable nanoparticle size (even less than 80nm) and high loading capacity of plasmid DNA. The microfluidizing process parameters were shown to affect the size of the liposomes. Higher operating pressures and passage for at least 10 times through the microfluidizer produced small liposomes with narrow size distribution. The liposomes were physically stable for several months at +4°C. In vivo distribution of the optimized liposome formulations in the rat eyes was investigated with confocal microscopy of the histological specimens. Transferrin was used as a targeting ligand directed to retinal pigment epithelium. Size dependent distribution of liposomes to different posterior segment tissues was seen. Liposomes with the diameter less than 80nm permeated to the retinal pigment epithelium whereas liposomes with the diameter of 100nm or more were distributed to the choroidal endothelium. Active targeting was shown to be necessary for liposome retention to the target tissue. In conclusion, these microfluidizer produced small liposomes in eye drops are an attractive option for drug delivery to the posterior segment tissues of the eye.

Expression and Regulation of LOXL1 and Elastin-related Genes in Eyes With Exfoliation Syndrome

Exfoliation syndrome (XFS) is a complex, late-onset disorder of the elastic fiber system and the most common identifiable cause of open-angle glaucoma. Strong genetic risk is conferred by the lysyl oxidase-like 1 (LOXL1) gene, but additional comodulating factors are necessary for the manifestation of the disease. The aim of this study was to establish a comprehensive expression profile of LOXL1 and elastic proteins in XFS eyes and to analyze their regulation in an in vitro cell culture system. Eyes with XFS with and without glaucoma, and normal control eyes were analyzed for major elastic fiber components (elastin, fibrillin-1, fibulin-4), and lysyl oxidase (LOX) enzymes by real-time PCR, immunohistochemistry, and electron microscopy. Cultured human Tenon's capsule fibroblasts were exposed to transforming growth factor-β1, IL-6, homocysteine, oxidative stress, hypoxia, or ultraviolet radiation, and changes in the expression of LOXL1 and elastic components of XFS material were assessed by real-time PCR, immunohistochemistry, and Western blotting. LOXL1 expression in anterior eye tissues was significantly increased in early XFS stages but was decreased in advanced stages as compared with controls. LOXL1 was also found to be a major component of XFS material and to colocalize with elastin, fibrillin-1, and fibulin-4, which were upregulated in parallel to LOXL1. In contrast, in most posterior segment tissues, LOXL1 and elastic fiber proteins displayed no differential expression. Interestingly, lamina cribrosa specimens of early and late XFS stages without and with glaucoma revealed a selective downregulation of LOXL1 and elastic fiber components on the mRNA and protein level, which was associated with pronounced ultrastructural alterations of the laminar elastic fiber network in XFS eyes. Treatment of cultured cells with XFS-associated pathogenetic stimuli induced a significant increase in the expression of LOXL1 and elastic proteins and resulted in their assembly into XFS-like fibrils in vitro. The findings support the notion that both genetic and nongenetic factors may cooperate in the stable accumulation of XFS aggregates and provide evidence for a XFS-specific elastinopathy of the lamina cribrosa, possibly rendering XFS eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development.

Targeted Administration into the Suprachoroidal Space Using a Microneedle for Drug Delivery to the Posterior Segment of the Eye

This study seeks to determine the intraocular pharmacokinetics of molecules and particles injected into the suprachoroidal space of the rabbit eye in vivo using a hollow microneedle. Suprachoroidal injections of fluorescein and fluorescently tagged dextrans (40 and 250 kDa), bevacizumab, and polymeric particles (20 nm to 10 μm in diameter) were performed using microneedles in New Zealand white rabbits. The fluorescence intensity within the eye was monitored in each animal using an ocular fluorophotometer to determine the distribution of the injected material in the eye over time as compared with intravitreal injection of fluorescein. Fundus photography and histology were performed as well. Molecules and particles injected near the limbus using a microneedle flowed circumferentially around the eye within the suprachoroidal space. By targeting the suprachoroidal space, the concentration of injected materials was at least 10-fold higher in the back of the eye tissues than in anterior tissues. In contrast, intravitreal injection of fluorescein targeted the vitreous humor with no significant selectivity for posterior versus anterior segment tissues. Half-lives in the suprachoroidal space for molecules of molecular weight from 0.3 to 250 kDa ranged from 1.2 to 7.9 hours. In contrast, particles ranging in size from 20 nm to 10 μm remained primarily in the suprachoroidal space and choroid for a period of months and did not clear the eye. No adverse effects of injection into the suprachoroidal space were observed. Injection into the suprachoroidal space using a microneedle offers a simple and minimally invasive way to target the delivery of drugs to the choroid and retina.

LOXL1 Deficiency in the Lamina Cribrosa as Candidate Susceptibility Factor for a Pseudoexfoliation-Specific Risk of Glaucoma

To test the hypothesis that a primary disturbance in lysyl oxidase-like 1 (LOXL1) and elastin metabolism in the lamina cribrosa of eyes with pseudoexfoliation syndrome constitutes an independent risk factor for glaucoma development and progression. Observational, consecutive case series. Posterior segment tissues obtained from 37 donors with early and late stages of pseudoexfoliation syndrome without glaucoma, 37 normal age-matched control subjects, 5 eyes with pseudoexfoliation-associated open-angle glaucoma, and 5 eyes with primary open-angle glaucoma (POAG). Protein and mRNA expression of major elastic fiber components (elastin, fibrillin-1, fibulin-4), collagens (types I, III, and IV), and lysyl oxidase crosslinking enzymes (LOX, LOXL1, LOXL2) were assessed in situ by quantitative real-time polymerase chain reaction, (immuno)histochemistry, and light and electron microscopy. Lysyl oxidase-dependent elastin fiber assembly was assessed by primary optic nerve head astrocytes in vitro. Expression levels of elastic proteins, collagens, and lysyl oxidases in the lamina cribrosa. Lysyl oxidase-like 1 proved to be the major lysyl oxidase isoform in the normal lamina cribrosa in association with a complex elastic fiber network. Compared with normal and POAG specimens, lamina cribrosa tissues obtained from early and late stages of pseudoexfoliation syndrome without and with glaucoma consistently revealed a significant coordinated downregulation of LOXL1 and elastic fiber constituents on mRNA and protein level. In contrast, expression levels of collagens and other lysyl oxidase isoforms were not affected. Dysregulated expression of LOXL1 and elastic proteins was associated with pronounced (ultra)structural alterations of the elastic fiber network in the laminar beams of pseudoexfoliation syndrome eyes. Inhibition of LOXL1 interfered with elastic fiber assembly by optic nerve head astrocytes in vitro. The findings provide evidence for a pseudoexfoliation-specific elastinopathy of the lamina cribrosa resulting from a primary disturbance in LOXL1 regulation and elastic fiber homeostasis, possibly rendering pseudoexfoliation syndrome eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development and progression.