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PURPOSE: VitreoDx is an experimental device enabling push-button collection of a neat vitreous liquid biopsy incidental to an intravitreal injection. We explored the ability of the device to collect a sample usable for proteomic biomarker discovery and testing. DESIGN: Pilot study using ex vivo human eyes. METHODS: Non-vitrectomized, human eyes from nine donors 75-91 years of age were refrigerated in BSS and used within 5 days of death. Four VitreoDx devices fitted with 25G needles, and four staked needle insulin syringes with 30G needles, were inserted at equal intervals through the pars plana of each eye and held in place by a fixture. The sampling mode of each VitreoDx device was triggered to attempt to acquire a liquid biopsy up to 70 μL. The plunger of each insulin syringe was retracted to attempt to obtain a liquid biopsy with a maximum volume of 50 μL. Samples acquired with the VitreoDx were extracted to polypropylene cryovials, refrigerated to -80 ºC, and sent for offsite proteomic analysis by proximity extension assay with a focus on panels containing approved and pipelined drug targets for neovascular disease and inflammatory factors. RESULTS: Of the attempted liquid biopsies with the novel 25G VitreoDx, 92% (66 of 72) resulted in successful acquisition (>25 μL) while 89% (64 of 72) attempted by a traditional 30G needle resulted in a successful acquisition. Sample volume sufficient for proteomics array analysis was acquired by the VitreoDx for every eye. Detectable protein was found for 151 of 166 unique proteins assayed in at least 25% of eyes sampled by VitreoDx. CONCLUSIONS: The high acquisition rate achieved by the prototype was similar to that achieved in previous clinical studies where a standard syringe was used with a 25G needle to biopsy vitreous fluid directly prior to standard intravitreal injection. Successful aspiration rates were likewise high for 30G needles. Together, these suggest that it is possible to routinely acquire liquid vitreous biopsies from patients who typically receive intravitreal injections with an injection device using a standard size needle without a vitreous cutter. Protein analysis shows that proteins of interest survive the sampling mechanism and may have potential to direct care in the future.

BACKGROUND: One of the core aims of the European Union of Medical Specialists is to harmonize training across Europe by creating European Training Requirements for all medical specialties including Ophthalmology. The theoretical part is already defined by the EBO, however as ophthalmology also includes surgical skills, we herein propose a surgical minimum curriculum for ophthalmology residents in Europe. METHODS: National and international ophthalmic training curricula which are publicly available in English were reviewed and compared. The final proposal was created from 5 criteria: 1. Disease prevalence; 2. Patient safety; 3. Case-trainee ratio; 4. Skill transfer; and 5. Technical difficulty. RESULTS: In total 7 different training curricula from across the world were compared. Among the surgical procedures, cataract surgery has the highest median number of procedures required to be completed during residency: 86 procedures (50-350). Followed by oculoplastics: 28 procedures (10-40) and panretinal photocoagulation: 27.5 procedures (10-49) Full procedural competence is proposed in 9 surgical skills, including YAG laser posterior capsulotomy, retinal argon laser, intravitreal injection, corneal foreign body removal, removal of corneal sutures, facial and periocular laceration repair, eyelid laceration repair, minor eyelid procedures, and punctal occlusion. These procedures are deemed essential and feasible for all ophthalmology residents in Europe to perform independently upon completion of their training. CONCLUSION: This proposal should be regarded as a recommendation based on comparable surgical curricula in use worldwide to establish standards across European countries and may serve as valuable insight to those responsible for compiling ETRs for ophthalmology, or their national curriculums.

Purpose: To improve awareness of delayed onset uveitis in patients with a history of intraocular tattoo ink injection.Results: A 47-year-old man underwent a scleral tattoo procedure during which there was inadvertent intraocular injection of tattoo ink into his right eye. He subsequently developed endophthalmitis, retinal detachment, and retinal necrosis. He was treated with intravitreal and oral antibiotics and underwent vitreoretinal surgical intervention. A vitreous specimen was obtained and demonstrated significantly elevated levels of several heavy metals. One month later, he developed an acute granulomatous anterior uveitis in the same eye that was managed with a combination of topical and perioperative intravitreal, intravenous, and oral corticosteroids.Conclusion: This case highlights the importance of monitoring patients with a history of intraocular tattoo ink injection for delayed onset uveitis in addition to retinal toxicity.

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.

Vitreous humor is a potential alternative matrix for postmortem toxicology drug screens when peripheral blood is unavailable. It is easily and reliably collected and may not suffer from the same postmortem redistribution as seen in blood. Here, we compared the concentrations of 7 acidic drugs (acetaminophen, ibuprofen, naproxen, salicylic acid, carbamazepine, phenobarbital and phenytoin) in peripheral blood and vitreous fluid collected in 89 autopsy cases. Analysis was done by high-performance liquid chromatography with diode-array detection. Overall, we found that vitreous drug concentrations were significantly lower than peripheral blood with median vitreous to peripheral blood (V/PB) ratios ranging from 0.0 to 0.6 (mean, 0.1-0.6). The correlations between the concentrations of over-the-counter analgesics in peripheral blood versus vitreous fluid were poor, with acetaminophen exhibiting the best linearity (R2 = 0.72). The antiepileptic drugs (carbamazepine, phenytoin and phenobarbital) exhibited good correlations between peripheral blood and vitreous humor, with all exhibiting an R2 ≥ 0.95. Overall, we have demonstrated the potential of vitreous fluid as an alternative matrix for the detection of select acidic drugs.

Vitreous humor is a potential alternative matrix for postmortem toxicology drug screens when peripheral blood is unavailable. It is easily and reliably collected and may not suffer from the same postmortem redistribution as seen in blood. Here, we compared the concentrations of 7 acidic drugs (acetaminophen, ibuprofen, naproxen, salicylic acid, carbamazepine, phenobarbital and phenytoin) in peripheral blood and vitreous fluid collected in 89 autopsy cases. Analysis was done by high-performance liquid chromatography with diode-array detection. Overall, we found that vitreous drug concentrations were significantly lower than peripheral blood with median vitreous to peripheral blood (V/PB) ratios ranging from 0.0 to 0.6 (mean, 0.1-0.6). The correlations between the concentrations of over-the-counter analgesics in peripheral blood versus vitreous fluid were poor, with acetaminophen exhibiting the best linearity (R2 = 0.72). The antiepileptic drugs (carbamazepine, phenytoin and phenobarbital) exhibited good correlations between peripheral blood and vitreous humor, with all exhibiting an R2 ≥ 0.95. Overall, we have demonstrated the potential of vitreous fluid as an alternative matrix for the detection of select acidic drugs.

PurposeThe aim of this study was to briefly review the clinical and diagnostic features of vitreoretinal lymphoma (VRL) and to introduce the recent introduction of metagenomic deep sequencing in this ocular lymphomatous disease.Design and methodsReview and description of the process of using metagenomic deep sequencing for ocular specimens at the Proctor Foundation, University of California, San Francisco, CA.ResultsVRL masquerades as a uveitis, but clinical signs of subretinal lesions, and vitritis should prompt the inclusion of VRL in a differential diagnosis. Imaging features such as hyporeflective infiltrative lesions between the retinal pigment epithelium and Bruch's membrane are compatible with VRL, but diagnosis requires satisfying specific cytopathological and immunohistochemical or molecular features. Diagnosis, then, is subject to the cellularity, viability, and volume of the specimen submitted for these tests. Metagenomic deep sequencing has the ability to detect numerous lymphoma-associated mutations and is able to utilize minute volume samples and cell-free nucleic acid, so is well-suited for ocular tissues.ConclusionsMetagenomic deep sequencing may offer an additional tool in the future with which to diagnose VRL.

PurposeTo evaluate the feasibility of using the Proximity Extension Assay (PEA) platform to detect biomarkers in vitreous and to compare the findings with results obtained with an electrochemiluminescent (ECL) sandwich immunoassay.MethodsVitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic controls were tested using two different proteomics platforms. Forty-one assays were completed with the ECL platform and 459 with the PEA platform. Spearman's rank correlation coefficient (rs ) was used to determine the direction and strength of the relationship between protein levels detected by both platforms.ResultsThree hundred sixty-six PEA assays detected the tested protein in at least 25% of samples, and the difference in protein abundance between PDR and controls was statistically significant for 262 assays. Seventeen ECL assays yielded a detection rate ≥ 25%, and the difference in protein concentration between PDR and controls was statistically significant for 13 proteins. There was a subset of proteins that were detected by both platforms, and for those the Spearman's correlation coefficient was higher than 0.8.ConclusionsPEA is suitable for the analysis of vitreous samples, showing a strong correlation with the ECL platform. The detection rate of PEA panels was higher than the panels tested with ECL. The levels of several proinflammatory and angiogenic cytokines were significantly higher in PDR vitreous compared to controls.Translational relevanceThis study provides new information on the yields of small-volume assays that can detect proteins of interest in ocular specimens, and it identifies patterns of cytokine dysregulation in PDR.

PurposeTo evaluate the feasibility of using the Proximity Extension Assay (PEA) platform to detect biomarkers in vitreous and to compare the findings with results obtained with an electrochemiluminescent (ECL) sandwich immunoassay.MethodsVitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic controls were tested using two different proteomics platforms. Forty-one assays were completed with the ECL platform and 459 with the PEA platform. Spearman's rank correlation coefficient (rs ) was used to determine the direction and strength of the relationship between protein levels detected by both platforms.ResultsThree hundred sixty-six PEA assays detected the tested protein in at least 25% of samples, and the difference in protein abundance between PDR and controls was statistically significant for 262 assays. Seventeen ECL assays yielded a detection rate ≥ 25%, and the difference in protein concentration between PDR and controls was statistically significant for 13 proteins. There was a subset of proteins that were detected by both platforms, and for those the Spearman's correlation coefficient was higher than 0.8.ConclusionsPEA is suitable for the analysis of vitreous samples, showing a strong correlation with the ECL platform. The detection rate of PEA panels was higher than the panels tested with ECL. The levels of several proinflammatory and angiogenic cytokines were significantly higher in PDR vitreous compared to controls.Translational relevanceThis study provides new information on the yields of small-volume assays that can detect proteins of interest in ocular specimens, and it identifies patterns of cytokine dysregulation in PDR.

PurposeTo develop an animal model of vitreous hemorrhage (VH) to explore the impact of surgical parameters on VH associated with insertion of the Port Delivery System with ranibizumab (PDS) implant.MethodsNinety eyes from 45 treatment-naive male Yucatan minipigs received PDS implant insertion or a sham procedure. The effect of prophylactic pars plana hemostasis, scleral incision length, scleral cauterization, surgical blade type/size, and viscoelastic usage on postsurgical VH was investigated.ResultsPostsurgical VH was detected in 60.0% (54/90) of implanted eyes. A systematic effect on VH was only detected for pars plana hemostasis before the pars plana incision. The percentage of eyes with VH was 96.6% (28/29) among eyes that did not receive prophylactic pars plana hemostasis and 42.4% (24/58) among eyes that did. There was no VH in eyes that received laser ablation of the pars plana using overlapping 1,000-ms spots; pars plana cautery or diathermy was less effective. The majority of all VH cases (83.3% [45/54]) were of mild to moderate severity (involving ≤25% of the fundus).ConclusionIn this minipig surgical model of VH, scleral dissection followed by pars plana laser ablation before pars plana incision most effectively mitigated VH secondary to PDS implant insertion.

The viscoelastic behavior of vitreous gel is due to the presence of biopolymers in its structure. Fluid properties of the vitreous is mainly the result of interactions between the characteristics of collagen type II and Hyaluronic Acid networks. Having a better understanding of the structure of each component and their changes during aging and various diseases such as diabetes can lead to better monitoring and treatment options. We study the effects of collagenase type II on 44 samples of porcine vitreous using an in situ rheological experiment in comparison with 18 eyes in a control group injected with Phosphate Buffered Saline Solution. We analyze the behavior of each component over time in both groups. We focus on the changes of viscosity and elasticity of the collagen network within the vitreous. The results of the analysis in this study show that the changes in the fluid properties of the vitreous after collagenase injection is driven by the structural alterations of the collagen network. Creep compliance values of the collagen network are significantly higher in the first group compared to the control group one hour and twenty-four hours after the injection. In contrast, creep compliance of the HA network shows no statistically significant change one hour after the injection in both groups. The results of the reported analysis of individual components in this study support the previous findings on the alterations within the vitreous structure in its entirety.

Despite high metabolic activity, the retina and optic nerve head lack traditional lymphatic drainage. We here identified an ocular glymphatic clearance route for fluid and wastes via the proximal optic nerve in rodents. β-amyloid (Aβ) was cleared from the retina and vitreous via a pathway dependent on glial water channel aquaporin-4 (AQP4) and driven by the ocular-cranial pressure difference. After traversing the lamina barrier, intra-axonal Aβ was cleared via the perivenous space and subsequently drained to lymphatic vessels. Light-induced pupil constriction enhanced efflux, whereas atropine or raising intracranial pressure blocked efflux. In two distinct murine models of glaucoma, Aβ leaked from the eye via defects in the lamina barrier instead of directional axonal efflux. The results suggest that, in rodents, the removal of fluid and metabolites from the intraocular space occurs through a glymphatic pathway that might be impaired in glaucoma.

Background/objectivesTo evaluate retinal lymphoma treatment at the University of California San Francisco.Subjects/methodsProspective observational audit. Patients were treated systemically, usually with: methotrexate, temozolomide, and rituximab induction; etoposide consolidation; and maintenance with lenalidomide or another immunomodulatory agent. Persistent disease was treated initially by ocular radiotherapy or intravitreal melphalan and latterly by vitrectomy.ResultsThe cohort comprised eight females and two males. The median age was 58 years (range, 38-73). Ocular manifestations were initially unilateral in four patients. Vitreous and subretinal infiltrates were initially present in 16 and 12 eyes, respectively, with three eyes having vision of 20/200 or worse. Four patients had a history of central nervous system (CNS) lymphoma. The median ophthalmic follow-up was 37 months (range, 8-56). Diagnostic and therapeutic vitrectomy were performed in 10 and 2 eyes, respectively. All patients had systemic chemotherapy and eight received maintenance immunotherapy. Four patients underwent ocular radiotherapy, bilaterally in two. One patient received bilateral intravitreal melphalan injections. Two eyes of four patients developed lymphoma during the study and two patients developed CNS disease. At study close, subretinal deposits were subtle in nine eyes and more prominent in two, whereas vitreous infiltrates were minimal in nine eyes, mild in one and moderate in one. The latest visual acuity was significantly worse than at presentation in two eyes and better in two. All patients were alive with no active CNS disease.ConclusionsSubretinal lymphomatous infiltrates respond to systemic chemotherapy with immunomodulatory maintenance, but dense vitreous infiltrates require therapeutic vitrectomy.

PurposeTo investigate the relationship between proangiogenic and inflammatory cytokines in concurrent vitreous, aqueous, and plasma samples from patients with proliferative diabetic retinopathy (PDR).MethodsVitreous, aqueous, and plasma samples were analyzed using multiplex immunoassay for 10 PDR-related cytokines (IL-6, IL-8, TNF-α, monocyte chemoattractant protein-1 [MCP-1], macrophage inflammatory protein-1β [MIP-1β], VEGF receptor 1 [Flt-1], placental growth factor [PlGF], VEGF-A, VEGF-C, VEGF-D). A total of 17 patients with PDR and 7 controls were included. The primary outcome was correlation of cytokines in vitreous, aqueous, and plasma. The secondary outcome was the comparison of cytokine levels in controls and diabetics with and without recent anti-VEGF injection.ResultsThe following factors were elevated in diabetics compared with controls: vitreous IL-6, IL-8, TNF-α, MCP-1, MIP-1β, PlGF, and VEGF-A; and aqueous IL-6, IL-8, PlGF, and VEGF-C (all P < 0.05). Vitreous and aqueous IL-8, PlGF, and VEGF-A were significantly correlated in patients with PDR (all P < 0.05). Plasma cytokines were not correlated with those in vitreous and aqueous (all P > 0.05). Vitreous and aqueous IL-6, IL-8, TNF-α, PlGF, and VEGF-A differed among controls and diabetics with and without recent anti-VEGF injection (all P < 0.05). In one-to-one comparisons, aqueous VEGF-A levels were lower in diabetic patients who had recent anti-VEGF injection compared with those who did not (P = 0.01).ConclusionsIn this proof-of-concept study, IL-8, VEGF-A, and PlGF demonstrated a strong correlation in vitreous and aqueous of patients with PDR. The aqueous may serve as a proxy for vitreous for some cytokines involved in PDR. Recent anti-VEGF injections decreased VEGF-A levels in aqueous, but did not significantly affect other cytokines, suggesting a role for other targeted therapies in PDR management.

Purpose:To investigate the relationship between proangiogenic and inflammatory cytokines in concurrent vitreous, aqueous, and plasma samples from patients with proliferative diabetic retinopathy (PDR). Methods:Vitreous, aqueous, and plasma samples were analyzed using multiplex immunoassay for 10 PDR-related cytokines (IL-6, IL-8, TNF-α, monocyte chemoattractant protein-1 [MCP-1], macrophage inflammatory protein-1β [MIP-1β], VEGF receptor 1 [Flt-1], placental growth factor [PlGF], VEGF-A, VEGF-C, VEGF-D). A total of 17 patients with PDR and 7 controls were included. The primary outcome was correlation of cytokines in vitreous, aqueous, and plasma. The secondary outcome was the comparison of cytokine levels in controls and diabetics with and without recent anti-VEGF injection. Results:The following factors were elevated in diabetics compared with controls: vitreous IL-6, IL-8, TNF-α, MCP-1, MIP-1β, PlGF, and VEGF-A; and aqueous IL-6, IL-8, PlGF, and VEGF-C (all P < 0.05). Vitreous and aqueous IL-8, PlGF, and VEGF-A were significantly correlated in patients with PDR (all P < 0.05). Plasma cytokines were not correlated with those in vitreous and aqueous (all P > 0.05). Vitreous and aqueous IL-6, IL-8, TNF-α, PlGF, and VEGF-A differed among controls and diabetics with and without recent anti-VEGF injection (all P < 0.05). In one-to-one comparisons, aqueous VEGF-A levels were lower in diabetic patients who had recent anti-VEGF injection compared with those who did not (P = 0.01). Conclusions:In this proof-of-concept study, IL-8, VEGF-A, and PlGF demonstrated a strong correlation in vitreous and aqueous of patients with PDR. The aqueous may serve as a proxy for vitreous for some cytokines involved in PDR. Recent anti-VEGF injections decreased VEGF-A levels in aqueous, but did not significantly affect other cytokines, suggesting a role for other targeted therapies in PDR management.

Proliferative vitreoretinopathy (PVR) is a significant threat for vision recovery from retinal detachment or ocular trauma. Currently, no approved pharmacological intervention to prevent PVR. Daunorubicin (DNR) and dexamethasone (DEX) were sequentially loaded into oxidized porous silicon (pSiO2) particles by covalent conjugation. The DNR + DEX-loaded particles, and control particles loaded with DNR only and DEX only were incubated with RPE-populated collagen for daily gel surface quantitation. Toxicity was monitored by ophthalmic examinations and histological evaluation 21 days after injection. At 3rd week following intravitreal injection, a localized retinal detachment (RD) was created by subretinal injection of Healon in all pretreated eyes in addition to 3 non-interventional control eyes. 10 µg of bromodeoxyuridine (BrdU) was injected into the vitreous 4 h before sacrifice on day 3 after RD induction. Retinal sections were stained for glial fibrillary green protein (GFAP) and BrdU to identify activated glial cells and retinal cell proliferation. The studies demonstrated that all three pSiO2 particle types were well tolerated in vivo. DNR alone and DNR + DEX combination formulations demonstrated equally strong suppression on gel contraction (least square mean area of the gel: control = 1.71 vs. 30DNR = 1.85 or 30/40Dual = 1.83, p

The Joslin Medalist Study characterized people affected with type 1 diabetes for 50 years or longer. More than 35% of these individuals exhibit no to mild diabetic retinopathy (DR), independent of glycemic control, suggesting the presence of endogenous protective factors against DR in a subpopulation of patients. Proteomic analysis of retina and vitreous identified retinol binding protein 3 (RBP3), a retinol transport protein secreted mainly by the photoreceptors, as elevated in Medalist patients protected from advanced DR. Mass spectrometry and protein expression analysis identified an inverse association between vitreous RBP3 concentration and DR severity. Intravitreal injection and photoreceptor-specific overexpression of RBP3 in rodents inhibited the detrimental effects of vascular endothelial growth factor (VEGF). Mechanistically, our results showed that recombinant RBP3 exerted the therapeutic effects by binding and inhibiting VEGF receptor tyrosine phosphorylation. In addition, by binding to glucose transporter 1 (GLUT1) and decreasing glucose uptake, RBP3 blocked the detrimental effects of hyperglycemia in inducing inflammatory cytokines in retinal endothelial and Müller cells. Elevated expression of photoreceptor-secreted RBP3 may have a role in protection against the progression of DR due to hyperglycemia by inhibiting glucose uptake via GLUT1 and decreasing the expression of inflammatory cytokines and VEGF.

Coccidioidomycosis is an invasive fungus found primarily in the soil of Southwestern United States, Mexico, and Central America. Primary disease mostly presents as a pulmonary disease although multiple organ systems can be affected through lymphohematogenous dissemination, with ocular seeding extremely rare. When present, the anterior segment structures are most commonly affected. Isolated choroid and/or vitreal disease has been reported infrequently. This is a case of chorioretinitis with vitreal involvement.

The number of blind and low vision persons in the US is projected to increase to 5.68 million by 2020. The eye diseases causing loss of vision are life-long, chronic, and often need protracted presence of therapeutics at the disease site to keep the disease in remission. In addition, multiple pathologies participate in the disease process and a single therapy seems insufficient to bring the disease under control and prevent vision loss. This study demonstrates the use of porous silicon (pSi) particles sequentially loaded with daunorubicin (DNR) and dexamethasone (DEX) to create a synergistic intravitreally injectable dual-drug delivery system. DEX targets chronic inflammation while DNR inhibits excessive cell proliferation as well as suppresses hypoxia-inducible factor 1 to reduce scarring. This pSi-based delivery system releases therapeutic concentrations of DNR for 100 days and DEX for over 165 days after a single dose. This intravitreal dual-drug delivery system is also well tolerated after injection into the rabbit eye model, attested by ocular biomicroscopy, ocular tonometry, electroretinography, and histology. This novel dual-drug delivery system opens an attractive modality for combination therapy to manage refractory chorioretinal diseases and further preclinical studies are warranted to evaluate its efficacy.

The number of blind and low vision persons in the US is projected to increase to 5.68 million by 2020. The eye diseases causing loss of vision are life-long, chronic, and often need protracted presence of therapeutics at the disease site to keep the disease in remission. In addition, multiple pathologies participate in the disease process and a single therapy seems insufficient to bring the disease under control and prevent vision loss. This study demonstrates the use of porous silicon (pSi) particles sequentially loaded with daunorubicin (DNR) and dexamethasone (DEX) to create a synergistic intravitreally injectable dual-drug delivery system. DEX targets chronic inflammation while DNR inhibits excessive cell proliferation as well as suppresses hypoxia-inducible factor 1 to reduce scarring. This pSi-based delivery system releases therapeutic concentrations of DNR for 100 days and DEX for over 165 days after a single dose. This intravitreal dual-drug delivery system is also well tolerated after injection into the rabbit eye model, attested by ocular biomicroscopy, ocular tonometry, electroretinography, and histology. This novel dual-drug delivery system opens an attractive modality for combination therapy to manage refractory chorioretinal diseases and further preclinical studies are warranted to evaluate its efficacy.

Blinding retinal diseases become more epidemic as the population ages. These diseases, such as diabetic retinopathy and macular edema, are of chronic nature and require protracted drug presence at the disease site. A sustained intravitreal porous silicon delivery system with dexamethasone (pSiO2-COO-DEX) was evaluated in a new rabbit model of proliferative vitreoretinopathy (PVR) in a real treatment design. In contrast to the pretreatment design model, pSiO2-COO-DEX was intravitreally injected into the eyes with active inflammation. Subretinal injection of vascular endothelial growth factor (VEGF) and Matrigel induced a late-onset vitreoretinal inflammation that gradually developed into PVR. This method mimics the human disease better than PVR induced by either intravitreal cell injection or trauma. The pSiO2-COO-DEX intervened eyes had minimal PVR, while balanced saline solution or free dexamethasone intervened eyes had significantly more PVR formation. In addition, adding VEGF to the Matrigel for subretinal injection induced greater inflammation and retinal neovascularization in comparison to only Matrigel injected under the medullary ray. Clinical and pathological examinations, including fundus fluorescein angiography and optical coherence tomography, confirmed these changes. In the current study, neither subretinal injection of Matrigel or subretinal injection of VEGF and Matrigel induced choroidal neovascularization. However, the current PVR model demonstrates a chronic course with moderate severity, which may be useful for drug screening studies.

PurposeTo identify changes induced by environmental tobacco smoke (ETS) in circulatory microRNA (miRNA) in plasma and ocular fluids of the Rhesus macaque and compare these changes to normal age-related changes. Tobacco smoke has been identified as the leading environmental risk factor for age-related macular degeneration (AMD).MethodsAll Rhesus macaques were housed at the California National Primate Research Center (CNPRC), University of California, Davis. Four groups of animals were used: Group 1 (1-3 years old), Group 2 (19-28 years old), Group 3 (10-16 years old), and Group 4 (middle aged, 9-14 years old). Group 4 was exposed to smoke for 1 month. Ocular fluids and plasma samples were collected, miRNAs isolated, and expression data obtained using Affymetrix miRNA GeneTitan Array Plates 4.0. Bioinformatics analysis was done on the Affymetrix Expression Console (EC), Transcriptome Analysis Software (TAS) using ANOVA for candidate miRNA selection, followed by Ingenuity Pathway Analysis (IPA).ResultsThe expression of circulatory miRNAs showed statistically significant changes with age and ETS. In the plasma samples, 45 miRNAs were strongly upregulated (fold change >±1.5, p

Blinding retinal diseases become more epidemic as the population ages. These diseases, such as diabetic retinopathy and macular edema, are of chronic nature and require protracted drug presence at the disease site. A sustained intravitreal porous silicon delivery system with dexamethasone (pSiO2-COO-DEX) was evaluated in a new rabbit model of proliferative vitreoretinopathy (PVR) in a real treatment design. In contrast to the pretreatment design model, pSiO2-COO-DEX was intravitreally injected into the eyes with active inflammation. Subretinal injection of vascular endothelial growth factor (VEGF) and Matrigel induced a late-onset vitreoretinal inflammation that gradually developed into PVR. This method mimics the human disease better than PVR induced by either intravitreal cell injection or trauma. The pSiO2-COO-DEX intervened eyes had minimal PVR, while balanced saline solution or free dexamethasone intervened eyes had significantly more PVR formation. In addition, adding VEGF to the Matrigel for subretinal injection induced greater inflammation and retinal neovascularization in comparison to only Matrigel injected under the medullary ray. Clinical and pathological examinations, including fundus fluorescein angiography and optical coherence tomography, confirmed these changes. In the current study, neither subretinal injection of Matrigel or subretinal injection of VEGF and Matrigel induced choroidal neovascularization. However, the current PVR model demonstrates a chronic course with moderate severity, which may be useful for drug screening studies.

PurposeTo identify changes induced by environmental tobacco smoke (ETS) in circulatory microRNA (miRNA) in plasma and ocular fluids of the Rhesus macaque and compare these changes to normal age-related changes. Tobacco smoke has been identified as the leading environmental risk factor for age-related macular degeneration (AMD).MethodsAll Rhesus macaques were housed at the California National Primate Research Center (CNPRC), University of California, Davis. Four groups of animals were used: Group 1 (1-3 years old), Group 2 (19-28 years old), Group 3 (10-16 years old), and Group 4 (middle aged, 9-14 years old). Group 4 was exposed to smoke for 1 month. Ocular fluids and plasma samples were collected, miRNAs isolated, and expression data obtained using Affymetrix miRNA GeneTitan Array Plates 4.0. Bioinformatics analysis was done on the Affymetrix Expression Console (EC), Transcriptome Analysis Software (TAS) using ANOVA for candidate miRNA selection, followed by Ingenuity Pathway Analysis (IPA).ResultsThe expression of circulatory miRNAs showed statistically significant changes with age and ETS. In the plasma samples, 45 miRNAs were strongly upregulated (fold change >±1.5, p

Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.

Proliferative vitreoretinopathy (PVR) is a blinding eye disease and there is no effective pharmacological measure to prevent PVR development. The difficulty comes from lack of potent antiproliferative agent and lack of sustained delivery to cover high-risk time window for PVR to develop. Lipid prodrug of PMEG, hexadecyloxypropyl 9-[(2-phosphonomethoxy)ethyl]guanine (HDP-PMEG), was prepared and was evaluated as a pharmacological adjuvant to surgical management of PVR. A dose-escalation study determined that the highest nontoxic dose for intravitreal use in pigmented rabbits was 3 µg per eye. The genotoxicity of HDP-PMEG was harnessed as a perioperative preventative measure against PVR in a rabbit eye model while the sustained intravitreal pharmacological effect was evaluated on a laser-induced fibrovascular model in rat eye. After intravitreal 3 µg, HDP-PMEG particles in the rabbit vitreous was visible for at least 6 weeks. A single 50-min intravitreal infusion of HDP-PMEG demonstrated significant inhibition of PVR formation when compared with the eyes infused with only BSS (BSS vs. HDP-PMEG: estimate = 1.14, OR = 3.1, p = .027). A single intravitreal 104 ng (equivalent to 3 µg for rabbit eye) of HDP-PMEG significantly inhibit laser-induced fibrovascular proliferation in rat eye by 55% (least square mean pixel, BSS = 4763569.5 vs. HDP-PMEG = 2148129.7, p

Proliferative vitreoretinopathy (PVR) is a blinding eye disease and there is no effective pharmacological measure to prevent PVR development. The difficulty comes from lack of potent antiproliferative agent and lack of sustained delivery to cover high-risk time window for PVR to develop. Lipid prodrug of PMEG, hexadecyloxypropyl 9-[(2-phosphonomethoxy)ethyl]guanine (HDP-PMEG), was prepared and was evaluated as a pharmacological adjuvant to surgical management of PVR. A dose-escalation study determined that the highest nontoxic dose for intravitreal use in pigmented rabbits was 3 µg per eye. The genotoxicity of HDP-PMEG was harnessed as a perioperative preventative measure against PVR in a rabbit eye model while the sustained intravitreal pharmacological effect was evaluated on a laser-induced fibrovascular model in rat eye. After intravitreal 3 µg, HDP-PMEG particles in the rabbit vitreous was visible for at least 6 weeks. A single 50-min intravitreal infusion of HDP-PMEG demonstrated significant inhibition of PVR formation when compared with the eyes infused with only BSS (BSS vs. HDP-PMEG: estimate = 1.14, OR = 3.1, p = .027). A single intravitreal 104 ng (equivalent to 3 µg for rabbit eye) of HDP-PMEG significantly inhibit laser-induced fibrovascular proliferation in rat eye by 55% (least square mean pixel, BSS = 4763569.5 vs. HDP-PMEG = 2148129.7, p

Current administration of ranibizumab and other therapeutic macromolecules to the vitreous and retina carries ocular risks, a high patient treatment burden, and compliance barriers that can lead to suboptimal treatment. Here we introduce a device that produces sustained release of ranibizumab in the vitreous cavity over the course of several months. Composed of twin nanoporous polymer thin films surrounding a ranibizumab reservoir, these devices provide release of ranibizumab over 16 weeks in vitro and 12 weeks in vivo, without exhausting the initial drug payload. Following implantation in vivo, devices were well-tolerated and showed no sign of immune response. This platform presents a potential solution to the challenge of delivering protein therapeutics to the vitreous and retina for sustained periods of time.

Current administration of ranibizumab and other therapeutic macromolecules to the vitreous and retina carries ocular risks, a high patient treatment burden, and compliance barriers that can lead to suboptimal treatment. Here we introduce a device that produces sustained release of ranibizumab in the vitreous cavity over the course of several months. Composed of twin nanoporous polymer thin films surrounding a ranibizumab reservoir, these devices provide release of ranibizumab over 16 weeks in vitro and 12 weeks in vivo, without exhausting the initial drug payload. Following implantation in vivo, devices were well-tolerated and showed no sign of immune response. This platform presents a potential solution to the challenge of delivering protein therapeutics to the vitreous and retina for sustained periods of time.

PurposeTo compare the real-time visualization of vitreoretino-choroidal structures using full-depth imaging (FDI) spectral domain optical coherence tomography (SD-OCT) and swept-source (SS)-OCT.MethodsFoveal scans using both FDI SD-OCT (Heidelberg Spectralis) and SS-OCT (Topcon Deep Range Imaging-OCT-1) were obtained in 40 normal eyes, 40 eyes with macular pathologies, and 40 eyes with glaucoma. Full-depth imaging SD-OCT images were obtained by manually enhancing the vitreoretinal interface first and then the choroid while averaging each OCT B-scan 100 times. Swept-source-OCT images were obtained by averaging each B-scan 96 times. After masking and randomly mixing the original OCT images, two independent physicians graded visualization of the premacular bursa, interdigitation zone line, and chorioscleral boundary, and also sharpness of choroidal structures.ResultsA real-time full-depth image of vitreoretino-choroidal structures was successfully achieved with FDI SD-OCT in 118 cases (98.3%) and with SS-OCT in 45 cases (37.5%, P < 0.001). Full-depth imaging SD-OCT imaging was superior to SS-OCT imaging in visualizing the anterior border of the premacular bursa in 109 eyes (90.8%), with average grading of 1.63 ± 0.53 for the FDI SD-OCT and 0.39 ± 0.52 for the SS-OCT (P < 0.001). Swept-source-OCT was similar to FDI SD-OCT in visualizing the chorioscleral boundary in 108 eyes (90.0%), with average grading of 1.81 ± 0.39 for the SS-OCT and 1.78 ± 0.38 for the FDI-OCT (P = 0.566). The visualization of the interdigitation zone line was identical in the 2 imaging instruments (P = 1.000). The sharpness of the choroidal structures was greater with SS-OCT than with FDI-OCT (P < 0.001).ConclusionManual double-enhancing FDI technique using SD-OCT provided a good compromise between vitreous and retinochoroidal structures visualization in real time during scanning procedure. In contrast, SS-OCT imaged well details of choroidal sublayers. Appropriate OCT technology and software should be selected according to its application in clinical settings.

PurposeTo compare the real-time visualization of vitreoretino-choroidal structures using full-depth imaging (FDI) spectral domain optical coherence tomography (SD-OCT) and swept-source (SS)-OCT.MethodsFoveal scans using both FDI SD-OCT (Heidelberg Spectralis) and SS-OCT (Topcon Deep Range Imaging-OCT-1) were obtained in 40 normal eyes, 40 eyes with macular pathologies, and 40 eyes with glaucoma. Full-depth imaging SD-OCT images were obtained by manually enhancing the vitreoretinal interface first and then the choroid while averaging each OCT B-scan 100 times. Swept-source-OCT images were obtained by averaging each B-scan 96 times. After masking and randomly mixing the original OCT images, two independent physicians graded visualization of the premacular bursa, interdigitation zone line, and chorioscleral boundary, and also sharpness of choroidal structures.ResultsA real-time full-depth image of vitreoretino-choroidal structures was successfully achieved with FDI SD-OCT in 118 cases (98.3%) and with SS-OCT in 45 cases (37.5%, P < 0.001). Full-depth imaging SD-OCT imaging was superior to SS-OCT imaging in visualizing the anterior border of the premacular bursa in 109 eyes (90.8%), with average grading of 1.63 ± 0.53 for the FDI SD-OCT and 0.39 ± 0.52 for the SS-OCT (P < 0.001). Swept-source-OCT was similar to FDI SD-OCT in visualizing the chorioscleral boundary in 108 eyes (90.0%), with average grading of 1.81 ± 0.39 for the SS-OCT and 1.78 ± 0.38 for the FDI-OCT (P = 0.566). The visualization of the interdigitation zone line was identical in the 2 imaging instruments (P = 1.000). The sharpness of the choroidal structures was greater with SS-OCT than with FDI-OCT (P < 0.001).ConclusionManual double-enhancing FDI technique using SD-OCT provided a good compromise between vitreous and retinochoroidal structures visualization in real time during scanning procedure. In contrast, SS-OCT imaged well details of choroidal sublayers. Appropriate OCT technology and software should be selected according to its application in clinical settings.

Alternative specimens have been occasionally considered as substitutes for whole blood for postmortem toxicology testing. We studied the applicability of vitreous humor, and evaluated whether it would be suitable to replace (or augment) whole blood for routine drug screening. Results showed that from 51 autopsy cases, we were able to identify an aggregate of 209 findings in whole blood compared with 169 in vitreous. The total number of compounds identified was 71 for whole blood and 60 for vitreous humor. Quantitative analysis showed that whole-blood concentrations of trazodone were several fold higher than vitreous humor concentrations (1.42 ± 0.57 vs. 0.15 ± 0.05 mg/L, respectively) and similar results were also obtained for diazepam (0.37 ± 0.06 vs. 0.13 ± 0.01, respectively). For other drugs such as oxycodone, hydrocodone and doxylamine, a trend suggesting higher concentrations in vitreous humor vs. whole blood was observed; however, this was not significant. Our results are consistent with the limited work of other investigators, and suggest that vitreous humor could be an appropriate matrix for drug screening in postmortem toxicology.

Alternative specimens have been occasionally considered as substitutes for whole blood for postmortem toxicology testing. We studied the applicability of vitreous humor, and evaluated whether it would be suitable to replace (or augment) whole blood for routine drug screening. Results showed that from 51 autopsy cases, we were able to identify an aggregate of 209 findings in whole blood compared with 169 in vitreous. The total number of compounds identified was 71 for whole blood and 60 for vitreous humor. Quantitative analysis showed that whole-blood concentrations of trazodone were several fold higher than vitreous humor concentrations (1.42 ± 0.57 vs. 0.15 ± 0.05 mg/L, respectively) and similar results were also obtained for diazepam (0.37 ± 0.06 vs. 0.13 ± 0.01, respectively). For other drugs such as oxycodone, hydrocodone and doxylamine, a trend suggesting higher concentrations in vitreous humor vs. whole blood was observed; however, this was not significant. Our results are consistent with the limited work of other investigators, and suggest that vitreous humor could be an appropriate matrix for drug screening in postmortem toxicology.

PurposeThe current study aims to evaluate a porous silicon-based drug delivery system meant for sustained delivery of dexamethasone (Dex) to the vitreous and retina.MethodsDexamethasone was grafted covalently into the pore walls of fully oxidized porous silicon particles (pSiO2-COO-Dex), which then was evaluated for the pharmacological effect of the payload on cultured ARPE19 cells before intravitreal injection. The Dex release profile was investigated in a custom designed dynamic dissolution chamber to mimic the turnover of vitreous fluid in rabbit eyes. Ocular safety, in vivo release, and pharmacodynamics were evaluated in rabbit eyes, and the human VEGF-induced rabbit retinal vascular permeability model.ResultsLoading efficiency of Dex was 69 ± 9 μg per 1 mg of the pSiO2-COO-Dex particles. Dynamic in vitro release demonstrated a sustained mode when compared to free Dex, with the drug half-life extended by 5 times. The released Dex was unaltered and biologically active. In vivo drug release in rabbit eyes revealed a mode similar to the release seen in vitro, with a vitreous half-life of 11 days. At 2 and 4 weeks after a single intravitreal injection of pSiO2-COO-Dex particles (mean 2.71 ± 0.47 mg), intravitreal 500 ng of VEGF did not induce significant retinal vessel dilation or fluorescein leakage, while these events were observed in the eyes injected with empty pSiO2 particles or with free Dex. The retinal vessel score from fluorescein angiography for the control eyes was double the score for the eyes injected with pSiO2-COO-Dex. No adverse reaction was observed for the eyes injected with drug-loaded pSi particles during the course of the study.ConclusionsThe porous silicon-based Dex delivery system (pSiO2-COO-Dex) can be administered safely into vitreous without toxicity. Dex release from the porous silicon particles was sustained for 2 months and was effective against VEGF-induced retinal vessel reaction.

PurposeThe current study aims to evaluate a porous silicon-based drug delivery system meant for sustained delivery of dexamethasone (Dex) to the vitreous and retina.MethodsDexamethasone was grafted covalently into the pore walls of fully oxidized porous silicon particles (pSiO2-COO-Dex), which then was evaluated for the pharmacological effect of the payload on cultured ARPE19 cells before intravitreal injection. The Dex release profile was investigated in a custom designed dynamic dissolution chamber to mimic the turnover of vitreous fluid in rabbit eyes. Ocular safety, in vivo release, and pharmacodynamics were evaluated in rabbit eyes, and the human VEGF-induced rabbit retinal vascular permeability model.ResultsLoading efficiency of Dex was 69 ± 9 μg per 1 mg of the pSiO2-COO-Dex particles. Dynamic in vitro release demonstrated a sustained mode when compared to free Dex, with the drug half-life extended by 5 times. The released Dex was unaltered and biologically active. In vivo drug release in rabbit eyes revealed a mode similar to the release seen in vitro, with a vitreous half-life of 11 days. At 2 and 4 weeks after a single intravitreal injection of pSiO2-COO-Dex particles (mean 2.71 ± 0.47 mg), intravitreal 500 ng of VEGF did not induce significant retinal vessel dilation or fluorescein leakage, while these events were observed in the eyes injected with empty pSiO2 particles or with free Dex. The retinal vessel score from fluorescein angiography for the control eyes was double the score for the eyes injected with pSiO2-COO-Dex. No adverse reaction was observed for the eyes injected with drug-loaded pSi particles during the course of the study.ConclusionsThe porous silicon-based Dex delivery system (pSiO2-COO-Dex) can be administered safely into vitreous without toxicity. Dex release from the porous silicon particles was sustained for 2 months and was effective against VEGF-induced retinal vessel reaction.

PurposeThe proteomic profile of vitreous from second-trimester human embryos and young adults was characterized using mass spectrometry and analyzed for changes in protein levels that may relate to structural changes occurring during this time. This vitreous proteome was compared to previous reports to confirm proteins already identified and reveal novel ones.MethodsVitreous from 17 human embryos aged 14 to 20 weeks gestation (WG) and from a 12-, a 14-, a 15-, and a 28-year-old was individually analyzed using tandem mass spectrometry-based proteomics. Peptide spectral count associations with embryonic age were assessed using a general linear model of fold changes and Spearman's rank correlation. Differences between embryonic and young adult vitreous proteomes were also compared. Immunohistochemistry was used to evaluate three proteins in five additional fetal (10-18 WG) human eyes.ResultsThere were 1217 proteins identified in fetal and young adult human vitreous, 206 after quantile normalization and variance filtering. In embryos, the peptide counts of 37 proteins changed significantly from 14 to 20 WG: 75.7% increased, 24.3% decreased. Immunohistochemistry confirmed the absence of clusterin and cadherin in 10 and 14 WG eyes and their presence at 18 WG. Comparing embryonic to young adult vitreous, 47 proteins were significantly higher or lower. A total of 768 proteins not previously identified in the literature are presented.ConclusionsProteins previously unreported in the human vitreous were identified. The human vitreous proteome undergoes significant changes during embryogenesis and young adulthood. A number of protein levels change considerably during the second trimester, with the majority decreasing.

ObjectiveIdentification of a particular cause of meningoencephalitis can be challenging owing to the myriad bacteria, viruses, fungi, and parasites that can produce overlapping clinical phenotypes, frequently delaying diagnosis and therapy. Metagenomic deep sequencing (MDS) approaches to infectious disease diagnostics are known for their ability to identify unusual or novel viruses and thus are well suited for investigating possible etiologies of meningoencephalitis.MethodsWe present the case of a 74-year-old woman with endophthalmitis followed by meningoencephalitis. MDS of her cerebrospinal fluid (CSF) was performed to identify an infectious agent.ResultsSequences aligning to Balamuthia mandrillaris ribosomal RNA genes were identified in the CSF by MDS. Polymerase chain reaction subsequently confirmed the presence of B. mandrillaris in CSF, brain tissue, and vitreous fluid from the patient's infected eye. B. mandrillaris serology and immunohistochemistry for free-living amoebas on the brain biopsy tissue were positive.InterpretationThe diagnosis was made using MDS after the patient had been hospitalized for several weeks and subjected to costly and invasive testing. MDS is a powerful diagnostic tool with the potential for rapid and unbiased pathogen identification leading to early therapeutic targeting.

ObjectiveIdentification of a particular cause of meningoencephalitis can be challenging owing to the myriad bacteria, viruses, fungi, and parasites that can produce overlapping clinical phenotypes, frequently delaying diagnosis and therapy. Metagenomic deep sequencing (MDS) approaches to infectious disease diagnostics are known for their ability to identify unusual or novel viruses and thus are well suited for investigating possible etiologies of meningoencephalitis.MethodsWe present the case of a 74-year-old woman with endophthalmitis followed by meningoencephalitis. MDS of her cerebrospinal fluid (CSF) was performed to identify an infectious agent.ResultsSequences aligning to Balamuthia mandrillaris ribosomal RNA genes were identified in the CSF by MDS. Polymerase chain reaction subsequently confirmed the presence of B. mandrillaris in CSF, brain tissue, and vitreous fluid from the patient's infected eye. B. mandrillaris serology and immunohistochemistry for free-living amoebas on the brain biopsy tissue were positive.InterpretationThe diagnosis was made using MDS after the patient had been hospitalized for several weeks and subjected to costly and invasive testing. MDS is a powerful diagnostic tool with the potential for rapid and unbiased pathogen identification leading to early therapeutic targeting.

PurposeTo evaluate the effect of nonenzymatic cross-linking (glycation) upon the permeability of the vitreous to small- and large-solute diffusion.MethodsVitreous from freshly excised porcine eyes was treated for 30 minutes with control or 0.01%, 0.1%, or 1% methylglyoxal (MG) solution. The efficacy of the glycation regimen was verified by measuring nonenzymatic cross-link density by fluorescence in the vitreous samples. Resistance to collagenase digestion as well as N(ε)-(carboxyethyl) lysine (CEL) content were also measured. The permeability coefficient for fluorescein and fluorescein isothiocyanate (FITC)-IgG diffusion through 3 mL of the vitreous samples was determined by using a custom permeability tester.ResultsVitreous cross-linking with MG treatment was confirmed by increased fluorescence, increased CEL concentration, and increased resistance to collagenase digestion. Vitreous glycation resulted in a statistically significant decrease in the permeability coefficient for fluorescein diffusion when either 0.1% or 1% MG solution was used (5.36 ± 5.24 × 10(-5) cm s(-1), P = 0.04; and 4.03 ± 2.1 × 10(-5) cm s(-1), P = 0.001; respectively, compared with control, 9.77 ± 5.45 × 10(-5) cm s(-1)). The permeability coefficient for diffusion of FITC-IgG between control (9.9 ± 6.37 × 10(-5) cm s(-1)) and treatment groups was statistically significant at all MG concentrations (0.01% MG: 3.95 ± 3.44 × 10(-5) cm s(-1), P = 0.003; 0.1% MG: 4.27 ± 1.32 × 10(-5) cm s(-1), P = 0.004; and 0.1% MG: 3.72 ± 2.49 × 10(-5) cm s(-1), P = 0.001).ConclusionsAdvanced glycation end-product (AGE) accumulation reduces vitreous permeability when glycation is performed in ex vivo porcine vitreous. The permeability change was more pronounced for the larger solute, suggesting a lower threshold for AGE-induced permeability changes to impact the movement of proteins through the vitreous when compared with smaller molecules.

PurposeProliferative vitreoretinopathy (PVR) is the most common cause of poor visual outcomes in association with retinal detachment surgeries and ocular trauma. Daunorubicin (DNR) has shown the strongest efficacy in proliferation inhibition in vitro. However, clinical studies have shown only mild effect owing to limitations of narrow therapeutic window and short vitreous half-life.MethodsThree milligrams of DNR-loaded particles were intravitreally injected into 18 pigmented rabbits, and vitreous samples were collected up to 84 days for analysis. Thirty-seven rabbits were used for a dose-escalation (1, 3, 6 mg) safety and efficacy study in a rabbit PVR model using a pretreatment design.ResultsLoading efficiency of DNR was 108.55 ± 12 μg per 1 mg particles. Eighty-four days of follow-up did not reveal any adverse reaction. Pharmacokinetic analysis demonstrated a vitreous half-life of 29 days with a maximum DNR concentration of 178 ng/mL and a minimum concentration of 29 ng/mL at day 84. Daunorubicin-loaded porous silicon (pSi) particles were dosed 8 to 9 weeks before PVR induction, and PVR severity score was dose dependent (Spearman ρ = -0.25, P = 0.0005). Proliferative vitreoretinopathy with tractional retinal detachment was 88% in the control group, 63% in the low-dose group, 14% in the medium-dose group, and 0% in the high-dose group (Cochran-Armitage Trend Test, Z = 8.99, ρ = -0.67, P < 0.0001).ConclusionsDaunorubicin-loaded pSi particles can safely reside in the vitreous for at least 3 months. The pSi-based delivery expanded the therapeutic window of DNR by a factor of 862 and drove down the minimum effective concentration by a factor of 175.

PurposeClinical evaluation of floaters lacks quantitative assessment of vitreous structure. This study used quantitative ultrasound (QUS) to measure vitreous opacities. Since floaters reduce contrast sensitivity (CS) and quality of life (Visual Function Questionnaire [VFQ]), it is hypothesized that QUS will correlate with CS and VFQ in patients with floaters.MethodsTwenty-two eyes (22 subjects; age = 57 ± 19 years) with floaters were evaluated with Freiburg acuity contrast testing (FrACT; %Weber) and VFQ. Ultrasonography used a customized probe (15-MHz center frequency, 20-mm focal length, 7-mm aperture) with longitudinal and transverse scans taken in primary gaze and a horizontal longitudinal scan through premacular vitreous in temporal gaze. Each scan set had 100 frames of log-compressed envelope data. Within each frame, two regions of interest (ROIs) were analyzed (whole-central and posterior vitreous) to yield three parameters (energy, E; mean amplitude, M; and percentage of vitreous filled by echodensities, P50) averaged over the entire 100-frame dataset. Statistical analyses evaluated E, M, and P50 correlations with CS and VFQ.ResultsContrast sensitivity ranged from 1.19%W (normal) to 5.59%W. All QUS parameters in two scan positions within the whole-central ROI correlated with CS (R > 0.67, P < 0.001). P50 in the nasal longitudinal position had R = 0.867 (P < 0.001). Correlations with VFQ ranged from R = 0.52 (P < 0.013) to R = 0.65 (P < 0.001).ConclusionsQuantitative ultrasound provides quantitative measures of vitreous echodensity that correlate with CS and VFQ, providing objective assessment of vitreous structure underlying the functional disturbances induced by floaters, useful to quantify vitreous disease severity and the response to therapy.

There still is an unmet need for a safe and sustained intravitreal drug delivery system. In this study we are proposing and characterizing a micelle based, clear-media intravitreal drug delivery system using the lipid derivatized nucleoside analog, hexadecyloxypropyl-cidofovir (HDP-CDV, CMX 001). HDP-CDV forms micelles in water and in vitreous supernatant with the critical micelle concentration of 19 μg/mL and 9 μg/mL, respectively at 37 °C. The formed micelles had the average size of 274.7 nm and the Zeta potential of -47.1 mV. Drug release study in the excised rabbit vitreous showed a sustained release profile with a half-life of 2.7 days. The micelle formulation of HDP-CDV demonstrated a good safety profile in two animal species (rabbit and guinea pig) following intravitreal injection. The sustained efficacy was tested in a pretreatment study design and the drug potency was tested in an ongoing herpes simplex virus (HSV-1) retinitis model. The pretreatment studies using single intravitreal injection and later HSV-1 infection revealed at least 9 weeks of vitreous presence and therapeutic level of HDP-CDV, with 71% eyes protection from infection. The treatment study demonstrated that intravitreal administration halted active HSV-1 retinitis in 80% of the infected eyes while cidofovir (CDV) treatment failed to suppress active HSV-1 retinitis. In summary, lipid derivatized nucleoside analogs can be formulated as a micelle intravitreal injection and provides a sustained drug release in vitreous for chronic retinal diseases.

PurposeVitreous liquefaction and subsequent posterior vitreous detachment can lead to several sight-threatening diseases, including retinal detachment, macular hole and macular traction syndrome, nuclear cataracts, and possibly, open-angle glaucoma. In this study, we tested the ability of three novel synthetic chondroitin sulfate proteoglycan mimics to preserve the structure and physical properties of enzymatically-degraded bovine vitreous.MethodsChondroitin sulfate proteoglycan mimics, designed to bind to type II collagen, hyaluronic acid, or both, were applied to trypsin- or collagenase-treated bovine vitreous in situ and in vitro. Rheology and liquefaction tests were performed to determine the physical properties of the vitreous, while Western blots were used to detect the presence and degradation of soluble collagen II (α1). Deep-etch electron microscopy (DEEM) identified the ultrastructure of mimic-treated and untreated enzyme-degraded bovine vitreous.ResultsProteoglycan mimics preserved the physical properties of trypsin-degraded bovine vitreous and protected against vitreous liquefaction. Although the collagen-binding mimic maintained the physical properties of collagenase-treated vitreous, liquefaction still occurred. Western blots indicated that the mimic provided only marginal protective ability against soluble collagen degradation. Deep-etch electron microscopy, however, showed increased density and isotropy of microstructural components in mimic-treated vitreous, supporting the initial result that vitreous structure was preserved.ConclusionsProteoglycan mimics preserved bovine vitreous physical properties after enzymatic degradation. These compounds may be useful in delaying or preventing the pathological effects of age-related, or enzymatically-induced, degradation of the vitreous body.

Dexamethasone is a glucocorticoid that is widely used in the ophthalmic arena. The recent FDA approved dexamethasone implant can provide a three month efficacy but with high rate of drug related cataract and high intraocular pressure (IOP). It seems that higher steroid in aqueous humor and around lens may be associated with these complications based on clinical fact that higher IOP was observed with intravitreal triamcinolone acetonide (TA) than with subtenon TA. We hypothesize that placing a sustained dexamethasone release system near back of the eye through a fine needle can maximize efficacy while mitigate higher rate of IOP rise and cataract. To develop a sustained intravitreal dexamethasone delivery system, porous silicon dioxide (pSiO2) microparticles were fabricated and functionalized with amines as well as carboxyl groups. Dexamethasone was conjugated to pSiO2 through the Steglich Esterification Reaction between hydroxyl of dexamethasone and carboxyl groups on the pSiO2. The drug loading was confirmed by Fourier transform infrared spectroscopy (FTIR) and loading efficiency was quantitated using thermogravimetric analysis (TGA). In vitro release was conducted for three months and dexamethasone was confirmed in the released samples using liquid chromatography-tandem mass spectrometry (LC/MS/MS). A pilot ocular safety and determination of vitreous drug level was performed in rabbit eyes. The drug loading study demonstrated that loading efficiency was from 5.96% to 10.77% depending on the loading reaction time, being higher with longer loading reaction time before reaching saturation around 7 days. In vitro drug release study revealed that dexamethasone release from pSiO2 particles was sustainable for over 90 days and was 80 days longer than free dexamethasone or infiltration-loaded pSiO2 particle formulation in the same setting. Pilot in vivo study demonstrated no sign of ocular adverse reaction in rabbit eyes following a single 3 mg intravitreal injection and free drug level at 2-week was 107.23 ± 10.54 ng/mL that is well above the therapeutic level but only around 20% level of dexamethasone released from OZURDEX(®) (dexamethasone intravitreal implant) in a rabbit eye model. In conclusion, dexamethasone is able to covalently load to the pSiO2 particles and provide sustained drug release for at least 3 months in vitro. Intravitreal injection of these particles were well tolerated in rabbit eyes and free drug level in vitreous at 2-week was well above the therapeutic level.

Daunorubicin (DNR) is an effective inhibitor of an array of proteins involved in neovascularization, including VEGF and PDGF. These growth factors are directly related to retina scar formation in many devastating retinal diseases. Due to the short vitreous half-life and narrow therapeutic window, ocular application of DNR is limited. It has been shown that a porous silicon (pSi) based delivery system can extend DNR vitreous residence from a few days to 3months. In this study we investigated the feasibility of altering the pore size of the silicon particles to regulate the payload release. Modulation of the etching parameters allowed control of the nano-pore size from 15nm to 95nm. In vitro studies showed that degradation of pSiO2 increased with increasing pore size and the degradation of pSiO2 was approximately constant for a given particle type. The degradation of pSiO2 with 43nm pores was significantly greater than the other two particles with smaller pores, judged by observed and normalized mean Si concentration of the dissolution samples (44.2±8.9 vs 25.7±5.6 or 21.2±4.2μg/mL, p

Seasonal hyperacute panuveitis (SHAPU) is a potentially blinding ocular disease occurring in Nepal that principally affects young children. Random amplification of partially purified vitreous fluid (VF)-derived nucleic acid revealed the presence of human anelloviruses in VF of SHAPU patients. In a comparative study of patients with different ocular pathologies, SHAPU patients were at highest risk of harboring anelloviruses in their eyes. The majority of SHAPU patients had multiple anelloviruses in their VF. The ocular anellovirus load in SHAPU and non-SHAPU patients did not differ and no SHAPU-specific anellovirus variant was detected. Analysis of paired serum and VF samples from SHAPU and non-SHAPU patients showed that the anellovirus detected in VF samples most likely originated from the systemic viral pool during viremia, potentially through breakdown of the blood-ocular barrier. The detection of anelloviruses in VF samples of uveitis patients, profoundly so in SHAPU patients, is imperative and warrants elucidation of its clinical significance.

Seasonal hyperacute panuveitis (SHAPU) is a potentially blinding ocular disease occurring in Nepal that principally affects young children. Random amplification of partially purified vitreous fluid (VF)-derived nucleic acid revealed the presence of human anelloviruses in VF of SHAPU patients. In a comparative study of patients with different ocular pathologies, SHAPU patients were at highest risk of harboring anelloviruses in their eyes. The majority of SHAPU patients had multiple anelloviruses in their VF. The ocular anellovirus load in SHAPU and non-SHAPU patients did not differ and no SHAPU-specific anellovirus variant was detected. Analysis of paired serum and VF samples from SHAPU and non-SHAPU patients showed that the anellovirus detected in VF samples most likely originated from the systemic viral pool during viremia, potentially through breakdown of the blood-ocular barrier. The detection of anelloviruses in VF samples of uveitis patients, profoundly so in SHAPU patients, is imperative and warrants elucidation of its clinical significance.

Fibronectin and laminin are clinically relevant plasmin receptors in the eye. Located at the vitreoretinal interface, they are cleaved by ocriplasmin (Microplasmin, ThromboGenics, Iselin, NJ), a novel ophthalmic medication. A series of clinical trials to study ocriplasmin for the treatment of vitreoretinal diseases such as vitreomacular traction, macular hole, and exudative age-related macular degeneration are underway. The results are promising and may impact patient care.

Fibronectin and laminin are clinically relevant plasmin receptors in the eye. Located at the vitreoretinal interface, they are cleaved by ocriplasmin (Microplasmin, ThromboGenics, Iselin, NJ), a novel ophthalmic medication. A series of clinical trials to study ocriplasmin for the treatment of vitreoretinal diseases such as vitreomacular traction, macular hole, and exudative age-related macular degeneration are underway. The results are promising and may impact patient care.