Single cell RNA sequencing confirms retinal microglia activation associated with early onset retinal degeneration.

Mutations in the Membrane-type frizzled related protein (Mfrp) gene results in an early-onset retinal degeneration associated with retinitis pigmentosa, microphthalmia, optic disc drusen and foveal schisis. In the current study, a previously characterized mouse model of human retinal degeneration carrying homozygous c.498_499insC mutations in Mfrp (MfrpKI/KI) was used. Patients carrying this mutation have retinal degeneration at an early age. The model demonstrates subretinal deposits and develops early-onset photoreceptor degeneration. We observed large subretinal deposits in MfrpKI/KI mice which were strongly CD68 positive and co-localized with autofluorescent spots. Single cell RNA sequencing of MfrpKI/KI mice retinal microglia showed a significantly higher number of pan-macrophage marker Iba-1 and F4/80 positive cells with increased expression of activation marker (CD68) and lowered microglial homeostatic markers (TMEM119, P2ry13, P2ry13, Siglech) compared with wild type mice confirming microglial activation as observed in retinal immunostaining showing microglia activation in subretinal region. Trajectory analysis identified a small cluster of microglial cells with activation transcriptomic signatures that could represent a subretinal microglia population in MfrpKI/KI mice expressing higher levels of APOE. We validated these findings using immunofluorescence staining of retinal cryosections and found a significantly higher number of subretinal Iba-1/ApoE positive microglia in MfrpKI/KI mice with some subretinal microglia also expressing lowered levels of microglial homeostatic marker TMEM119, confirming microglial origin. In summary, we confirm that MfrpKI/KI mice carrying the c.498_499insC mutation had a significantly higher population of activated microglia in their retina with distinct subsets of subretinal microglia. Further, studies are required to confirm whether the association of increased subretinal microglia in MfrpKI/KI mice are causal in degeneration.

Effective treatment of retinal neovascular leakage with fusogenic porous silicon nanoparticles delivering VEGF-siRNA.

Aim: To evaluate an intravitreally injected nanoparticle platform designed to deliver VEGF-A siRNA to inhibit retinal neovascular leakage as a new treatment for proliferative diabetic retinopathy and diabetic macular edema. Materials & methods: Fusogenic lipid-coated porous silicon nanoparticles loaded with VEGF-A siRNA, and pendant neovascular integrin-homing iRGD, were evaluated for efficacy by intravitreal injection in a rabbit model of retinal neovascularization. Results: For 12 weeks post-treatment, a reduction in vascular leakage was observed for treated diseased eyes versus control eyes (p = 0.0137), with a corresponding reduction in vitreous VEGF-A. Conclusion: Fusogenic lipid-coated porous silicon nanoparticles siRNA delivery provides persistent knockdown of VEGF-A and reduced leakage in a rabbit model of retinal neovascularization as a potential new intraocular therapeutic.

A sustained dual drug delivery system for proliferative vitreoretinopathy.

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 < .05). Eyes pretreated with pSiO2-DNR + DEX exhibited the least GFAP activation (least square mean intensity mm-2: Dual = 4.03, DNR = 7.76, Dex = 16.23, control = 29.11, p < .05) and BrdU expression (Mean number of BrdU positive cells per mm of retina: Dual = 2.77, DNR = 4.58, Dex = 4.01, control = 6.16, p < .05). The synergistic effect of a sustained release pSiO2-DNR/DEX showed promise for the prevention of PVR development while reducing the necessary therapeutic concentration of each drug.

Single subconjunctival injection formulation using sol-gel mesoporous silica as a controlled release system for drop-free post-cataract surgery care.

PURPOSE: To develop a mesoporous silica drug delivery system and target drop-free care after cataract surgery with a single subconjunctival injection. SETTING: Laboratory. DESIGN: Experimental animal study. METHODS: Ketorolac was infiltration-loaded into sol-gel mesoporous silica particles encapsulated with poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) using a layer-by-layer adsorption technique (SG-Ket-LBL). The formulation was subjected to an in vitro and in vivo drug release study in addition to ocular toxicology evaluation. RESULTS: Thermogravimetric analysis revealed that the drug loading efficiency was 4.4% for the SG-Ket-LBL particles. The in vivo safety study demonstrated that the formulation was well tolerated after subconjunctival injection and aqueous humor pharmacokinetics showed sustained therapeutic drug release for the targeted time window of 6 to 8 weeks. CONCLUSIONS: Findings indicated that sol-gel mesoporous silica could be used as a drug carrier for subconjunctival administration. The tested formulation, SG-Ket-LBL, provided therapeutic ketorolac for 6 to 8 weeks, which might be used for a single subconjunctival injection to replace nonsteroidal anti-inflammatory drug eyedrops after cataract surgery.

Intravitreal safety profiles of sol-gel mesoporous silica microparticles and the degradation product (Si(OH)4).

Mesoporous silica has attracted significant attention in the drug delivery area; however, impurities can be a source of toxicity. The current study used commercial microparticles produced at large scale in a well-controlled environment. Micrometer sized mesoporous silica particles were acquired through a commercial vendor and pore structures were characterized by SEM. The three silica particle formulations had a diameter of 15 micrometers and three different pore sizes of 10 nm, 30 nm, and 100 nm. The fourth formulation had particle size of 20-40 micrometers with 50 nm pores. Before in vivo tests, an in vitro cytotoxicity test was conducted with silicic acid, derived from the sol-gel particles, on EA.hy926 cells. Low concentration (2.5 µg/mL) of silicic acid showed no cytotoxicity; however, high concentration (25 µg/mL) was cytotoxic. In vivo intravitreal injection demonstrated that 15 um silica particles with 10 nm pore were safe in both rabbit and guinea pig eyes and the particles lasted in the vitreous for longer than two months. Formulations of with larger pores demonstrated variable localized vitreous cloudiness around the sol-gel particle depot and mild inflammatory cells in the aqueous humor. The incidence of reaction trended higher with larger pores (10 nm: 0%, 30 nm: 29%, 50 nm: 71%, 100 nm: 100%, p < .0001, Cochran Armitage Trend Test). Sol-gel mesoporous silica particles have uniform particle sizes and well-defined pores, which is an advantage for implantation via a fine needle. Selected formulations may be used as an intraocular drug delivery system with proper loading and encapsulation.

Acute Rabbit Eye Model for Testing Subretinal Prostheses.

PURPOSE: Subretinal prostheses are a novel technology for restoring useful vision in patients with retinitis pigmentosa or age-related macular degeneration. We characterize the surgical implantation technique and functional time window of an acute rabbit eye model for testing of human subretinal prostheses. METHODS: Retinal prostheses were implanted subretinally in 26 rabbits using a two-step technique. Fundus imaging, fluorescein fundus angiography, and optical coherence topography (OCT) were conducted postoperatively from days 1 to 21 to monitor prosthesis positioning and retinal anatomic changes. RESULTS: Successful implantation and excellent retina apposition were achieved in 84.6% of the rabbits. OCTs showed the overlying retina at full thickness for the first 2 days after implantation. Histology confirmed intact inner layers of the overlying retina until day 3. Progressive atrophy of the overlying retina was revealed by repeated OCTs; approximately 40% of the retina thickness remained on postoperative days 5 and 6. CONCLUSIONS: The two-step implantation technique works well for the rabbit eye model with human prostheses. Rabbit retina may be used for acute electrophysiologic testing of a retinal prosthesis, but is unsuitable for chronic studies due to the merangiotic retina and its limited time window of validity. TRANSLATIONAL RELEVANCE: The improved efficacy in prosthesis surgery using this technique will circumvent the challenges in animal models that provide human-like features critical for the transition into human clinical trials.

The Effects of a Targeted History Question on Patient-Triage Nurse Communication.

Fast tracks are widely used in emergency departments to increase patient throughput as annual visits continue to rise in the United States. A modified triage process known as QuickLook, which omits patients' past medical history, is used in some hospitals to further increase throughput. This article discusses the effects of QuickLook on patient placement, reviews the role of past medical history in triage, and discusses the impact of integrating a targeted history question into the QuickLook process of an emergency department in Arizona.

Porous silicon based intravitreal platform for dual-drug loading and controlled release towards synergistic therapy.

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.

New model of proliferative vitreoretinopathy in rabbit for drug delivery and pharmacodynamic studies.

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.

A Novel Approach of Daunorubicin Application on Formation of Proliferative Retinopathy Using a Porous Silicon Controlled Delivery System: Pharmacodynamics.

PURPOSE: Proliferative 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. METHODS: Three 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. RESULTS: Loading 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). CONCLUSIONS: Daunorubicin-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.