Dendrimer-based targeted intravitreal therapy for sustained attenuation of neuroinflammation in retinal degeneration.

Retinal neuroinflammation, mediated by activated microglia, plays a key role in the pathogenesis of photoreceptor and retinal pigment epithelial cell loss in age-related macular degeneration and retinitis pigmentosa. Targeted drug therapy for attenuation of neuroinflammation in the retina was explored using hydroxyl-terminated polyamidoamine (PAMAM) dendrimer-drug conjugate nanodevices. We show that, upon intravitreal administration, PAMAM dendrimers selectively localize within activated outer retinal microglia in two rat models of retinal degeneration, but not in the retina of healthy controls. This pathology-dependent biodistribution was exploited for drug delivery, by covalently conjugating fluocinolone acetonide to the dendrimer. The conjugate released the drug in a sustained manner over 90 days. In vivo efficacy was assessed using the Royal College of Surgeons (RCS) rat retinal degeneration model over a four-week period when peak retinal degeneration occurs. One intravitreal injection of 1 µg of FA conjugated to 7 µg of the dendrimer was able to arrest retinal degeneration, preserve photoreceptor outer nuclear cell counts, and attenuate activated microglia, for an entire month. These studies suggest that PAMAM dendrimers (with no targeting ligands) have an intrinsic ability to selectively localize in activated microglia, and can deliver drugs inside these cells for a sustained period for the treatment of retinal neuroinflammation.

Intravitreous delivery of the corticosteroid fluocinolone acetonide attenuates retinal degeneration in S334ter-4 rats.

PURPOSE: To study the neuroprotective properties of low-dose, sustained-release intravitreous fluocinolone acetonide (FA) in transgenic S334ter-4 rats. METHODS: S334ter-4 rats aged 4 weeks were divided into four groups: 0.5 microg/d FA-loaded intravitreous drug delivery implant (IDDI); 0.2 microg/d FA-loaded IDDI; inactive IDDI; and unoperated controls. Electroretinography (ERG) was performed before surgery and every 2 weeks after surgery for 8 weeks. When the rats were 12 weeks of age, outer nuclear layer (ONL) and inner nuclear layer (INL) thicknesses were measured. Microglial cell counts were obtained from retinal wholemounts labeled for Iba-1. RESULTS: At the end of the study, unoperated and inactive IDDI-implanted rats demonstrated 50% to 60% reductions in ERG amplitudes compared with those recorded at 4 weeks (P < 0.001 for both groups). FA 0.2-microg/d animals demonstrated 15% amplitude attenuation, while FA 0.5-microg/d animals showed 30% reduction. ONL thickness in FA 0.2-microg/d-treated eyes was 25.8% +/- 2.3% higher than in control group eyes (P < 0.001) and 30.0% +/- 2.1% higher than in inactive IDDI-implanted eyes (P < 0.001). In FA 0.5-microg/d-treated eyes, ONL thickness was 22.4% +/- 2.8% higher than in control group eyes (P < 0.001) and 22.3% +/- 3.7% higher than in inactive IDDI-implanted eyes (P < 0.01). No statistically significant difference was observed between the two control groups. No statistically significant difference between the two FA-treated groups was found. FA-treated groups demonstrated significantly fewer activated microglial cells than control groups. CONCLUSIONS: Chronic intravitreous infusion of FA preserves ONL cell morphology and ERG a- and b-wave amplitudes and reduces retinal neuroinflammation in S334ter rats. Based on these findings, the synthetic corticosteroid FA may promise a therapeutic role in patients with retinal degeneration.

Toll-like receptor 2 ligand-induced protection against bacterial endophthalmitis.

BACKGROUND: Activation of innate immunity plays a key role in determining the outcome of an infection. Here, we investigated whether Toll-like receptors (TLRs) are involved in retinal innate response and explored the prophylactic use of TLR2 ligand in preventing bacterial endophthalmitis. METHODS: C57BL/6 mice were given intravitreal injections of Pam3Cys, a synthetic ligand of TLR2, or vehicle (phosphate-buffered saline) 24 h prior to Staphylococcus aureus inoculation. The severity of endophthalmitis was graded by slit lamp, electroretinography, histological examinations, and determination of bacterial load in the retina. The expression of cytokines/chemokines and cathelicidin-related antimicrobial peptide was assessed by enzyme-linked immunosorbent assay and Western blot, respectively. RESULTS: Intravitreal injections of Pam3Cys up-regulated TLR2 expression in the retina of C57BL/6 mice, and Pam3Cys pretreatment significantly improved the outcome of S. aureus endophthalmitis, preserved retinal structural integrity, and maintained visual function as assessed by electroretinography in C57BL/6 mice. Furthermore, Pam3Cys pretreatment activated retinal microglia cells, induced the expression of cathelicidin-related antimicrobial peptide, and remarkably reduced the bacterial load. CONCLUSIONS: This is the first report that highlights the existence and role of TLR2 in retinal innate immune response to S. aureus infection and suggests that modulation of TLR activation provides a novel prophylactic approach to prevent bacterial endophthalmitis.

Photoreceptor neuroprotection in RCS rats via low-dose intravitreal sustained-delivery of fluocinolone acetonide.

PURPOSE: To study the neuroprotective effects of intravitreal fluocinolone acetonide (FA) in Royal College of Surgeons (RCS) rats. METHODS: Five-week-old RCS rats were divided into four groups: 0.5 microg/d FA-loaded intravitreal drug-delivery implant (IDDI); 0.2 microg/d FA-loaded IDDI; inactive IDDI; and nonsurgical control. Electroretinography (ERG) and intraocular pressure (IOP) measurements were performed before surgery and weekly thereafter. Thicknesses of the retinal outer (ONL) and inner (INL) nuclear layers were evaluated at 9 weeks of age. ED-1-labeled activated microglia were counted. Total microglial cell counts were made by using Iba-1 antibody labeling. RESULTS: At 9 weeks, control groups demonstrated an 80% reduction in ERG amplitudes (P < 0.001 for both groups). FA-treated groups demonstrated no statistically significant attenuation of ERG amplitudes at the end of the study, compared with the initial ERGs. Intraocular pressure (IOP) remained normal in all groups. ONL thickness in FA 0.2 microg/d-treated eyes was 2.1 +/- 0.5 times greater than in nonsurgical eyes (P < 0.001) and 3.4 +/- 0.7 times greater than in inactive IDDI-treated eyes (P < 0.0001). In FA 0.5 microg/d-treated eyes, ONL thickness was 1.5 +/- 0.1 times higher than in nonsurgical controls (P < 0.05) and 2.4 +/- 0.4 times higher than in inactive IDDI-treated eyes (P < 0.01). INL thickness was not different among groups. FA-treated eyes demonstrated significantly fewer activated microglia (P < 0.001) and overall number of microglia in the photoreceptor and outer debris zone layers (P < 0.001), compared with control groups. CONCLUSIONS: Chronic intravitreal infusion of FA is neuroprotective in RCS rats, preserves ONL morphology and ERG amplitudes and reduces retinal neuroinflammation. These findings may have a therapeutic role in human photoreceptor cell degenerations.

Chronic intravitreous infusion of ciliary neurotrophic factor modulates electrical retinal stimulation thresholds in the RCS rat.

PURPOSE: To determine whether the sustained intravitreous delivery of CNTF modulates cortical response thresholds to electrical retinal stimulation in the RCS rat model of retinal degeneration. METHODS: Animals were assigned to four groups: untreated, nonsurgical control and infusion groups of 10 ng/d CNTF, 1 ng/d CNTF, and PBS vehicle control. Thresholds for electrically evoked cortical potentials (EECPs) were recorded in response to transcorneal electrical stimulation of the retina at p30 and again at p60, after a three-week infusion. RESULTS: As the retina degenerated over time, EECP thresholds in response to electrical retinal stimulation increased. Eyes treated with 10 ng/d CNTF demonstrated significantly greater retinal sensitivity to electrical stimulation when compared with all other groups. In addition, eyes treated with 1 ng/d CNTF demonstrated significantly greater retinal sensitivity than both PBS-treated and untreated control groups. CONCLUSIONS: Retinal sensitivity to electrical stimulation was preserved in animals treated with chronic intravitreous infusion of CNTF. These data suggest that CNTF-mediated retinal neuroprotection may be a novel therapy that can lower stimulus thresholds in patients about to undergo retinal prosthesis implantation. Furthermore, it may maintain the long-term efficacy of these devices in patients.

Localization of multifocal electroretinogram abnormalities to the lesion site: findings in a family with Best disease.

OBJECTIVE: To determine the association between multifocal electroretinogram (mfERG) abnormalities and macular lesions, as shown by retinal photography and optical coherence tomography (OCT), in a 3-generation family with vitelliform macular dystrophy. METHODS: Five family members were examined using OCT, mfERG, and retinal photography. To localize mfERG abnormalities in relation to retinal findings, we overlaid the mfERG trace arrays on the retinal images and aligned the mfERGs and OCT images in the 180 degrees meridian. RESULTS: Family members had typical macular lesions, normal full-field ERGs, and reduced electro-oculogram light-dark ratios. The OCT images demonstrated variable lesion severity. Some individuals with good vision and normal-appearing fundi showed OCT abnormalities of the choroid and retinal pigment epithelium. The overlay technique revealed that the depressed mfERGs corresponded with the lesions detected by OCT and retinal photography. The latencies of mfERG components in the 2 central stimulus rings in our patients were often prolonged. CONCLUSIONS: The mfERG abnormalities matched the localization of the macular lesions in our patients. The latencies of the mfERG N1 and P1 components in the first 2 concentric stimulus rings were often significantly (>2 SDs) delayed, an observation that has not been previously reported, to our knowledge.