Response of Diabetic Macular Edema to Anti-VEGF Medications Correlates with Improvement in Macular Vessel Architecture Measured with OCT Angiography.

Purpose: Improvements in best-corrected visual acuity (BCVA) and central subfield thickness (CST) have been well documented after intravitreal injection of anti-VEGF medications in diabetic macular edema (DME); however, their effect on the vasculature of the macula in diabetic retinopathy (DR) remains poorly understood. Our aim was to explore the effect of intravitreal injection of anti-VEGF on parameters of retinal vascular microstructure in DR with OCT angiography (OCTA). Design: Retrospective study of adult patients with DME that were treated with anti-VEGF intravitreal injections at the University of Illinois at Chicago between 2017 and 2022. Participants: Forty-one eyes from 30 patients with nonproliferative or proliferative DR with a mean age of 58.83 ± 11.71 years, mean number of intravitreal injections of 2.8 ± 1.4, and mean follow-up of 6.5 ± 1.7 months. Methods: ImageJ was employed to measure parameters of retinal vascular microstructure in OCTA images, which included perfusion density, vessel-length density (VLD), vessel diameter, and foveal avascular zone (FAZ) characteristics (area, perimeter, and circularity). Student t tests and analysis of variance were used to determine statistical significance. Main Outcome Measures: A primary analysis was performed comparing the mean of each parameter of all patients as a single group at the beginning and end of the study period. A subgroup analysis was then performed after stratifying patients based on visual improvement, change in CST, prior injection history, and number of injections. Results: Eyes demonstrated statistical improvement in BCVA logarithm of the minimum angle of resolution score and CST after anti-VEGF treatment. Primary analysis showed a reduction in the vessel diameter of the superficial and deep retinal vasculature, as well as an increase in the circularity of the FAZ within the superficial retinal vasculature after anti-VEGF treatment. Subgroup analysis revealed that eyes with improvement in BCVA exhibited reduced vessel diameter in the superficial retinal vasculature and that eyes with the largest decrease in CST displayed increased perfusion density and VLD in the deep retinal vasculature. Conclusions: Intravitreal injection of anti-VEGF agents to treat DME improved parameters of retinal vascular microstructure on OCTA over a period of 3 to 9 months, and this effect was most pronounced in eyes that experienced improvement in BCVA and CST. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Occult retinopathy following treatment of Hepatitis C with glecaprevir/pibrentasvir (Mavyret).

BACKGROUND/PURPOSE: Medication-induced ocular toxicity is an important consideration in the differential diagnosis of unexplained visual disturbance. We present a case of visual disturbance after starting treatment with glecaprevir/pibrentasvir (Mavyret), a therapy for Hepatitis C virus approved by the FDA in 2017. METHODS: A 50-year-old male with no significant ocular history experienced bilateral visual disturbance, including visual field and acuity loss, shortly after initiating treatment with Mavyret for Hepatitis C. Examination of the anterior and posterior segments was unremarkable, and no abnormalities could be identified on multimodal imaging of the eye and brain, including MRI, SD-OCT, and fundus autofluorescence. Extensive testing for inflammatory, infectious, nutritional, and genetic etiologies for optic neuropathy and retinopathy was negative. RESULTS: Electrophysiology testing was pursued to narrow the broad differential diagnosis. Full-field electroretinography and multi-focal electroretinography detected deficiencies in the rod and cone visual pathways and attenuated electrophysiologic responses in the fovea. Pattern electroretinography and visually-evoked potentials demonstrated macula dysfunction. Taken together, electrophysiologic data suggested diffuse retinal dysfunction, which was most pronounced in the macula. CONCLUSIONS: Given the temporal relationship between Mavyret administration and vision loss in our patient, and the absence of an underlying cause after extensive evaluation, we propose that Mavyret may be associated with a toxic occult retinopathy characterized by panretinal dysfunction without clinically apparent structural findings.

Binocular benefit following monocular subretinal AAV injection in a mouse model of autosomal dominant retinitis pigmentosa (adRP).

Autosomal dominant retinitis pigmentosa (adRP) is frequently caused by mutations in RHO, the gene for rhodopsin. In previous experiments in dogs with the T4R mutation in RHO, an AAV2/5 vector expressing an shRNA directed to human and dog RHO mRNA and an shRNA-resistant human RHO cDNA (AAV-RHO820-shRNA820) prevented retinal degeneration for more than eight months following injection. It is crucial, however, to determine if this RNA replacement vector acts in a mutation-independent and species-independent manner. We, therefore, injected mice transgenic for human P23H RHO with this vector unilaterally at postnatal day 30. We monitored their retinal structure by using spectral-domain optical coherence tomography (SD-OCT) and retinal function using electroretinography (ERG) for nine months. We compared these to P23H RHO transgenic mice injected unilaterally with a control vector. Though retinas continued to thin over time, compared to control injected eyes, treatment with AAV-RHO820-shRNA820 slowed the loss of photoreceptor cells and the decrease in ERG amplitudes during the nine-month study period. Unexpectedly, we also observed the preservation of retinal structure and function in the untreated contralateral eyes of AAV-RHO820-shRNA820 treated mice. PCR analysis and western blots showed that a low amount of vector from injected eyes was present in uninjected eyes. In addition, protective neurotrophic factors bFGF and GDNF were elevated in both eyes of treated mice. Our finding suggests that using this or similar RNA replacement vectors in human gene therapy may provide clinical benefit to both eyes of patients with adRP.

Modulation of Retinal Inflammation Delays Degeneration in a Mouse Model of Geographic Atrophy.

The advanced form of AMD, geographic atrophy, is associated with increased RPE oxidative stress and chronic inflammation. Here we evaluated the effects of delivering an anti-inflammatory viral gene by an AAV-vector in a mouse model of geographic atrophy. We measured changes in retinal function, structure, and morphology over nine months with electroretinography, optical coherence tomography, and fundoscopy, respectively. In addition, we used retinal tissue to quantify changes in markers of inflammation by multiplex ELISA, RT-qPCR, and immunofluorescence staining. Our AAV significantly delayed the loss of retinal function and structure and decreased retinal inflammation compared to the control AAV treatment. Our results suggest that modulating retinal inflammation could significantly slow the progression of geographic atrophy.

Automated segmentation and analysis of retinal microglia within ImageJ.

Microglia are immune cells of the central nervous system capable of distinct phenotypic changes and migration in response to injury. These changes most notably include the retraction of fine dendritic structures and adoption of a globular, phagocytic morphology. Due to their characteristic responses, microglia frequently act as histological indicators of injury progression. While algorithms seeking to automate microglia counts and morphological analysis are becoming increasingly popular, few exist that are adequate for use within the retina and manual analysis remains prevalent. To address this, we propose a novel segmentation routine, implemented within FIJI-ImageJ, to perform automated segmentation and cell counting of retinal microglia. We show that our routine could perform cell counts with accuracy similar to manual observers using the I307N Rho model. Tracking cell position relative to retinal vasculature, we observed population migration towards the photoreceptor layer beginning 12 h post light damage. Using feature selection with Chi2 and principal component analysis, we resolved cells along a morphological gradient, demonstrating that extracted features were sufficiently descriptive to capture subtle morphological changes within cell populations in I307N Rho and Balb/c TLR2-/- retinal degeneration models. Taken together, we introduce a novel automated routine capable of efficient image processing and segmentation. Using data retrieved following segmentation, we perform morphological analysis simultaneously on whole populations of cells, rather than individually. Our algorithm was built entirely with open-source software, for use on retinal microglia.

Sectoral activation of glia in an inducible mouse model of autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of blinding disorders caused by diverse mutations, including in rhodopsin (RHO). Effective therapies have yet to be discovered. The I307N Rho mouse is a light-inducible model of autosomal dominant RP. Our purpose was to describe the glial response in this mouse model to educate future experimentation. I307N Rho mice were exposed to 20,000 lx of light for thirty minutes to induce retinal degeneration. Immunofluorescence staining of cross-sections and flat-mounts was performed to visualize the response of microglia and Müller glia. Histology was correlated with spectral-domain optical coherence tomography imaging (SD-OCT). Microglia dendrites extended between photoreceptors within two hours of induction, withdrew their dendrites between twelve hours and one day, appeared ameboid by three days, and assumed a ramified morphology by one month. Glial activation was more robust in the inferior retina and modulated across the boundary of light damage. SD-OCT hyper-reflectivity overlapped with activated microglia. Finally, microglia transiently adhered to the RPE before which RPE cells appeared dysmorphic. Our data demonstrate the spatial and temporal pattern of glial activation in the I307N Rho mouse, and correlate these patterns with SD-OCT images, assisting in interpretation of SD-OCT images in preclinical models and in human RP.

Expression of a CARD Slows the Retinal Degeneration of a Geographic Atrophy Mouse Model.

Age-related macular degeneration (AMD) has been linked to oxidative damage and para-inflammation, an activation of inflammasome signaling in the retinal pigment epithelium (RPE) and the underlying choriocapillaris. Herein, we tested the efficacy of a gene-delivered caspase-1 inhibitor in controlling the retinal degeneration observed in two models of RPE-choroid oxidative damage. In an acute model of oxidative stress (NaIO3 injection), eyes pre-treated with the sGFP-TatCARD (trans-activator of transcription; caspase activation and recruitment domain) vector demonstrated a recovery of retinal function and partial protection of RPE structure 1 month after damage, in contrast with control-treated eyes. In a model of chronic oxidative stress (RPE-specific deletion of Sod2), eyes treated with the sGFP-TatCARD vector after the onset of degeneration had a significantly slower decline in retinal function when compared to control-treated eyes. Earlier treatment of this model with the same adeno-associated virus (AAV) vector resulted in a greater protection of RPE function in eyes treated with the TatCARD when compared to control-treated eyes. Our results demonstrate that intravitreal delivery of sGFP-TatCARD reduces inflammation and can protect the retina from both acute and sustained oxidative damage within the RPE and choroid. Therefore, gene therapy with a cell-penetrating inflammasome inhibitor such as CARD may stem the progression of AMD.

Co-Delivery of a Short-Hairpin RNA and a shRNA-Resistant Replacement Gene with Adeno-Associated Virus: An Allele-Independent Strategy for Autosomal-Dominant Retinal Disorders.

Recombinant adeno-associated virus (rAAV) has become an important gene delivery vector for the treatment of inherited retinal degenerative diseases. Many of the mutations leading to retinal degeneration are inherited in an autosomal-dominant pattern and can produce toxic gain-of-function and/or dominant-negative effects. Here we describe an allele-independent gene therapy strategy with rAAV to treat autosomal-dominant retinal degenerative diseases. In this methodology, we co-deliver a short-hairpin RNA (shRNA) to inhibit expression of both the toxic and (WT) copies of the gene as well as an shRNA-resistant cDNA for functional gene replacement with a rAAV.

Clinically Relevant Outcome Measures for the I307N Rhodopsin Mouse: A Model of Inducible Autosomal Dominant Retinitis Pigmentosa.

Purpose: The I307N rhodopsin (Rho) mouse is a light-inducible model of autosomal dominant retinitis pigmentosa (adRP) that may be useful in testing therapies. We investigated the time-course of retinal changes of the I307N Rho mouse with spectral-domain optical coherence tomography (SD-OCT). Methods: SD-OCT was performed up to day 30 after light damage; electroretinography (ERG) was employed to evaluate photoreceptor function. We utilized ImageJ to analyze reflectivity of the retina. We used light and electron microscopy to assess retinal organization. We stained synaptophysin and zonula occludins-1 with immunohistochemistry to determine injury to the plexiform layers and retinal pigment epithelium (RPE). We performed lectin staining to evaluate retinal blood vessels. Results: Retinal degeneration increased with longer exposures to light. An increase in retinal thickness was detected by SD-OCT on day 1 after light challenge followed by loss of the outer nuclear layer (ONL) by day 8. Degeneration was most severe in the nasal and inferior retina. Hyper-reflectivity on SD-OCT developed as early as 1 day after light exposure. Disorganization of the ONL, condensation of photoreceptor chromatin, disruption of the outer limiting membrane, and disarray of outer segments were associated with the hyper-reflectivity. Retraction of the outer plexiform synapses and resorption of the subretinal detachment contributed to retinal thinning. The RPE remained intact, whereas atrophied major retinal vessels were evident after light damage. Conclusions: Our time-course analysis of retinal degeneration in the I307N Rho mouse with SD-OCT and other outcome measures should enable the use of the mouse model in preclinical efficacy studies and mechanistic studies.

Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector.

Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP.

A cell penetrating peptide from SOCS-1 prevents ocular damage in experimental autoimmune uveitis.

We describe an immunosuppressive peptide corresponding to the kinase inhibitory region (KIR) of the intracellular checkpoint protein suppressor of cytokine signaling 1 (SOCS-1) that binds to the phospho-tyrosine containing regions of the tyrosine kinases JAK2 and TYK2 and the adaptor protein MAL, and thereby inhibits signaling downstream from these signaling mediators. The peptide, SOCS1-KIR, is thus capable of downregulating overactive JAK/STAT or NF-kB signaling in somatic cells, including those in many compartments of the eye. Attachment of poly-arginine to this peptide (R9-SOCS1-KIR) allows it to penetrate the plasma membrane in aqueous media. R9-SOCS1-KIR was tested in ARPE-19 cells and was found to attenuate mediators of inflammation by blocking the inflammatory effects of IFNγ, TNFα, or IL-17A. R9-SOCS1-KIR and also protected against TNFα or IL-17A mediated damage to the barrier properties of ARPE-19 cells, as evidenced by immunostaining with the tight junction protein, zona occludin 1 (ZO-1), and measurement of transepithelial electrical resistance (TEER). Experimental autoimmune uveitis (EAU) was generated in B10. RIII mice using a peptide of interphotoreceptor retinal binding protein (IRBP161-180) as immunogen. Topical administration of R9-SOCS1-KIR, 2 days before (prophylactic), or 7 days after immunization (therapeutic) protected ocular structure and function as seen by fundoscopy, optical coherence tomography (OCT), and electroretinography (ERG). The ability R9-SOCS1-KIR to suppress ocular inflammation and preserve barrier properties of retinal pigment epithelium makes it a potential candidate for treatment of autoimmune uveitis.

Neuroinflammation in Retinitis Pigmentosa, Diabetic Retinopathy, and Age-Related Macular Degeneration: A Minireview.

The eye is an immuno-privileged organ. However, certain diseases such as uveitis are intrinsically linked to inflammation. In several retinal degenerative diseases, there is a unique damage at the onset of the disease, but evidence suggests that chronic and low-grade inflammatory processes play an important role in their progression. Studies have identified similar signaling pathways and changes in resident immune cells within the retina among these diseases. Herein, we will discuss some of these studies and propose how understanding this inflammatory response could aid in the development of therapies.

Acute heart failure with cardiomyocyte atrophy induced in adult mice by ablation of cardiac myosin light chain kinase.

AIMS: Under pressure overload, initial adaptive hypertrophy of the heart is followed by cardiomyocyte elongation, reduced contractile force, and failure. The mechanisms governing the transition to failure are not fully understood. Pressure overload reduced cardiac myosin light chain kinase (cMLCK) by ∼80% within 1 week and persists. Knockdown of cMLCK in cardiomyocytes resulted in reduced cardiac contractility and sarcomere disorganization. Thus, we hypothesized that acute reduction of cMLCK may be causative for reduced contractility and cardiomyocyte remodelling during the transition from compensated to decompensated cardiac hypertrophy. METHODS AND RESULTS: To mimic acute cMLCK reduction in adult hearts, the floxed-Mylk3 gene that encodes cMLCK was inducibly ablated in Mylk3(flox/flox)/merCremer mice (Mylk3-KO), and compared with two control mice (Mylk3(flox/flox) and Mylk3(+/+)/merCremer) following tamoxifen injection (50 mg/kg/day, 2 consecutive days). In Mylk3-KO mice, reduction of cMLCK protein was evident by 4 days, with a decline to below the level of detection by 6 days. By 7 days, these mice exhibited heart failure, with reduction of fractional shortening compared with those in two control groups (19.8 vs. 28.0% and 27.7%). Severely convoluted cardiomyocytes with sarcomeric disorganization, wavy fibres, and cell death were demonstrated in Mylk3-KO mice. The cardiomyocytes were also unable to thicken adaptively to pressure overload. CONCLUSION: Our results, using a new mouse model mimicking an acute reduction of cMLCK, suggest that cMLCK plays a pivotal role in the transition from compensated to decompensated hypertrophy via sarcomeric disorganization.

Targeting the Nrf2 Signaling Pathway in the Retina With a Gene-Delivered Secretable and Cell-Penetrating Peptide.

PURPOSE: Oxidative stress has been linked to several ocular diseases, initiating an inflammatory response that increases tissue injury. The Nrf2 transcription factor regulates expression of antioxidant genes and is tightly regulated by Kelch-Like ECH-Associated Protein 1 (Keap-1). We evaluate the antioxidant and anti-inflammatory properties of an adeno-associated virus (AAV) vector delivering an Nrf2-derived peptide that binds Keap-1. METHODS: The sequence of the Nrf2 peptide was fused to a cell-penetrating peptide (Tat-peptide) sequence (TatNrf2mer). The effects of lentiviral-delivered TatNrf2mer were studied in vitro. Transcript (quantitative [q] RT-PCR) and protein levels (ELISA and immunofluorescence) were quantified. Cell viability was measured by MTT and Cell Titer assays. The AAV vectors were packaged with the TatNrf2mer fused to secretable green fluorescent protein (GFP) under the control of the small chicken ß actin promoter. The protective effects of this vector were evaluated in a model of RPE oxidative injury and in a mouse model of uveitis after intravitreal injection. RESULTS: Expression of TatNrf2mer peptide induced antioxidant gene expression, blocked IL-1ß secretion, and protected cells from oxidative injury. In mice, TatNrf2mer expression partially protected photoreceptor function based on ERG responses and optical coherence tomography measurements in the sodium iodate (NaIO3) model. Furthermore, sGFP-TatNrf2mer expression decreased IL-1ß and IL-6 in the NaIO3-treated mice, and resulted in a 54% decrease in the number of inflammatory cells in the vitreous body of the endotoxin-induced uveitis mouse model. CONCLUSIONS: The intravitreally delivered AAV-TatNrf2mer has antioxidant and anti-inflammatory effects in widely-used models of ocular injury, suggesting it also could be useful in ocular diseases associated with oxidative stress and inflammasome activation.