Clinical Pharmacology Perspective on Development of Adeno-Associated Virus Vector-Based Retina Gene Therapy.

Adeno-associated virus (AAV) vector-based gene therapy is an innovative modality being increasingly investigated to treat diseases by modifying or replacing defective genes or expressing therapeutic entities. With its unique anatomic and physiological characteristics, the eye constitutes a very attractive target for gene therapy. Specifically, the ocular space is easily accessible and is generally considered "immune-privileged" with a low risk of systemic side effects following local drug administration. As retina cells have limited cellular turnover, a one-time gene delivery has the potential to provide long-term transgene expression. Despite the initial success with voretigene neparvovec (Luxturna), the first approved retina gene therapy, there are still challenges to be overcome for successful clinical development of these products and scientific questions to be answered. The current review paper aims to integrate published experience learned thus far for AAV-based retina gene therapy related to preclinical to clinical translation; first-in-human dose selection; relevant bioanalytical assays and strategies; clinical development considerations including trial design, biodistribution and vector shedding, immunogenicity, transgene expression, and pediatric populations; opportunities for model-informed drug development; and regulatory perspectives. The information presented herein is intended to serve as a guide to inform the clinical development strategy for retina gene therapy with a focus on clinical pharmacology.

A phase II, multicenter, open-label, randomized trial of pegfilgrastim for patients with alcohol-associated hepatitis.

Background: In trials conducted in India, recombinant granulocyte colony stimulating factor (GCSF) improved survival in alcohol-associated hepatitis (AH). The aim of this trial was to determine the safety and efficacy of pegfilgrastim, a long-acting recombinant GCSF, in patients with AH in the United States. Methods: This prospective, randomized, open label trial conducted between March 2017 and March 2020 randomized patients with a clinical diagnosis of AH and a Maddrey discriminant function score ≥32 to standard of care (SOC) or SOC+pegfilgrastim (0.6 mg subcutaneously) on Day 1 and Day 8 (clinicaltrials.gov NCT02776059). SOC was 28 days of either pentoxifylline or prednisolone, as determined by the patient's primary physician. The second injection of pegfilgrastim was not administered if the white blood cell count exceeded 30,000/mm3 on Day 8. Primary outcome was survival at Day 90. Secondary outcomes included the incidence of acute kidney injury (AKI), hepatorenal syndrome (HRS), hepatic encephalopathy, or infections. Findings: The study was terminated early due to COVID19 pandemic. Eighteen patients were randomized to SOC and 16 to SOC+pegfilgrastim. All patients received prednisolone as SOC. Nine patients failed to receive a second dose of pegfilgrastin due to WBC > 30,000/mm3 on Day 8. Survival at 90 days was similar in both groups (SOC: 0.83 [95% confidence interval [CI]: 0.57-0.94] vs. pegfilgrastim: 0.73 [95% CI: 0.44-0.89]; p > 0.05; CI for difference: -0.18-0.38). The incidences of AKI, HRS, hepatic encephalopathy, and infections were similar in both treatment arms and there were no serious adverse events attributed to pegfilgrastim. Interpretation: This phase II trial found no survival benefit at 90 days among subjects with AH who received pegfilgrastim+prednisolone compared with subjects receiving prednisolone alone. Funding: was provided by the United States National Institutes of Health and National Institute on Alcohol Abuse and Alcoholism U01-AA021886 and U01-AA021884.

Pediatric orbital cellulitis: Case report of an unusual etiology and pathogen.

Orbital cellulitis is an uncommon condition with risks to sight and life. As a complication of maxillofacial injuries, the literature suggests this is only possible with fractures or direct inoculation, and there are no reports to the contrary. Here, we make the first report of a possible etiology by which orbital cellulitis developed in a 14-year-old boy even without skin breach or bony fractures; as well as a rare causative pathogen. He presented with facial abscess and progressive orbital cellulitis after blunt facial trauma, requiring functional endoscopic sinus surgery with needle aspiration of facial abscess externally. Cultures showed growth of Streptococcus constellatus/Parvimonas micra, and he received further antibiotics with full recovery.The pathophysiology of orbital cellulitis in this patient is attributed to vascular congestion and local pressure from maxillofacial contusion and maxillary hemoantrum, with impaired paranasal sinus ventilation encouraging anaerobic bacterial growth. Further progression led to facial abscess formation and intraorbital spread with orbital cellulitis. The pediatric demographic is injury-prone, and self-reporting of symptoms can be delayed. Hence, increased suspicion of complicated injuries and orbital cellulitis may be required when managing maxillofacial contusions so that prompt treatment can be given.

Cooling Anesthesia for Intravitreal Injection: Results of the Prospective Open-Label, Dose-Ranging COOL-1 Trial.

PURPOSE: To evaluate the safety and efficacy of a novel medical device to provide cooling anesthesia to the eye as local anesthesia for intravitreal injections. STUDY DESIGN: First in human, open-label study of 43 subjects assessed at three different doses: -10°C for 20 seconds (group 1), -15°C for 15 seconds (group 2), and -15°C for 20 seconds (group 3). Main outcome measures were safety and pain of injection using a numeric rating scale (NRS). RESULTS: Cooling anesthesia did not result in any serious ocular adverse events. One grade 1 adverse event was a vasovagal response during cooling administration which resolved immediately after cooling. Mean NRS scores at the time of injection for each group ranged from 2.5 to 4.3 There was a statistically significant difference between pain scores of the 3 groups at injection in aggregate but not in pairwise comparisons (P value = 0.047). There was a statistically significant decrease in pain from injection to 5 minutes post injection in all groups (P value = 0.00008, 0.003, 0.0005 for groups 1, 2, 3, respectively) as well as from 5 minutes to 24-48 hours (P value = 0.00001, 0.018, and 0.0545 for groups 1, 2, 3, respectively). CONCLUSION: The rapid cooling anesthesia device was well tolerated for achieving local anesthesia among patients receiving intravitreal injections with no serious ocular adverse events.

Advances in Retinal Oximetry.

Similar to other organs, the retina relies on tightly regulated perfusion and oxygenation. Previous studies have demonstrated that retinal blood flow is affected in a variety of eye and systemic diseases, including diabetic retinopathy, age-related macular degeneration, and glaucoma. Although measurement of peripheral oxygen saturation has become a standard clinical measurement through the development of pulse oximetry, developing a noninvasive technique to measure retinal oxygen saturation has proven challenging, and retinal oximetry technology currently remains inadequate for reliable clinical use. Here, we review current strategies and approaches, as well as several newer technologies in development, and discuss the future of retinal oximetry.

Research Funding, Income, and Career Satisfaction Among Clinician-Scientists in Ophthalmology in the United States.

PURPOSE: The purpose of this study was to characterize clinician-scientists in ophthalmology and identify factors associated with successful research funding, income, and career satisfaction. DESIGN: Cross-sectional study. METHODS: A survey was conducted of clinician-scientists in ophthalmology at US academic institutions between April 17, 2019, and May 19, 2019. Collected information including 1) demographic data; 2) amount, type, and source of startup funding; first extramural grant; and first R01-equivalent independent grant; 3) starting and current salaries; and 4) Likert-scale measurements of career satisfaction were analyzed using multivariate regression. RESULTS: Ninety-eight clinician-scientists in ophthalmology were surveyed across different ages (mean: 48 ± 11 years), research categories, institutional types, geographic regions, and academic ranks. Median startup funding ranged from $50-99k, and median starting salaries ranged from $150-199k. A majority of investigators (67%) received their first extramural award from the National Eye Institute, mainly through K-award mechanisms (82%). The median time to receiving their first independent grant was 8 years, mainly through an R01 award (70%). Greater institutional startup support (P = .027) and earlier extramural grant success (P = .022) were associated with earlier independent funding. Male investigators (P = .001) and MD degreed participants (P = .008) were associated with higher current salaries but not starting salaries. Overall career satisfaction increased with career duration (P = .011) but not with earlier independent funding (P = .746) or higher income (P = .300). CONCLUSIONS: Success in research funding by clinician-scientists in ophthalmology may be linked to institutional support and earlier acquisition of extramural grants but does not impact academic salaries. Nevertheless, career satisfaction among clinician-scientists improves with time, which is not necessarily influenced by research or financial success.

SGLT2 Inhibitor-Induced Low-Grade Ketonemia Ameliorates Retinal Hypoxia in Diabetic Retinopathy-A Novel Hypothesis.

Diabetic retinopathy (DR) is a well-recognized microvascular complication of diabetes. Growing evidence suggests that, in addition to retinal vascular damage, there is significant damage to retinal neural tissue in DR. Studies reveal neuronal damage before clinically evident vascular lesions and DR is now classified as a neurovascular complication. Hyperglycemia causes retinal damage through complex metabolic pathways leading to oxidative stress, inflammation, vascular damage, capillary ischemia, and retinal tissue hypoxia. Retinal hypoxia is further worsened by high oxygen consumption in the rods. Persistent hypoxia results in increases in vascular endothelial growth factor (VEGF) and other pro-angiogenic factors leading to proliferative DR/macular edema and progressive visual impairment. Optimal glucose control has favorable effects in DR. Other treatments for DR include laser photocoagulation, which improves retinal oxygenation by destroying the high oxygen consuming rods and their replacement by low oxygen consuming glial tissue. Hypoxia is a potent stimulator of VEGF, and intravitreal anti-VEGF antibodies are effective in regressing macular edema and in some studies, retinal neovascularization. In this review, we highlight the complex pathophysiology of DR with a focus on retinal oxygen/fuel consumption and hypoxic damage to retinal neurons. We discuss potential mechanisms through which sodium-glucose cotransporter 2 (SGLT2) inhibitors improve retinal hypoxia-through ketone bodies, which are energetically as efficient as glucose and yield more ATP per molecule of oxygen consumed than fat, with less oxidative stress. Retinal benefits would occur through improved fuel energetics, less hypoxia and through the anti-inflammatory/oxidative stress effects of ketone bodies. Well-designed studies are needed to explore this hypothesis.

BILATERAL ENTEROCOCCUS FAECALIS ENDOPHTHALMITIS WITH MULTIPLE RECURRENCES.

PURPOSE: To describe the first case report of a bilateral recurrent Enterococcus faecalis endophthalmitis postcataract surgery. METHODS: Case report with a description of the timeline, diagnosis, and management of a patient with bilateral recurrent E. faecalis endophthalmitis. RESULTS: An 89-year-old man presented 6 weeks' postcataract surgery with pain, tearing, and blurred vision in the left eye. B-scan ultrasonography revealed vitritis and cultures postvitrectomy grew E. faecalis. There was gradual improvement in vision postintravitreal vancomycin administration. Four years later, the patient experienced another episode of E. faecalis endophthalmitis in the right eye postcataract extraction, followed by several additional episodes in both eyes posttreatment. CONCLUSION: Enterococcus faecalis is a rare but highly virulent cause of endophthalmitis that may remain sequestered in the capsular bag, despite aggressive treatment. Even after recurrent episodes, early vitrectomy and aggressive antibiotic therapy may prove to be effective in preventing vision loss.

Assessment of Respiratory Droplet Transmission During the Ophthalmic Slit-Lamp Exam: A Particle Tracking Analysis.

PURPOSE: The global COVID-19 pandemic has resulted in a renewed focus on the importance of personal protective equipment (PPE) and other interventions to decrease spread of infectious diseases. Although several ophthalmology organizations have released guidance on appropriate PPE for surgical procedures and ophthalmology clinics, there is limited experimental evidence that demonstrates the efficacy of various interventions that have been suggested. In this study, we evaluated high-risk aspects of the slit-lamp exam and the effect of various PPE interventions, specifically the use of a surgical mask and a slit-lamp shield. DESIGN: Experimental simulation study. METHODS: This was a single-center study in a patient simulation population. This study examined the presence of particles in the air near or on a slit-lamp, a simulated slit-lamp examiner, or a simulated patient using a fluorescent surrogate of respiratory droplets. RESULTS: Simulated coughing without a mask or slit-lamp shield resulted in widespread dispersion of fluorescent droplets during the model slit-lamp examination. Coughing with a mask resulted in the most significant decrease in droplets; however, particles still escaped from the top of the mask. Coughing with the slit-lamp shield alone blocked most of forward particle dispersion; however, significant distributions of respiratory droplets were found on the slit-lamp joystick and table. Coughing with both a mask and slit-lamp shield resulted in the least dispersion to the simulated examiner and the simulated patient. Scanning electron microscopy demonstrated particle sizes of 3-100 µm. CONCLUSIONS: Masking had the greatest effect in limiting spread of respiratory droplets, whereas slit-lamp shields and gloves also contributed to limiting exposure to droplets from SARS-CoV-2 during slit-lamp examination.

Implementing the new normal in ophthalmology care beyond COVID-19.

The COVID-19 pandemic has altered the clinical landscape immeasurably. The need to physical distance requires rethinking how we deliver ophthalmic care. Within healthcare, we will need to focus our resources on the five T's: Utilising technology, multidisciplinary clinical teams with wide professional talents need to work efficiently to reduce patient contact time. With regular testing, this will allow us to reduce the risk further. We also must acknowledge the explosion of different modalities to train our future ophthalmologists and the global challenges and advantages that these bring. Finally, we must not forget the psychological impact that this pandemic will have on ophthalmologists and ancillary staff, and need to have robust mechanisms for support.

Elovl2 Is Required for Robust Visual Function in Zebrafish.

Omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) play critical roles in membrane stability and cell signaling within the retina. ELOVL2 (Elongation of Very Long Chain Fatty Acids-Like 2), an elongase involved in the synthesis of long chain polyunsaturated fatty acids (LC-PUFAs), has recently been implicated in regulating aging in the mammalian retina. In this work, we characterize the expression and function of elovl2 in the retina development in embryonic zebrafish. Whole mount in situ hybridization shows elovl2 is expressed in the Muller glia in embryonic and adult zebrafish. Lipidomics analysis of elovl2 crispants whole embryos at day 2 and eyes at day 7 demonstrated significant changes in lipids composition, especially on the level of lipids containing docosahexaenoic acid (DHA). Histological analysis of zebrafish lacking elovl2 revealed increased retinal thickness compared to controls at day 7 without gross disruptions of the retinal architecture. Finally, elovl2 crispants showed differences in the visual motor reflex light off (VMR-OFF) at day 7 compared to controls. In sum, inactivation of elovl2 in zebrafish embryos caused changes in lipid composition and in visual behavior, further confirming the important role of LC-PUFAs in healthy vision.

ELOVL2: Not just a biomarker of aging.

DNA methylation of the ELOVL2 (Elongation Of Very Long Chain Fatty Acids-Like 2) promoter is one of the most robust molecular biomarkers for chronological age, but whether ELOVL2 plays a functional role in aging has not been explored. ELOVL2 encodes a transmembrane protein involved in the synthesis of very long polyunsaturated fatty acids (VLC-PUFAs). These fatty acids play important roles in retinal biology and photoreceptor renewal, key processes implicated in age-related eye diseases such as age-related macular degeneration (AMD). Here, we summarize our work deciphering the role of ELOVL2 in the eye emphasizing the potential functional role of age-related DNA methylation in the pathophysiology of AMD.

Assessing the Physiological Relevance of Cough Simulators for Respiratory Droplet Dispersion.

Various breathing and cough simulators have been used to model respiratory droplet dispersion and viral droplets, in particular for SARS-CoV-2 modeling. However, limited data are available comparing these cough simulations to physiological breathing and coughing. In this study, three different cough simulators (Teleflex Mucosal Atomization Device Nasal (MAD Nasal), a spray gun, and GloGermTM MIST) that have been used in the literature were studied to assess their physiologic relevance. Droplet size, velocity, dispersion, and force generated by the simulators were measured. Droplet size was measured with scanning electron microscopy (SEM). Slow-motion videography was used to 3D reconstruct and measure the velocity of each simulated cough. A force-sensitive resistor was used to measure the force of each simulated cough. The average size of droplets from each cough simulator was 176 to 220 µm. MAD Nasal, the spray gun, and GloGermTM MIST traveled 0.38 m, 0.89 m, and 1.62 m respectively. The average velocities for the MAD Nasal, spray gun, and GloGermTM MIST were 1.57 m/s, 2.60 m/s, and 9.27 m/s respectively, and all yielded a force of <0.5 Newtons. GloGermTM MIST and the spray gun most closely resemble physiological coughs and breathing respectively. In conclusion, none of the simulators tested accurately modeled all physiologic characteristics (droplet size, 3-D dispersion velocity, and force) of a cough, while there were various strengths and weaknesses of each method. One should take this into account when performing simulations with these devices.

Potential of Ocular Transmission of SARS-CoV-2: A Review.

PURPOSE OF REVIEW: to provide a prospective on the current mechanisms by which SARS-CoV-2 enters cells and replicates, and its implications for ocular transmission. The literature was analyzed to understand ocular transmission as well as molecular mechanisms by which SARS-CoV-2 enters cells and replicates. Analysis of gene expression profiles from available datasets, published immunohistochemistry, as well as current literature was reviewed, to assess the likelihood that ocular inoculation of SARS-CoV-2 results in systemic infection. RECENT FINDINGS: The ocular surface and retina have the necessary proteins, Transmembrane Serine Protease 2 (TMPRSS2), CD147, Angiotensin-Converting Enzyme 2 (ACE2) and Cathepsin L (CTSL) necessary to be infected with SARS-CoV-2. In addition to direct ocular infection, virus carried by tears through the nasolacrimal duct to nasal epithelium represent a means of ocular inoculation. SUMMARY: There is evidence that SARS-CoV-2 may either directly infect cells on the ocular surface, or virus can be carried by tears through the nasolacrimal duct to infect the nasal or gastrointestinal epithelium.

Quality of Life Assessment of Severely Visually Impaired Individuals Using Aira Assistive Technology System.

Purpose: To assess patient-reported quality of life outcome improvements in severely visually impaired (SVI) individuals using the Aira system over a 1-year follow-up period. Methods: Aira is an on-demand assistive technology designed for SVI. Aira subscribers were recruited and administered the validated 28-item Impact of Vision Impairment-Very Low Vision Questionnaire by phone before starting Aira with follow-ups at 3 months and 1 year. Total score and validated subset scores of activities of daily living, mobility, and safety (ADLMS) and emotional well-being (EWB) were assessed. Pearson correlation analyses and paired t-tests were used to examine the data. Results: Fifty participants (mean, age, 52.5 years; 25 males, 25 females) were recruited with a mean of 401 ± 66.3 days to follow-up. The initial total score (mean, 53.1 ± 18.9) significantly improved at 1 year (mean, 63.1 ± 16.2; P = 0.0002). The initial ADLMS score (mean, 30.7 ± 11.3) significantly improved at 1 year (mean, 37.2 ± 10.7; P = 0.001). The initial EWB score (mean, 22.5 ± 8.5) significantly improved at 1 year (mean, 25.9 ± 8.0; P = 0.0001). There was no significant difference between the 3-month and 1-year total (P = 0.972), ADLMS (P = 0.897), and EWB scores (P = 0.700). There was a significant correlation between minutes used and improvement in total (r = 0.371; P = 0.009), ADLMS (r = 0.302; P = 0.035), and EWB (r = 0.439; P = 0.002) scores. Conclusions: Aira use significantly improves Impact of Vision Impairment-Very Low Vision total, ADLMS, and EWB scores for SVI individuals at 3 months. This improvement is sustained at the 1-year follow-up and correlated with total minutes used. Translational Relevance: Aira technology may provide sustained improvement in quality of life for SVI, and further study to evaluate the usefulness of this technology to assist SVI may be beneficial.

Targeting of the NRL Pathway as a Therapeutic Strategy to Treat Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal dystrophy (IRD) with a prevalence of 1:4000, characterized by initial rod photoreceptor loss and subsequent cone photoreceptor loss with accompanying nyctalopia, visual field deficits, and visual acuity loss. A diversity of causative mutations have been described with autosomal dominant, autosomal recessive, and X-linked inheritance and sporadic mutations. The diversity of mutations makes gene therapy challenging, highlighting the need for mutation-agnostic treatments. Neural leucine zipper (NRL) and NR2E3 are factors important for rod photoreceptor cell differentiation and homeostasis. Germline mutations in NRL or NR2E3 leads to a loss of rods and an increased number of cones with short wavelength opsin in both rodents and humans. Multiple groups have demonstrated that inhibition of NRL or NR2E3 activity in the mature retina could endow rods with certain properties of cones, which prevents cell death in multiple rodent RP models with diverse mutations. In this review, we summarize the literature on NRL and NR2E3, therapeutic strategies of NRL/NR2E3 modulation in preclinical RP models, as well as future directions of research. In summary, inhibition of the NRL/NR2E3 pathway represents an intriguing mutation agnostic and disease-modifying target for the treatment of RP.

Outcomes Comparison of the Veterans' Choice Program With the Veterans Affairs Health Care System for Hepatitis C Treatment.

BACKGROUND: The US Department of Veterans Affairs (VA) has been stressed by the large number of veterans requiring direct-acting antiviral (DAA) medications for hepatitis C virus (HCV) treatment. The Veterans Choice Program provides VA patients more options to receive treatment. This study compared the experience of veterans who received HCV treatment through the Veterans Choice Program and those that received treatment at the VA Loma Linda Healthcare System (VALLHCS) in fiscal year (FY) 2016. METHODS: A chart review was performed on all veterans referred by VALLHCS to Choice for HCV treatment during FY 2016, and matched to veterans who received treatment at VALLHCS. Data collected included Fibrosis-4 score (Fib-4), platelet count, days elapsed between time of referral and time of appointment (wait time), rate of sustained virologic response at 12 weeks (SVR12), reason for treatment failure, and cost effectiveness. RESULTS: One hundred veterans were referred to Choice; 71 were seen at least once by a Choice provider, and 61 completed a treatment course. Mean Fib-4 and platelet count was 1.9 and 228,000 for the Choice population and 3.4 and 158,000 for the VALLHCS population, respectively. There was no difference in SVR12 rate. Mean wait time was 42 days for Choice vs 29 days for VALLHCS (P < .001). Choice health care providers incurred a mean $8,561.40 in additional costs per veteran seen. CONCLUSIONS: While treatment success rates were similar between Choice and VALLHCS, the degree of liver fibrosis was more advanced in the VALLHCS population. The wait time for care was longer with Choice compared with a direct referral within the VA. While Choice offers a potential solution to providing care for veterans, the current program has unique problems that must be considered.

Multiexon deletion alleles of ATF6 linked to achromatopsia.

Achromatopsia (ACHM) is an autosomal recessive disease that results in severe visual loss. Symptoms of ACHM include impaired visual acuity, nystagmus, and photoaversion starting from infancy; furthermore, ACHM is associated with bilateral foveal hypoplasia and absent or severely reduced cone photoreceptor function on electroretinography. Here, we performed genetic sequencing in 3 patients from 2 families with ACHM, identifying and functionally characterizing 2 mutations in the activating transcription factor 6 (ATF6) gene. We identified a homozygous deletion covering exons 8-14 of the ATF6 gene from 2 siblings from the same family. In another patient from a different family, we identified a heterozygous deletion covering exons 2 and 3 of the ATF6 gene found in trans with a previously identified ATF6 c.970C>T (p.Arg324Cys) ACHM disease allele. Recombinant ATF6 proteins bearing these exon deletions showed markedly impaired transcriptional activity by qPCR and RNA-Seq analysis compared with WT-ATF6. Finally, RNAscope revealed that ATF6 and the related ATF6B transcripts were expressed in cones as well as in all retinal layers in normal human retina. Overall, our data identify loss-of-function ATF6 disease alleles that cause human foveal disease.

Anesthesia for Intravitreal Injection: A Systematic Review.

BACKGROUND: The intravitreal injection has become one of the most commonly performed procedures in ophthalmology; however, there is no standardized approach to anesthesia during the procedure. The goal of this systematic review is to review approaches to anesthesia for intravitreal injection and look at comparative efficacy between these different anesthetics. METHODS: A systematic review of literature was performed in the MEDLINE, PubMed, Cochrane Library, and Clinicaltrials.gov databases using the key words "anesthesia", "pain management", and "intravitreal injection". Of the initial 239 search matches, 30 articles were found to be relevant to the topic. 18 studies were excluded as they did not include primary data or did not include the visual analog scale as a primary outcome. The remaining 12 articles were assessed to look at the comparative efficacy of anesthesia and adverse events. RESULTS: The anesthesia techniques reported include topical methods such as anesthetic eyedrops, anesthetic gels, and anesthetic-soaked pledgets as well as subconjunctival injection of anesthetic. Ultimately, no single anesthetic or delivery mechanism was shown to be superior to the others in a statistically significant way and adverse events were largely insignificant. Limitations of these studies include relatively small sizes of the studies, as well as the lack of masking which may introduce bias. CONCLUSION: In the current literature, no type of anesthetic method was found to be superior to another for intravitreal injection. Future studies in this area may lead to new insights into the efficacy of different forms of intravitreal anesthesia.