An optimized method for retrograde labelling and quantification of rabbit retinal ganglion cells.

Rabbits are a commonly used animal model in glaucoma research, but their application has been limited by the techniques used to assess optic nerve injury (ONI). Our study devised an optimized method for retrograde labelling and analysing rabbit retinal ganglion cells (RGCs). This method involved improvements over the conventional method regarding the stereotaxic device, the positioning of superior colliculi, the target of axonal tracer delivery, and the visualization and analysis of labelled RGCs. To evaluate its efficacy, eight New Zealand White rabbits were divided into naïve and ONI groups. Unilateral limbal buckling surgery was performed in each animal of the ONI group to induce chronic ocular hypertension (OHT). The animals of both groups were injected with indocyanine green (ICG) into the interstice between the superior colliculus and occipital lobe on each side of the brain, and their eyes were examined by confocal scanning laser ophthalmoscopy (CSLO) after 48 h. The acquired images were then analysed to quantify the number of ICG-labelled RGCs in these eyes and their loss induced by OHT. To verify the identity and changes of the labelled RGCs, the retinas of the rabbits were subjected to immunofluorescence analyses. In addition, three animals were subjected to a second ICG labelling after 12 months to determine the influence of this procedure on the long-term viability of the labelled RGCs. Our results showed that ICG-labelled RGCs were detected by CSLO throughout the retinas of all animals. These RGCs showed a distinctly higher density below the ONH and were defective in sectorial areas in OHT eyes. Their average number in the cell counting area was 3989.2 ± 414.2 and 4023.3 ± 603.4 in the right and left eyes, respectively, of the naïve animals and 2590.9 ± 1474.2 and 3966.7 ± 24.0 in the OHT and non-OHT eyes, respectively, of the ONI animals. Immunofluorescence analyses showed positive staining with Brn3a and RBPMS in the ICG-labelled RGCs and sectorial defects of the cells in the OHT eyes, similarly as observed by CSLO. The second ICG labelling after 12 months in three animals showed no appreciable changes in RGC density compared with the first one. In summary, the optimized method of rabbit RGC retrograde labelling is reliable and accurate in both naïve and ONI animals and offers an approach for longitudinal observation of RGCs in the same eyes, which suggests its potential as a powerful tool for glaucoma and optic nerve research.

Repeated retinal photocoagulation in monkeys for the optimization of a laser-induced choroidal neovascularization model.

The laser-induced choroidal neovascularization (CNV) model in nonhuman primates has played a critical role in the development of new therapies for neovascular age-related macular degeneration. The widespread use of this model, however, has been limited by its high costs, mainly due to the lower efficiency of animal use. We optimized the CNV model by administering repeated photocoagulation treatments to the same eye of each animal, and preliminarily evaluated this model using an assessment of the efficacy of an anti-vascular endothelial growth factor (VEGF) agent to address this problem. Seven rhesus monkeys were included and divided into two groups, which were named the laser-only and laser-bevacizumab groups. Each animal underwent 3 retinal photocoagulation sessions in the same eye at 4-week intervals to induce CNV. Three weeks after the first laser treatment, the animals in the laser-bevacizumab group were administered an intravitreal injection of bevacizumab. Fluorescein angiography (FA) was performed in all animals at multiple time points within 12 weeks to assess the severity and development of CNV following each laser treatment. The laser lesions produced in each photocoagulation session were analysed separately using grading and densitometry methods, and CNV severity was represented by the CNV incidence and the mean integrated fluorescence intensity (MIFI), respectively. Our results showed that in the animals in the laser-only group, the average CNV incidence rates were 62.5%, 42% and 50% at 2 weeks after each laser treatment, and the average MIFI values (x105) were 3.83 ±â€¯2.36, 2.66 ±â€¯1.42 and 2.52 ±â€¯0.18, respectively. No significant differences were found among treatments. After week 2, the CNVs progressed or regressed continuously over 2-6 weeks before stabilization, and the time course of CNV development in each animal was generally the same after each photocoagulation session. In the laser-bevacizumab group, however, the average CNV incidence rates of each laser treatment at week 2 were 50%, 0 and 37.5%, respectively, and the average MIFI values were 3.79 ±â€¯0.47, 1.09 ±â€¯0.35 and 2.37 ±â€¯1.35, respectively. The differences between treatments 1 and 2 were statistically significant. Meanwhile, the CNVs induced by laser treatment 1, which progressed during weeks 2-3, were reduced after bevacizumab administration. The average CNV incidence decreased from 50% at week 3 to 4.2% at week 4, and the average MIFI decreased from 4.62 ±â€¯1.15 to 1.76 ±â€¯0.81, both of which were statistically significant. On the other hand, the CNVs induced by treatments 2 and 3 did not show any significant changes over time. Our study demonstrated that repeated retinal photocoagulation in the monkey eye produces relatively consistent CNVs, which can be used to assess the efficacies of anti-angiogenic agents more efficiently.

Impact of Acute Ocular Hypertension on Retinal Ganglion Cell Loss in Mice.

Purpose: To assess the correlation between intraocular pressure (IOP) levels and retinal ganglion cell (RGC) loss across different fixed-duration episodes of acute ocular hypertension (AOH). Methods: AOH was induced in Thy1-YFP-H transgenic mice by inserting a needle connected to a saline solution container into the anterior chamber. Thirty-one groups were tested, each comprising three to five mice exposed to IOP levels ranging from 50 to 110 mm Hg in 5/10 mm Hg increments for 60/90/120 minutes and a sham control group. The YFP-expressing RGCs were quantified by confocal scanning laser ophthalmoscopy, whereas peripapillary ganglion cell complex thickness was measured using spectral-domain optical coherence tomography. Changes in RGC count and GCCT were determined from values measured 30 days after AOH relative to baseline (before AOH). Results: In the 60-minute AOH groups, RGC loss varied even when IOP was increased up to 110 mm Hg (36.8%-68.2%). However, for longer durations (90 and 120 minutes), a narrow range of IOP levels (60-70 mm Hg for 90-minute duration; 55-65 mm Hg for 120-minute duration) produced a significant difference in RGC loss, ranging from <25% to >90%. Additionally, loss of YFP-expressing RGCs was comparable to that of total RGCs in the same retinas. Conclusions: Reproducible RGC loss during AOH depends on precise durations and IOP thresholds. In the current study, the optimal choice is an AOH protocol set at 70 mm Hg for a duration of 90 minutes. Translational Relevance: This study can assist in determining the optimal duration and intensity of IOP for the effective utilization of AOH models.

Novel loci for ocular axial length identified through extreme-phenotype genome-wide association study in Chinese populations.

PURPOSE: To investigate genetic loci associated with ocular axial length (AL) in the Chinese population. METHODS: A genome-wide association study meta-analysis was conducted in totalling 2644 Chinese individuals from 3 cohorts: the Guangzhou cohort (GZ, 537 high myopes and 151 hyperopes), Wenzhou cohort (334 high myopes and 6 hyperopes) and Guangzhou Twin Eye Study (1051 participants with normally distributed AL). Functional mapping was performed to annotate the significant signals, possible tissues and cell types by integrating available multiomics data. Logistic regression models using AL-associated SNPs were constructed to predict three AL status in GZ. RESULTS: Two novel loci (1q25.2 FAM163A and 7p22.2 SDK1) showed genome-wide significant associations with AL, together explaining 29.63% of AL variance in GZ. The two lead SNPs improved the prediction accuracy for AL status, especially for hyperopes. The frequencies of AL decreasing (less myopic) alleles of the two SNPs were lowest in East Asians as compared with other populations (rs17370084: f EAS=0.03, f EUR=0.24, f AFR=0.05; rs73046501: f EAS=0.06, f EUR=0.07, f AFR=0.20), which was in line with the global distribution of myopia. The cerebral cortex and gamma-aminobutyric acidergic interneurons showed possible functional involvement in myopia development, and the galactose metabolic pathways were significantly enriched. CONCLUSION: Our study identified two population-specific novel loci for AL, expanding our understanding of the genetic basis of AL and providing evidence for a role of the nervous system and glucose metabolism in myopia pathogenesis.

Navigating the uncertainty: A novel taxonomy of vaccine hesitancy in the context of COVID-19.

Vaccine hesitancy remains a significant and evolving public health challenge. The COVID-19 pandemic has created a unique decision context with significant uncertainty caused by the novelty of the disease being targeted, unfamiliarity with the vaccines being offered, misinformation, and strong handed government measures. In an effort to extend our understanding of vaccine hesitancy to the high uncertainty decision environment presented by COVID-19, we present a novel taxonomy of the determinants of vaccine hesitancy, based on an inductive analysis of qualitative data gathered during the COVID-19 pandemic. We report on focus group data from a purposive sample of 18 Canadians with varying sociodemographic characteristics and COVID-19 vaccination attitudes. An inductive thematic analysis of this data reveals eight core themes related to vaccine hesitancy: values, trust, social environment, personal anecdotes, environmental fluctuation, prior knowledge, perceived risk & systems of care. We explore these core themes as well as 25 sub-themes, contrasting them with previous models of vaccine hesitancy and suggesting potential strategies for public health professionals.

Identification of novel loci influencing refractive error in East Asian populations using an extreme phenotype design.

The global "myopia boom" has raised significant international concerns. Despite a higher myopia prevalence in Asia, previous large-scale genome-wide association studies (GWASs) were mostly based on European descendants. Here, we report a GWAS of spherical equivalent (SE) in 1852 Chinese Han individuals with extreme SE from Guangzhou (631 < -6.00D and 574 > 0.00D) and Wenzhou (593 < -6.00D and 54 > -1.75D), followed by a replication study in two independent cohorts with totaling 3538 East Asian individuals. The discovery GWAS and meta-analysis identify three novel loci, which show genome-wide significant associations with SE, including 1q25.2 FAM163A, 10p11.22 NRP1/PRAD3, and 10p11.21 ANKRD30A/MTRNR2L7, together explaining 3.34% of SE variance. 10p11.21 is successfully replicated. The allele frequencies of all three loci show significant differences between major continental groups (P < 0.001). The SE reducing (more myopic) allele of rs10913877 (1q25.2 FAM163A) demonstrates the highest frequency in East Asians and much lower frequencies in Europeans and Africans (EAS = 0.60, EUR = 0.20, and AFR = 0.18). The gene-based analysis additionally identifies three novel genes associated with SE, including EI24, LHX5, and ARPP19. These results provide new insights into myopia pathogenesis and indicate the role of genetic heterogeneity in myopia epidemiology among different ethnicities.

Effects of corneal crosslinking on corneal shape stabilization after orthokeratology.

Orthokeratology (Ortho-K) works to reshape cornea and is the only non-surgical way to enable vision without corrective aids. However, its effect is only temporary, and successful stabilization requires ongoing Ortho-K wear to maintain the reshaping effect. Corneal crosslinking (CXL) is a commonly-used technique in clinical practice to stabilize corneal shape in keratoconic eyes. However, whether or not CXL can stabilize corneal shape after Ortho-K in normal cornea has not been reported. Therefore, this proof-of-concept study using 2 rhesus monkeys aimed to determine the efficacy of the combined procedure. One monkey wore Ortho-K bilaterally for 24 hours, and the other from 6 pm to 8 am for 7 days. The left eyes of both monkeys underwent CXL after Ortho-K while the contralateral eye served as control. Results showed a gradual regression of corneal shape in all eyes with or without CXL. However, eyes underwent CXL regressed more slowly than the control eyes. The control eyes and the CXL treatment eye in the 7-day Ortho-K monkey regressed completely at last, while the CXL treatment eye in the 24 h Ortho-K monkey maintained a corneal flattening of -1.48 D 27 days after procedure. These findings suggest CXL can slow the regression of Ortho-K for a short duration, but cannot sustain its effect according to the current protocol.

Long-term therapeutic effect in nonhuman primate eye from a single injection of anti-VEGF controlled release hydrogel.

Wet age-related macular degeneration (wet-AMD) is a leading cause of irreversible blindness. Current treatment of AMD requires monthly intravitreal injection, which is difficult to be implemented in many parts of the world. In recent years, controlled release of anti-vascular endothelial growth factor (VEGF) therapeutics has attracted intense research interest aiming to reduce the injection frequency to one or two times per year. In this study, we evaluated the in vivo pharmacokinetics and the long-term therapeutic efficacy of an in situ hydrogel encapsulating an anti-VEGF antibody in nonhuman primates. We show that after a single injection of anti-VEGF controlled release hydrogel, a relatively constant concentration of drug can be maintained in the monkey eye for at least 5 months and the dose was sufficient for the treatment of recurrent choroidal neovascularization induced by repeat laser photocoagulation in monkeys. Our result suggested that when formulated into a controlled release formulation, a single dose of anti-VEGF may be sufficient for a half-year treatment and controlled release may be a suitable strategy to reduce the injection frequency in the treatment of AMD in human.

Chronic ocular hypertension in rabbits induced by limbal buckling.

AIM: To establish a rabbit model of chronic ocular hypertension (OHT) by limbal buckling. METHODS: Eighteen New Zealand White rabbits were involved and divided into three groups. A latex encircling band of 20, 25 or 35 mm was implanted behind the limbus in the right eye of each animal. The intraocular pressure (IOP) was monitored for 8 weeks, after which optic nerve damage was evaluated by fundus photography, optical coherence tomography (OCT) retrograde labelling and histology. Meanwhile, the anterior chamber angle (ACA) was examined by OCT and gonioscopy. RESULTS: OHT was induced in all animals after surgery. The IOP peaked at 38.0±3.7, 32.0±3.9 and 24.1±6.5 mm Hg in groups 1, 2 and 3, respectively, and remained elevated for 22, 25 and 39 days on average, respectively. The elevated IOPs showed good consistency within 2 weeks, although the durations of high IOP varied moderately. The area ratio between the optic cup and disc (cup to disc area) was increased in 73% of the treated eyes, and the average changes were 0.10±0.13, 0.11±0.08 and 0.09±0.02 in groups 1, 2 and 3, respectively. The depth of the optic cup was also increased in the treated eyes, and the density of the retinal ganglion cells was reduced. Additionally, the ACA showed a dynamic change with IOP after the latter was reduced by paracentesis. CONCLUSION: Limbal buckling provides an effective method of producing chronic OHT and glaucomatous optic neuropathy in rabbits.

Assessment of laser induction of Bruch's membrane disruption in monkey by spectral-domain optical coherence tomography.

PURPOSE: Laser-induced choroidal neovascularisation is a widely used model for age-related macular degeneration. The success rates of induction have been relatively low in large animals such as monkeys. Our study aimed to investigate the laser-induced damages to the Bruch's membrane of monkeys using the spectral-domain optical coherence tomography (OCT). METHODS: Laser photocoagulation was performed in the posterior and peripheral fundus of a rhesus monkey using a 532 nm laser. The lesions were examined by fundus photography and spectral-domain OCT immediately after the procedure. Fluorescein angiography was performed after 3 and 4 weeks in the animal to assess the development of choroidal neovascularisation. RESULTS: A total of 44 lesions were produced in both eyes of the animal. Subretinal bubbles with or without haemorrhage were observed at 41 spots during the procedure. Spectral-domain OCT showed that laser damages varied considerably among lesions and the disruption of the Bruch's membrane could be visualised at 23 spots on the OCT images. Leakage of fluorescein was only observed after 3 and 4 weeks within the macular area at lesions where Bruch's membrane disruptions had been detected by OCT. CONCLUSIONS: The presence of subretinal bubbles with haemorrhage is not an accurate indicator for successful disruption of the Bruch's membrane. Instead, spectral-domain OCT provides a better alternative to assess the retinal damages to the Bruch's membrane during laser induction of choroidal neovascularisation in monkeys.