Background: Rods and cones are critical for light detection. Although there has been considerable work done in elucidating the molecular mechanisms involved in rod development, not much is known about how the cone cell fate decision is made by the multipotent retinal progenitor cells during development. Analysis of the promoter regions of Nrl and trβ2, rod and cone differentiation factors respectively, revealed DNA binding motifs of two POU-domain containing transcription factors, Pou2f1 and Pou2f2. Preliminary experiments showed that Pou2f1/2 are expressed during the peak of cone genesis in the embryonic retina. Therefore, we hypothesize that Pou2f1/2 specify cone cell fate in the developing retina.
Methods: We used immunofluorescence and in situ hybridization to establish the spatiotemporal expression of Pou2f1/2 during retinogenesis. We performed in vivo electroporation in post-natal mice to misexpress Pou2f1/2 and used antibodies specific to proteins expressed in cones such as Rxrγ and S-opsin to count cones. Using ex vivo electroporation of embryonic retinal explants, we knocked out Pou2f1 and Pou2f2 using CRISPR/Cas9 gRNAs at the peak of cone production window. Finally, we transfected post-natal retinal explants with a combination of regulatory elements of Nrl or thrb with control backbone vector, Pou2f1 or Pou2f2 using electroporation.
Results: We found that Pou2f1/2 are expressed in retinal progenitor cells in the developing retina and subsequently in the differentiated cones. Pou2f1/2 misexpression outside the cone genesis window led to an increase in cones at the expense of rods. Pou2f1/2 indel knockouts generated by CRISPR/Cas9 gRNAs led to a decrease in cones and a converse increase in rods. Finally, we found that Pou2f1/2 activate the cis-regulatory module (CRM) of the thrb gene and repress the activity of the CRM of Nrl.
Conclusions: These results uncover novel players that establish the complex gene regulatory network for cone photoreceptor fate specification in the retinal progenitor cells. We anticipate that this work should help us devise improved replacement therapies in the future utilizing stem cells for retinal degenerative diseases such as aged-related macular degeneration (AMD) and Stargardt’s disease.
Background: Decrease of ocular blood flow has been linked to the pathogenesis of ocular diseases such as glaucoma and age-related macular degeneration. Current methods that measure the pulsatile blood flow have major limitations, including the assumption that ocular rigidity is the same in all eyes. Our group has recently developed a new method to measure the pulsatile choroidal volume change by direct visualization of the choroid with OCT imaging and automated segmentation. Our goal in this study is to describe the distribution of PCBF in a healthy Caucasian population.
Methods: Fifty-one subjects were recruited from the Maisonneuve-Rosemont Hospital Ophthalmology Clinic and underwent PCBF measurement in one eye. The distribution of PCBF in healthy eyes was assessed.
Results: The distribution of PCBF among the healthy eyes was found to be 3.94±1.70 μL with this technique.
Conclusions: This study demonstrates the normal range of PCBF values obtained in a healthy Caucasian population. This technique could be used for further investigation of choroid pulsatility and to study glaucoma pathophysiology.
Background: Zellweger spectrum disorder (ZSD) is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly. Retinopathy leading to blindness is one of the major handicaps faced by affected individuals, but treatment for this is supportive only. To test whether we could improve visual function in ZSD, we performed a proof-of-concept trial for PEX1 gene augmentation therapy using the Pex1-G844D mouse model, which bears the equivalent to a common human mutation. This model exhibits a gradual decline in scotopic ffERG response, an always residual photopic ffERG response, diminished visual acuity, and cone and bipolar cell anomalies.
Methods: We administered subretinal injections of a PEX1-containing viral vector (AAV8.CMV.hPEX1.HA) to 2 mouse cohorts of 5 or 9 weeks of age. A GFP-containing vector was used as a control in the contralateral eye of each animal. Efficient expression of the virus was confirmed by retinal histology/immunohistochemistry, and its ability to recover peroxisome import was confirmed in vitro. Preliminary ffERG and optokinetic (OKN) analyses were performed on a subset of animals at 8, 16, and 20 weeks after gene delivery. Final ffERG and OKN measures were performed when each cohort reached 32 weeks of age (23 or 27 weeks post injection).
Results: Preliminary ffERG and OKN analyses at 8 weeks post injection showed mildly better retinal response and visual acuity, respectively, in the PEX1-injected eyes, as did ffERG analysis when each cohort reached 25 weeks of age (16 or 20 weeks after gene delivery). This effect was more pronounced in the cohort treated at 5 weeks of age, when ffERG response is highest in Pex1-G844D mice. At 32 weeks of age, the ffERG response in the PEX1-injected eyes was double that of GFP-injected eyes, on average, though there was no change in OKN. Furthermore, in PEX1-injected eyes the photopic ffERG response improved over time, and the decline in scotopic b-wave amplitude was ameliorated compared to un-injected eyes.
Conclusions: AAV8.CMV.hPEX1.HA was subretinally delivered into the left eye of 5- and 9-week-old Pex1-G844D retina. Successful expression of the protein with no gross histologic side effect was observed. Neither the injection, nor exposure to the AAV8 capsid or the transgenic protein negatively altered the ERG or OKN response. At 5–6 months after gene delivery, therapeutic vector-treated eyes showed improved ERG compared to control eyes, on average, in both the “prevention” and “recovery” cohorts. This implies clinical potential of gene delivery to improve vision in patients with ZSD. Retinal immunohistochemistry (to visualize retinal cell types) and biochemical analyses will be performed on treated and untreated retinas, and may inform the mechanism of ERG improvement.
Background: Retinal pigment epithelium (RPE) is vital for the homeostasis of the subretina including photoreceptors and choroid. Interestingly, our previous results suggested that the recently discovered lactate receptor GPR81 is abundantly expressed in RPE. To date, only one previous study has shown that activating GPR81 could enhance DNA repair by activating HDAC1. Consequently, we investigated whether GPR81 exhibits epigenetic modification in the subretina by using GPR81?/? mice.
Methods: GPR81?/? mice and wide type littermates were generated on a background of C57BL/6J mice. The thicknesses of their choroid were evaluated by immunohistochemistry. Meanwhile, Q-PCR, western blot and choroid sprout assay were performed. In vitro, primary retinal pigment epithelium (pRPE) cells were isolated from mice, and cultured for treatments.
Results: The thickness of choroid was reduced in GPR81?/? mice compared to GPR81+/+ mice, suggesting that GPR81 is important for the integrity of choroid. In the choroid sprout assay, lactate treated RPE/choroid complex showed a significant increase in angiogenesis compared to controls while lactate treated KO RPE/choroid complex showed no difference compared to their controls. For Q-PCR, most of the genes screened elevated their expression in GPR81?/? mice compared to WT mice, suggesting epigenetic modification may exist, which were confirmed by histone acetylation and HDACs activity assay.
Conclusions: Taking together, the lactate receptor GPR81 in RPE is very important for maintaining homeostasis of the subretina. This novel discovery sheds new light on the relationship between metabolism and epigenetic modification.
Background: We investigated the role of beta-adrenergic receptor (B-AR) on choroidal neovascularization (CNV) in an animal model of age-related macular degeneration in mice.
Methods: The angiogenic effect of the B-AR was evaluated in retinal pigment epithelium (RPE)-choroid explants from C57Bl6 mice stimulated with propranolol or isoproterenol (10 μM) (respectively antagonist and agonist of the B-AR) during 24 h. Conversely, a classic choroidal neovascularization (CNV) model induced by laser burn in C57Bl6 mice (8 weeks) was used to assess the anti-angiogenic effect of propranolol. In this experiment, mice were treated with intraperitoneal propranolol (6 mg/kg/d) or vehicle (saline solution) daily for 10 days, starting on day 4 after laser burn and until sacrifice (day 14). Immunostaining analysis on retinal flatmounts and cryosections were performed to determine the surface of CNV, the distribution of B-AR and the number and morphology of microglia/macrophages associated with CNV. To explore if the antiangiogenic effect of propranolol involved the modulation of the inflammatory microenvironment associated with CNV, we used RPE primary cells, J774 macrophages cell line and polarized M1 and M2 bone marrow-derived macrophage (BMDM). Choroidal explants treated with conditioned media (CM) from J774 or polarized M1/M2 BMDM pre-treated with propranolol to confirm the anti-angiogenic effect of propranolol. Expression of angiogenic factors was evaluated by RT PCR and Elisa.
Results: The expression and distribution of the B-1, B-2 and B-3 adrenergic receptors were localized in the choroid and RPE cells. The stimulation of RPE-choroid explants with isoproterenol increased CNV compared to vehicle, while propranolol decreased CNV. In vivo, propranolol inhibited significantly the levels of VEGF and CNV growth in laser burn model compared to the vehicle. Additionally, the treatment with propranolol decremented the number of activated (amoeboid shape) microglia/macrophages but surprisingly, the number of non-activated microglia/macrophages around the CNV was higher than with the vehicle treatment. In vitro, propranolol modulated the angiogenic balance in macrophages promoting anti-angiogenic factors expression, especially with M2 BMDM. CM from macrophages pre-treated with propranolol reduced CNV on choroidal explants.
Background: Diabetic macular edema (DME) is a leading cause of severe visual impairments in older and the working-age population. An important target of current therapy is vascular endothelial growth factor (VEGF), which plays a role in the pathogenesis of DME by inducing angiogenesis and increasing vascular permeability. Currently available anti-VEGF agents include off-label use of Bevacizumab, which has been shown to be effective in the treatment of DME. However, many patients with DME do not respond or demonstrate only a partial response to this agent. As of November 2016, the Canadian Health authorities approved Aflibercept as an anti-VEGF agent for treatment of DME, and the patients who are non-responders to Bevacizumab are switched to this non-off label medication. We aimed to investigate the anatomical and functional visual changes associated with response to Aflibercept in a real-life Canadian population of Bevacizumab non-responders.
Methods: A retrospective review of chronic DME patients refractory to bevacizumab treatment who were switched to Aflibercept was done. Best-corrected visual acuity (BCVA), Intraocular pressure (IOP), central subfield thickness (CST), average macular thickness, and total macular volume were extracted at the visit prior to switching to Aflibercept (baseline) as well as the first, second and third follow-up visits after switching. Anatomical and functional visual changes were compared using Generalized Estimating Equations and the association between variables was tested using Pearson correlation test with significance set at P<0.05.
Results: Twenty-six eyes with mean age of 63 were included. Average CST at baseline was 421.5±116.1 μm and the number of Bevacizumab injections received prior to switching was 15.3±8.0. No significant changes were observed in terms of BCVA and IOP, from baseline to any of the follow-ups. Switching to Aflibercept significantly improved CST, average macular thickness, and total macular volume. From baseline to the first follow-up visit, CST decreased from 421.5±116.1 to 333.0±91.2 μm (P=0.001) and average macular thickness reduced from 344.6±74.9 to 322.2±60.5 μm (P=0.008). Similarly, total macular volume decreased from 12.4±2.7 to 11.6±2.2 μm3, measured at baseline and the first follow-up (P=0.007). No further improvements were observed from the first follow-up to the subsequent ones. The median CST value at baseline (378 μm) was used to classify the patients into low and high CST groups. We observed that those with higher CST at baseline (>378 μm) showed a trend for improvements in visual acuity (P=0.058). Pearson correlation test confirmed the association between higher CST at baseline and better visual outcomes in response to switching to Aflibercept (P=0.018).
Conclusions: Our data evidenced significant anatomical improvements in macula, which did not translate to immediate functional vision improvements. Bevacizumab non-responders with higher CST might also gain visual acuity and benefit functionally from switching to Aflibercept.
