AB082.Gold nanoparticles as a new drug vector for glaucoma therapy

Background:Glaucoma is an optical neuropathy affecting over 67 million people in the world.Efficiency of current active molecules,as travoprost(hydrophobic)is limited when administered by ophthalmic drops.Indeed,more than 99.9%is discarded due to multiple factors including lacrimal drainage.Low retention time of drugs at the cornea leads to their poor penetration.The aim of the project is to develop a drug delivery system allowing the drug penetration through biological barriers.Our hypothesis is that a drug delivery system based on gold nanoparticles should enhance the efficiency of the drugs.The main objective is to study the encapsulation ability of gold nanoparticles towards travoprost.The specific objectives are(I)the synthesis and characterizations of gold nanoparticles;(II)the establishment of the encapsulation protocol;(III)the method development of the separation of free and encapsulated drugs and;(IV)the quantification of the encapsulated drugs.Methods:Gold nanoparticles were synthesized by a new method developed in our laboratory.An encapsulation protocol was settled using aqueous conditions at 37℃.The separation of free and encapsulated drugs was performed with magnetic beads.The quantification of the encapsulated drugs was then performed by high performance liquid chromatography and confirmed by UV-visible spectroscopy.Results:Gold nanoparticles of 28±1 nm were synthesized and purified according to our new experimental conditions.The encapsulation protocol lasts 5 days in the optimised conditions.The separation method involving magnetic beads was optimized to get rid of non-specific interactions.The travoprost was incubated with the nanoparticles until the reach of equilibrium in solution.Conclusions:We showed that active molecules used for glaucoma therapy,as travoprost,can be encapsulated in gold nanoparticles.Further analysis will allow identifying the encapsulation properties of various gold nanoparticles,differing by their size,shape and chemical surface.These data suggest the possible improvements.

AB091.A novel method for the measurement of pulsatile choroidal blood flow based on video-rate OCT imaging and automated segmentation of the choroid:description and reproducibility

Background:Over the years,a variety of non-invasive techniques have been developed to allow the measurement of blood flow in living human eyes.However,none of the existing techniques has yet been adopted in clinical practice due to their limitations and lack of standardization.Moreover,no reliable technique is currently available to measure the pulsatile choroidal blood flow(PCBF).We propose a novel method based on video-rate optical coherence tomography(OCT)imaging and automated segmentation to measure the pulsatile component of choroidal blood flow in vivo,and demonstrate its repeatability.Methods:Adapted from our earlier work(Beaton et al.),this method uses video-rate OCT with enhanced depth imaging and automated segmentation of the choroid to measure the pulsatile choroidal volume change.Imaging is carried out at the fundus for less than a minute at 7 Hz.In each frame,choroidal thickness(CT)is measured by a segmentation algorithm based on graph cuts using an edge-probability weighting scheme.The algorithm computes the CT change corresponding to choroidal filling over the time-series and subsequently derives the pulsatile choroidal volume change through an approximate model of the eye.Fifty-eight subjects were recruited from the Maisonneuve-Rosemont Hospital and PCBF was measured twice in one eye within the same session and by a single examiner.Repeatability was assessed using the Bland-Altman plot and Intraclass correlation coefficient as calculated with SPSS.Results:Two measurements of PCBF were successfully obtained for each eye using our technique.The average measures ICC for choroidal volume change was 0.929(95%CI,0.881,0.958),showing good to excellent repeatability.The Bland-Altman plot and Pearson coefficient(r=0.840,P<0.001)showed agreement and a strong correlation respectively between intra-session measurement of OR in all examined eyes.Conclusions:This study confirms the high repeatability of pulsatile choroidal blood flow measurements obtained with our optical method,allowing further investigation of blood flow in ocular diseases such as glaucoma and AMD.

AB089.Impact of WNK1 inhibition on corneal wound healing using a model of human tissue-engineered cornea

Background:Because of its superficial anatomical localization,the cornea is particularly vulnerable to abrasive forces and various traumas,which can lead to significant visual impairments.Upon injury of the corneal epithelium,there are important changes that occur in the composition of the underlying extracellular matrix(ECM).Those changes are perceived by the integrins that recognize the ECM components as their ligand and activate different intracellular signalling pathways,ultimately leading to reepithelialisation and reorganization of the injured epithelium,both of which are necessary in order to restore the visual properties of the cornea.The goal of this study was to analyse the impact of the pharmacological inhibition of specific signal transduction mediators of integrin-dependant signalling pathways on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas(hTECs)as in vitro models.Methods:hTECs were produced by the self-assembly approach and wounded with a 8-mm diameter biopsy punch.Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays.The wounded tissues were then incubated with the WNK1 inhibitor WNK463 and wound healing was monitored over a period of 6 days.Control corneas were incubated with the vehicle alone(DMSO).The impact of WNK1 inhibition on hCECs monolayers was determined using a scratch wound assay.Results:Gene profiling analyses and protein kinases arrays revealed important alterations in the expression and activity of several mediators from the integrin-dependent signalling pathways in response to the ECM changes taking place during corneal wound healing.Among these,WNK1 is considerably activated through phosphorylation during corneal wound healing.The pharmacological inhibition of WNK1 by WNK463 significantly reduced the dynamic of corneal wound closure in our hTECs and hCECs monolayers compared to their respective negative controls.Conclusions:These results allowed the identification of WNK1 kinase as an important player for a proper healing of the cornea.Also,these results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing and they may lead to the identification of new therapeutic targets in the field of corneal wounds.

AB021.Discovery of a new role for the WNK1 kinase in corneal wound healing

Background:Damage to the corneal epithelium triggers important changes in the composition of the extracellular matrix(ECM)to which the basal human corneal epithelial cells(hCECs)attach.These changes are perceived by integrins,a family of trans-membrane receptors that activate different intracellular signalling pathways,ultimately leading to re-epithelialization of the injured epithelium.Our goal is to study the impact of the pharmacological inhibition/activation of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas(hTECs)as in vitro models.Methods:hTECs were produced by the self-assembly approach and wounded with a 8-mm biopsy punch.Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays.The wounded tissues were then incubated either with the lysine deficient protein kinase 1(WNK1)inhibitor WNK463,the WNK1 indirect agonist AM1241,or with the vehicle alone(DMSO;negative control)and wound healing was monitored for 6 days.The impact of WNK1 inhibition/activation on hCECs monolayers was determined using scratch wound assays.Results:Gene profiling analyses and protein kinases arrays revealed that expression and activity of several mediators from the integrin-dependent signalling pathways were altered in response to the ECM changes taking place during corneal wound healing.Phosphorylation of the WNK1 kinase turned out to be the most striking activation event occurring during wound healing.Since the pharmacological inhibition of WNK1 by WNK463 significantly reduced the rate of corneal wound closure in our hTECs and hCECs monolayers compared to their respective negative controls,we believe that the pharmacological activation of WNK1 could turn out to be an interesting avenue to accelerate corneal wound closure.Conclusions:These results will contribute to a better understanding of the cellular and molecular mechanisms involved in corneal wound healing.Furthermore,they identified a new function for the WNK1 kinase in corneal wound healing and might lead to the identification of a new therapeutic target in the field of corneal wounds.

Annals of Eye Science, 2018 7 © Annals of Eye Science. All rights reserved. Ann Eye Sci 2018;3:AB007 aes.amegroups.com

Background:The goal of this study was to engineer an epithelialized and endothelialized pigmented choroidal substitute using the self-assembly approach of tissue engineering.Methods:Cells from human choroids were isolated and cultured.Culture purity was assessed using immunostaining(CD31,HMB45,vimentin,keratins 8/18).To engineer the choroid,fibroblasts were cultured in the presence of serum and ascorbic acid to promote extracellular matrix(ECM)assembly.Endothelial cells,melanocytes or RPE cells were separately seeded on the stromal substitutes.Choroidal substitutes were further characterized by histology,mass spectrometry,immunostaining,and compared to native human choroids.Results:The technique used to isolate choroidal cells yielded pure cultures of fibroblasts,melanocytes and vascular endothelial cells.The stromal substitutes engineered using the self-assembly approach were composed of collagen(types I,VI,XII and XIV),proteoglycans(decorin,lumican)and other ECM proteins.Protein expression was confirmed using immunostaining.Endothelial cells spontaneously assembled into capillary-like structures and vascular networks when cocultured with fibroblast-containing ECM sheets.Conclusions:This study shows that the self-assembly approach of tissue engineering can be used to reconstruct a choroid using native cells.This model represents a unique tool to better understand the crosstalk between the different choroidal cell types and cell-ECM interactions.

AB047.Quebec database of clinical data and biological material for research on uveal melanoma

Background:Sharing biological material and clinical data from patients with uveal melanoma.Methods:Uveal melanoma is the most common intraocular malignancy in the adult population.Because uveal melanoma is primarily a sporadic cancer and familial cases are rare,it is difficult to prevent or detect it.Despite effective treatment of ocular tumors,more than 50%of patients develop incurable liver metastases mainly in the 5-10 years following the detection of the primary tumor.This cancer is relatively rare and the obtained biopsies are very small.About 20 samples are taken each year in Quebec.This provincial infrastructure is made of biological material from donors with uveal melanoma and a large clinical database.Collected tumor biopsies are used for culturing cell lines and the creation of a DNA/RNA library used for genomic and genetic studies.Results:This infrastructure plays an important role in the achievement of various research programs for a better understanding of genetic and environmental factors involved in the development of melanoma and the spread of metastasis.It allows collaboration with other researchers at a provincial,national and international level in order to make progress in basic and clinical research on uveal melanoma.Conclusions:The biological material and clinical data of this infrastructure are available upon request to VHRN members whose research project was approved by the ethics committee of the institution.

AB032.The effect of eyemasks on reducing stress associated with retinopathy of prematurity screening

Background:Retinopathy of prematurity(ROP)is a disorder of retinal development in the low birthweight preterm infant.Eye screening is routinely performed for infants at risk of developing this disorder.While these examinations help prevent blindness,they can be physiologically stressful for infants,with changes in oxygen saturation,blood pressure and heart rate occurring during the exam and increased apneic episodes reported the 24-48 hours period afterward.The cause of these increased apneic episodes is not currently known.Our Background is to evaluate the effect of decreasing light simulation during mydriasis using an eye mask on the frequency of stressful episodes after ROP screening.Methods:Multi-centre randomized clinical study.This study was approved by hospital ethics boards at all sites.After informed consent was obtained,infants with a birthweight<1,500 g or gestational age of≤32 weeks and scheduled for their first ROP screening were randomized to receive either standard of care or a phototherapy mask during pupil dilation in addition to routine care.Dilated retinal exams were performed by retinal surgeons and fellows.The primary outcome was the frequency of any desaturation,bradycardic event,or apneic event 12 hours following the examination,compared to a baseline rate 12 hours prior to the exam.Heart rate,respiratory rate and oxygen saturation were recorded for up to 48 hours following the examination and compared to baseline.Results:A total of 51 infants were examined;28 randomized to the masked group and 23 to the control group.Ten and 13 infants were on ventilator support at the time of examination in each group,respectively.There was a 57.7%decrease in the total number of all stressful events in the masked group compared to controls in the 12-hour post exam period(rate ratio 0.42,95%CI,0.2-0.9,P=0.024).There was a 61.3%decrease in the number of bradycardic events in the masked group compared to controls(RR 0.39,95%CI,0.2-1.0,P=0.042).Heart rate was significantly higher in both groups after the exam(Mean HR:164.67 bpm post vs.157.3 bpm pre;P=0.04),with no difference in between groups(Effect by group P=0.31).There was no significant difference seen in either group in respiratory rate or oxygen saturation at 2 or 4 hours after the ROP examination compared to baseline.Risk factors that were associated with increased stress included younger gestational age(RR=1.3295%CI,1.2-1.5 per week),lower birthweight[RR=1.39(1.2-1.5)per 100 g],ventilator support around the time of exam[RR=2.67(1.3-5.6)],diagnosis of intraventricular hemorrhage[RR=3.78(1.9-7.3)],and hyponatremia[RR=3.42(1.8-6.6)].No adverse events occurred while using eye masks.Conclusions:The infants who wore a phototherapy mask during pupillary dilation had lower rates of stressful episodes following screening for retinopathy of prematurity,particularly lower episodes of bradycardia.

AB027.Nogo-A neutralisation improves vision recovery after retinal injury

Background:In glaucoma and after an ischemic injury of the retina,excessive activation of N-Methyl-D-Aspartate receptors,a type of glutamatergic receptors,induces the death of retinal ganglion cells and an irreversible vision loss.The painless loss of retinal cells does not allow for a swift diagnostic and treatment of retinal damages.There is no efficient therapy to improve retinal functions in this case.In order to develop new therapeutic approaches for retinal injury,we propose,in this study,to stimulate neuronal plasticity of the visual system by neutralising the glial protein,Nogo-A.The inhibitory action of this protein on axonal regeneration is well known in spinal cord injuries but not in the visual system.We thus studied the function of Nogo-A in vision recovery in mice.Methods:Nogo-A activities were chronically blocked by deleting its gene in KO mice and acutely,by intravitreal injections of an antibody known as 11C7.Inner retina lesions were done by injection of 0.5 or 5 nmol of NMDA in the vitreous humor.A PBS buffer was administered in control animals.The visual system functions were accessed with an optokinetic test in awake mice,by electroretinography(ERG)in the eye and visual evoked potentials in the visual cortex.Cell survival of retinal ganglion cells,amacrine and bipolar cells was evaluated on histological sections by immunofluorescence.Changes in expression of Nogo-A,its receptors and neuronal plasticity associated molecules were observed by Western blot and q-PCR.Results:At NMDA doses of≤0.5 nmol,Nogo-A KO mice show a recovery of optokinetic responses much faster than in WT mice.Surprisingly,a single injection of the antibody,11C7 was sufficient to improve VEPs of NMDA injured animals as compared to control antibody.Furthermore,ERGs showed that a dose of 0.5 nmol induced retinal lesions limited to the ganglion cell layer,with significant changes to the VEPs but without influencing photoreceptors and inner nuclear layer cells functions.However,5 nmol of NMDA affected the survival of inner nuclear layer cells and reduced by~50%their activity.Conclusions:Our results show that the neutralisation of Nogo-A can improve visual functions after injury to the inner retina.Inhibition of Nogo-A could thus be an efficient way to treat pathologies like glaucoma.

AB093.Ocular tissues bank for vision health research

Background:This infrastructure delivers biological material necessary for several research projects to Vision Health Research Network investigators(VHRN).Methods:Héma-Québec is the organism in charge obtaining consent and retrieving donor eyes for patient treatment or for research.In Quebec City,donor eyes are sent to the eye bank of the“Centre Universitaire d’Ophtalmologie”(CUO)of Saint-Sacrement hospital.Technicians at the eye bank evaluate the quality of the tissues.Those unfit for graft are transferred to the infrastructure where the coordinator encodes samples prior to their distribution.Results:Between 2013 and 2017,27 fundamental investigators,clinical investigators and collaborators supported by 60 students,trainees and laboratory assistants used this infrastructure to move forward their projects.Since 2013,results from those projects generated 21 scientific publications and 232 presentations.The infrastructure helped VHRN investigators obtain near 4 million dollars in grants from many organisms(CIHR,NSERC,Foundations,etc.).These grants allowed recruitment and formation of highly qualified personnel.Last year(April 2016 to March 2017),189 corneas and 23 eyes transited through the infrastructure.Conclusions:This infrastructure is available for all investigators that are members of the VHRN.Many original projects have been elaborated thanks to the human ocular tissues provided by this infrastructure.These projects will advance our knowledge in vision health.A better understanding of eye functions will lead to new treatments for eye diseases.