Hip joint center localisation: A biomechanical application to hip arthroplasty population

AIM: To determine hip joint center(HJC) location on hip arthroplasty population comparing predictive and functional approaches with radiographic measurements.METHODS: The distance between the HJC and the mid-pelvis was calculated and compared between the three approaches. The localisation error between the predictive and functional approach was compared using the radiographic measurements as the reference. The operated leg was compared to the non-operated leg.RESULTS: A significant difference was found for the distance between the HJC and the mid-pelvis when comparing the predictive and functional method. The functional method leads to fewer errors. A statistical difference was found for the localization error between the predictive and functional method. The functional method is twice more precise.CONCLUSION: Although being more individualized, the functional method improves HJC localization and should be used in three-dimensional gait analysis.

用以评估痴呆胆碱能通路上重要部位白质高信号的新视觉等级量表

Background and Purpose -One possible mechanism of cognitive decline in individuals with subcortical vascular disease is disruption of cholinergic fibers by ischemic lesions, such as strategically located white matter hyperintensities (WMH). The authors applied a new MRI visual rating scale to assess WMH within cholinergic pathways in patients with Alzheimer Disease (AD) and subcortical ischemic microvascular disease. Methods -Subjects included 60 AD patients with and with out WMH, matched for age, as well as 15 control subjects. A visual rating scale was developed based on published immunohistochemical tracings of the cholinergic pathways in humans. On 4 selected axial images, the severity of WMH in the chol inergic pathways was rated on a 3-point scale for ten regions, identified with major anatomical landmarks. A published, consensus-derived, general WMH scale w as also applied. All subjects underwent standardized neuropsychological testing. Results -The Cholinergic Pathways HyperIntensities Scale showed reliability an d was validated with volumetry of strategic WMH. After accounting for age and ed ucation in a multiple linear regression model, The Cholinergic Pathways Hyperint ensities Scale ratings were associated with impaired performance on the Mattis D ementia Rating Scale (r=0.40; P=0.02) and accounted for 12%of the variance (cor rected r2). A similar model was not significant for general WMH scores. Conclusi ons -The new MRI rating scale for WMH in cholinergic pathways is reliable and s hows stronger correlations with cognitive performance than a general WMH rating scale in AD with WMH. This new rating scale provides indirect evidence that loca lization of WMH within neurotransmitter systems may contribute to cognitive decl ine.

Total dysphagia after short course of systemic corticotherapy:Herpes simplex virus esophagitis

A 72 year-old female developed a herpetic esophagitis after 3 d of oral corticotherapy for an acute exacerbation of chronic obstructive pulmonary disease,presenting as odynophagia and total dysphagia.Biospies were taken during a first esophagogastroduodenoscopy(EGD) and the patient was referred to the thoracic surgery service with a presumptive diagnosis of esophageal cancer.A second EGD was planned for dilatation,but by that time the stenosis was completely resolved.The biopsies taken during the first EGD revealed multiple herpetic viral inclusions and ulcerations without any dysplasia or neoplasia.In front of a severe esophageal stenosis,one must still exclude the usual differential diagnosis peptic stenosis and cancer.Visualization of endoscopic lesions can suggest the diagnosis but must be promptly confirmed by biopsy,viral culture or polymerase chain reaction.Although immune systemic effects of corticotherapy are well known and herpetic esophagitis occurs most frequently in immunocompromised individuals,this case emphasizes the importance of clinical awareness concerning short courses of corticotherapy for immunocompetent individuals.This article discusses the reactivation process of herpetic infection in this context and addresses its diagnostic and therapeutic issues.

Dendritic cells and the extracellular matrix:A challenge for maintaining tolerance/homeostasis

The importance of the extracellular matrix(ECM) in contributing to structural,mechanical,functional and tissue-specific features in the body is well appreciated. While the ECM was previously considered to be a passive bystander,it is now evident that it plays active,dynamic and flexible roles in shaping cell survival,differentiation,migration and death to varying extents depending on the specific site in the body. Dendritic cells(DCs) are recognized as potent antigen presenting cells present in many tissues and in blood,continuously scrutinizing the microenvironment for antigens and mounting local and systemic host responses against harmful agents. DCs also play pivotal roles in maintaining homeostasis to harmless self-antigens,critical for preventing autoimmunity. What is less understood are the complex interactions between DCs and the ECM in maintaining this balance between steady-state tissue residence and DC activation during inflammation. DCs are finely tuned to inflammationinduced variations in fragment length,accessible epitopes and post-translational modifications of individual ECM components and correspondingly interpret these changes appropriately by adjusting their profiles of cognate binding receptors and downstream immune activation. The successful design and composition of novel ECMbased mimetics in regenerative medicine and other applications rely on our improved understanding of DCECM interplay in homeostasis and the challenges involved in maintaining it.

AB085.E-beam sterilization of recombinant human collagen-phosphorylcholine corneal implants for transplantation

Background:The sterilization of corneal implants composed of carbodiimide crosslinked recombinant human collagen type III(RHCIII)and phosphorylcholine polymers(RHCIII-MPC)is constrained by the biochemical properties of RHCIII.Early human trials used 1%chloroform in 0.1 M phosphate buffered saline(C-PBS),but require a stringent wash procedure with antibiotics to remove the chloroform.Irradiation with gamma or electron-beam(e-beam)allows a chemical-free sterilization method,but may result in crosslinking or denaturation.Here,electron-beam irradiation is evaluated as a sterilization method for RHCIII-MPC implants.Methods:Dose-finding study:RHCIII-MPC were cast in round,350µm thick,12 mm diameter molds for corneal implants and 0.5 mm thick dumbbell-shaped molds for mechanical testing.The hydrogels received an irradiation dose of 17,19,or 21 kGy and unirradiated controls were stored in C-PBS,n=3 per group.The hydrogels were tested for sterility and endotoxin,optical and mechanical properties,biodegradation,free radicals,and cell compatibility.Clinical evaluation in rabbits:RHCIII-MPC implants were e-beamed at 17 kGy or kept in C-PBS.One implant from each group was implanted into the right cornea of each rabbit by deep anterior lamellar keratoplasty,n=4 animals per group.Animals underwent preoperative and 6-month post-operative in vivo confocal microscopy(IVCM)to check nerve count and ingrowth of keratocytes.Corneal grafts and controls were assessed via histology and immunohistochemistry.Results:Dose finding study:hydrogels were sterile at all irradiation doses with no evidence of free radicals.There were no significant differences in optical or mechanical properties between the treatment groups and controls.All hydrogels supported cell growth.The 19 and 21 kGy implants had high collagenase degradation for 21 hours until they stabilized,whereas the 17 kGy and C-PBS implants had gradual degradation until 48 hours.Clinical results:the rabbits did not experience post-surgical inflammatory reactions and full epithelial coverage of the implants occurred within the first week of surgery for all animals.Mild neovascularization occurred in all animals,but resolved by 6-month follow-up.A mild 0.5-1.0 grade subepithelial haze was observed in all rabbits,but the implanted grafts remained transparent.Re-innervation occurred in all grafts with no significant differences between sterilization methods.All regenerated corneas had mucin production and were positive for cytokeratin 3 and 12.Grafted and control corneas were negative for macrophages and blood vessels.Conclusions:E-beam sterilization is a safe and effective form of sterilization for RHCIII-MPC implants.

AB018.Ocular hypertension promotes early mitochondrial fragmentation in retinal endothelial cells in a mouse model of glaucoma

Background:Retinal endothelial cells are very active and contribute to the integrity of the neurovascular unit.Vascular dysfunction has been proposed to contribute to the pathogenesis of glaucoma.Here,we evaluated the hypothesis that ocular hypertension triggers mitochondrial alterations in endothelial cells impairing the integrity of the blood retinal barrier(BRB).Methods:Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of EndoMito-EGFP mice,a strain expressing green fluorescent protein selectively in the mitochondria of endothelial cells.Capillary density,mitochondrial volume,and the number of mitochondrial components were quantified in 3D-reconstructed images from whole-mounted retinas using Imaris software.Dynamin-related protein(DRP-1),mitofusin-2(MFN-2)and optic atrophy-1(OPA-1)expression were assessed by western blot analysis of enriched endothelial cells.Mitochondrial structure was evaluated by transmission electron microscopy(TEM)and oxygen consumption rate was monitored by Seahorse analysis.The integrity of the BRB was evaluated by quantifying Evans blue leakage.Results:Our data demonstrate that two and three weeks after ocular hypertension induction,the total mitochondria volume in endothelial cells decreased from 0.140±0.002µm3 from non-injured retinas to 0.108±0.005 and 0.093±0.007µm3,respectively in glaucomatous eyes(mean±S.E.M,ANOVA,P<0.001;N=6/group).Frequency distribution showed a substantial increase of smaller mitochondria complexes(<0.5µm3)in endothelial cells from glaucomatous retinas.Significant upregulation of DRP-1 was found in vessels isolated from glaucomatous retinas compared to the intact retinas,while MFN-2 and OPA-1 expression was not affected.Structural alteration in endothelial cell mitochondria was confirmed by TEM,which were accompanied by a 1.93-fold reduction in the oxygen consumption rate as well as 2.6-fold increase in vasculature leakage in glaucomatous retinas(n=3-6/group).In addition,this model did not trigger changes in the density of the vascular network,suggesting that mitochondrial fragmentation was not due to endothelial cell loss.Conclusions:This study shows that ocular hypertension leads to early alterations in the dynamic of endothelial cell mitochondria,contributing to vascular dysfunction in glaucoma.

AB020. 3D scaffolds for optic nerve regeneration

Background:Regeneration of nerves or nerve bundles is problematic mainly due to issues of nerves finding their target tissues.A very clear example of this is the lack of treatments for traumatic injury to the optic nerve,something that is associated with surgery or trauma to the skull.Nerve guides have been used to support this for the better part of the last century,unfortunately the clinical improvements in patients receiving this sort of treatment is poor.Large improvements to the type of nerve guides used are needed to make this a viable solution for repair.It has been shown that electrostimulation of cells on conductive polymers can have positive effects on nerve regeneration.There are several material innovations that improve on speed of nerve regeneration;conductive polymer coatings being one example.There are constant improvements on solutions for nerve regeneration in many fields,unfortunately combining these different solutions is often slow.We combine electrospinning,3D printing and surface modification.Electrospinning allows control over fibrous structures.We tune the surface properties using conductive polymer coatings.The conductive fibrous structure can be integrated in a larger 3D printed scaffold that takes the role of guiding the nerve bundle.Methods:For manufacture of aligned fibers,PCL in chloroform was electrospun on a rotating mandrel.Random fibers were collected on a flat stationary collector.Dip coating was performed by submerging the fibrous scaffold in a solution of PEDOT:PSS in water and isopropanol.An outer layer of PEDOT:tosylate was added using vapor phase polymerization(VPP).3D printing was performed using an ink consisting of 0.25%alginate and 8.75%gelatin.The ink was cross-linked after printing using 0.4%CaCl2.Cell cultures were performed using chick dorsal root ganglia and a mouse neuroblastoma cell line.Ganglia and cells were seeded on the fibrous scaffolds.Electrostimulation was performed using a custom set up at constant DC current and slow pulsed DC current(1 min on off cycle)Materials were imaged using scanning electron microscopy.Cell cultures were stained using ICC and imaged with fluorescence microscopy.Results:All of the materials supported cell growth and neurite extension to some degree.The materials that were coated with PEDOT:tosylate and a combination of PEDOT:tosylate+PEDOT:PSS outperformed the PSS only group.Stimulation with a slow pulsed or constant DC current increased neurite extension on the negative pole,while there was inhibition of neurite growth on the opposite pole.The 3D printed outer layer serves as a biocompatible,bioactive support and guide for the bundle of neurites.Conclusions:The nerve guides can guide nerve growth.The 3D printed scaffold is cell friendly.The construct allows electrostimulation to increase speed of regeneration.

AB041.A novel IL-1 receptor modulator prevents photoreceptor loss in a model of age-related macular degeneration

Background:The objective of this study is to evaluate the implications of interleukin-1β(IL-1β)in photoreceptor degeneration using a model of blue light in rodents.Methods:CD-1 mice(12-16 weeks-old)were exposed to blue LED light(6000 lux at 450 nm)for 1 hour and then sacrificed at day 3 post-illumination.Mice were intraperitoneally treated or not with a peptide antagonist of the IL-1βreceptor,named Rytvela(or 101.10)twice per day until sacrifice.Several markers related to the inflammatory process such as F4/80,NLRP3,Caspase-1,IL-1βand glial fibrillary acidic protein(GFAP)were evaluated by immunohistochemistry.Photoreceptor cell death was assessed by TUNEL assay and Caspase-3 immunofluorescence.Results:Immunofluorescence experiments revealed an infiltration of positive F4/80 cells(microglia and macrophages)into the subretinal space in mice exposed to blue light,which was significantly(P<0.01)abrogated with Rytvela treatment.Co-localization of NLRP3,Caspase-1,and IL-1βwith F4/80 positive cells was clearly detected in the subretinal space,suggesting that these inflammatory cells are the main source of IL-1β.Interestingly,GFAP immunoreactivity,a marker of stress in Müller cells,was augmented in retinas exposed to the blue light,and reduced with Rytvela administration.The TUNEL assay showed that Rytvela prevents photoreceptor apoptosis in the retina of mice exposed to blue light.Likewise,co-culture of retinal explants with LPS-ATP activated bone marrow-derived macrophages resulted in a high number of TUNEL positive photoreceptors,which was reduced by treatment with Rytvela.Conclusions:These results show that Rytvela attenuated the inflammatory response and prevented the death of photoreceptors in a model of dry AMD.Modulation of IL-1βsignaling would be a useful therapeutic avenue for dry AMD,for which no approved treatment currently exists.

AB025.Machine learning applied to the oxygen induced retinopathy model

Background:The oxygen induced retinopathy rodent model is widely used,notably for the assessment of developmental dystrophies in preclinical studies of vascular retinal diseases.Typically,the quantification of vessel tufts and avascular regions is computed manually from flat mounted retinas imaged using fluorescent probes that highlight the vascular network.However,such manual measurements are time-consuming and hampered by user variability and bias,thus a rapid and objective alternative is required.Methods:We employ a machine learning approach to segment and characterize vascular tufts.The proposed quantitative retinal vascular assessment(QuRVA)technique uses quadratic discrimination analysis and morphological techniques to provide reliable measurements of vascular density and pathological vascular tuft regions,devoid of user intervention within seconds.Our algorithms allow also delineating the whole vasculature network,and identifying and analyzing avascular regions.Results:Our first experiment shows the high degree of error and variability of manual segmentations.In consequence,we developed a set of algorithms to perform this task automatically.We benchmark and validate the results of our analysis pipeline using the consensus of several manually curated segmentations using commonly used computer tools.We describe the method,provide details for reproducing the algorithm,and validate all aspects of the analysis.Conclusions:Manual and semi-automated procedures for tuft detection present strong fluctuations among users,demonstrating the need for fast and unbiased tools in this highly active research field with tremendous implications for basic research and industry.

AB042.microRNA-96-based therapy protect microvasculature against oxygen-induced retinopathy:a novel uncovered property of miR-96 in vascular repair

Background:Ischemic retinopathies(IRs)are ocular disorders associated to microvascular degeneration leading to visual impairments and blindness.microRNA(miRNAs)are a family of non-coding RNAs that regulate a wide range of gene expression involved in various biological process such blood vessel development and pathological NV.However,the post-transcriptional modulation of miRs and especially,their specific functions in the eyes during IRs remain to be evaluated.We aim to evaluate the potential role of miR-96 on microvascular degeneration in a rat model of oxygen-induced retinopathy(OIR).Methods:In vivo:next generation sequencing(NSG)was used to perform a complete miRNAs profiling in the retina and choroid from OIR and normoxia(CTL)rats.To evaluate the effects of miR-96 on microvasculature,OIR animals were treated with a miR-96 mimic(1 mg/kg)or a control-miR by intravitreal injection before hyperoxia-exposure(80%O2).Immunostaining analysis of retinal flatmounts and cryosections was used to explore the microvascular effects of miR-96.In vitro:Human Retinal Microvascular Endothelial Cells(HRMVEC)were subjected or not to hyperoxia(80%O2)and transfected with 50 nM of miR-96 mimic or antagomir-96.Angiogenic assay was performed(tube formation and migration)and molecular analysis evaluated by qRT-PCR and western blot.Results:NSG and qRT-PCR analyses identified miR-96 as one of most highly expressed miRNAs in retina and choroid during development.However,miR-96 showed a strong downregulation in OIR rats,and also in HRMVEC subjected to hyperoxia.In HRMVEC,we found that miR-96 regulates positively the expression of the key pro-angiogenic factors VEGF,FGF-2 and ANG-2.To better explore the role of miR-96 on HRMVEC angiogenic activity,we performed a gain/loss of function study.Similarly,to hyperoxia exposure,we observed a robust angiogenic impairment(tube formation and migration)on HMRVEC transfected with an antagomiR-96.Interestingly,overexpression of miR-96 completely recued the basal phenotype of HRMVEC and protected against hyperoxia-induced endothelial dysfunction.In vivo,intravitreal injection of miR-96 mimic(1 mg/kg)in OIR rats significantly restored retinal vascular density and choroidal tightness/sprouting hability.This was accompanied by the restoration in the physiological levels of VEGF,FGF-2 and ANG-2.Conclusions:This is the first study showing that reduced expression of miR-96 in OIR conditions lead to a reduction of VEGF/FGF/ANG-2 signaling,and inneficient post-ischemic revascularization in retinal/choroidal tissues.Intravitreal supplementation of miR-96 using a miR mimic could constitute a novel therapeutic strategy to improve vascular repair in IRs.

AB025.One-year outcomes of trabecular micro-bypass stents with concomitant cataract surgery in primary angle closure glaucoma

Background:Trabecular microbypass stents have allowed improved multidirectional flow with good efficacy and safety profile in primary open-angle glaucoma(OAG).The efficacy of these devices in primary angle closure glaucoma has been understudied.We aimed to assess the one-year postoperative outcomes following implantation of trabecular micro-bypass stents with concomitant cataract surgery in angle-closure glaucoma patients.Methods:We evaluated the baseline clinical characteristics and the 12-month outcomes of patients with mild to severe primary angle-closure glaucoma who underwent cataract surgery with implantation of either a first generation trabecular microbypass stent(iStent group)or two second generation trabecular microbypass stents(iStent-inject group).The primary outcomes included intraocular pressure(IOP)and anti-glaucoma medication use.The secondary outcomes were success rate(defined by IOP between 5-18 mmHg with IOP reduction of at least 20%)and visual acuity.Results:A total of 83 eyes(58 from the iStent and 25 from the iStent-inject group)were included with an average age of 68.9±8.6 and 67.6±8.3 years,respectively.All eyes had mild to severe angle-closure glaucoma.At one-year follow-up,the IOP decreased by 21%(from 18.8±4.5 mmHg)and 25%(from 18.7±3.6 mmHg),in each group respectively(P<0.001).Additionally,the medication burden dropped by 52%and 50%at one-year follow-up(P<0.001).The 12-month success rate was 45%in the iStent group compared to 64%in the iStent-inject group(P=0.086)and visual acuity remained stable.Conclusions:The present study provides clinically relevant,real-world data on the utility of iStent and iStent inject with cataract surgery in angle-closure glaucoma-a population that has been understudied in the world of trabecular micro-bypass stents.Our data demonstrated efficacy of these stents in reducing intraocular pressure and medication burden among patients with primary angle closure glaucoma.

AB046. The retinoblastoma model for translational research

Background:Our national collaborative research initiative is proposing to develop a common infrastructure for Rb research.We are proposing a novel in vivo Rb model using human Rb cells line.Methods:The rabbit model has advantages over the mouse models:(I)the larger eye size of rabbits,similar to the human infant eye,permits a more accurate injection of the drugs and evaluation of methods of targeted intraocular drug delivery;(II)the rabbit model demonstrated similar fundus appearance and pathologic features to human Rb,including vitreous seeds of viable tumor when the retinal tumor is mid-sized,which are usually found in the late stage in mouse models.The lack of ability to eliminate vitreous seeds is a major reason of current treatment failures in Group C and D tumors;therefore,the rabbit model of Rb may be used as a model to evaluate the effectiveness and various routes of drug delivery.Results:This is an implementation of an infrastructure for evaluating therapeutic targets.In addition,this finding enables a variety of pharmacokinetic studies,pharmacodynamic and toxicology studies for new therapeutic agents.Conclusions:This infrastructure meets the growing concern of practitioners and researchers in the field.The common facility is easily accessible to all VHRN members on request,including requests from other sectors.

Nuclear factor erythroid 2-related factor 2 a master regulator of retinal vascular regeneration

In this issue of PNAS,Wei and colleagues demonstrate that the Nuclear factor erythroid 2-related factor 2(Nrf2),a well-known cell-intrinsic cytoprotective factor and critical regulator of the anti-oxidant response plays an important function in reparative angiogenesis by suppressing the antiangiogenic effects of Semaphorin 6A(Sema6A),a membrane-associated guidance molecule expressed

AB026.Ocular rigidity is correlated with glaucomatous structural damage

Background:The rigidity of the corneoscleral shell is an important biomechanical property which could be relevant in the pathophysiology of open-angle glaucoma(OAG).This study aims to evaluate the relationship between ocular rigidity(OR)and glaucomatous damage as represented by structural optical coherence tomography(OCT)-based parameters such as retinal nerve fiber layer(RNFL)and ganglion cell layer+inner plexiform layer(GCL+IPL)thicknesses.These parameters characterize the retinal layers that contain neuronal structures that form the optic nerve.Methods:Sixty-six subjects(37 with early OAG,11 with moderate to advanced OAG,16 healthy)were recruited in this study.OR measurements were carried out using a non-invasive clinical method developed by our group.As described in Beaton et al.(2015),this method,which is based on Friedenwald’s equation,involves video-rate OCT imaging and automated choroidal segmentation,as well as dynamic contour tonometry to calculate the OR coefficient.RNFL and macular GCL thicknesses were acquired using the Cirrus SD-OCT(Carl-Zeiss Meditec,Dublin,CA,USA).Correlations between OR and structural parameters in all 66 eyes were assessed using SPSS.Results:Significant correlations were found between OR and the average GCL+IPL thickness(r=0.355,P=0.004)as well as the minimum GCL+IPL thickness(r=0.340,P=0.006).Direct correlations were also found between OR and RNFL thickness in the inferior quadrant(r=0.258,P=0.036)and inferior clock hour(r=0.313,P=0.011).Conclusions:In this study,we found a positive correlation between structural OCT-based parameters and OR,perhaps indicating more structural damage in less rigid eyes.These findings could provide insight unto the pathophysiology of OAG.Further investigation is warranted to confirm the role of OR in glaucoma and elucidate whether there is a subgroup of patients for which OR plays a greater role.

AB008.Cellular senescence and retinal angiogenesis

Pathological retinal neovascularization is the hallmark of primary blinding diseases across all age groups,yet surprisingly little is known about the causative factors.These diseases include diabetic retinopathy and retinopathy of prematurity where progressive decay of retinal vasculature yields zones of neural ischemia.These avascular zones and the hypoxic neurons and glia that reside in them are the source of pro-angiogenic factors that mediate destructive pre-retinal angiogenesis.Central neurons such as retinal ganglion cells(RGCs),which are directly apposed to degenerating vasculature in ischemic retinopathies,require stable metabolic supply for proper function.However,we unexpectedly found that RGCs are resilient to hypoxia/ischemia and a generally compromised metabolic supply and instead of degenerating,trigger protective mechanisms of cellular senescence.Paradoxically,while potentially favoring neuronal survival,the senescent state of RGCs is incompatible with vascular repair as they adopt a senescence-associated secretory phenotype(SASP)that provokes release of a secretome of inflammatory cytokines that drives paracrine senescence and further exacerbates pathological angiogenesis.The mechanisms that lead to retinal cellular senescence and dormancy as well as the therapeutic potential of targeting these pathways will be discussed.

AB023.Characterization of a novel anti-angiogenic protein for the treatment of exudative age-related macular degeneration

Background:Age-related macular degeneration(AMD)is a leading cause of blindness in Canada.The exudative(wet)form of AMD accounts for approximately 15%of AMD patients,but is responsible for the majority of severe vision loss associated with the disease.Wet AMD is characterized by choroidal neovascularization,the abnormal growth of blood vessels from the choroid into the sub-retinal space.Current therapies for exudative AMD directly target and inhibit the vascular endothelial growth factor(VEGF)signaling pathway,a signaling axis that promotes endothelial cell survival.While initially effective at restoring visual acuity,recent studies suggest that chronic use of anti-VEGF therapies can lead to further vision impairment through off-target effects on photoreceptors and other non-vascular tissues.These off-target effects of anti-VEGF therapy highlights the need for alternative treatments for this increasingly common disease.We have recently identified a novel anti-angiogenic protein,AAP1 that inhibits retinal angiogenesis during development.As a regulator of the vasculature,our current work aims to characterize the function of AAP1 in endothelial cells and determine the potential of AAP1 as a therapy for exudative AMD.Methods:To address our aims,we used various in vivo and in vitro models of normal and pathological vascular growth.Mice were injected intravitreally with AAP1 to investigate its effects on developmental and pathological angiogenesis.Using HUVECs,we employed immunofluorescent quantifications to determine the impact of AAP1 on sprouting angiogenesis by measuring cellular proliferation,apoptosis and migration.To investigate the signaling events that mediate the actions of AAP1,we examined key signaling pathways involved in angiogenesis by western blotting and qPCR.Results:We evaluated the role of AAP1 as a regulator of angiogenesis during retinal development and mouse models of AMD.Our pilot data show that AAP1 prevents angiogenesis in vitro and in vivo,and that it can also inhibit pathological neovascularization in experimental models of AMD.In spite of its anti-angiogenic effects,our data show that AAP1 does not adversely affect photoreceptors.Both in vitro and in vivo systems showed a decrease in cellular division,while apoptosis was not affected in response to AAP1 treatment.While cellular migration was reduced in AAP1-treated HUVECs,cellular polarity was not affected.Finally,gene and protein expression of key angiogenic factors were modified in response to AAP1.Conclusions:AAP1 is a potent regulator of angiogenesis.However,in contrast to anti-VEGF agents,our data suggests that AAP1 does not adversely affect the photoreceptors-highlighting the therapeutic potential of this protein.Further data generated from our studies characterizing the mechanism of AAP1 action may lead to a novel treatment option for AMD patients preventing vision loss and improving their quality of life.

The Vision Health Research Network Annual Research Day,and International Symposium on Retinal and Choroidal Angiogenesis(Montreal)

The Vision Health Research Network of Québec(the Network)was established in 1995 and funded by the Fonds de la recherche du Québec en santé(FRQS).In the past 23 years,it has been the major venue to foster collaborations within Quebec’s research community and beyond.The Network aims to increase research capacity,improve infrastructure and enhance competitiveness of Quebec’s vision research on the international stage.Focusing on key matters proposed by Canadian health policy,the Network strikes to improve visual outcomes among patients.

AB087.Corneal phenotype of a Slc4a11 knockout murine model for congenital hereditary endothelial dystrophy

Background:Congenital hereditary endothelial dystrophy(CHED)is characterized by blindness at birth or in early infancy resulting from bilateral corneal opacification,and is linked to mutation in the Slc4a11 gene.A Slc4a11 knockout(KO)mouse,generated by gene deletion(Vithana et al.Nat Genet 2006),was acquired in order to study this disease.To confirm the phenotype of this Slc4a11 KO mouse model as a function of age,using the wild type(WT)mouse as a control.Methods:Genotyping was performed by PCR(REDExtract-N-AmpTM Tissue PCR Kit,Sigma-Aldrich,Oakville,ON).Slc4a11 WT and KO mice populations aged from 5 to 50 weeks were studied(n=5 animals per age group;5-year age intervals).Slit lamp examination,anterior segment-ocular coherence tomography(OCT930SR;Thorlabs,Inc.,Newton,NJ),corneal endothelial cell staining,and scanning(SEM)and transmission(TEM)electron microscopy were used to assess the morphological and cellular differences between the two groups.The expression of basolateral membrane transporter NaBC1 within the corneal endothelium was also assessed using immunohistochemistry.Results:Diffuse and progressive corneal opacification was observed at the slit lamp in the Slc4a11 KO mice,starting at 10 weeks.The central corneal thickness(CCT)also increased progressively as a function of time.In comparison,Slc4a11 WT corneas remained clear over the entire study period.Early TEM results showed vacuole degeneration of the corneal endothelium in the 15-week KO mouse,which was not seen in the same age WT mouse.Conclusions:The corneal phenotype of this Slc4a11 KO mouse is representative of the clinical manifestations of CHED in human subjects,confirming the usefulness of this model for studying pathophysiology and therapeutic alternatives for Slc4a11-associated corneal dystrophies.

AB021.The effect of anti-VEGF on retinal inflammation and its relationship with the Kinin system in a rat model of laser-induced choroidal neovascularization

Background:The neovascular aged-related macular degeneration(AMD)is the leading cause of legal blindness in the elderly.It is presently treated by anti-VEGF intravitreal injection in order to stop the neovascularization.In seeking of more efficient treatments to prevent retinal damage,it has been proposed that the kinin-kallikrein system(KKS),a key player in inflammation,could be involved in AMD etiology.However,the role of kinin receptors and their interaction with VEGF in AMD is poorly understood.Methods:In order to address this question,choroidal neovascularization(CNV)was induced in the left eye of Long-Evans rat.After laser induction,anti-VEGF or IgG control were injected into the vitreal cavity.Gene expression was measured by qRT-PCR,retinal adherent leukocytes were labelled with FITC-Concanavalin A lectin,vascular leakage by the method of Evans blue and cellular localisation by immunohistochemistry.Results:The number of labelled adherent leucocytes was significantly increased in laser-induced CNV compared to the control eye.This was significantly reversed by one single injection of anti-VEGF.Extravasation of Evans blue dye was significantly increased in laser-induced CNV eyes compared to control eyes and partially reversed by one single injection of anti-VEGF or by R954 treatment.The mRNA expression of inflammatory mediators was significantly increased in the retina of CNV rats.Immunodetection of B1R was significantly increased in CNV eyes.B1R immunolabeling was detected on endothelial and ganglion cells.Conclusions:This study is the first to highlight an effect of the kinin/kallikrein system in a model of CNV that could be reduced by both anti-VEGF therapy and topically administered B1R antagonist R-954.

AB022.Biosynthetic implants for corneal regeneration in patients at high risk of rejecting donor transplantation

Background:Patients with inflammation or severe corneal pathology are often at high risk of rejecting the human donor corneas that they receive during transplantation.Our goal was to determine whether cell-free implants incorporating phosphorylcholine-based polymer,2-methacryloyloxyethyl phosphorylcholine(MPC),which has inflammation suppressing properties,can support repair and regeneration of ulcerated,high-risk corneas.Methods:Interpenetrating networks of recombinant human collagen and MPC(RHC-MPC)were fabricated into corneal implants in a certified and monitored cleanroom.An open-label,first-in-human observational study was conducted following ISO 14971 and 14155:2011,the Declaration of Helsinki and relevant laws of Ukraine and India,after respective ethical approval and trial registration.Seven unilaterally blind patients,aged 36 to 76 years old,diagnosed with conditions putting them at high risk of rejecting conventional corneal transplantation,and capable of providing informed written consent were grafted and followed up for an average of two years.However,RHC like native collagen is a large macromolecule and difficult to process and is non-customisable.Hence,small,customisable analogs to collagen comprising collagen-like peptides(CLP)conjugated to polyethylene glycol(PEG)were developed.These CLP-PEG analogs like RHC were combined with MPC into implants and tested in mini-pig models with alkali burned corneas,a high-risk condition.Results:One patient had an unrelated infection that necessitated re-grafting and was excluded from the study.The RHC-MPC implants in the remaining six patients were stably integrated throughout the entire observational period.There was regeneration of the cornea epithelium and stroma.Significant vision improvement was observed in in patients with damaged corneas due to infection.By two weeks post-operation,RHC-MPC implanted corneas of patients with active ulcers were free from the symptoms of pain,irritation and photophobia.Over the two-year follow-up period,sensitivity to touch improved,suggesting that the implants were able to promote nerve regeneration.The results seen in the clinical trial were reproduced in corneal grafts comprising MPC and the CLP-PEG collagen analog.Both CLP-PEG-MPC and control CLP-PEG only implants promoted regeneration of corneal epithelium,stroma and nerves.However,the alkali-burned corneas grafted with CLP-PEG-MPC implants retained the thickness of their healthy contralateral controls.Control corneas with CLP-PEG implants without the MPC however,were significantly thicker.Conclusions:These results demonstrate that collagen or its synthetic analog comprising CLP-PEG,can promote regeneration.The incorporation of MPC appears to suppress inflammation in pathologies that constitute conditions with a high-risk for graft rejection.Regeneration was able to occur in inflamed corneas as evidenced by CLP-PEG only grafts,but long-term follow-up is needed to determine if the chronic inflammation may influence graft failure over time.