Background:Primary open-angle glaucoma(POAG)is a genetically complex disorder caused primarily by gene-gene interactions.To identify these interactions,we studied the CA family,a large French-Canadian pedigree in whic...Background:Primary open-angle glaucoma(POAG)is a genetically complex disorder caused primarily by gene-gene interactions.To identify these interactions,we studied the CA family,a large French-Canadian pedigree in which the myocilin K423E mutation(MYOCK423E)causes autosomal dominant glaucoma with diagnoses ranging from juvenile-onset OAG(JOAG)to late adult-onset POAG in the heterozygotes(HTZ).To explain this extreme variability,we hypothesized that a second gene,called a modifier,was interacting with MYOC,the primary disease gene.Our goals were(I)to map the modifier on the human genome and;(II)to characterize the symptoms affected genetically by the modifier.These symptoms are called endophenotypes.Methods:Three hundred seventy-five CA members were studied using four quantitative endophenotypes:age of maximal intra-ocular pressures(IOPmax),IOPs progression,progression of cup to disk ratios and age-at-onset(AAO)defined as age at which ocular hypertension(OHT)was first detected with IOP≥22 mmHg.Genome-wide linkage analysis was performed by genotyping 408 genetic markers in 184 CA members.An unbiased pedigree-based algorithm was designed to identify the individuals who were double-mutants,i.e.,these individuals carried one MYOCK423E mutation(i.e.,they were HTZ,affected or not)and they also carry simultaneously a DNA mutation within the modifier.Results:Out of the 375 CA family members investigated,156 were HTZ for the MYOCK423E mutation.120 HTZ were affected with OAG or OHT with treatment while the remaining 36 HTZ were asymptomatic.AAO ranged from 7 to 63 years old;4 individuals over 50 years old were still asymptomatic.OHT preceded optic nerve damage in>98%of the HTZ carriers,confirming that AAO reflected the true severity of the disorder.The modifier showed strong inherited effects on 2 of the 4 endophenotypes:AAO and IOPmax.We next mapped with very high confidence the modifier locus for AAO at chromosome 20q13.Saturation genotyping with additional markers refined the locus to a 9 to 10 centimorgan interval,or about 10 million DNA nucleotides,between D20S857 and D20S832.The locus was named modifier of glaucoma 1(MOG1).When comparing the AAOs of the double mutants versus the median of the AAOs of the MYOCK423E HTZ who carried a wild-type(normal)MOG1 gene and were 1st cousins or closer with the double mutant under investigation,we observed that MOG1 delayed the ages at onset by an average of 8 to 10 years in the double mutants.Conclusions:The MOG1 locus encodes a DNA element that delays the onset of glaucoma by an average of 8-10 years by hampering the first manifestations of OHT.This research will lead to the development of new therapeutic targets for glaucoma.These treatments should prevent optic nerve damage by maintaining IOPs within the normal range.展开更多
Background: There are few studies on the cost of glaucoma management in developing country, especially in Togo, there are no data on the cost of POAG management. Aims: To determine the annual direct cost of the manage...Background: There are few studies on the cost of glaucoma management in developing country, especially in Togo, there are no data on the cost of POAG management. Aims: To determine the annual direct cost of the management of POAG, to evaluate the annual economic weight of the management of POAG and to determine the factors associated with the annual economic weight of the management. Methods: We conducted a retrospective and descriptive study over a period of 12 months from January 1 to December 31, 2019 based on the records of patients followed for POAG in AFIA Eye Clinic in Lomé-Togo. The annual direct cost was defined by the sum of the costs of consultations, explorations and treatments. We defined the direct cost per patient and per year and related to the average annual income. It was said to be catastrophic at 20% or more of the estimated annual income. Chi 2 and Fisher tested the comparison of proportions. We conducted univariate and multivariate logistic regression to search correlations. Results: During the study period, 150 patient records were included. The average age was 47.24 ± 17.09 years and the sex ratio was 0.82. The cost of the diagnosis was 112.18 ± 22.26 €. The average cost of consultations was 19.46 ± 11.35 € and that of explorations was 92.71 ± 10.91 €. The annual cost of treatment per patient was 165.52 ± 110.16 €. The annual global direct cost of POAG management per patient was 277.69 ± 132.42 €. Compared to the annual income of 1166.29 €, the economic weight of the glaucoma management was 23.8%. This direct cost was catastrophic for 32.1% of patients in the study (44/150 of people with no care). Compared to the guaranteed inter-professional minimum wage (SMIG) of 640.30 €, the economic direct cost weight was 43.3%. Risk factors significantly associated with the direct cost were age over 40 (OR = 1.05 and p = 0.032), liberal profession (OR = 4.72 and p = 0.04), the absence of health insurance (OR = 6.68 and p = 0.017) and the use carbonic anhydrase inhibitors (OR = 7.4 and p = 0.012) and prostaglandin analogues (OR of 38.2 and p Conclusion: This first study on the direct cost of POAG management in Lomé showed that the economic burden glaucoma represents for the patient, his family and society. The data from this study will allow health decision-makers to adopt strategies to mitigate the effects of glaucoma on the economy.展开更多
Background:Cell transfection has the potential to transform gene therapy,potentially enabling the treatment of genetic diseases.We are developing a cell transfection approach that uses laser pulses and gold nanopartic...Background:Cell transfection has the potential to transform gene therapy,potentially enabling the treatment of genetic diseases.We are developing a cell transfection approach that uses laser pulses and gold nanoparticles to induce transient holes in the cell membrane,thus allowing external material to pass into the cell before the membrane is healed.Our previous research has demonstrated the feasibility of perforating the cell membrane in vitro by irradiating(λ=532 nm,τ=6 ns)cancer cells previously incubated with gold-nanoparticles(100 nm).Our ultimate goal is to develop an optofluidic probe(needle-like)capable of performing simultaneous and precise delivery of a perforation mixture(nanoparticles and perforation indication dye)and laser pulses in vivo.We will present the initial validation of the technology with in vitro perforation of cancer cells.Methods:MDA-MB-231 cells were cultured in DMEM+GlutaMax(9%FBS,1%P/S).The medium was changed to Leibovitz’s L-15 for experimentation.100 nm gold nanoparticles and a perforation indicator dye(calcein,green dye)were placed on cells by pumping them through the optofluidic tip.The optofluidic tip was coupled to a nanosecond laser(λ=532 nm,τ=6 ns).Cells were irradiated with 5 laser pulses(Energy:15-38μJ)through the optrode tip at a distance of 50μm.Successful cell uptake was indicated by calcein uptake.Cells were incubated in 0.5%CO_(2) for one hour following irradiation.Then,propidium iodide(PI,red dye)was added to investigate cell death.The quantification of the perforation efficiency and viability was performed with fluorescence microscopy.Results:We have been able to successfully inject cells by pumping the perforation mixture through an optofluidic tip.The early quantitative results indicate that injection efficiencies are near 35%at the optimal fluence with cell viability near 90%.These efficiencies are similar to our previous results involving pre-incubation of cells with the perforation mixture.Conclusions:Our results show the feasibility of cell transfection using a particularly designed optofluidic probe(needle-like)for light and liquid delivery.The method does not involve pre-incubation of cells with the preformation mixture and thus brings the technology closer to in vivo applications.展开更多
Background:Our previous studies revealed that Nogo-A gene ablation improved visual function recovery after retinal injury.Moreover,Nogo-A expression is highly expressed in the healthy retina.Its physiological role in ...Background:Our previous studies revealed that Nogo-A gene ablation improved visual function recovery after retinal injury.Moreover,Nogo-A expression is highly expressed in the healthy retina.Its physiological role in retinal function is not known.The purpose of this current study was to determine the effects of acute Nogo-A silencing on retinal neuron structure and function in physiological conditions.Methods:Nogo-A silencing was done by intravitreal injection of adeno-associated virus serotype 2.2 containing a short hairpin RNA sequence(AAV2.2 shRNA-Nogo-A)and a GFP reporter gene in adult C57BL/6J mice.As control,an empty AAV2.2 vector was used.Infection of retinal cells was followed by fluorescent fundoscopy.Changes in Nogo-A expression were analysed by Western blotting in whole retinal lysates.Electroretinography was used to monitor retinal activity.The assessment of optokinetic reflex(OKR)allowed to follow visual acuity in unrestrained mice.Immunofluorescence on histological sections using the following cell markers,i.e.,RNA-binding protein with multiple splicing(RBPMS)and sex-determining region Y-box 2(Sox-2)allowed to visualize retinal ganglion cells(RGCs)and Müller glia respectively.Results:GFP fluorescence revealed efficient AAV2.2 transfection in the ganglion cell layer and the inner nuclear layer 30 days after viral injection.By Western blotting,Nogo-A expression was decreased by~75%in AAV2.2-shRNA-Nogo-A-treated retinae(n=3)as compared to the control mice(n=3).Strikingly,AAV2.2-shRNA-Nogo-A-injected animals(n=10)had a visual acuity reduction of 43.7%as compared to control(n=7),60 days after transfection.Electroretinography(ERG)b-wave and a-wave amplitudes were also decreased by~35%and 24.4%respectively relative to controls.After two months of transfection,RBPMS-positive RGCs were reduced by~30%in AAV2.2-shRNA-Nogo-A(n=4)compared to non-injected contralateral eyes(n=4).The number of Sox2-expressing Müller cells was not affected after Nogo-A knockdown.Conclusions:Nogo-A gene silencing in the retina has deleterious effects on the mouse retinal structure and function,suggesting an important role for Nogo-A in retinal physiology.展开更多
Background:Cells are influenced by their environment.In vivo,the corneal endothelium is subjected to intraocular pressure(IOP).The purpose of this project was to evaluate in vitro,the effect of the IOP on the formatio...Background:Cells are influenced by their environment.In vivo,the corneal endothelium is subjected to intraocular pressure(IOP).The purpose of this project was to evaluate in vitro,the effect of the IOP on the formation of tight junctions in the corneal endothelium.Methods:Cultivated corneal endothelial cells(P2-P3;n=6 populations)were seeded on devitalized on corneas(n=10 pairs).Native corneas and devitalized corneas were respectively used as positive(n=2 pairs)and negative controls(n=3 pairs).Corneas were placed in artificial anterior chambers and subjected to a hydrostatic pressure between 0.3 and 0.4 psi during 4-5 days.Unpressured control corneas were maintained in cell culture dishes.Pictures of the corneas were taken following the experiment to assess stromal transparency.Morphology,corneal thickness and distribution of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporter were evaluated by electron microscopy,histological staining and immunofluorescences.Results:Pressure treated corneas were more transparent than the controls.Thickness was accordingly reduced by 38.4%±4.9%for cultivated endothelium and 32.2%±2.7%for native endothelium.Negative controls change in transparency and thickness were marginal.Pressure treated cells showed none or at most marginal difference in morphology and expression of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporters and failed to recreate a phenotype similar to native corneas.Pressure however increased cortical localisation of the protein ZO-1 in both cultivated and native endothelium.Conclusions:These results suggest that anterior chamber hydrostatic pressure may enhance endothelial functionality by modulating the distribution of tight junction’s proteins.展开更多
Background:Age-related macular degeneration(AMD)is the second Canadian cause in visual deficiency.AMD is characterized by the death of photoreceptors and retinal pigmented epithelium(RPE)in the macular region of the r...Background:Age-related macular degeneration(AMD)is the second Canadian cause in visual deficiency.AMD is characterized by the death of photoreceptors and retinal pigmented epithelium(RPE)in the macular region of the retina,leading to the loss of central vision.Epidemiologic studies suggest an association between lifetime sun exposure and the probability to develop AMD even though mechanisms are unknown.Sunlight is made of about 30%of high-energy visible(HEV)light(blue light),the most energetic wavelength reaching the retina.These wavelengths can be absorbed by lipofuscin,an age pigment accumulating in RPE cells.Lipofuscin principal component is N-retinylidene-N-retinylethanolamine(A2E).Many research teams showed that absorption of HEV light by A2E in RPE cells at non-physiological doses produces free radicals and leads to cell death.Our earlier work shows that when A2E-loaded RPE cells are irradiated with HEV light at physiological doses,the same light does not lead to oxidative stress as measured by telomere and mitochondrial integrity.Our hypothesis is that HEV light,at physiological doses,modify or convert A2E in derived produces,inhibiting its photo-oxidant effect.Methods:In vitro,we irradiated A2E with HEV light with or without antioxidants and with varying irradiation regimen to observe the UV-Visible spectrum of A2E.In cellulo,we loaded ARPE-19 cells with A2E and irradiated cells at physiological levels for 4 consecutive days.We then observed A2E fluorescence using a fluorescence microscope with nucleus counterstaining with DRAQ5.Results:HEV light leads to the disappearance of A2E characteristic UV-Visible spectrum and the apparition of a new product suggesting that HEV light modifies A2E.Nor oxidation and irradiation regimen seem to have an impact in A2E’s conversion by HEV light.We observed and progressive diminution of A2E fluorescence in cellulo during physiological irradiations.Conclusions:The loss of A2E photo-oxidation capacities by HEV light seems to be caused by its conversion by HEV light.We suggest that HEV light,at physiological doses,may be protective rather than photo-toxic.The next step would be to identify A2E’derived produces and their cell toxicity.展开更多
Background:Lesion to the retinal pigment epithelium(RPE)is a crucial event in age-related macular degeneration(AMD)development.Although the pathogenesis of this complex disease is poorly understood,sunlight exposure a...Background:Lesion to the retinal pigment epithelium(RPE)is a crucial event in age-related macular degeneration(AMD)development.Although the pathogenesis of this complex disease is poorly understood,sunlight exposure and smoking are major environmental risk factors associated with AMD.High-energy visible blue light(HEV;400-500 nm)is the most energetic and potentially harmful solar wavelengths reaching adults retina.On the other hand,RPE cells can be exposed to a large range of pollutants from cigarette smoke,with polycyclic aromatic hydrocarbons(PAH)being among the most toxic.Some PAH from cigarette smoke can absorb HEV light.This led us hypothesize that in RPE cells,the combination of PAH and HEV could synergize to exacerbate the stress caused by either factor alone.We thus investigate the combined effect of PAH and HEV light in RPE cells.Methods:Confluent RPE immortalized cells(ARPE19)were exposed to nanomolar concentrations of benzo[a]pyrene(BaP)or indeno[1,2,3-cd]pyrene(IcdP).While IcdP efficiently absorbs HEV wavelengths,BaP,the most studied PAH,does not significantly absorb HEV light and was used as a control.BaP or IcdP contaminated ARPE19 were then irradiated with increasing sub-lethal doses of HEV light(150-500 J/cm2)using a setup that mimics the light spectrum normally reaching the retina.Cytotoxicity,apoptosis and reactive oxygen species(ROS)generation were assessed in each condition.Results:In presence of low concentrations of IcdP,sub-lethal amounts of HEV light trigger,in a dose-dependent way,up to 70%of apoptotic cell death.Co-exposure to IcdP and HEV also leads to a synergistic ROS generation in ARPE19 cells,thus inducing oxidative stress.None of these effects were observed with BaP.Efficient inhibition of ROS production by specific antioxidants only decreases death by 20%in cells simultaneously exposed to both IcdP and HEV light.Conclusions:Low concentrations of IcdP synergize with HEV light to induce phototoxicity in ARPE19 cells.An increased oxidative stress results from the interaction between both agents and partially explains the enhanced HEV phototoxicity in IcdP contaminated ARPE19 cells.This suggests that another major mechanism is involved in the synergetic toxicity.For smokers,this synergy between HEV and PAH may accelerate RPE cells loss and contribute to their greater risk of developing AMD.展开更多
Background:The goal of this project was to analyze the relationship between cell morphology and proteases/proteases inhibitors(PIs)secretion profile in fuchs endothelial corneal dystrophy(FECD)corneal endothelial cell...Background:The goal of this project was to analyze the relationship between cell morphology and proteases/proteases inhibitors(PIs)secretion profile in fuchs endothelial corneal dystrophy(FECD)corneal endothelial cells(CECs).Methods:Cell morphology was determined using a circularity index(4π×area/perimeter2)for each CECs population extracted from surgical FECD specimens(N=2)and healthy Eye bank corneas(N=3).CECs were cultured 28 days post-confluency.Supernatant was collected and analysed using Proteome Profiler Array detecting 35 proteases and 32 PIs(R&D Systems).Proteome signal was analyzed using Image Studio Lite and correlated with the population’s circularity index.Results:Calculation of circularity index reported different morphologies among FECD populations(0.59±0.18 and 0.64±0.17)and healthy populations(0.44±0.18,0.66±0.13 and 0.71±0.11).Proteome arrays revealed the presence of 10 proteases(ADAMTS1,Cathepsin A,B,D,and X/Z/P,DPPIV/CD26,MMP-2,3 and 12,uPA/Urokinase)and 10 PIs(Protease Nexin II,Cystatin B and C,EMMPRIN/CD147,Latexin,Lipocalin-1,Serpin E1,TFPI,TFPI-2,TIMP-1,2 and 4).Healthy and FECD specimens showed similar variation patterns according to morphology for secretion of ADAMTS1,MMP-3 and 12.However,opposing patterns between healthy and FECD populations were observed for Cathepsin B and D.Moreover,some proteins did not show variation according to phenotype in healthy CECs,but did in FECD CECs:Cathepsin A,Cystatin C,TFPI-2 and total TIMPs.For the other proteins,secretion did not vary according to morphology or no specific pattern was distinguishable.Conclusions:To conclude,our results suggest that cell phenotype is linked to the secretion of certain proteases/PIs in both groups.However,there seems to be differences in secretion of particular proteases and PIs between FECD and healthy specimens as morphology did not have a similar influence.These differences might initiate an imbalance between proteases and PIs explaining the irregular thickening of the Descemet membrane seen in FECD.展开更多
Aims: To describe the progression of Primary open angle glaucoma (POAG) on Optical Coherence Tomography (OCT) of the optic nerve head and retinal nerve fiber layers (RNFL). Method: We conducted a descriptive retrospec...Aims: To describe the progression of Primary open angle glaucoma (POAG) on Optical Coherence Tomography (OCT) of the optic nerve head and retinal nerve fiber layers (RNFL). Method: We conducted a descriptive retrospective study from January 1, 2015 to December 31, 2019, a period of 5 years from the files of patients followed for POAG and having carried out at least two OCT examinations of the optic nerve head (ONH), one automated visual field and Intraocular pression (IOP). The variables studied were: age, sex, mean IOP, glaucoma stage, progression of ONH parameters, and progression of RNFL parameters. Results: During the period, 112 eyes of 56 patients were included. The mean age was 48.96 ± 16.57 [12 - 83] years with a sex-ratio of 1.33 (32 M/27 F). The mean IOP was 21 ± 4.54 [10 - 36] mm Hg. According to the mean deviation (MD) of the visual field, 98 eyes or 87.5% were stage 1 of POAG, 10 eyes or 8.9% at stage 2 and 4 eyes or 3.6% at stage 3. The mean time between the 1st and 2nd OCT examination was 28.91 ± 11.07 [6 - 56] months, corresponding to an average of 2.18 OCT per patient in 5 years of follow-up. There was an average increase of 6.2% of the Cup area and an increase in the vertical Cup/Disc ratio of 1.79% per year. The thinning average of neuro-retinal ring area was 1.64% per year. The RNFL thickness had decreased on average by 4.28 μ or 0.93% per year. The lower quadrant had the highest fiber loss with 1.08% per year followed by the upper quadrant with a loss of 1.05% per year. Conclusion: OCT of the ONH and RNFL proves to be an essential tool in the follow-up of POAG. A subsequent study taking into account the OCT of the macular ganglion complex will enable to study its contribution in the follow-up of glaucomatous patients in the same population.展开更多
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...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.展开更多
Background:Uveal melanoma(UM)is the most common primary eye tumor in adults,and the most frequent site of malignant transformation of the melanocytes after the skin.It spreads to the liver in half of the cases,and the...Background:Uveal melanoma(UM)is the most common primary eye tumor in adults,and the most frequent site of malignant transformation of the melanocytes after the skin.It spreads to the liver in half of the cases,and the death rate following the report of metastasis is 92%at 2 years.Hepatic tropism of UM cannot simply be explained by the blood circulatory system organization,and illustrates the“seed and soil”hypothesis that describes an interaction between tumor cells(seed)and a specific microenvironment(soil).We decided to focus our study on the synergic interaction between UM cells and hepatic stellate cells(HSC),whose role has been previously described in the metastatic progression of colon and pancreatic cancers.Furthermore,HSC have been found surrounding UM liver metastasis,and the UM secretome contains activating cytokines of hepatic stromal cells.Our hypothesis is that HSC provide a specific microenvironment in the liver enhancing the growth of UM cells and increasing their therapeutic resistance.Using an in vitro 3D model and an original xenograft mouse model,we aim to decipher the mechanisms of UM metastatic progression,in order to elaborate new therapeutic strategies.Methods:First,using an agar coating,spheroids were generated with UM cells and were allowed to grow for 72 h.These tumor spheroids were then embedded in Matrigel and the HSC conditioned medium was used to evaluate the impact of the HSC secretome on UM invasion.Next,an original in vivo xenograft mouse model was generated,in which metastatic UM cells were injected alone or with human HSC in the spleen of immunodeficient mice.This model allows us to evaluate in 3-6 weeks the metastatic potential of each cell population,and thus to determine the cooperation between HSC and UM cells in the liver.Results:The HSC conditioned medium increased the invasion of UM spheroids compared to non-conditioned medium in our in vitro model.In addition,UM cells inoculated in the mouse spleen alone or with human HSC were able to metastasize to the liver,and the host HSC were also recruited by UM metastases.Conclusions:Our preliminary results strongly suggest that the secretome of HSC provides a permissive microenvironment for UM metastatic progression.We now have to confirm these results by characterizing the secretome of HSC,in order to identify cytokines or growth factors that increase the invasion of the liver by UM.Our models can be used to test the efficacy of new therapeutic strategies targeting the UM microenvironment.展开更多
Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression ...Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression by non-hematopoietic cells is scarce. In the present study, gene and protein expression of this integrin subunit was characterized in proliferating and quiescent human RPE cells. Immunofluorescent studies confirm that the α4 subunit is expressed in vitro by RPE cells, a result that has been validated by immunofluorescence and FACS analyses. The accumulation of the α4 integrin at cell-cell junctions in post-confluent RPE cell cultures negatively correlated with the level of expression of the mRNA transcript. Accordingly, transient transfection analyses reveal that the α4 promoter activity is considerably reduced when RPE cells form a confluent monolayer. Moreover, transfection of recombinant constructs bearing 5’-deletions of the α4 promoter segment allows the localization of strong negative regulatory elements on the -76 to -300 region of the α4 gene suggesting that its expression is intimately linked to the proliferative state of primary cultured RPE cells.展开更多
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 obtaini...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.展开更多
文摘Background:Primary open-angle glaucoma(POAG)is a genetically complex disorder caused primarily by gene-gene interactions.To identify these interactions,we studied the CA family,a large French-Canadian pedigree in which the myocilin K423E mutation(MYOCK423E)causes autosomal dominant glaucoma with diagnoses ranging from juvenile-onset OAG(JOAG)to late adult-onset POAG in the heterozygotes(HTZ).To explain this extreme variability,we hypothesized that a second gene,called a modifier,was interacting with MYOC,the primary disease gene.Our goals were(I)to map the modifier on the human genome and;(II)to characterize the symptoms affected genetically by the modifier.These symptoms are called endophenotypes.Methods:Three hundred seventy-five CA members were studied using four quantitative endophenotypes:age of maximal intra-ocular pressures(IOPmax),IOPs progression,progression of cup to disk ratios and age-at-onset(AAO)defined as age at which ocular hypertension(OHT)was first detected with IOP≥22 mmHg.Genome-wide linkage analysis was performed by genotyping 408 genetic markers in 184 CA members.An unbiased pedigree-based algorithm was designed to identify the individuals who were double-mutants,i.e.,these individuals carried one MYOCK423E mutation(i.e.,they were HTZ,affected or not)and they also carry simultaneously a DNA mutation within the modifier.Results:Out of the 375 CA family members investigated,156 were HTZ for the MYOCK423E mutation.120 HTZ were affected with OAG or OHT with treatment while the remaining 36 HTZ were asymptomatic.AAO ranged from 7 to 63 years old;4 individuals over 50 years old were still asymptomatic.OHT preceded optic nerve damage in>98%of the HTZ carriers,confirming that AAO reflected the true severity of the disorder.The modifier showed strong inherited effects on 2 of the 4 endophenotypes:AAO and IOPmax.We next mapped with very high confidence the modifier locus for AAO at chromosome 20q13.Saturation genotyping with additional markers refined the locus to a 9 to 10 centimorgan interval,or about 10 million DNA nucleotides,between D20S857 and D20S832.The locus was named modifier of glaucoma 1(MOG1).When comparing the AAOs of the double mutants versus the median of the AAOs of the MYOCK423E HTZ who carried a wild-type(normal)MOG1 gene and were 1st cousins or closer with the double mutant under investigation,we observed that MOG1 delayed the ages at onset by an average of 8 to 10 years in the double mutants.Conclusions:The MOG1 locus encodes a DNA element that delays the onset of glaucoma by an average of 8-10 years by hampering the first manifestations of OHT.This research will lead to the development of new therapeutic targets for glaucoma.These treatments should prevent optic nerve damage by maintaining IOPs within the normal range.
文摘Background: There are few studies on the cost of glaucoma management in developing country, especially in Togo, there are no data on the cost of POAG management. Aims: To determine the annual direct cost of the management of POAG, to evaluate the annual economic weight of the management of POAG and to determine the factors associated with the annual economic weight of the management. Methods: We conducted a retrospective and descriptive study over a period of 12 months from January 1 to December 31, 2019 based on the records of patients followed for POAG in AFIA Eye Clinic in Lomé-Togo. The annual direct cost was defined by the sum of the costs of consultations, explorations and treatments. We defined the direct cost per patient and per year and related to the average annual income. It was said to be catastrophic at 20% or more of the estimated annual income. Chi 2 and Fisher tested the comparison of proportions. We conducted univariate and multivariate logistic regression to search correlations. Results: During the study period, 150 patient records were included. The average age was 47.24 ± 17.09 years and the sex ratio was 0.82. The cost of the diagnosis was 112.18 ± 22.26 €. The average cost of consultations was 19.46 ± 11.35 € and that of explorations was 92.71 ± 10.91 €. The annual cost of treatment per patient was 165.52 ± 110.16 €. The annual global direct cost of POAG management per patient was 277.69 ± 132.42 €. Compared to the annual income of 1166.29 €, the economic weight of the glaucoma management was 23.8%. This direct cost was catastrophic for 32.1% of patients in the study (44/150 of people with no care). Compared to the guaranteed inter-professional minimum wage (SMIG) of 640.30 €, the economic direct cost weight was 43.3%. Risk factors significantly associated with the direct cost were age over 40 (OR = 1.05 and p = 0.032), liberal profession (OR = 4.72 and p = 0.04), the absence of health insurance (OR = 6.68 and p = 0.017) and the use carbonic anhydrase inhibitors (OR = 7.4 and p = 0.012) and prostaglandin analogues (OR of 38.2 and p Conclusion: This first study on the direct cost of POAG management in Lomé showed that the economic burden glaucoma represents for the patient, his family and society. The data from this study will allow health decision-makers to adopt strategies to mitigate the effects of glaucoma on the economy.
文摘Background:Cell transfection has the potential to transform gene therapy,potentially enabling the treatment of genetic diseases.We are developing a cell transfection approach that uses laser pulses and gold nanoparticles to induce transient holes in the cell membrane,thus allowing external material to pass into the cell before the membrane is healed.Our previous research has demonstrated the feasibility of perforating the cell membrane in vitro by irradiating(λ=532 nm,τ=6 ns)cancer cells previously incubated with gold-nanoparticles(100 nm).Our ultimate goal is to develop an optofluidic probe(needle-like)capable of performing simultaneous and precise delivery of a perforation mixture(nanoparticles and perforation indication dye)and laser pulses in vivo.We will present the initial validation of the technology with in vitro perforation of cancer cells.Methods:MDA-MB-231 cells were cultured in DMEM+GlutaMax(9%FBS,1%P/S).The medium was changed to Leibovitz’s L-15 for experimentation.100 nm gold nanoparticles and a perforation indicator dye(calcein,green dye)were placed on cells by pumping them through the optofluidic tip.The optofluidic tip was coupled to a nanosecond laser(λ=532 nm,τ=6 ns).Cells were irradiated with 5 laser pulses(Energy:15-38μJ)through the optrode tip at a distance of 50μm.Successful cell uptake was indicated by calcein uptake.Cells were incubated in 0.5%CO_(2) for one hour following irradiation.Then,propidium iodide(PI,red dye)was added to investigate cell death.The quantification of the perforation efficiency and viability was performed with fluorescence microscopy.Results:We have been able to successfully inject cells by pumping the perforation mixture through an optofluidic tip.The early quantitative results indicate that injection efficiencies are near 35%at the optimal fluence with cell viability near 90%.These efficiencies are similar to our previous results involving pre-incubation of cells with the perforation mixture.Conclusions:Our results show the feasibility of cell transfection using a particularly designed optofluidic probe(needle-like)for light and liquid delivery.The method does not involve pre-incubation of cells with the preformation mixture and thus brings the technology closer to in vivo applications.
文摘Background:Our previous studies revealed that Nogo-A gene ablation improved visual function recovery after retinal injury.Moreover,Nogo-A expression is highly expressed in the healthy retina.Its physiological role in retinal function is not known.The purpose of this current study was to determine the effects of acute Nogo-A silencing on retinal neuron structure and function in physiological conditions.Methods:Nogo-A silencing was done by intravitreal injection of adeno-associated virus serotype 2.2 containing a short hairpin RNA sequence(AAV2.2 shRNA-Nogo-A)and a GFP reporter gene in adult C57BL/6J mice.As control,an empty AAV2.2 vector was used.Infection of retinal cells was followed by fluorescent fundoscopy.Changes in Nogo-A expression were analysed by Western blotting in whole retinal lysates.Electroretinography was used to monitor retinal activity.The assessment of optokinetic reflex(OKR)allowed to follow visual acuity in unrestrained mice.Immunofluorescence on histological sections using the following cell markers,i.e.,RNA-binding protein with multiple splicing(RBPMS)and sex-determining region Y-box 2(Sox-2)allowed to visualize retinal ganglion cells(RGCs)and Müller glia respectively.Results:GFP fluorescence revealed efficient AAV2.2 transfection in the ganglion cell layer and the inner nuclear layer 30 days after viral injection.By Western blotting,Nogo-A expression was decreased by~75%in AAV2.2-shRNA-Nogo-A-treated retinae(n=3)as compared to the control mice(n=3).Strikingly,AAV2.2-shRNA-Nogo-A-injected animals(n=10)had a visual acuity reduction of 43.7%as compared to control(n=7),60 days after transfection.Electroretinography(ERG)b-wave and a-wave amplitudes were also decreased by~35%and 24.4%respectively relative to controls.After two months of transfection,RBPMS-positive RGCs were reduced by~30%in AAV2.2-shRNA-Nogo-A(n=4)compared to non-injected contralateral eyes(n=4).The number of Sox2-expressing Müller cells was not affected after Nogo-A knockdown.Conclusions:Nogo-A gene silencing in the retina has deleterious effects on the mouse retinal structure and function,suggesting an important role for Nogo-A in retinal physiology.
文摘Background:Cells are influenced by their environment.In vivo,the corneal endothelium is subjected to intraocular pressure(IOP).The purpose of this project was to evaluate in vitro,the effect of the IOP on the formation of tight junctions in the corneal endothelium.Methods:Cultivated corneal endothelial cells(P2-P3;n=6 populations)were seeded on devitalized on corneas(n=10 pairs).Native corneas and devitalized corneas were respectively used as positive(n=2 pairs)and negative controls(n=3 pairs).Corneas were placed in artificial anterior chambers and subjected to a hydrostatic pressure between 0.3 and 0.4 psi during 4-5 days.Unpressured control corneas were maintained in cell culture dishes.Pictures of the corneas were taken following the experiment to assess stromal transparency.Morphology,corneal thickness and distribution of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporter were evaluated by electron microscopy,histological staining and immunofluorescences.Results:Pressure treated corneas were more transparent than the controls.Thickness was accordingly reduced by 38.4%±4.9%for cultivated endothelium and 32.2%±2.7%for native endothelium.Negative controls change in transparency and thickness were marginal.Pressure treated cells showed none or at most marginal difference in morphology and expression of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporters and failed to recreate a phenotype similar to native corneas.Pressure however increased cortical localisation of the protein ZO-1 in both cultivated and native endothelium.Conclusions:These results suggest that anterior chamber hydrostatic pressure may enhance endothelial functionality by modulating the distribution of tight junction’s proteins.
文摘Background:Age-related macular degeneration(AMD)is the second Canadian cause in visual deficiency.AMD is characterized by the death of photoreceptors and retinal pigmented epithelium(RPE)in the macular region of the retina,leading to the loss of central vision.Epidemiologic studies suggest an association between lifetime sun exposure and the probability to develop AMD even though mechanisms are unknown.Sunlight is made of about 30%of high-energy visible(HEV)light(blue light),the most energetic wavelength reaching the retina.These wavelengths can be absorbed by lipofuscin,an age pigment accumulating in RPE cells.Lipofuscin principal component is N-retinylidene-N-retinylethanolamine(A2E).Many research teams showed that absorption of HEV light by A2E in RPE cells at non-physiological doses produces free radicals and leads to cell death.Our earlier work shows that when A2E-loaded RPE cells are irradiated with HEV light at physiological doses,the same light does not lead to oxidative stress as measured by telomere and mitochondrial integrity.Our hypothesis is that HEV light,at physiological doses,modify or convert A2E in derived produces,inhibiting its photo-oxidant effect.Methods:In vitro,we irradiated A2E with HEV light with or without antioxidants and with varying irradiation regimen to observe the UV-Visible spectrum of A2E.In cellulo,we loaded ARPE-19 cells with A2E and irradiated cells at physiological levels for 4 consecutive days.We then observed A2E fluorescence using a fluorescence microscope with nucleus counterstaining with DRAQ5.Results:HEV light leads to the disappearance of A2E characteristic UV-Visible spectrum and the apparition of a new product suggesting that HEV light modifies A2E.Nor oxidation and irradiation regimen seem to have an impact in A2E’s conversion by HEV light.We observed and progressive diminution of A2E fluorescence in cellulo during physiological irradiations.Conclusions:The loss of A2E photo-oxidation capacities by HEV light seems to be caused by its conversion by HEV light.We suggest that HEV light,at physiological doses,may be protective rather than photo-toxic.The next step would be to identify A2E’derived produces and their cell toxicity.
文摘Background:Lesion to the retinal pigment epithelium(RPE)is a crucial event in age-related macular degeneration(AMD)development.Although the pathogenesis of this complex disease is poorly understood,sunlight exposure and smoking are major environmental risk factors associated with AMD.High-energy visible blue light(HEV;400-500 nm)is the most energetic and potentially harmful solar wavelengths reaching adults retina.On the other hand,RPE cells can be exposed to a large range of pollutants from cigarette smoke,with polycyclic aromatic hydrocarbons(PAH)being among the most toxic.Some PAH from cigarette smoke can absorb HEV light.This led us hypothesize that in RPE cells,the combination of PAH and HEV could synergize to exacerbate the stress caused by either factor alone.We thus investigate the combined effect of PAH and HEV light in RPE cells.Methods:Confluent RPE immortalized cells(ARPE19)were exposed to nanomolar concentrations of benzo[a]pyrene(BaP)or indeno[1,2,3-cd]pyrene(IcdP).While IcdP efficiently absorbs HEV wavelengths,BaP,the most studied PAH,does not significantly absorb HEV light and was used as a control.BaP or IcdP contaminated ARPE19 were then irradiated with increasing sub-lethal doses of HEV light(150-500 J/cm2)using a setup that mimics the light spectrum normally reaching the retina.Cytotoxicity,apoptosis and reactive oxygen species(ROS)generation were assessed in each condition.Results:In presence of low concentrations of IcdP,sub-lethal amounts of HEV light trigger,in a dose-dependent way,up to 70%of apoptotic cell death.Co-exposure to IcdP and HEV also leads to a synergistic ROS generation in ARPE19 cells,thus inducing oxidative stress.None of these effects were observed with BaP.Efficient inhibition of ROS production by specific antioxidants only decreases death by 20%in cells simultaneously exposed to both IcdP and HEV light.Conclusions:Low concentrations of IcdP synergize with HEV light to induce phototoxicity in ARPE19 cells.An increased oxidative stress results from the interaction between both agents and partially explains the enhanced HEV phototoxicity in IcdP contaminated ARPE19 cells.This suggests that another major mechanism is involved in the synergetic toxicity.For smokers,this synergy between HEV and PAH may accelerate RPE cells loss and contribute to their greater risk of developing AMD.
文摘Background:The goal of this project was to analyze the relationship between cell morphology and proteases/proteases inhibitors(PIs)secretion profile in fuchs endothelial corneal dystrophy(FECD)corneal endothelial cells(CECs).Methods:Cell morphology was determined using a circularity index(4π×area/perimeter2)for each CECs population extracted from surgical FECD specimens(N=2)and healthy Eye bank corneas(N=3).CECs were cultured 28 days post-confluency.Supernatant was collected and analysed using Proteome Profiler Array detecting 35 proteases and 32 PIs(R&D Systems).Proteome signal was analyzed using Image Studio Lite and correlated with the population’s circularity index.Results:Calculation of circularity index reported different morphologies among FECD populations(0.59±0.18 and 0.64±0.17)and healthy populations(0.44±0.18,0.66±0.13 and 0.71±0.11).Proteome arrays revealed the presence of 10 proteases(ADAMTS1,Cathepsin A,B,D,and X/Z/P,DPPIV/CD26,MMP-2,3 and 12,uPA/Urokinase)and 10 PIs(Protease Nexin II,Cystatin B and C,EMMPRIN/CD147,Latexin,Lipocalin-1,Serpin E1,TFPI,TFPI-2,TIMP-1,2 and 4).Healthy and FECD specimens showed similar variation patterns according to morphology for secretion of ADAMTS1,MMP-3 and 12.However,opposing patterns between healthy and FECD populations were observed for Cathepsin B and D.Moreover,some proteins did not show variation according to phenotype in healthy CECs,but did in FECD CECs:Cathepsin A,Cystatin C,TFPI-2 and total TIMPs.For the other proteins,secretion did not vary according to morphology or no specific pattern was distinguishable.Conclusions:To conclude,our results suggest that cell phenotype is linked to the secretion of certain proteases/PIs in both groups.However,there seems to be differences in secretion of particular proteases and PIs between FECD and healthy specimens as morphology did not have a similar influence.These differences might initiate an imbalance between proteases and PIs explaining the irregular thickening of the Descemet membrane seen in FECD.
文摘Aims: To describe the progression of Primary open angle glaucoma (POAG) on Optical Coherence Tomography (OCT) of the optic nerve head and retinal nerve fiber layers (RNFL). Method: We conducted a descriptive retrospective study from January 1, 2015 to December 31, 2019, a period of 5 years from the files of patients followed for POAG and having carried out at least two OCT examinations of the optic nerve head (ONH), one automated visual field and Intraocular pression (IOP). The variables studied were: age, sex, mean IOP, glaucoma stage, progression of ONH parameters, and progression of RNFL parameters. Results: During the period, 112 eyes of 56 patients were included. The mean age was 48.96 ± 16.57 [12 - 83] years with a sex-ratio of 1.33 (32 M/27 F). The mean IOP was 21 ± 4.54 [10 - 36] mm Hg. According to the mean deviation (MD) of the visual field, 98 eyes or 87.5% were stage 1 of POAG, 10 eyes or 8.9% at stage 2 and 4 eyes or 3.6% at stage 3. The mean time between the 1st and 2nd OCT examination was 28.91 ± 11.07 [6 - 56] months, corresponding to an average of 2.18 OCT per patient in 5 years of follow-up. There was an average increase of 6.2% of the Cup area and an increase in the vertical Cup/Disc ratio of 1.79% per year. The thinning average of neuro-retinal ring area was 1.64% per year. The RNFL thickness had decreased on average by 4.28 μ or 0.93% per year. The lower quadrant had the highest fiber loss with 1.08% per year followed by the upper quadrant with a loss of 1.05% per year. Conclusion: OCT of the ONH and RNFL proves to be an essential tool in the follow-up of POAG. A subsequent study taking into account the OCT of the macular ganglion complex will enable to study its contribution in the follow-up of glaucomatous patients in the same population.
文摘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.
文摘Background:Uveal melanoma(UM)is the most common primary eye tumor in adults,and the most frequent site of malignant transformation of the melanocytes after the skin.It spreads to the liver in half of the cases,and the death rate following the report of metastasis is 92%at 2 years.Hepatic tropism of UM cannot simply be explained by the blood circulatory system organization,and illustrates the“seed and soil”hypothesis that describes an interaction between tumor cells(seed)and a specific microenvironment(soil).We decided to focus our study on the synergic interaction between UM cells and hepatic stellate cells(HSC),whose role has been previously described in the metastatic progression of colon and pancreatic cancers.Furthermore,HSC have been found surrounding UM liver metastasis,and the UM secretome contains activating cytokines of hepatic stromal cells.Our hypothesis is that HSC provide a specific microenvironment in the liver enhancing the growth of UM cells and increasing their therapeutic resistance.Using an in vitro 3D model and an original xenograft mouse model,we aim to decipher the mechanisms of UM metastatic progression,in order to elaborate new therapeutic strategies.Methods:First,using an agar coating,spheroids were generated with UM cells and were allowed to grow for 72 h.These tumor spheroids were then embedded in Matrigel and the HSC conditioned medium was used to evaluate the impact of the HSC secretome on UM invasion.Next,an original in vivo xenograft mouse model was generated,in which metastatic UM cells were injected alone or with human HSC in the spleen of immunodeficient mice.This model allows us to evaluate in 3-6 weeks the metastatic potential of each cell population,and thus to determine the cooperation between HSC and UM cells in the liver.Results:The HSC conditioned medium increased the invasion of UM spheroids compared to non-conditioned medium in our in vitro model.In addition,UM cells inoculated in the mouse spleen alone or with human HSC were able to metastasize to the liver,and the host HSC were also recruited by UM metastases.Conclusions:Our preliminary results strongly suggest that the secretome of HSC provides a permissive microenvironment for UM metastatic progression.We now have to confirm these results by characterizing the secretome of HSC,in order to identify cytokines or growth factors that increase the invasion of the liver by UM.Our models can be used to test the efficacy of new therapeutic strategies targeting the UM microenvironment.
文摘Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression by non-hematopoietic cells is scarce. In the present study, gene and protein expression of this integrin subunit was characterized in proliferating and quiescent human RPE cells. Immunofluorescent studies confirm that the α4 subunit is expressed in vitro by RPE cells, a result that has been validated by immunofluorescence and FACS analyses. The accumulation of the α4 integrin at cell-cell junctions in post-confluent RPE cell cultures negatively correlated with the level of expression of the mRNA transcript. Accordingly, transient transfection analyses reveal that the α4 promoter activity is considerably reduced when RPE cells form a confluent monolayer. Moreover, transfection of recombinant constructs bearing 5’-deletions of the α4 promoter segment allows the localization of strong negative regulatory elements on the -76 to -300 region of the α4 gene suggesting that its expression is intimately linked to the proliferative state of primary cultured RPE cells.
文摘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.
