Therapeutics for the chorioretina

脉络膜视网膜的治疗

• 批准号: 10706108
• 负责人: SOFIA P BECERRA
• 金额: $ 52.51万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10706108
• 关键词: Adult; Affect; Affinity; Age; Anatomy; Angiography; Animal Model; Animals; Apoptosis; Apoptotic; BCL2 gene; BODIPY; Binding; Biological Assay; CRISPR/Cas technology; Cations; Cell Death; Chest; Cholesterol; Choroid; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Color; Confocal Microscopy; DNA Nucleotidylexotransferase; Deformity; Detection; Disease; Electroretinography; Evaluation; Exhibits; Eye; Eyedrops; Fatty Acids; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Fundus; Genes; Glycosaminoglycans; Goals; Heart; Histology; Image; Immunofluorescence Immunologic; Immunofluorescence Microscopy; Knock-out; Knockout Mice; Label; Lipids; Mass Spectrum Analysis; Membrane; Methods; Microscope; Modeling; Modification; Molecular Profiling; Mouse Strains; Mus; Necrosis; Nonesterified Fatty Acids; Optical Coherence Tomography; Organ; Peptides; Phosphatidylserines; Phosphodiesterase Inhibitors; Phospholipase; Phospholipase A2; Phospholipids; Photoreceptors; Plasma; Positioning Attribute; Process; Property; Proteins; Proteomics; Recombinants; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Role; Serpins; Stains; Technology; Texas red; Therapeutic; Time; Tissues; Transferase; Transmission Electron Microscopy; Triglycerides; Vision; Visualization; Zinc; attenuation; base; extracellular; in vivo; in vivo fluorescence imaging; mimicry; neurotrophic factor; pigment epithelium-derived factor; prevent; receptor; screening; zaprinast

To develop a method to evaluate photoreceptor cell death in mice, we continued to optimize non-invasive in vivo fluorescence imaging of dying photoreceptor cells using the rd10 mouse, which is an animal model of retinal degeneration. Phosphatidylserine (PS) externalization, an early molecular signature for cell death by apoptosis and necrosis, was followed transiently with the phosphatidylserine-binding conjugate of Bis(zinc(II)-dipicolylamine (Zn-DPA) with Texas-red (PSVue-550) in the fundus of mice by fluorescence fundoscopy. To determine a time point for the maximum PS externalization in the fundus, the fluorescent probe was administered daily as eyedrops to rd10 and rd10 x Serpinf1-/- mice of 15-25 days of age followed by evaluation of fluorescence in their fundi. To determine the effects of neurotrophic factors on photoreceptor survival, eyedrops of PEDF peptide 17-merH105A were administered daily to rd10 and rd10 x Serpinf1-/- mice at 15-24 days of age. At end point, the probe was administered as eyedrops to evaluate fluorescence in their fundi. Electroretinography (ERG) was performed to evaluate visual function of the treated animals. Enucleated whole eyes were processed for histology. Detection of anti-apoptotic B-cell lymphoma 2 (Bcl2) and pro-apoptotic BCL2 associated X (BAX) markers was performed by immunofluorescence and confocal microscopy. We continued optimizing retinal explant cultures prepared from adult wild type C57BL/6J mice to screen retino-protective PEDF peptides. Zaprinast, an inhibitor of phosphodiesterases, was included in the medium of the explant cultures to obtain a mimicry of retinal cell death ex vivo as in the retinal degeneration rd10 animal model. Photoreceptor cell death was assessed in whole retinal mounts by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and PSVue 550 staining followed by fluorescence and confocal microscopy visualization. We used the zaprinast-retinal explant model for screening a set of recombinant PEDF protein versions with modifications of selected neutral or anionic residues into cationic residues in positions that affect glycosaminoglycan affinity (collaboration with H Chu). The modified PEDF versions were assayed for retinal survival activity ex vivo and had comparable cytoprotective properties as the unmodified PEDF in the assay. To investigate the role of PEDF-R in photoreceptors, we continued to characterize mouse strains in which the gene was constitutively knocked out using CRISPR-CAS-9 technology (in collaboration with L Dong). CRISPR Pnpla2-/- knock-out mouse lines were generated. Pnpla2 expression was followed by RT-PCR in dissected mouse retinas. PEDF-R protein was detected by immunofluorescence of retina tissue sections. Phospholipase A2 activity was determined in extracts from dissected mouse retina. Three- and seven-month-old mice and their littermate controls of two lines were evaluated. Organs of the thorax were dissected and imaged using a LEICA microscope for anatomical evaluation revealing that mice from the two Pnpla2-/- mouse lines had hearts that were enlarged and whiter color than its control littermates. Plasma was collected for the determination of free fatty acids, triglycerides, cholesterol levels. Plasma free fatty acids and triglycerides of Pnpla2-/- mice were lower and cholesterol was higher than of littermate Pnpla2+/+ controls. Fundoscopy was performed on the Pnpla2-/- and Pnpla2+/- mice of both lines. Electroretinography (ERG) showed attenuation in the a-wave in the Pnpla2-/- and Pnpla2+/- mice for both lines relative to littermate controls Pnpla2+/+. Optical coherence tomography angiography (OCT-A) was also performed. Enucleated whole eyes were processed for histology, immunofluorescence, confocal microscopy, and transmission electron microscopy. The retinas had abnormalities with deformities in the photoreceptors. Protein extracts from dissected retinas and RPE/choroid of Pnpla2+/+, Pnpla2+/- and Pnpla2-/- mice prepared for mass spectrometry for proteomics and protein profiles were analyzed in collaboration with L Jenkins. Retinal flat mounts were prepared and stained for lipids using with BODIPY. Retinas and RPE/choroid from enucleated eyes of Pnpla2+/+, Pnpla2+/- and Pnpla2-/- mice were dissected, and extracts were prepared for analyses of phospholipid and fatty acid composition by mass spectrometry (in collaboration with MP Agbaga). We administered eyedrops of the fluorescent probe PSVue to identify cell death in the retina of the Pnpla2-KO mice in vivo. The fluorescent stain was evaluated by fluorescence fundoscopy. Dissected retinas from the mice were sectioned and cell death was evaluated by TUNEL using fluorescence microscopy. We cross-bred of Serpinf1-/- mice with selected Pnpla2-KO mice to generate double Serpinf1-/- x Pnpla2-KO mice. The organs of the thorax of the double KO mice were dissected and imaged using a LEICA microscope for anatomical evaluation generated. The retinas were evaluated by fundoscopy, ERG, and OCT-A. Enucleated whole eyes of the double KO mice were processed for and evaluated by histology, and transmission electron microscopy.

为了开发一种评估小鼠感光细胞死亡的方法，我们继续使用RD10小鼠（是视网膜变性的动物模型）优化垂死感光细胞的无创体内荧光成像。磷脂酰丝氨酸（PS）外部化是凋亡和坏死细胞死亡的早期分子特征，随后与BIS（锌（II） - 二氨基氨基胺（Zn-DPA）的磷脂酰丝氨酸结合结合物与Texas-Red（psvue-550）中的流动性产生的磷酸化（II） - 二氨基氨基胺（ZN-DPA）的菌丝的最高含量（ZIN） - 二氨基氨基胺（Zn-DPA）的含量为单位的泡沫中的含量（ZIN） - 在眼底的外部化，每天将荧光探针作为15-25天的RD10和RD10 X SERPINF1 - / - 小鼠，然后评估其基金中的荧光，以确定神经营养性因素对光疗中的pedfore and dealeded and decter to pedfipe 17 merhs105a的影响。 SERPINF1 - / - 在终点时，将探针作为荧光量在其基金中评估荧光，以评估治疗动物的视觉功能。 （BAX）通过免疫荧光和共聚焦显微镜进行标记。 我们继续优化从成年野生型C57BL/6J小鼠制备的视网膜外植体培养物，以筛选视网膜保护性PEDF肽。 Zaprinast是一种磷酸二酯酶的抑制剂，被包括在外植体培养基的介质中，以获得视网膜变性RD10动物模型中的视网膜细胞死亡的模仿。通过末端脱氧核苷酸转移酶Dutp nick End标记（TUNEL）和PSVUE 550染色，然后在整个视网膜架上评估感光细胞死亡，然后进行荧光和共聚焦显微镜可视化。我们使用Zaprinast-Retinal Epplant模型来筛选一组重组PEDF蛋白版本，并将选定中性或阴离子残基修改为阳离子残基，以影响糖胺聚糖亲和力（与H CHU协作）的位置。测定了修饰的PEDF版本的视网膜存活活性，并具有可比的细胞保护特性作为分析中未修饰的PEDF。 为了研究PEDF-R在光感受器中的作用，我们继续表征小鼠菌株，其中使用CRISPR-CAS-9技术（与L Dong合作）将基因被组成式地淘汰。生成CRISPR PNPLA2 - / - 敲除鼠标线。 PNPLA2表达在解剖的小鼠视网膜中为RT-PCR。通过视网膜组织切片的免疫荧光检测PEDF-R蛋白。在解剖小鼠视网膜提取物中确定磷脂酶A2活性。评估了三个月和七个月大的小鼠及其两条线的同立静脉对照。使用Leica显微镜进行解剖学评估，解剖胸腔器官并成像，这表明来自两种PNPLA2 - / - 小鼠系的小鼠具有比其对照同窝仔相比的心脏，其心脏被增大且鲜艳。收集血浆以测定游离脂肪酸，甘油三酸酯，胆固醇水平。 PNPLA2 - / - 小鼠的血浆无血浆脂肪酸和甘油三酸酯较低，胆固醇高于同窝型PNPLA2+/+对照。在两条线的PNPLA2 - / - 和PNPLA2 +/-小鼠上进行了基础镜检查。电子模拟图（ERG）在PNPLA2 - / - 和PNPLA2 +/-小鼠的A波中显示衰减，相对于同窝脚同窝控制PNPLA2+/+。还进行了光学相干层析成像血管造影（OCT-A）。将全眼的全眼处理以进行组织学，免疫荧光，共聚焦显微镜和透射电子显微镜。视网膜具有光感受器中畸形的异常。与L Jenkins合作分析了PNPLA2+/+的RPE/RPE/脉络膜，PNPLA2+/+，PNPLA2 +/-和PNPLA2 - / - 小鼠，用于用于蛋白质组学和蛋白质谱的质谱法。制备视网膜平坦的坐骨，并使用Bodipy染色脂质。解剖PNPLA2+/+，PNPLA2 +/-和PNPLA2 - / - 小鼠的视网膜和RPE/脉络膜，并通过质谱法（与MP Agbaga合作）准备了提取物，并准备提取提取物以分析磷脂和脂肪酸组成的分析。我们管理了荧光探针PSVUE的眼药水，以鉴定体内PNPLA2-KO小鼠视网膜中的细胞死亡。通过荧光基础镜检查评估荧光染色。切除了来自小鼠的解剖视网膜，并使用荧光显微镜通过TUNEL评估细胞死亡。我们将Serpinf1 - / - 小鼠与选定的PNPLA2-KO小鼠交叉，生成双serpinf1 - / - x pnpla2-ko小鼠。解剖双KO小鼠的胸腔器官，并使用Leica显微镜进行解剖学评估。通过基础镜，ERG和OCT-A评估视网膜。通过组织学和透射电子显微镜处理并评估双KO小鼠的全眼。