Pérez de Lara María J, Guzmán-Aránguez Ana, de la Villa Pedro, Díaz-Hernández Juan Ignacio, Miras-Portugal María Teresa, Pintor Jesús
Departamento de Bioquímica y Biología Molecular IV, Facultad Óptica, Universidad Complutense de Madrid, Madrid, Spain.
Departmento de Biología de Sistemas, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, Spain.
Mol Vis. 2015 Sep 2;21:1060-70. eCollection 2015.
To study retinal extracellular ATP levels and to assess the changes in the vesicular nucleotide transporter (VNUT) expression in a murine model of glaucoma during the development of the disease.
Retinas were obtained from glaucomatous DBA/2J mice at 3, 9, 15, and 22 months together with C57BL/6J mice used as age-matched controls. To study retinal nucleotide release, the retinas were dissected and prepared as flattened whole mounts and stimulated in Ringer buffer with or without 59 mM KCl. To investigate VNUT expression, sections of the mouse retinas were evaluated with immunohistochemistry and western blot analysis using newly developed antibodies against VNUT. All images were examined and photographed under confocal microscopy. Electroretinogram (ERG) recordings were performed on the C57BL/6J and DBA/2J mice to analyze the changes in the electrophysiological response; a decrease in the scotopic threshold response was observed in the 15-month-old DBA/2J mice.
In the 15-month-old control and glaucomatous mice, electrophysiological changes of 42% were observed. In addition, 50% increases in the intraocular pressure (IOP) were observed when the pathology was fully established. The responses in the retinal ATP net release as the pathology progressed varied from 0.32±0.04 pmol/retina (3 months) to 1.10±0.06 pmol/retina (15 months; threefold increase). Concomitantly, VNUT expression was significantly increased during glaucoma progression in the DBA/2J mice (58%) according to the immunohistochemical and western blot analysis.
These results may indicate a possible correlation between retinal dysfunction and increased levels of extracellular ATP and nucleotide transporter. These data support an excitotoxicity role for ATP via P2X7R in glaucoma. This modified cellular environment could contribute to explaining the functional and biochemical alterations observed during the development of the pathology.
研究青光眼小鼠模型在疾病发展过程中视网膜细胞外ATP水平,并评估囊泡核苷酸转运体(VNUT)表达的变化。
从3、9、15和22月龄的青光眼DBA/2J小鼠获取视网膜,同时获取作为年龄匹配对照的C57BL/6J小鼠的视网膜。为研究视网膜核苷酸释放,将视网膜解剖并制备成扁平全层标本,在含或不含59 mM KCl的林格缓冲液中进行刺激。为研究VNUT表达,使用新开发的针对VNUT的抗体,通过免疫组织化学和蛋白质印迹分析对小鼠视网膜切片进行评估。所有图像均在共聚焦显微镜下检查和拍照。对C57BL/6J和DBA/2J小鼠进行视网膜电图(ERG)记录以分析电生理反应变化;在15月龄的DBA/2J小鼠中观察到暗视阈值反应降低。
在15月龄的对照小鼠和青光眼小鼠中,观察到42%的电生理变化。此外,当病变完全形成时,眼压(IOP)升高了50%。随着病变进展,视网膜ATP净释放量从0.32±0.04 pmol/视网膜(3个月)变化到1.10±0.06 pmol/视网膜(15个月;增加了三倍)。同时,根据免疫组织化学和蛋白质印迹分析,在DBA/2J小鼠青光眼进展过程中,VNUT表达显著增加(58%)。
这些结果可能表明视网膜功能障碍与细胞外ATP水平和核苷酸转运体增加之间可能存在关联。这些数据支持ATP通过P2X7R在青光眼中发挥兴奋性毒性作用。这种改变的细胞环境可能有助于解释在病变发展过程中观察到的功能和生化改变。