Simpson Jennifer, Nien Chyong Jy, Flynn Kevin, Jester Brian, Cherqui Stephanie, Jester James
Department of Biochemistry, Jawaharlal Nehru Medical College, Maharashtra, India.
Mol Vis. 2011;17:2212-20. Epub 2011 Aug 17.
The purpose of this study was to assess the ability of quantitative in vivo confocal microscopy to characterize the natural history and detect changes in crystal volume in corneas from a novel animal model of cystinosis, the cystinosin (Ctns(-/-)) mouse.
Two Ctns(-/-) mice and one C57Bl/6 mouse were examined at each of the following time points: 2, 3, 5, 7, 10, 12, and 14 months of age. In vivo confocal microscopy scans were performed in 4 different regions of the cornea per eye. After, animals were sacrificed and cornea blocks evaluated for cell morphology using phalloidin and lymphocytic infiltration using CD45 antibodies by ex vivo confocal microscopy. Cystine crystal content in the cornea was measured by calculating the pixel intensity of the crystals divided by the stromal volume using Metamorph Image Processing Software.
Corneal crystals were identified in Ctns(-/-) eyes beginning at 3 months of age and increased in density until 7-12 months, at which time animals begin to succumb to the disease and corneas become scarred and neovascularized. Older Ctns(-/-) mice (7 months and older) showed the presence of cell infiltrates that stained positively for CD45 associated with progressive keratocyte disruption. Finally, at 12 months of age, decreased cell density and endothelial distortion were detected.
Confocal microscopy identified corneal crystals starting at 3 month old Ctns(-/-) eyes. Cystine crystals induce inflammatory and immune response with aging associated with loss of keratocyte and endothelial cells. These findings suggest that the Ctns(-/-) mouse can be used as a model for developing and evaluating potential alternative therapies for corneal cystinosis.
本研究旨在评估定量体内共聚焦显微镜对一种新型胱氨酸贮积症动物模型胱氨酸转运蛋白（Ctns(-/-)）小鼠角膜中晶体自然病程的特征描述能力以及检测晶体体积变化的能力。
在以下每个时间点检查两只Ctns(-/-)小鼠和一只C57Bl/6小鼠：2、3、5、7、10、12和14月龄。对每只眼睛的角膜4个不同区域进行体内共聚焦显微镜扫描。之后，处死动物，通过离体共聚焦显微镜使用鬼笔环肽评估角膜块的细胞形态，使用CD45抗体评估淋巴细胞浸润情况。使用MetaMorph图像处理软件通过计算晶体的像素强度除以基质体积来测量角膜中的胱氨酸晶体含量。
在3月龄的Ctns(-/-)小鼠眼中开始发现角膜晶体，其密度一直增加到7 - 12月龄，此时动物开始死于该疾病，角膜出现瘢痕和新生血管。年龄较大的Ctns(-/-)小鼠（7月龄及以上）显示存在CD45染色阳性的细胞浸润，伴有进行性角膜细胞破坏。最后，在12月龄时，检测到细胞密度降低和内皮细胞变形。
共聚焦显微镜在3月龄的Ctns(-/-)小鼠眼中识别出角膜晶体。随着年龄增长，胱氨酸晶体引发炎症和免疫反应，伴有角膜细胞和内皮细胞的丧失。这些发现表明，Ctns(-/-)小鼠可作为开发和评估角膜胱氨酸贮积症潜在替代疗法的模型。
