In vitro detection of neuronal programmed cell death by ultrahigh resolution optical coherence tomography

在细胞死亡开始之前在体内检测细胞功能障碍对于我们了解在体内启动细胞死亡的过程是至关重要的。到目前为止，我们在这一领域的大部分知识都来自于对培养中保持的细胞的分析。虽然这产生了许多有价值的见解，但我们还无法将这些变化转化为那些发生在组织中的变化，也无法在体内证明组织中细胞之间的相互作用。最近，高分辨率光学相干断层摄影术已经发展成为一种用于在体内分析组织的技术，其分辨率在亚细胞水平(？lt；5微米)。该技术可应用于对任何可获得光学访问的组织进行分析。因此，OCT被开发用于皮肤组织、膀胱和肠道的分析。最近，它被用来检测皮层中发生的变化，这些变化发生在对周围刺激的神经可塑性反应中。OCT在眼部结构分析方面取得了最大的发展。对于分析眼睛前部(晶状体和角膜)的眼睛结构，OCT提供了前所未有的亚细胞级别的分辨率。最近，超高分辨率OCT已经发展到监测健康和疾病中发生的视网膜结构和功能的变化。与组织学准备的相关性表明，UHR-OCT可以检测视网膜边界的细微差异，并可用于在细胞水平上探测视网膜结构。事实上，现在通过关注视网膜的特定区域来监测活组织随着时间的推移的活动是可能的。视网膜是从光学角度研究神经组织变化的理想模型系统它在体内是独一无二的，可以进行活体成像，现在，我们第一次能够在细胞水平上成像神经元，并绘制它们在健康和疾病中的响应图。在加的夫，我们最近成立了一个生物成像小组，开发用于体内生物结构分析的高分辨率OCT。我们已经证明了以亚细胞分辨率成像是可能的，但到目前为止，我们还不知道这是否可以应用于分析整个组织的变化。做到这一点的能力对于我们理解整个组织在应对压力(如新陈代谢损伤，如缺氧和缺血)时可能发生的变化至关重要。在这项研究中，我们建议使用UHR OCT来检测视网膜神经组织(特别是视网膜神经节细胞)在缺氧(低氧水平)或低血糖(低糖水平)等条件下发生的变化，这些条件已知会导致这些细胞通过凋亡(神经系统中最常见的非炎性细胞死亡形式)死亡。UHR OCT的分辨率是这样的，我们应该能够检测到对维持细胞健康至关重要的线粒体-亚细胞器的变化)。当线粒体受损时，它们会引发细胞变化，导致细胞凋亡。我们假设，这将有可能检测到这些变化，以开发分析光学信号的技术，使我们能够检测到容易发生凋亡的细胞。这个项目分析的一个重要部分是，我们将能够预测那些将经历凋亡的细胞。我们将首先在培养的视网膜神经节细胞中进行这一分析，然后将其应用于培养中保持的视网膜组织。通过将光学变化与我们将在经过处理的组织中观察到的(图像分析后获得的)相关联，我们将能够开发用于体内发生的细胞变化的替代光学测量方法。从长远来看，我们预测高分辨率将是了解组织水平上的细胞变化的重要资源，这些变化发生在一系列组织病理中。

The in vivo detection of cellular dysfunction prior to the onset of cell death is critical to developing our inderstadning of the processes that initiate cell death in vivo. To date much of our knolwdge in this area has been derived from the analysis of cells maintained in culture. While this has generated many valuable insights we havenot been able to translate these change to those occuring in tissues or to demonstrate the interaction, in vivo between cells in tissue. Recently high resolution optical coherence tomogrpahy has been developed as a technique for the in vivo analysis of tissue a resolution at the subcellular level (<5um). This technique can be applied to the analysis of any tissue for which optical access is available. Thus OCT haas been developed for the analysis of dermal tissues, bladder and bowel. More recently it has been used to detect changes in the cortex that occur in response to neuroplasticity in response to peripheral stimuli. OCT has seen its greatest development in the analysis of ocular structures. For the analysis of ocular structure at the fron of the eye (the lens and cornea) OCT has provided unprecedented subcellular levels of resolution. More recently ultrahigh resolution OCT has been development to montor changes in retinal structurre and function that occur in health and disease. Correlation with histological preparations has shown that UHR-OCT can detect subtle differences in retinal boundaries and can be used to probe retinal structure at the cellular level. Indeed it is now possible, by focusing on selected areas of the retina to monitor activity in living tissue over time. The retina is an ideal model system in which to study changes in neural tissue From the point of view of optic access it is unique in the body in allowing in vivo imaging and now, for the first time we are able to image neurons at the cellular level and to chart their repsonse in health and disease. At Cardiff we have recently established a Bioimaging Group to develop hight resolution OCT for the in vivo analysis of biological structures. We have shown that it is possible to image at subcellular resolution but as yet wedo not know whether this can be applied to the analysis of changes in whole tissues. The ability to do this is critical to our understanding of the changes that can occur in whole tissues in response to stresses such as metabolicc insults such as hypoxia and ischaemia. In this study we propose using UHR OCT to detect changes in retinal neural tissue (and in particular retinal ganglion cells) that occur in repsonse to condicitions such as hypoxia (low oxygen levels) or hypoglycaemia (low sugar levels) that are know to result in the death of these cells by apoptosis (the most common form of non-inflammatory cell death in the nervous system). The resoltuon of UHR OCT is such that we should be able to detect changes in mitochondira- subcellular organelles that are critical to maintaining the health of the cell). When mitochondria are compromised they can initiated cellular changes that result in apoptosis. We hypothessie that is will be possible to detect these changes in to develop techniques for the analysis of optical signatures that will allow us to detect cells that are predisposed to apoptosis. An importat part of the analysis in this project is that we will be able to predict those cells that will undergo apoptosis. We will first conduct this analysis in cultured retinal ganglion cells and then apply this to retinal tissue that is maintained in culture. By correlating optical changes wiith those that we will oberve in processed tissue (obtained following image analysis) we will be able to develop surrgoate optical measures for the cellular changes that occur in vivo. In the long term we predict that high resolution will be an important resource to understand cellular changes at the tissue level that occur ina range of tissue pathologies.