Imaging and Quantification of Myelin Integrity After Injury With Spectral Confocal Reflectance Microscopy
Imaging and Quantification of Myelin Integrity After Injury With Spectral Confocal Reflectance Microscopy
复制标题
DOI：
10.3389/fnmol.2019.00275
复制
发表时间：
2019-11-19
影响因子：
4.8
通讯作者：
Xiao, Junhua
中科院分区：
文献类型：
--
作者：
Gonsalvez, David G.;Yoo, SangWon;Xiao, Junhua
开发一种高通量方法来量化脱髓鞘疾病临床前模型中髓鞘完整性的程度，将增强我们识别髓鞘修复新疗法的能力。鉴于髓鞘形成的电子显微镜和免疫组化分析的技术局限性，我们利用了一种新的成像技术，光谱共聚焦反射（SCoRe）显微镜。SCoRe利用了致密髓鞘的光学反射特性，允许在cuprizone诱导的中枢脱髓鞘模型的过程中量化致密髓鞘的完整性。我们将SCoRe成像应用于固定的冷冻脑切片。对照小鼠的SCoRe分析鉴定了在铜腙施用和恢复期间胼胝体髓鞘形成的增加，表明此时髓鞘形成的正常发育过程正在进行。重要的是，与年龄匹配的对照小鼠相比，对经受cuprizone的小鼠的分析鉴定了胼胝体的喙部和尾部中的紧密髓鞘的显著减少。SCoRe显微镜还允许脱髓鞘病变中髓鞘碎片的量的可视化和定量。将SCoRe成像与免疫组织化学相结合，我们定量了IBA-1+小胶质细胞内髓鞘碎片的量，发现11%的髓鞘碎片共定位于小胶质细胞中，而与胼胝体区域无关，绝大多数碎片在小胶质细胞外。总之，我们已经证明，SCoRe显微镜是一种有效和强大的工具，可以对健康或体内损伤后的致密髓鞘完整性进行定量和定性分析，证明其未来在脱髓鞘疾病临床前动物模型中的高通量评估和髓鞘修复治疗疗效筛选中的应用。
Developing a high-throughput approach to quantify the extent of myelin integrity in preclinical models of demyelinating diseases will enhance our capacity to identify novel therapies for myelin repair. In light of the technical limitations of electron microscopy and immunohistochemical analyses of myelination, we have utilized a novel imaging technique, spectral confocal reflectance (SCoRe) microscopy. SCoRe takes advantage of the optically reflective properties of compact myelin, allowing the integrity of compact myelin to be quantified over the course of the cuprizone-induced model of central demyelination. We applied SCoRe imaging on fixed frozen brain sections. SCoRe analysis of control mice identified an increase in corpus callosum myelination during the period of cuprizone administration and recovery, suggesting that the normal developmental processes of myelination are ongoing at this time. Importantly, analysis of mice subjected to cuprizone identified a significant reduction in compact myelin in both rostral and caudal corpus callosum compared to age-matched control mice. SCoRe microscopy also allowed the visualization and quantification of the amount of myelin debris in demyelinating lesions. Combining SCoRe imaging with immunohistochemistry, we quantified the amount of myelin debris within IBA-1+ microglia and found that 11% of myelin debris colocalized in microglia irrespective of the callosal regions, with the vast majority of debris outside of microglia. In summary, we have demonstrated that SCoRe microscopy is an effective and powerful tool to perform both quantitative and qualitative analyses of compact myelin integrity in health or after injury in vivo, demonstrating its future application in high-throughput assessments and screening of the therapeutic efficacy of myelin repair therapies in preclinical animal models of demyelinating diseases.