Intraoperative confocal microscopy for quantitative delineation of low-grade glio

描述(申请人提供)：近年来，由5-氨基乙酰丙酸(5-ALA)诱导的PpIX荧光提供对比的荧光图像引导手术(FICS)已被证明可以改善胶质瘤患者的手术结果。然而，这些广域成像方法有几个局限性。例如，很难准确地 根据胶质瘤组织中荧光强度的细微变化来确定手术边缘，胶质瘤组织是弥漫的，在肿瘤和正常之间缺乏明显的过渡。此外，宽视野(低分辨率)方法，如Figs和MRI，提供的像素强度代表许多细胞的平均值，导致检测弥漫性胶质瘤边缘稀疏肿瘤细胞群的能力减弱。这一问题在低级别胶质瘤中加剧，在低级别胶质瘤中，5-ALA诱导的PpIX荧光仅在罕见的增殖细胞群中产生，无法通过广野无花果检测到。最近，参与该项目的Nader Sanai博士展示了术中细胞分辨率共聚焦显微镜可以用来显示接受5-ALA治疗的低级别胶质瘤患者的稀疏荧光细胞。这一发现的意义非常重大，因为据报道， 低级别胶质瘤的大体全切除(GTR)相对较低(14%至46%)，这表明需要改进图像引导技术。在这里，我们建议对PI先前开发的手持式术中共焦显微镜进行优化，以可视化表达5-ALA诱导的PpIX的低级别胶质瘤细胞，并证明这种实时定量体内成像技术与MRI和侵袭性组织病理学之间的相关性，用于边缘评估。该项目汇集了一支久经考验的合作者团队，其中包括斯坦福大学的共同研究员Olav Solgaard，以开发一种优化的光学切片显微镜，该显微镜结合了高度坚固的微电子机械系统(MEMS)扫描仪和PI实验室最近发布的消色差(波长敏捷)光学设计。临床前、体外和体内成像将使用高度逼真和浸润性的脑胶质瘤模型进行，以证明表达PPIX荧光的细胞密度与实际肿瘤细胞密度和组织学分级之间的关联能力。临床研究将利用巴罗神经研究所(BNI)的联合调查员Nader Sanai的现有专业知识，使用大视野无花果和术中共焦显微镜对胶质瘤患者的PpIX荧光进行研究。基于BNI已建立和批准的方案，将进行一项探索性临床研究，以将PpIX标记细胞的密度与组织病理学、术中神经导航(术中神经导航)和术后MRI相关联。这些初步研究的结果将证明未来的临床调查是合理的，以评估患者的预后，如体积切除范围、总存活率和神经发病率。

DESCRIPTION (provided by applicant): In recent years, fluorescence image-guided surgery (FIGS) with contrast provided by 5-aminolevulinic acid (5- ALA)-induced PpIX fluorescence has been demonstrated to improve surgical outcomes for glioma patients. However, there are several limitations to these wide-field imaging methods. For example, it is difficult to accurately determine a surgical margin based on subtle variations in fluorescence intensity in glioma tissues, which are diffuse and lack a distinct transition between tumor and normal. Furthermore, wide-field (low- resolution) approaches, such as FIGS and MRI, provide pixel intensities that represent an average value from many cells, resulting in a diminished ability to detect the sparse tumor cell populations at the margins of diffuse gliomas. This problem is exacerbated in low-grade gliomas, where 5-ALA-induced PpIX fluorescence is only generated in rare proliferating cell populations and is undetectable via wide-field FIGS. Recently, Dr. Nader Sanai, a Co-Investigator on this project, has shown that intraoperative cellular-resolution confocal microscopy can be used to visualize the sparse fluorescent cells in low-grade glioma patients treated with 5-ALA. The implications of this finding are highly significant since reported rates of gross-total resection (GTR) have been relatively low for low-grade gliomas (14% to 46%), suggesting a need for improved image-guidance techniques. Here, we propose to optimize a hand-held intraoperative confocal microscope, previously developed by the PI, to visualize low-grade glioma cells that express 5-ALA-induced PpIX, and to demonstrate that this real-time quantitative in vivo imaging technique correlates with MRI and invasive histopathology for margin assessment. This project brings together a proven team of collaborators, including Co-Investigator Olav Solgaard at Stanford University, to develop an optimized optical-sectioning microscope that incorporates a highly robust MicroElectroMechanical System (MEMS) scanner and an achromatic (wavelength-agile) optical design recently published by the PI's lab. Preclinical ex vivo and in vivo imaging will be performed with a highly realistic and infiltrative at model of glioma to demonstrate the ability to correlate the density of cells expressing PpIX fluorescence vs. actual tumor-cell density and histological grade. Clinical studies will leverage the existing expertise of Co-Investigator Nader Sanai, at Barrow Neurological Institute (BNI), with both wide-field FIGS and intraoperative confocal microscopy of PpIX fluorescence in glioma patients. Based on established and approved protocols at BNI, an exploratory clinical study will be performed to correlate the density of PpIX-labeled cells with histopathology, pre-operative MRI (intraoperative neuronavigation), and post- operative MRI. The results of these preliminary studies will justify future clinical investigations to assess patient outcomes such as volumetric extent of resection, overall survival, and neurological morbidity.