Real-time multiphoton microscopy of periprostatic tissue architecture

背景资料：在过去的15年里，美国有超过300万患者被诊断患有前列腺癌;大约100万人接受了根治性前列腺切除术。这种手术的成功是通过完全切除癌腺和保留控制性功能的神经来衡量的。这些神经是微小的，与前列腺包膜上的脂肪细胞和血管组织混合在一起。有时，神经周围的空间被癌细胞用作逃跑路线。因此，不能将癌细胞与神经区分开可导致癌症的不完全切除和/或由于神经的损伤或切除而导致的术后阳痿。这项提案的目标是为外科医生提供真实的时间组织识别工具，这些工具可以更好地区分正常细胞和癌细胞，并准确地识别和保存相邻的神经，而手术正在进行中。 假设：多光子显微镜（MPM）方法可用于真实的实时准确地可视化和区分前列腺癌与相邻的神经纤维。 具体目标： 具体目标1：我们将测试的假设，MPM/SHG使实时成像具有足够的分辨率，在大鼠前列腺组织的歧视。培训期将伴随新鲜安乐死大鼠的切除组织的离体成像（目的1.1），随后是活的麻醉大鼠的体内成像（目的1.2）;将所有组织鉴定结果与组织学分析获得的结果进行比较。 具体目标2：我们将测试的假设，MPM/SHG成像可用于活体成像的前列腺和相关组织在大鼠前列腺癌模型。将再次对结果进行量化，并与通过相同组织的组织病理学分析获得的结果进行比较。 具体目标3：我们将通过在大鼠活体MPM/SHG成像后进行安全性评估，检验使用MPM/SHG的实时成像不会对海绵体神经或相关组织造成损伤的假设。通过MPM/SHG成像的神经的持续功能将使用勃起活动的标准测量与未成像的神经进行定量比较。 研究设计：该应用程序汇集了机器人椎间盘切除术（AT），MPM，SHG和高级光学显微镜（SM，FRM，WWW）的专家。我们将标准化我们的成像条件，并在大鼠模型中通过MPM/SHG成像测试我们识别所有相关组织类型（前列腺包膜、海绵体神经、前列腺泡细胞、脂肪、动脉和静脉）的能力。我们建议通过两个步骤实现这一目标：从安乐死大鼠中切除的组织的体外成像和麻醉大鼠中的体内成像。所有组织的身份将通过同一标本的标准组织学分析来确认，并且将评估MPM识别的统计可靠性。我们还将通过在成像后6周进行海绵体神经刺激来检验活体动物的MPM/SHG成像不会对前列腺组织或神经造成不可逆损伤的假设。将不使用外源性染料，这将使该技术未来通过MP内窥镜在人体中的术中使用更加直接。 结束点：到本资助期结束时，我们预计将有足够的初步数据，准备通过我们的合作者目前正在开发的多光子内窥镜的术中使用，将这些技术转化为人类受试者。我们的研究结果将作为在实际临床环境中测试这种MPM内窥镜的基础 影响力：我们预计，这些研究，涉及动物和人类手术标本，将导致手术过程中适用的方法的发展。我们设想术中实时成像将大大减少根治性盆腔肿瘤切除术的不良后果，这项技术也可以扩展到其他保留神经的盆腔肿瘤手术，如。根治性膀胱切除术、结直肠切除术和妇科盆腔手术以及腹膜后淋巴结清扫术。实现的动态高分辨率光学图像可以使癌症外科医生在手术过程中更好地区分组织形态，从而改善患者的功能结果和生活质量。

Background: In the last 15 years, over 3 million patients in the USA have been diagnosed with prostate cancer; approximately 1 million have undergone radical prostatectomy. The success of this surgery is measured by complete removal of the cancer-harboring gland and preservation of nerves that control sexual function. These nerves are minute and intermingle with fat cells and vascular tissue on the prostate capsule. Occasionally, the space around the nerves is used as an escape route by cancerous cells. Thus the inability to differentiate cancerous cells from nerves can result in the incomplete removal of the cancer and/or postoperative impotence due to the damage or excision of the nerves. The goal of this proposal is to provide surgeons access to real time tissue recognition tools that can better differentiate normal cells from cancerous ones and accurately identify and save the adjacent nerves, all while the surgery is in progress. Hypothesis: Multiphoton Microscopy (MPM) methods can be used to accurately visualize and differentiate prostate cancer from adjacent nerve fibers in real time. Specific Aims: Specific aim 1: We will test the hypothesis that MPM/SHG enables real-time imaging with sufficient resolution for tissue discrimination in the rat prostate. A training period will be accompanied by ex-vivo imaging of excised tissue from freshly euthanized rats (aim 1.1), followed by in vivo imaging of live, anesthetized rats (aim 1.2); all tissue identification results will be compared to those obtained by histological analysis. Specific aim 2: We will test the hypothesis that MPM/SHG imaging can be used for intravital imaging of the prostate and associated tissue in a rat prostate cancer model. Results will again be quantified and compared to those obtained by histopathological analysis of the same tissue. Specific aim 3: We will test the hypothesis that live imaging using MPM/SHG will not cause damage to the cavernous nerve or associated tissue by performing a safety assessment following intravital MPM/SHG imaging in rats. Continued function of nerves imaged by MPM/SHG will be quantitatively compared to unimaged nerves using a standard measure of erectile activity. Study Design: This application brings together experts in robotic prostatectomy (AT), MPM, SHG, and advanced optical microscopy(SM, FRM, WWW). We will standardize our imaging conditions and test our ability to identify all relevant tissue types (prostatic capsule, cavernous nerve, prostatic acinar cells, fat, arteries, and veins)by MPM/SHG imaging in a rat model. We propose to achieve this aim in two steps: Ex vivo imaging of tissue excised from euthanized rats and in vivo imaging in anesthetized rats. The identity of all tissues will be confirmed by standard histological analysis of the same specimen, and the statistical reliability of MPM identification will be assessed.We will extend these studies to a rat prostate cancer model. We will also test thehypothesis that MPM/SHG imaging in a live animal does not cause irreversible damage to the prostatic tissue or nerves by performing cavernous nerve stimulation six weeks after imaging. No exogenous dyes will be used, which will make the future translation of this technology to intra-operative use in humans via MP endoscopy much more straightforward. Endpoints: By the end of this funding period, we expect to have sufficient preliminary data to be ready to translate these techniques to human subjects via introperative use of a multiphoton endoscope currently being developed by our collaborators. Our findings will serve as a foundation for testing this MPM endoscope in actual clinical settings Impact: We anticipate that these studies, involving both animals and human surgical specimens, will lead to the development of methodologies applicable during surgery. We envision that live imaging during surgery will greatly reduce negative outcomes of radical prostatectomies.This technology may also be extended to other nerve-sparing pelvic oncologic surgeries, eg. radical cystectomy, extirpative colorectal and gynaecologic pelvic procedures, and retroperitoneal lymph node dissection. The dynamic, high-resolution optical images achieved could empower cancer surgeons to better distinguish tissue morphology during surgery, leading to improved functional outcomes and quality of life for their patients.