Real-time multiphoton microscopy of periprostatic tissue architecture

背景：在过去的15年里，美国有300多万患者被诊断出患有前列腺癌；大约100万人接受了根治性前列腺癌切除术。这种手术的成功是通过彻底切除携带癌症的腺体和保留控制性功能的神经来衡量的。这些神经很细小，与前列腺囊上的脂肪细胞和血管组织混合在一起。偶尔，神经周围的空间会被癌细胞用作逃生路线。因此，不能将癌细胞与神经区分开来，可能会导致肿瘤切除不全和/或由于神经受损或切除而导致术后阳萎。这项提议的目标是为外科医生提供实时组织识别工具，这些工具可以更好地区分正常细胞和癌细胞，并准确地识别和挽救邻近的神经，所有这些都在手术过程中进行。 假设：多光子显微镜(MPM)方法可以用来实时准确地观察和区分前列腺癌与邻近的神经纤维。 具体目标： 具体目标1：我们将测试这一假设，即MPM/SHG能够以足够的分辨率在大鼠前列腺中进行组织分辨的实时成像。培训期间，将对新安乐死大鼠的切除组织进行体外成像(Aim 1.1)，然后是活体、麻醉大鼠的体内成像(Aim 1.2)；所有组织识别结果将与组织学分析结果进行比较。 具体目标2：我们将测试MPM/SHG成像可用于在大鼠前列腺癌模型中对前列腺及相关组织进行活体成像的假设。结果将再次量化，并与相同组织的组织病理学分析结果进行比较。 具体目标3：我们将在大鼠活体MPM/SHG成像后进行安全性评估，以检验使用MPM/SHG的实时成像不会对海绵体神经或相关组织造成损害的假设。MPM/SHG成像的神经的持续功能将使用勃起活动的标准测量方法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前列腺切除术(AT)、MPM、SHG和高级光学显微镜(SM、FRM、WWW)方面的专家。我们将标准化我们的成像条件，并测试我们通过MPM/SHG成像在大鼠模型中识别所有相关组织类型(前列腺囊、海绵体神经、前列腺腺泡细胞、脂肪、动脉和静脉)的能力。我们建议通过两个步骤来实现这一目标：对安乐死大鼠切除的组织进行体外成像和对麻醉大鼠进行体内成像。所有组织的一致性将通过对同一标本的标准组织学分析来确认，并将评估MPM鉴定的统计可靠性。我们将把这些研究扩展到大鼠前列腺癌模型。我们还将通过在成像后六周进行海绵体神经刺激来检验这一假设，即在活体动物中进行MPM/SHG成像不会对前列腺组织或神经造成不可逆转的损害。不会使用外源染料，这将使这项技术未来通过MP内窥镜在人类手术中使用变得更加简单。 终点：到这个资金阶段结束时，我们预计将有足够的初步数据，准备通过介入使用我们的合作者目前正在开发的多光子内窥镜将这些技术转换到人类受试者身上。我们的发现将为在实际临床环境中测试这种MPM内窥镜奠定基础 影响：我们预计这些涉及动物和人类手术标本的研究将导致在手术中适用的方法学的发展。我们预计，手术中的实时成像将大大减少根治性前列腺切除术的负面结果。这项技术也可能推广到其他保留神经的盆腔肿瘤手术，例如。根治性膀胱切除术，结直肠和妇科盆腔手术，腹膜后淋巴结清扫术。所获得的动态、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.