Name: an in vivo murine sciatic nerve model of perineural invasion
Uploaded: 2018-04-23T13:00:00.000+00:00
Duration: 9 min 55 s
Description: Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

作者承认分子细胞学设施和纪念斯隆凯特林癌症中心的动物设施提供的技术服务。这项工作得到了 NIH 赠款 CA157686 (R.J.) 和 P30 CA008748 (纪念斯隆凯特林癌症中心支助补助金) 的支持。

所有的程序与动物的对象是批准的机构动物护理和使用委员会在纪念斯隆凯特林癌症中心。
1. 肿瘤细胞的制备
<ol><li>收获亚汇合 Panc02-H7 细胞与0.25% 胰蛋白酶为5分钟在37°c。收集15毫升离心管中的细胞。 注: 细胞生长在 T-225 烧瓶中, 它包含大约 12 x 106细胞每瓶在80% 融合和4毫升胰蛋白酶/烧瓶使用。</li>
	<li>离心机在900克的细胞在 4°c 5 分钟, 通过 resuspending 细胞颗粒在1毫升的 PBS (由吹打上下两次), 并转移悬浮到1.5 毫升离心管冰。</li>
	<li>再次离心细胞在900克在 4°c 5 分钟, 并放弃上清, 不干扰颗粒。把颗粒细胞放在冰上直到注射。</li>
</ol>2. 小鼠的制备与手术
注: 本研究使用8周大、雄性和雌性 C57BL/6J 小鼠。手术条件遵循本机构的 IACUC 规则。器械消毒, 手术工作表面消毒, 动物消毒, 外科医生佩戴无菌手套。
<ol><li>在手术前一天, 麻醉老鼠使用2% 异氟醚, 然后用一把薄剃刀或化学脱毛剂将皮毛沿着股骨的长度移除。</li>
	<li>在手术当天, 麻醉在诱导腔内使用 2% isofluorane 的老鼠。通过脚趾捏刺激和缺乏反应来确认麻醉。</li>
	<li>在眼部应用兽医软膏, 防止麻醉时的干燥。</li>
	<li>将麻醉的老鼠放在腹侧, 用低过敏的胶带轻轻地固定每一个肢体, 使其在肢体中产生轻度的张力。麻醉是维持使用异氟醚提供通过一个精确的蒸发器和鼻锥。</li>
	<li>用 Betadine 清洁注射部位, 再用70% 酒精。重复此过程两次。确保没有松动的头发留在手术领域。</li>
	<li>做一个1厘米切口与小剪刀约2毫米以下和平行的股骨。在侧面用镊子缩回皮肤, 露出下面的肌肉。</li>
	<li>坐骨神经向臀大肌和股二头肌肌肉深部延伸。用小剪刀将这两块肌肉沿筋膜平面分开, 露出坐骨神经下面。用钝夹层释放周围肌肉的神经。</li>
	<li>绘制 3 ul 的癌细胞 (约5万细胞) 从颗粒成 10 ul 注射器。 注: 另一种做法是, 将 PBS 的 3 ul 作为控件绘制。</li>
	<li>将一个小金属铲下的神经在注射点的支持。在可视化的解剖显微镜下, 将针插入神经对金属底座, 使针与神经平行, 在插入时尽可能。小心不要刺穿神经的背部。在整个过程中尽量减少对神经的处理。</li>
	<li>在5秒内慢慢注入神经。在注射区形成灯泡表明注射良好。然后把针放在原地3秒, 然后轻轻地取出针。保持针到位为 3 s 最小化细胞回流的神经。 注: 可对远端神经或脊髓进行注射。在一组实验中保持一致是很重要的。如果细胞外泄, 动物不应纳入实验分析。有经验, 这些事件是非常罕见的。</li>
	<li>把神经放回原来的位置。用上覆的肌肉遮住神经。用适当的镇痛治疗小鼠, 然后用5-0 尼龙缝线闭合皮肤。</li>
	<li>在恢复期间, 将鼠标单独放在干净的笼子里观察, 直到它完全从麻醉中苏醒。 注意: 动物恢复全意识需要5-15 分钟。动物没有被留在无人看管, 直到它恢复了足够的意识, 以维持胸骨卧床。在完全恢复之前, 动物不会返回到其他动物的公司。此后, 每24小时至少评估一次恢复72小时。</li>
</ol>3. 坐骨神经提取
<ol><li>在术后女人天 #7, 弄死与CO2 的鼠标。将鼠标放在腹侧, 用别针稳定远端四肢。</li>
	<li>去除注射肢体和躯干背面的皮肤。</li>
	<li>使用钝夹层, 将坐骨神经深深暴露在肌肉上。</li>
	<li>髂骨和骶骨之间的神经路线。要获得脊髓区的神经, 请将两根骨头分开。首先在坐骨神经所在的狭窄区域插入闭合剪刀, 然后在按住鼠标的同时打开剪刀。在解剖过程中保持坐骨神经的完整性和细腻。</li>
	<li>仔细解剖坐骨神经远端到股骨端, 下部到脊髓。</li>
	<li>注意: 侵入神经非常脆弱, 容易在紧张或强力处理下折断。</li>
	<li>先割下它的远端, 才能收获神经。小心抬起神经, 同时从相邻的组织中释放它。切断神经在近端, 尽可能接近其退出从脊柱。</li>
	<li>用游标卡尺记录下入侵的总长度。这种宏观估计只是指示性的。</li>
</ol>4. 神经处理和量化
<ol><li>将解剖神经嵌入 OCT 化合物中。确保将神经纵向和尽可能平坦的模具底部。</li>
	<li>通过标记字母 P 和 D, 在卡带上显示神经的近端和远端。</li>
	<li>如果不立即切片, 将嵌入的神经放在干冰的顶端。样品可以保存在摄氏-80 摄氏度几个星期。</li>
	<li>部分样品使用恒温器切片在5微米厚度和地方部分在玻璃幻灯片。如有可能, 每张幻灯片应符合2神经节。表示神经的近端。</li>
	<li>使用 H & E 染色着色幻灯片12。</li>
	<li>用可提供高分辨率数字数据的滑动扫描仪对染色的神经部分进行数字扫描。</li>
	<li>使用成像软件, 通过单击测量距离按钮、入侵区域或其他所需参数来量化入侵的长度。为了很好地估计入侵的长度, 使用同一神经的多节 (2 到 4)。</li>
</ol>

<ol>
	<li>Liebig, C., Ayala, G., Wilks, J. A., Berger, D. H., Albo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+in+cancer.">Perineural invasion in cancer.</a> Cancer. 115 (15), 3379-3391 (2009).</li><li>Bapat, A. A., Hostetter, G., Von Hoff, D. D., Han, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+and+associated+pain+in+pancreatic+cancer.">Perineural invasion and associated pain in pancreatic cancer.</a> Nat Rev Cancer. 11 (10), 695-707 (2011).</li><li>Deborde, S., Wong, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Schwann+cells+facilitate+cancer+progression+in+nerves.">How Schwann cells facilitate cancer progression in nerves.</a> Cell Mol Life Sci. 341 (177-186), 1236361-1236416 (2017).</li><li>Abiatari, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+transcriptome+signature+of+perineural+invasion+in+pancreatic+carcinoma.">Consensus transcriptome signature of perineural invasion in pancreatic carcinoma.</a> Mol Cancer Ther. 8 (6), 1494-1504 (2009).</li><li>Ayala, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+dorsal+root+ganglia+and+human+prostate+cell+line+interaction:+redefining+perineural+invasion+in+prostate+cancer.">In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer.</a> Prostate. 49 (3), 213-223 (2001).</li><li>Gil, Z., Cavel, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102 (2), 107-118 (2010).</li><li>Deborde, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cells+induce+cancer+cell+dispersion+and+invasion.">Schwann cells induce cancer cell dispersion and invasion.</a> J Clin Invest. 126 (4), 1538-1554 (2016).</li><li>Pour, P. M., Egami, H., Takiyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+growth+and+metastases+of+induced+pancreatic+cancer+in+relation+to+the+prognosis+and+its+clinical+implications.">Patterns of growth and metastases of induced pancreatic cancer in relation to the prognosis and its clinical implications.</a> Gastroenterology. 100 (2), 529-536 (1991).</li><li>Eibl, G., Reber, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+xenograft+nude+mouse+model+for+perineural+invasion+and+recurrence+in+pancreatic+cancer.">A xenograft nude mouse model for perineural invasion and recurrence in pancreatic cancer.</a> Pancreas. 31 (3), 258-262 (2005).</li><li>Stopczynski, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroplastic+changes+occur+early+in+the+development+of+pancreatic+ductal+adenocarcinoma.">Neuroplastic changes occur early in the development of pancreatic ductal adenocarcinoma.</a> Cancer Res. 74 (6), 1718-1727 (2014).</li><li>Gil, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve-sparing+therapy+with+oncolytic+herpes+virus+for+cancers+with+neural+invasion.">Nerve-sparing therapy with oncolytic herpes virus for cancers with neural invasion.</a> Clin Cancer Res. 13 (21), 6479-6485 (2007).</li><li>Cardiff, R. D., Miller, C. H., Munn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+hematoxylin+and+eosin+staining+of+mouse+tissue+sections.">Manual hematoxylin and eosin staining of mouse tissue sections.</a> Cold Spring Harb Protoc. 2014 (6), 655-658 (2014).</li><li>Cremer, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+N-CAM+gene+in+mice+results+in+size+reduction+of+the+olfactory+bulb+and+deficits+in+spatial+learning.">Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and deficits in spatial learning.</a> Nature. 367 (6462), 455-459 (1994).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+(CCL2-CCR2)+signaling+axis+mediates+perineural+invasion.">The chemokine (CCL2-CCR2) signaling axis mediates perineural invasion.</a> Mol Cancer Res. 13 (2), 380-390 (2015).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFR&#945;1+released+by+nerves+enhances+cancer+cell+perineural+invasion+through+GDNF-RET+signaling.">GFR&#945;1 released by nerves enhances cancer cell perineural invasion through GDNF-RET signaling.</a> P Natl Acad Sci USA. , 02944 (2014).</li></ol>

作者没有什么可透露的。

在本协议中, 我们描述了一个在体内小鼠神经入侵模型, 它允许量化的坐骨神经侵袭的胰腺癌细胞。该模型能够研究神经入侵的分子机制。成功的实验使用这项技术需要谨慎的方法, 在三关键步骤的过程中: 1) 注射癌细胞 (步骤 2.7, 2.8), 2) 提取的侵入神经 (步骤 3.4) 和 3) 处理收获的神经 (步骤 4.1)。
注射应该非常小心, 以避免刺穿神经后壁, 导致癌细胞的损失。一旦针到位, 注射应以缓慢和稳定的速度超过5秒, 以防止水压从注射点向前推进癌细胞太远。注射后, 将针放在神经后, 再增加3秒, 将减少回流和癌细胞渗漏。在坐骨神经提取过程中, 解剖必须非常温和, 因为侵入的坐骨神经非常脆弱。癌症侵袭扰乱了坐骨神经正常的细胞组织。尽管侵入神经的直径与非注射或非侵入神经相比呈加厚, 但他们更容易折断甚至轻微的伸展和操纵。在 OCT 中植入离体神经进行加工时, 保证神经完全平放在模具上是非常重要的。这个步骤允许切片的样本, 以捕获整个神经的长度, 以进行适当的分析。通常需要修剪神经上的实体肿瘤, 以防止肿瘤的神经部分从霉菌的底部增加神经。神经的整个长度必须完全地被放置对模子。
在使用此协议时可以考虑许多修改。当一个小金属铲不能被放置在坐骨神经下, 一双宽钳的表面可以代替。钳位可以用来在插入后放置针到位, 虽然这种方法的缺点是减少了对注射的看法和可能粉碎神经, 如果使用过度的力量。另一种方法是使用细钳解除神经, 然后打开镊子伸展神经。这种方法在注射过程中更容易执行, 同时提供张力和稳定, 但可能会损害神经过度伸展的神经。注射可以在近端或远端进行, 但我们发现, 在这两种情况下, 癌症侵袭的方向通常是朝向脊髓。因此, 我们倾向于远端注射, 以进一步减少可能推动癌细胞向脊髓的水力力, 并混淆后来的入侵长度措施。我们建议在一组实验中, 在注射方向的选择上保持一致是很重要的。
虽然我们提出一个自体移植的模型, 一个类似的技术可以用于移植的人癌细胞 immunodeficient 小鼠, 只有轻微的变化。在裸鼠中移植可能需要更长的时间的癌症侵袭, 以产生类似长度的神经入侵, 如在自体模型。也可以使用不同的癌细胞系。PNI 不仅在胰腺癌中流行, 而且在头颈、前列腺和皮肤癌中也很常见。我们的实验室成功地在坐骨神经中注射了人胰腺癌细胞系 MiaPaCa-2 和 Panc17,14,15裸鼠。
可能的不良事件包括伤口裂开, 因为小鼠偶尔会咀嚼他们的缝线, 导致伤口张开。在这种情况下, 伤口需要重新缝合。我们每天检查伤口的前3天。小鼠也可能在术后轻微跛行, 可能是由于手术过程中的疼痛以及他们的坐骨神经与癌症的侵袭。这通常不够严重, 需要止痛药。
这个模型有局限性。在手术过程中, 一定程度的神经伸展和捏, 可能造成神经损伤。此外, 很难控制细胞进入神经的确切数量。此外, 这个模型仅限于研究已经侵入神经的细胞。它不是研究神经与肿瘤细胞在原发肿瘤中相互作用的模型。使用坐骨神经与胃肠道神经的主要缺点是坐骨神经不是胰腺癌的转移部位。神经纤维的组成和性质的差异会影响入侵。然而, 胰腺神经的可接近性和非常小的大小不允许这样的研究。
我们对初学者的主要建议是在计划的切口部位周围清除毛发, 这样就可以很容易地识别出地标, 并确保在开始注射前有一个稳定的位置。经过多次注射, 表演者应该能够得到100% 的动物神经入侵。
这个模型允许研究癌症和神经在动物模型中的相互作用。它可以应用于广泛的肿瘤研究, 通过使用基因修饰小鼠, 癌细胞, 或两者兼而有之。对肿瘤侵袭神经的药理治疗也可以通过比较治疗和对照组之间的侵袭长度和面积进行测试。这些不同的可能的应用使得这个模型成为研究 PNI 的一个非常多才多艺和强大的工具。

神经形成特定的肿瘤微环境, 刺激癌症的生长和迁移1,2,3。神经入侵 (PNI) 是癌细胞在神经和周围侵袭的过程。它可能被认为是一种独特的转移途径, 因为癌症入侵从起源的地方沿神经。PNI 有多种癌症类型, 包括胰腺、前列腺、头 & 颈部、唾液腺、宫颈癌和结直肠癌, 发病率从22% 到 100%1,2不等。PNI 与疼痛相关, 预后较差, 存活率更差1,2。
神经入侵的发展模式对于阐明这一过程的细胞和分子机制, 以及检测候选治疗剂以减少 PNI 是至关重要的。体外方法研究癌症和神经之间的相互作用, 包括与神经外植体的癌细胞共培养4, 背根脊神经5,6,7, 或特定细胞从神经微环境例如雪旺细胞7。然而,体内方法在生理上是相关的, 包括使用癌症小鼠模型, 在这种模式中, 癌症已经被诱导或移植, 并具有占整个神经微环境的优势。在胰腺或前列腺癌的原位模型中, PNI 报告了8,9,10 , PNI 的发病率可能会被记录下来, 但是由于这些器官神经的体积小, 很难看到整个神经, 因此量化 PNI 的程度。我们在这里描述的模型是一个在体内模型中的 PNI, 其中癌细胞通过一个简单的手术程序11注入到小鼠的坐骨神经中。异位移植在神经内侵袭脊髓。从注射部位到脊髓的神经侵入的长度可以测量, 以及神经内的肿瘤体积。重要的是, 侵入神经也可以收集各种化验, 包括显微, 分子分析。可以对多种癌细胞进行检测, 并使用特定化合物进行基因修饰或治疗的宿主老鼠。这一强有力的化验允许肿瘤细胞和宿主微环境被修改, 以调查的机制, PNI。

<table><tbody><tr><td>Mouse</td><td></td><td></td><td>Number and age variable depending on experimental needs</td></tr><tr><td>Cell culture media (PBS, Trypsin, and DMEM+10% FBS)</td><td>Any</td><td></td><td>Steps 1.1, 1.2, 1.3.</td></tr><tr><td>Conical centrifuge tube, 50 mL</td><td>Falcon</td><td>352098</td><td>Step 1.1</td></tr><tr><td>Microcentrifuge tube 1.5 mL</td><td>Axygen</td><td>MCT-150-C-S</td><td>Step 1.2</td></tr><tr><td>Electric razor</td><td>WAHL</td><td>9962</td><td>Step 2.1. Can be substituted with commercial hair removal agent</td></tr><tr><td>Isoflurane, 250 mL</td><td>Baxter</td><td>1001936060</td><td>Step 2.2</td></tr><tr><td>Hypoallergenic surgical tape</td><td>3M Blenderm</td><td>70200419342</td><td>Step 2.3</td></tr><tr><td>Betadine Swapsticks</td><td>PDI</td><td>SKU 41350</td><td>Step 2.4</td></tr><tr><td>Webcol Alcohol Preps</td><td>Covidien</td><td>5110</td><td>Step 2.4</td></tr><tr><td>Sterile surgical tools (scissors and forceps)</td><td></td><td></td><td>Steps 2.4, 2.5, 3.3, 3.4, 3.5</td></tr><tr><td>10 &amp;mu;L Hamilton syringe</td><td>Hamilton</td><td>80308</td><td>Steps 2.7, 2.8</td></tr><tr><td>Steel Micro spatula</td><td>Fisher Scientific</td><td>S50823</td><td>Step 2.7</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>Step 2.7</td></tr><tr><td>Bupivacine, 1 g</td><td>Enzo Life Sciences</td><td>BML-NA139-0001</td><td>Step 2.9. Reconstitute to 0.5%</td></tr><tr><td>5-0 Nylon suture</td><td>Ethicon</td><td>698H</td><td>Step 2.9</td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>VWR</td><td>25608-930</td><td>Step 4.1</td></tr><tr><td>Tissue-Tek Cryomold Molds</td><td>VWR</td><td>25608-916</td><td>Step 4.1</td></tr></tbody></table>

an in vivo murine sciatic nerve model of perineural invasion

癌细胞通过称为神经侵袭 (PNI) 的过程侵入神经, 癌细胞在神经微环境中增殖和迁移。这种类型的入侵是由多种类型的癌症所表现出来的, 而且在胰腺癌中也经常出现。小鼠胰腺内神经纤维的显微大小使 PNI 在原位鼠模型中的研究更加困难。在这里, 我们描述一个异位的在体内模型的 PNI, 我们注射自体胰腺癌细胞系 Panc02-H7 到小鼠坐骨神经。在该模型中, 麻醉小鼠的坐骨神经被暴露并注入癌细胞。癌细胞从注射点到脊髓的神经向下部侵袭。侵入的坐骨神经, 然后提取和处理与 OCT 的冰冻切片。H & E 和免疫荧光染色这些部分允许量化的入侵程度和变化的蛋白质表达。该模型可应用于 PNI 的多种研究, 考虑了其通用性。使用不同基因修饰的小鼠和/或不同类型的癌细胞, 可以调查 PNI 和不同癌症类型的细胞和分子机制。此外, 治疗药物对神经侵袭的影响可以通过对这些小鼠的治疗来研究。

该方法描述了胰腺癌细胞移植到小鼠坐骨神经中的外科植入, 以建立一个可量化的神经入侵的体内模型。图 1说明了坐骨神经和注射部位的解剖位置。图 2显示了裸鼠的两个坐骨神经。PBS 注入神经 (左) 可与 MiaPaCa-2 癌细胞 (右) 注入的神经进行比较。癌细胞注入的神经被肿瘤浸润, 比用 PBS 注入的神经更厚、更暗。
图 3显示了在野生型和 NCAM 小鼠中, 胰腺癌细胞 Panc02H7 坐骨神经侵袭的可量化差异。NCAM 鼠是粘附分子神经细胞黏附分子1不足13的小鼠。使用成像软件量化了入侵的长度和面积。NCAM 鼠的侵袭性明显降低了7。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56857/56857fig1.jpg"> 图 1: 鼠标坐骨神经和注射部位.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56857/56857fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56857/56857fig2.jpg"> 图 2: 两个小鼠的坐骨神经解剖.一个 PBS 注入神经 (左) 和神经注射 MiaPaCa-2 癌细胞 (右) 显示。黑色箭头显示注射部位。 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56857/56857fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56857/56857fig3.jpg"> 图 3: H & E 在野生型和 NCAM 小鼠中注射 Panc02H7 癌细胞的坐骨神经纵断面.白色箭头表示注射部位和脊髓近端。黑色箭头表示入侵的长度与相应的值 (mm)。缩放栏: 5 毫米 (顶部图像) 和0.2 毫米 (底部图像)。根据入侵的长度和面积来测量神经入侵的数量。数据表示平均值 n = 8 (NCAM1), n = 7 (NCAM1), ** p < 0.005, *** p < 0.0005 使用 t 测试。(Deborde et al的结果, 20167)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56857/56857fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

神经入侵的体内小鼠坐骨神经模型

我们描述了一个体内小鼠神经入侵的模型, 注射自体胰腺癌细胞进入坐骨神经。该模型允许定量的神经入侵程度, 并支持调查的细胞和分子机制的神经入侵。

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

an-in-vivo-murine-sciatic-nerve-model-of-perineural-invasion

Nanyang Technological University

Research

JoVE Journal

Cancer Research

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

12.0K 次观看.  Memorial Sloan Kettering Cancer Center. 我们描述了一个体内小鼠神经入侵的模型, 注射自体胰腺癌细胞进入坐骨神经。该模型允许定量的神经入侵程度, 并支持调查的细胞和分子机制的神经入侵。

视频: An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

在头颈部鳞状细胞癌的神经浸润模型

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

在癌神经侵袭的体外模型:背根神经节型号

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

神经科学

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Vivo模型中的小鸡胆膜模型,用于评估头颈部癌症的围神经入侵

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

医学

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

<meta charset="utf8"></head><body>改良的小鼠神经性疼痛备用神经损伤手术模型

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

改良的小鼠神经性疼痛备用神经损伤手术模型

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Establishing a Three-dimensional Co-culture Model of Murine Dorsal Root Ganglia and Cancer Cells: An In Vitro Model to Study Perineural Cancer Cell Invasion

Source: Huyett, P. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55043" target="_blank">A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the procedure for developing an in vitro perineural cancer cell invasion model by co-culturing mouse dorsal root ganglion and neurotropic cancer cells. The model can be utilized to conduct cancer assays, producing rapid and reproducible results.

建立小鼠背根神经节和癌细胞的三维共培养模型:研究神经周围癌细胞侵袭的体外模型

Source: Huyett, P. et al. A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma. J. Vis. Exp. (2017)
In this video, we demonstrate the ...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

Assays & techniques

Murine Sciatic Nerve Model of Perineural Invasion or PNI: Extraction and Processing of Sciatic Nerve to Understand Invasion by Cancer Cells

Source: Deborde, S. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56857" target="_blank">An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.</a> J. Vis. Exp. (2018)
In this video, we describe the procedure to establish a murine model of perineural invasion by injecting cancer cells into the sciatic nerve. The model can help gain an understanding of the magnitude of nerve invasion for subsequent assessment of anti-cancer drugs.

神经周围浸润或 PNI 的小鼠坐骨神经模型:坐骨神经的提取和加工以了解癌细胞的侵袭

Source: Deborde, S. et al. An In Vivo Murine Sciatic Nerve Model of Perineural Invasion. J. Vis. Exp. (2018)
In this video, we describe the procedure to ...

Cancers of the Nervous System

Ex vivo studies

Murine Perineural Invasion Model: Generating an In Vivo Model to Simulate Perineural Invasion in Mouse Sciatic Nerve

Source: Deborde, S. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56857" target="_blank">An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.</a> J. Vis. Exp. (2018)
In this video, we develop an in vivo mouse model for perineural invasion by injecting pancreatic cancer cells into the sciatic nerve. This model helps quantify the extent of invasion and study the interactions between cancer cells and nerves.

小鼠神经周围浸润模型:生成体内模型以模拟小鼠坐骨神经中的神经周围浸润

Source: Deborde, S. et al. An In Vivo Murine Sciatic Nerve Model of Perineural Invasion. J. Vis. Exp. (2018)
In this video, we develop an in vivo mouse ...

神经入侵的体内小鼠坐骨神经模型

摘要

探索更多视频

此视频中的章节

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

在头颈部鳞状细胞癌的神经浸润模型

疲劳的小鼠模型的外围照射引起

在癌神经侵袭的体外模型:背根神经节型号

Vivo模型中的小鸡胆膜模型,用于评估头颈部癌症的围神经入侵

改良的小鼠神经性疼痛备用神经损伤手术模型

改良的小鼠神经性疼痛备用神经损伤手术模型

建立小鼠背根神经节和癌细胞的三维共培养模型:研究神经周围癌细胞侵袭的体外模型

神经周围浸润或 PNI 的小鼠坐骨神经模型:坐骨神经的提取和加工以了解癌细胞的侵袭

小鼠神经周围浸润模型:生成体内模型以模拟小鼠坐骨神经中的神经周围浸润