成人耳皮肤全安装共焦显微术: 一种研究神经血管分支形态发生和免疫细胞分布的模型系统
Summary
在这里, 我们描述了一个高分辨率全安装成像方法在整个成年小鼠耳皮肤, 这使我们能够想象分支形态发生和模式的周围神经和血管, 以及免疫细胞分布。
Abstract
在这里, 我们提出了一个完整的成人耳皮肤成像技术的协议, 以研究全面的三维神经血管分支形态发生和模式, 以及免疫细胞分布的细胞水平。对成人组织周围神经和血管解剖结构的分析, 为了解创伤愈合等病理条件下的功能性神经血管接线和神经血管变性提供了一些见解。作为一个高度信息的模型系统, 我们把我们的研究集中在成人耳皮肤, 这是容易被解剖。我们的简单和可重现的协议提供了一个准确的描述整个皮肤的细胞成分, 如周围神经 (感觉轴突, 交感神经轴突, 和雪旺细胞), 血管 (内皮细胞和血管平滑肌细胞) 和炎症细胞。我们相信这项议定书将为调查不同病理条件下成人耳皮肤的形态学异常和周围神经和血管的炎症铺平道路。
Introduction
皮肤由三层组成: 表皮、真皮和皮下组织。它已被作为一个模型系统来研究干细胞的维持, 分化和形态发生的发展, 以及再生, 肿瘤发生和炎症的成人。皮肤有丰富的血管化和支配, 使周围神经系统和血管系统的发育协调良好。
我们以前已经展示了一个完整的胚胎皮肤成像技术与多重标记研究完整的周围神经和血管, 包括其蜂窝组件1,2,3,4: 血管中的感觉轴突、交感神经轴突、神经的雪旺细胞、内皮细胞、毛细血管和血管平滑肌细胞 (VSMCs)。在血管生成过程中, 一个主要的毛细管网络经过密集的血管重塑, 发展成为一种分层的血管分支网络。在发育的真皮/皮下组织, 动脉分支与周围的感觉神经和静脉形成相邻的动脉。在分层的血管网络被 VSMCs 彻底覆盖后, 交感神经延伸沿和支配大口径血管1,5,6。尽管发展中的神经和血管系统之间的密切联系具有重要意义, 但一个主要问题是解决成人不同病理情况下神经血管网络的发生情况。三维高分辨率成像是必要的, 以了解发病机制, 以及解剖可识别的分支形态发生和模式。
组织切片染色法分析成年小鼠皮肤的神经细胞和血管形态。其他研究还使用了整个影像的皮肤来可视化周围神经和血管, 除了毛囊, 皮脂腺, 和 arrector 毛肌肉7,8,9。然而, 成人皮肤的厚度使得它很难分析整个深度的皮肤。
在本研究中, 我们开发了一种新的高分辨率的成人耳皮肤的全安装成像, 以克服这些挑战。耳朵皮肤是容易接近的解剖和后续整个安装成像的皮肤在其整个深度。因此, 它是一个简单的和高度重复的方法, 可用于比较三维结构的周围神经和血管系统的皮肤, 与综合量化测量。结果表明, 成人皮肤中保留了大口径血管对周围感觉和交感神经的定位。该协议的目标是可视化分支形态发生, 以及周围神经和血管的模式, 以及在各种情况下, 如炎症和成年小鼠模型的细胞水平上的免疫细胞分布。再生.
Protocol
Representative Results
Discussion
本协议描述成人耳皮肤的全芒 immunonohistochemical 成像, 用于分析神经血管结构和免疫细胞分布。我们认为, 这种方法有许多实验优势, 研究人员的分支形态发生和模式的周围神经和血管, 以及三维分布的皮肤成分, 包括免疫细胞和头发毛囊.图像的结果可以用成像软件进行量化, 进一步定量分析。
正确地制备耳部皮肤对本议定书的成功至关重要。首先, euthanizing 后应仔细解剖耳朵皮…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们感谢吉尔的实验室管理和技术支持, j. 霍金斯和国家卫生研究院 (NIH) 的工作人员50建立了援助老鼠护理的动物设施, 以及里德和 f. Baldrey 的行政援助。还感谢 s Motegi 和 m. Udey 分享他们的耳朵皮肤解剖协议, n. 烧伤的编辑帮助, 以及干细胞和神经血管生物学实验室成员的技术帮助和深思熟虑的讨论。KAITOKU 是日本促进科学协会 (jsp) 的支持。这项工作得到了国家心脏、肺和血液研究所 (HL005702-11 到 Y.M.) 的校内研究计划的支持。
Materials
| 10 x Phosphate Buffered Saline | KD Medical | RGE-3210 | PBS, without Ca2+/Mg2+ |
| Hank’s Balanced Salt Solution | Gibco | 14025-092 | HBSS, with Ca2+/Mg2+ |
| 16% Paraformaldehyde | Electron Microscopy Sciences | 15710 | PFA, fixative, diluted in PBS |
| Triton X-100 | Sigma | X100 | Detergent |
| Normal goat serum | Gibco | 16210064 | Component of blocking/washing buffer |
| Normal donkey serum | Jackson Immuno research | 017-000-121 | Component of blocking/washing buffer |
| Curved fine tweezers | Dumont | RS-5047 | |
| Curved tweezers | Integra Miltex Vantage | V918-782, V918-784 | |
| Filter Unit 0.45 mm | Thermo Scientific | 157-0045 | For filtration |
| 1 mL syringe | Coviden | 8881501400 | For filtration |
| Syringe filter Unit 0.22 mm | Millex-GV | SLGVR04NL | For filtration |
| ProLong Gold | Thermo Scientific | P36934 | Anti-fade mounting medium |
| Nail Polish | Electron Microscopy Sciences | 72180 | For sealing |
| Dissecting microscope | Leica | MZ95 | |
| Confocal microscope | Leica | TCS SP5 | |
| Photoshop CC 2017 | Adobe | Graphics editor software | |
| Illustrator CC 2017 | Adobe | Graphics editor software | |
| Image J | NIH | Image processing software | |
| Anti-PECAM-1 (CD31) antibody | Millipore | MAB1398Z | Hamster IgG, vascular endothelial cell marker, 1:300 |
| Anti-PECAM-1 (CD31) antibody | BD Pharmingen | 553369 | Rat IgG2a kappa, vascular endothelial cell marker, 1:300 |
| Anti-aSMA antibody conjugated with cy-3 | Sigma | C6198 | Mouse IgG2a, vascular smooth muscle cell marker, 1:500 |
| Anti-EphB1 antibody | Santa Cruz | sc-9319 | Goat polyclonal, venous endothelial cell marker, 1:100 |
| Anti-neuron-specific Class III b-tubulin (Tuj1) | Abcam | AB18207 | Tuj1, Rabbit polyclonal IgG, pan-axonal marker, 1:500 |
| Anti-Tuj1 antibody | Covance | MMS-435P | Mouse IgG2a, pan-axonal marker, 1:500 |
| Anti-MBP antibody | Abcam | AB40390 | Rabbit polyclonal IgG, myelination marker, 1:200 |
| Anti-Tyrosine Hydroxylase antibody | Chemicon | AB152 | Rabbit polyclonal, sympathetic neuron marker, 1:500 |
| Anti-Peripherin antibody | Chemicon | AB1530 | Rabbit polyclonal, peripheral neuron marker, 1:1000 |
| Anti-CD11b antibody | Bio-Rad | MCA74G | Rat IgG2b, inflammatory cell marker (macrophages), 1:50 |
| Anti-CD45 antibody | Thermo Fisher Scientific | 14-0451-85 | Rat IgG2b kappa, pan-hematopoietic cell marker, 1:500 |
| Anti-CD3 antibody | Bio-Rad | MCA1477T | Rat IgG1, immune cell marker, 1:100 |
| Anti-CD45R (B220) antibody | Thermo Fisher Scientific | 14-0452 | Rat IgG2a kappa, inflammatory cell marker, 1:200 |
| Anti-GFP antibody | Thermo Fisher Scientific | A11122 | Rabbit polyclonal, 1:300 |
| Anti-GFP antibody | Abcam | Ab13970 | Chicken polyclonal, 1:500 |
| Anti-b-gal antibody | Cappel | 55976 | Rabbit polyclonal, 1:5000 |
| Anti-RFP antibody | Abcam | Ab62341 | Rabbit polyclonal, 1:300 |
| Goat anti-rabbit IgG (H+L) Alexa 488 | Thermo Fisher Scientific | A11034 | Rabbit polyclonal secondary antibody, 1:250 |
| Goat anti-hamster IgG (H+L) Alexa 647 | Jackson Immuno research | 127-605-160 | Hamster polyclonal secondary antibody, 1:250 |
| Goat anti-rat IgG (H+L) Alexa 594 | Jackson Immuno research | 112-585-167 | Rat polyclonal secondary antibody, 1:250 |
| Goat anti-mouse IgG2a Alexa 633 | Thermo Fisher Scientific | A21136 | Mouse IgG2a secondary antibody, 1:250 |
Referencias
- Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin. Cell. 109, 693-705 (2002).
- Mukouyama, Y. S., James, J., Nam, J., Uchida, Y. Whole-mount confocal microscopy for vascular branching morphogenesis. Methods Mol Biol. 843, 69-78 (2012).
- Li, W., Mukouyama, Y. S. Whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo. J Vis Exp. , (2011).
- Yamazaki, T., et al. Tissue Myeloid Progenitors Differentiate into Pericytes through TGF-beta Signaling in Developing Skin Vasculature. Cell Rep. 18, 2991-3004 (2017).
- Mukouyama, Y. S. Vessel-dependent recruitment of sympathetic axons: looking for innervation in all the right places. J Clin Invest. 124, 2855-2857 (2014).
- Li, W., et al. Peripheral nerve-derived CXCL12 and VEGF-A regulate the patterning of arterial vessel branching in developing limb skin. Dev Cell. 24, 359-371 (2013).
- Chang, H., Wang, Y., Wu, H., Nathans, J. Flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology. J Vis Exp. , e51749 (2014).
- Salz, L., Driskell, R. R. Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin. J Vis Exp. , (2017).
- Liakath-Ali, K., et al. Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen. Nat Commun. 5, 3540 (2014).
- Gunawan, M., et al. The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin. Nat Immunol. 16, 505-516 (2015).
- Avci, P., et al. Animal models of skin disease for drug discovery. Expert Opin Drug Dis. 8, 331-355 (2013).
- Jin, H., He, R., Oyoshi, M., Geha, R. S. Animal models of atopic dermatitis. J Invest Dermatol. 129, 31-40 (2009).
- Wagner, E. F., Schonthaler, H. B., Guinea-Viniegra, J., Tschachler, E. Psoriasis: what we have learned from mouse models. Nat Rev Rheumatol. 6, 704-714 (2010).
- Nunan, R., Harding, K. G., Martin, P. Clinical challenges of chronic wounds: searching for an optimal animal model to recapitulate their complexity. Dis Model Mech. 7, 1205-1213 (2014).
- O’Brien, P. D., Sakowski, S. A., Feldman, E. L. Mouse models of diabetic neuropathy. ILAR J. 54, 259-272 (2014).
- Yamazaki, T., et al. Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models. Sci Rep. 8, (2018).
