在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤
Summary
这个协议描述了在使用磁铁的夹紧小鼠耳部皮肤缺血再灌注（IR）模型的诱导。使用定制的活体成像模型，我们研究的体内炎症反应再灌注后。这项技术的发展背后的基本原理是延长白细胞如何皮肤缺血再灌注损伤反应的认识。
Abstract
Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).
Introduction
时，有一个短暂缺氧由于随后组织（再灌注）的随后重新氧合血流量（缺血）的阻塞发生缺血再灌注损伤（IRI）。在皮肤中，缺血 – 再灌注（IR）被认为是起作用的因素，压力性溃疡的病理生理学，其中长期卧床易患长期医院病人受伤的一个。在这些患者中，无论是在皮肤和下面的肌肉经常暴露于施加在骨突出的区域重压力，导致局部损伤的是，如果不进行治疗，可能会变得坏死1。
参与了IRI的损失是双重的。期间缺血，血管的闭塞导致的氧气输送的急剧下降到组织。这导致的ATP和pH值，其中失活参与细胞代谢ATP酶的减少。反过来，细胞钙含量秒杀，并强调或损坏Ç厄尔发生凋亡或坏死2。细胞内的内容，或者损害相关分子模式（湿气）的发布，像HMGB1，有助于炎症反应3。再灌注过程中出现的第二个侮辱。虽然氧和pH水平在再灌注期间恢复，这会导致产生的活性氧（ROS），从而导致细胞内脂质，DNA和蛋白质的氧化。因此，促炎介质被激活，从而衬托涉及免疫细胞向炎症部位2招募次级炎症反应。而生化事件导致对炎症反应的级联已经很好地描述，所述免疫细胞活动的空间和时间调节都不能很好地理解。
在这里，我们描述了如何使用简单的磁性定位鼠耳皮肤一个强大的IR模式。再加上多光子活体成像（MP-IVM），我们建立了一个模型来研究再灌注发生后发生的体内炎症反应。开发和利用这种技术的基本原理是试图实时了解两者间质和浸润细胞对IR的反应。
使用夹紧技术在皮肤侧面的IR的现有模型是高度侵入性的，因为它们需要在皮肤侧面钢板的手术植入，使其低于理想免疫学研究4。类似的非侵入性的夹紧技术已在小鼠皮肤侧翼5,6进行了描述。但是，由于在该方法中，活体成像成分的掺入，我们改为选择耳朵皮肤作为目标的IR部位，因为它绕过由于呼吸运动和成像7,8-期间提供稳定性。此外，跨越间质白细胞亚群是耳朵皮肤和皮肤侧面之间是相同的，虽然数字和比例可能会略有变化9。从而，耳朵皮肤表示理想成象部位。
此外，从这些IRI模型检索大多数数据是有限的，以宏观评价（溃疡分级）和端点炎症指标10微观分析。利用该模型，嗜中性粒细胞中的荧光报道小鼠的皮肤再灌注后的细胞反应的实时可视化被启用。以前发表的活耳成像模型被利用8与另外的修饰（ 图1，图2）。
Protocol
Representative Results
Discussion
意义
IR为皮肤褥疮的主要原因之一。褥疮的早期阶段（I和II）描述的人的皮肤的状态（相对于底层皮下组织和肌肉）。然而，免疫病因的了解仍然缺乏。在这里，我们存在于小鼠耳部皮肤一个简单而强大的IR模式，以消除这一差距。我们通过夹紧两个磁铁和随后的小鼠耳研究磁铁去除（再灌注）后，下游的免疫反应模拟缺血。通过使用磁铁来产生一个固定的时间周期上每个耳朵?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Thomas Graf for providing us with the LysM-eGFP mice.
Materials
| Mice strains | |||
| Lysozyme-GFP C57BL/6 | Thomas Graf, Center for Genomic Regulation | ||
| C57BL/6-C2J | Jackson Laboratories | 000058 | To be crossed with Lysozyme-GFP to generate albino Lysozyme-GFP for skin imaging |
| Name | Company | Catalog Number | Comments |
| Reagents | |||
| PBS | |||
| Viaflex 0.9% (wt/vol) saline | Baxter Healthcare | F8B1323 | |
| Ketamine (100 mg ml−1 ketamine hydrochloride | Parnell | Ketamine is a controlled drug and all relevant local regulations should be followed | |
| Ilium Xylazil-20 (20 mg ml−1 xylazine hydrochloride) | Troy Laboratories | Xylazil-20 is a controlled drug and all relevant local regulations should be followed. | |
| Evans blue (10 mg ml−1 in PBS or saline) | Sigma-Aldrich | 46160 | |
| Ultrapurified water | |||
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Insulin syringe with needle | BD | 328838 | |
| Transfer pipettes | Biologix Research Company | 30-0135 | |
| 3M paper masking tape | 3M | 2214 | |
| Deckglaser microscope cover glass (22 mm × 32 mm) | Paul Marienfeld | 101112 | |
| Curved splinter forceps | Aesculap, B. Braun Melsungen | BD312R | |
| Veet hair removal cream | Reckitt Benckiser | ||
| Medical cotton-tipped applicators | Puritan Medical Products Company | 806-WC | |
| C-fold towels | Kimberly-Clark | 20311 | |
| Kimwipes delicate task wipes | Kimtech Science | 34155 | |
| Gold-plated, N42-grade neodymium magnets, 12mm in diameter and 2mm thick | first4magnets | F656S | |
| Plastic guide, 10cm by 1.5cm (polyvinyl chloride material) | fold in half lengthwise, bind with masking tape and slot magnet in | ||
| High vacuum grease | Dow Corning | ||
| Name | Company | Catalog Number | Comments |
| Microscope | |||
| TriM Scope II single-beam two-photon microscope | LaVision BioTec | ||
| Tunable (680–1,080 nm) Coherent Chameleon Ultra II One Box Ti:sapphire laser (≥3.3 W at 800 nm; pulse length of 140 fs, 80 MHz repetition rate) | Coherent | ||
| Water-dipping objectives (20×, NA = 1.0) | Olympus | XLUMPLFLN20xW | |
| Name | Company | Catalog Number | Comments |
| Miscroscope filter and mirror sets (for imaging GFP, SHG, Evans Blue) | |||
| 495 long-pass | Chroma | T495LPXR | |
| 560 lomg-pass | Chroma | T560LPXR | |
| 475/42 band-pass | Semrock | FF01-475/42-25 | |
| 525/50 band-pass | Chroma | ET525/50m | |
| 655/40 band-pass | Chroma | NC028647 | |
| Name | Company | Catalog Number | Comments |
| Skin-imaging stage platform (refer to diagram for assembly) | |||
| A metal base plate (126 mm × 126 mm × 1 mm) | |||
| A brass platform for the ear (79 mm × 19 mm; 1 mm thickness at side, 0.5 mm thickness in the middle; Fig. 1) with slit (1.7 mm × 1 mm; 1.5 mm away from long edge) | |||
| Two plastic blocks (10 mm in height)—for heat insulation | |||
| Curved holder, for positioning the control thermistor on the ear platform | |||
| Interface cable CC-28 with DIN connector and thermistors, one for the temperature control and the other for the temperature monitor | (Warner Instruments (Harvard Apparatus) | 640106 | connect the interface cable to both resistive heater blocks set at 35°C |
| Resistive heater blocks RH-2 | (Warner Instruments (Harvard Apparatus) | 640274 | Resistive heater blocks can heat the brass ear platform up to over 100 °C within minutes. Ensure that the control thermistor has been properly secured in the holder in order to avoid overheating. |
| Temperature controller TC-344B for the ear platform | (Warner Instruments (Harvard Apparatus) | 640101 | |
| Temperature controller TR-200 for mouse heating pad | Fine Science Tools | 21052-00 | Unit is no longer for sale. Ask manufacturer for alternatives |
| Power supply for TR-200 | Fine Science Tools | 21051-00 | Unit is no longer for sale. Ask manufacturer for alternatives |
| Heating pad | Fine Science Tools | 21060-00 | Unit is no longer for sale. Ask manufacturer for alternatives. |
| Animal rectal probe | Fine Science Tools | 21060-01 | Unit is no longer for sale. Ask manufacturer for alternatives. After connecting the rectal probe and heating pad to the temperature controller TR-200, set the temperature to 37 °C |
| Name | Company | Catalog Number | Comments |
| Coverslip holder | |||
| 2 plastic rods, 1 cm in diameter, 10 cm in length | |||
| 1 plastic adaptor with holes drilled to accommodate rods (refer to diagram) | |||
| 3 plastic tightening screws for keeping plastic rods in place | |||
| 1 metal plate, 6 cm x 2.5 cm, with a 2 cm square cut at 1 end, 2 mm edge away from short edge | |||
| 1 pair of nut and bolt for attaching metal plate to plastic rod | |||
| 1 acrylic base (4 cm x 5 cm x 1.5 cm) with magnet to hold coverslip holder on skin-imaging stage platform. 1 rod is permanently fixed onto base. | |||
| Name | Company | Catalog Number | Comments |
| Imaging analysis software | |||
| Imaris v8.1.2 | Bitplane | ||
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