大鼠模型中的尿道狭窄诱导后进行颊粘膜移植尿道成形术
Summary
在本方案中，在Wistar大鼠中开发了尿道狭窄诱导，然后用颊粘膜移植物进行尿道重建。进行逆行尿道造影和激光多普勒评估，验证尿道重建（狭窄形成后）和移植物放置。
Abstract
尿道重建是泌尿科医生的一个重要专业领域。当需要尿道移植时，颊粘膜被认为是最佳选择，尽管在某些情况下，它不合适或需要优化以修复给定的狭窄。因此，开发创新程序并评估其在实验模型中的假定成功对于满足临床需求至关重要。为此，本研究描述了一种方案，其中通过电烙术诱导Wistar大鼠尿道狭窄。1 周后，使用从下唇采集的颊粘膜移植物进行尿道重建，并以腹侧高嵌方式放置。逆行尿道造影显示，与狭窄诱导后的相应值相比，尿道成形术后的尿道直径显着改善。此外，使用激光多普勒通过血液灌注分析评估移植物的位置。正如预期的那样，深蓝色区域对应于非血管化的颊粘膜移植物。该手术可以成功地模拟尿道损伤和组织调节的正常病理生理过程，以及使用颊粘膜移植物以可重复的方式进行尿道重建，并可作为未来基于组织工程或尿道移植物的研究基础。
Introduction
尿道重建是泌尿外科医生在狭窄、创伤或先天性缺损的情况下处理尿道损伤的主要挑战1.出于治愈目的，尿道成形术是大多数患者的首选治疗方法，长尿道缺损（>2 cm）和前尿道缺损需要某种形式的替代尿道成形术2。许多组织已被用作尿道替代品，包括生殖器或生殖器外区域、膀胱壁粘膜或广泛的颊粘膜2 的全层或裂层皮肤移植物。颊粘膜移植物有几个优点，例如来自潮湿和无毛的环境、易于收获、抗感染、厚上皮、降低假性憩室形成的可能性以及薄层，允许早期吸收和接种3.与皮瓣相比，移植物没有血液供应，取决于受体的血管床才能存活4.
移植物或皮瓣的动物模型已被广泛用于开发或改进手术技术，研究和了解组织生理学、潜在机制和失败原因，以及评估创新治疗策略5,6。虽然较大的动物有助于技术执行，但啮齿动物，即大鼠和小鼠，更容易处理和维护，对疾病有抵抗力，更具成本效益，而且重要的是，具有研究分子机制的工具，这对于测试创新疗法至关重要5,6。在大鼠中使用不同的组织，即皮肤、骨骼、肌肉6、血管5，甚至实体器官7，已经描述了几种皮瓣和移植物模型。然而，在小鼠模型中，对用于尿道重建或组织工程的移植物的研究很少。
然而，转化科学的进步依赖于模仿疾病的动物模型。到目前为止，由于尿道重建在其狭窄后立即进行，因此尚未解决局部病理生理学环境。在此，本研究旨在使用颊粘膜移植物在局部病理生理学环境中进行尿道重建。为此，在重建前 1 周诱导尿道狭窄。该实验模型在大鼠身上进行，可以测试创新疗法，并在未来研究其分子机制和临床优势。
Protocol
所有动物手术均根据指令 2010/63/EU 进行。这些程序得到了机构动物福利机构的批准，该机构获得了葡萄牙动物保护主管当局DGAV的许可（许可证号为0421/000/000/2021）。本研究使用12-14周龄的雄性Wistar Han IGS（Crl：WI（Han）大鼠（400-500g）。这些动物是从商业来源获得的（见 材料表）。 1.溶液的制备 麻醉液用 2.3 mL 美托咪定（1 mg/mL;0.715 mg/kg 体…
Representative Results
共12只雄性Wistar大鼠，体重400-500 g，12-14周龄，用于尿道狭窄诱导。1 周后进行了逆行尿道造影 （RUG1）8，证实了该技术的成功。在狭窄诱导水平上以毫米为单位测量尿道直径。在此之后，在大鼠尿道的腹面进行颊粘膜移植物的尿道成形术。在尿道成形术后14天，将相同的大鼠提交到第二次逆行尿道造影（RUG2），并在移植物放置水平上以毫米为单位测量尿道直径。平均 RUG1 和 RUG2 ?…
Discussion
颊黏膜移植物的尿道成形术是尿道重建的主要基石。然而，应开发创新程序来优化已经描述的程序，并建立新的程序，例如组织工程材料和生物移植物，以减少并发症和发病率。已经发表了几种程序来建立临床前模型和定义手术技术。Souza 等人 1 进行了一项包括 12 只新西兰兔子的研究。进行腹侧纵向皮肤切开，将尿道从白膜中动员出来，然后切除尿道背段，形成缺损。同时，从…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢北里斯本大学医院中心放射科主任João Leitão和北里斯本大学医院中心放射科技术员Catia Fernandes在实现尿道造影方面的合作。
Materials
| Atipamezole | OrionPharma | ANTISEDAN (atipamezole) is indicated for the reversal of the sedative and analgesic effects. | |
| Buprenorphine | RichterPharma | Buprenorphine is a derivative of the opioid alkaloid thebaine that is a more potent (25-40 times) and longer lasting analgesic than morphine. | |
| Carl Zeiss Opmi-1 FC Surgical Stereo Microscope | Carl Zeiss Microscopy, Germany | OPMI 1 FC from ZEISS symbolizes quality, precision and reliability. The manual system is easy to use and delivers high fidelity images with the legendary ZEISS optics. | |
| Carprofen | Zoetis | Carprofen is a non-steroidal anti-inflammatory drug (NSAID) of the propionic acid class. | |
| Catheter 22 G. x 1'' | B Braun | Introcan Safety | IV Catheter of fluorinated ethylene propylene (FEP) with firmer construction for arterial access. |
| Enrofloxacin | Bayer | Enrofloxacin is a fluoroquinolone antibiotic. | |
| Fentanyl | Braun | 3644960 | Fentanyl is a powerful synthetic opioid analgesic. |
| Flumazenil | Fresenius Kabi | Benzodiazepine antagonist is used for the complete or partial reversal of the sedative effects caused by benzodiazepines. | |
| High temperature cautery | Fiab | F7244 | Disposable cautery, sterile, high temperature (1200 °C), 28 mm fine tip. |
| Instillagel gel | Farco-pharma | Cellulose-based lubricant with local anaesthetic and disinfecting properties. | |
| Iopromide | Bayer | Non-ionic injectable contrast medium, with iodine. | |
| Laser Doppler imaging system (perfusion imager moorLDI2-HIR and dedicated software) | MoorLDI-V6.0, Moor Instruments Ltd, Axminster, UK | 5710 | The angiogenesis models uses Laser Doppler imaging to assess blood perfusion in the hind limbs, one of which is ligated surgically. Dedicated measurement and comparison software allows the definition of regions of interest for blood flow assessment on the ischaemic versus non-ischaemic limb to establish a "reperfusion ratio" which can be assessed as often as needed and over a number of days on the same subject. |
| Lubrithal Eye Gel | Dechra | Eye gel used in animals for prevention of dry eyes during anaesthesia. Lubricating and moisturising action on cornea and conjunctiva. | |
| Male Wistar Han IGS (Crl:WI(Han) rats | Charles River Laboratories, Spain | Twelve to fourteen-week-old | |
| Metedomidin | Virbac | 037/01/07RFVPT | Medetomidine is a synthetic drug used as surgical anesthetic. |
| Midazolam | Labesfal | Benzodiazepine medication is used for anesthesia and procedural sedation. | |
| Monoplan Angiography System | Philips Medical Systems | Azurion 7 M20 | A stationary diagnostic fluoroscopic x-ray system specifically designed to optimize the capability of users to visually and quantitatively evaluate the anatomy and function of blood vessels of the heart, brain and other organs, as well as the lymphatic system. |
| Mosquito forceps | Henry Schein | 102-4346 | Hartman-Mosquito Hemostatic Forcep Curved 3-1/2" Stainless Steel |
| Needle Holder | Henry Schein | 100-2570 | Needle holder Mayo-Hegar, stainless steel, 14 cm |
| Ophtalmic Needle Holder | Asico | AE-6143 | Needle holder barranquer most delicate without lock |
| Pentobarbital Sodium | Ecuphar | Pentobarbital Sodium is the sodium salt of pentobarbital used for euthanasia. | |
| Pointed Forceps | Aesculap | BD335R | Microforceps, 0.30 mm tip |
| Polysorb 6.0 | Medtronic (Covidien) | UL-101 | Coated Synthetic Absorbable Suture aimed to reduce the inflammatory reaction in tissues, followed by gradual encapsulation of the suture by fibrous connective tissue. |
| Providone-Iodine | Mylan | Povidone-iodine 10% is an antiseptic drug, used as a disinfectant before and after surgery. | |
| Scalpel Blade nº11 | B Braun | BB511 | Carbon steel, sterile |
| Spring Scissor | Henry Schein | 600-4826 | Surgical scissors 31 castroviejo |
| Surgical Forceps | Aesculap | BD33R | Microforceps, 0.20mm tip |
| Surgical Scissor | Aesculap | MA873R | Micro Iris Scissor, curved shrap tips |
| SurgiPro 7.0 | Medtronic (Coviden) | VP-702-X | Non-Absorbable Monofilament Polypropylene Suture indicated for use in general soft tissue ligation. |
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Cite This Article
de Oliveira, P. S., Rocha, F., Vala, I. S., de Oliveira, P., Ministro, A., Santos, S. C. R. Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model. J. Vis. Exp. (194), e65094, doi:10.3791/65094 (2023).