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Denervation of Mouse Lower Hind Limb by Sciatic and Femoral Nerve Transection
通过坐骨和股神经横切对小鼠下后肢进行去神经试验   

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Abstract

The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. Reports from salamander models of appendage regeneration (Singer, 1952; Singer, 1947; Kumar et al., 2007), and of human clinical skin and nail problems associated with spinal cord injury patients (Stover et al., 1994) suggest that appendage regeneration may have an important nerve component. To explore this question, we have generated hind limb tissues devoid of nerve supply. This protocol, combined with multi-color ‘Rainbow’ reporter mouse lines permits single cell clonal analysis and genetic lineage tracing studies in the absence of nerve supply (Rinkevich et al., 2014), exposing nerve requirements on cellular replacement and differentiation during tissue growth, maintenance, and regeneration.

Keywords: Denervation(失神经支配), Peripheral Nervous System(外周神经系统), Tissue Maintenance(组织维修), Limb Regeneration(肢体再生)

Materials and Reagents

  1. Gauze pad
  2. Sterile drapes
  3. 4-0 plain gut suture, 18”, PS-4 needle (Ethicon)
  4. Handheld cautery: high temperature cautery fine tip (Bovie Medical, model: AA01 )
  5. Wild-type CD-1 mice (8-12 weeks old) (Charles River Laboratories International)
  6. Isoflurane (isothesia) (Henry Schein Animal Health)
  7. Veterinary ointment (Dechra, Puralube® Ophthalmic Ointment)
  8. 10% povidone iodine prep solution (Dynarex, catalog number: 1413 )
  9. 0.3 mg/ml buprenorphine (Buprenex) (Reckitt Benckiser)

Equipment

  1. Electric hair clippers
  2. Scissors
  3. CO2 chamber
  4. Far infrared warming pad (Kent Scientific)

Procedure

  1. Procedure description
    1. Ensure that adequate institutional animal care approvals are in place for the denervation procedure. Induce anesthesia with 2-3% inhaled isoflurane. Apply ophthalmic ointment to both eyes in order to prevent desiccation of the cornea during surgery.
    2. Use the toe pinch maneuver to ensure that the depth of anesthesia is adequate.
    3. Prior to starting the surgery, administer 0.1 mg/kg buprenorphine subcutaneously.
    4. Sciatic Nerve denervation
      1. Place the mouse in prone position on a clean, absorbent surface on top of a regulated heating pad.
      2. Remove hair from the posterior thigh and lower back of the mouse with electric clippers (Figure 1).


        Figure 1. The posterior surgical site for sciatic nerve exposure. Shaving an area of adequate size prevents contamination of the surgical field.

      3. Aseptically prepare the shaved skin with povidone iodine solution and drape with sterile drapes. Make sure to maintain sterile technique during the entire surgery. Antibiotics are not needed as long as the operative field remains sterile.
      4. Using sterile technique, make an incision parallel with the femur in the skin of the dorsal thigh. Use small sharp scissors to spread the tissue underneath the skin in order to mobilize the skin away from the underlying muscle. It is helpful to use self-securing skin hooks to open the operative field.
      5. The femur will be visible within the muscle (Figure 2; arrowheads indicate the femur.). Using the sharp scissors, carefully divide the muscle parallel to and just inferior to the femur. This will reveal the sciatic nerve. (Figure 3; arrowheads indicate the sciatic nerve.). The skin hooks can be inserted into the muscle to improve exposure of the nerve.


        Figure 2. Exposure of the femur. The sciatic nerve is not initially visible but is located inferior and deep to the surface of the femur seen here.


        Figure 3. Exposure of the sciatic nerve. Exposing the nerve requires delicate dissection with fine scissors.

      6. Obtain denervation by cutting away the 5 mm section of the sciatic nerve that is in the center of the operative field (Figure 4; P, proximal end. D, distal end). Removing 5 mm of the nerve rather than simply cutting prevents healing and regeneration of the nerve.


        Figure 4. Division of the sciatic nerve. Removing 5 mm of the nerve prevents regeneration.

      7. Cauterize the cut end of the proximal nerve with the hand-held cautery for 1 sec to further prevent regeneration. (In other words, cauterize the end of the nerve that is still attached to the spine, rather than the end attached to the distal limb.).
      8. If there is any bleeding, hold pressure with a gauze to obtain hemostasis. Close the skin incision with several interrupted 4-0 absorbable sutures (Figure 5).


        Figure 5. Closure of the posterior incision. Sutures should be spaced closely enough that no gaps remain in the incision.

    5. Femoral nerve denervation 
      1. Place the mouse in supine position on a clean, absorbent surface on top of a regulated heating pad.
      2. Shave the hair from the lower abdomen, groin, and entire thigh on the side to be operated on (Figure 6).


        Figure 6. The anterior surgical site for femoral nerve access. Shave hair past the midline and halfway to the anterior limbs.

      3. Aseptically prepare the shaved skin with povidone iodine solution and drape with sterile drapes. Make sure to maintain sterile technique during the entire surgery. Antibiotics are not needed as long as the operative field remains sterile.
      4. Make an incision parallel to the femur extending almost to the mouse’s midline. Using small sharp scissors, spread underneath the skin in order to loosen it from the underlying muscle.
      5. The femoral nerve, artery, and vein will be immediately visible running as a bundle parallel to the femur (Figure 7; arrowheads indicate the bundle.). Using the small sharp scissors with delicate movements, free the proximal part of the femoral nerve from the tissue surrounding it (Figure 8; arrowheads indicate the proximal part of the femoral nerve draped over the scissors.). Cut away a 5 mm segment of the nerve near the abdominal wall and use the hand-held cautery to cauterize for 1 sec the cut end of the proximal part of the nerve.


        Figure 7. Exposure of the femoral nerve and artery. Separating the proximal femoral nerve from the artery and vein requires very delicate dissection using fine scissors.


        Figure 8. Exposure of the proximal femoral nerve. The portion of the nerve to be divided is seen draped over the scissors. Be careful to avoid shearing the femoral blood vessels.

      6. Be careful not to disturb the femoral artery and vein. If bleeding from these vessels is accidentally caused, apply firm pressure with a gauze pad. If the bleeding cannot be stopped after 5 min of holding pressure, the animal should be euthanized immediately. Our typical method of euthanasia is to place the still-anesthetized mouse into a CO2 chamber until respirations cease, and to then perform cervical dislocation. However, it is best to consult with an institutional veterinarian before starting the procedure to determine the preferred method for euthanasia.
      7. Close the skin incision with several interrupted 4-0 absorbable sutures.

  2. Post-procedure care monitoring
    1. After surgery, allow the mouse to recover in a paper-lined cage on top of a heating pad. Monitor the respiratory rate and pattern, response to stimuli, and ability to resume normal activity every 15 min until fully awake (Figure 9). Once the mouse is fully recovered from anesthesia and is ambulatory, place it into a clean home cage. Make sure to clean most of the iodine off of the skin and fur using warm water before the mouse is awake.


      Figure 9. A mouse recovering from surgery. The mouse is placed onto clean paper towels to avoid aspiration of cage bedding. Monitor the mouse until it is able to ambulate.

    2. After the inhaled anesthetic is stopped, the mouse’s respiratory rate is expected to return to the normal rate of 80-200 and it should begin responding to stimuli within 5 min and resuming normal activity within 10 min. A mouse whose respiratory rate stays consistently below 60 respirations per minute, who does not respond to stimuli after 5 min, or who does resume normal activity after 10 min may not recover and should be considered for euthanasia. 

  3. Early euthanasia criteria
    For one week after surgery, monitor the mice daily for their general appearance, activity level, weight loss, signs of infection, and development of a large fluid-filled cyst in the region. If these signs develop, consult with a veterinarian or euthanize the animal.

  4. Procedure endpoint
    Based on the requirements of the experiment, the mice are followed for varying amounts of time before being euthanized and the limb harvested. In our group we wait 8-12 weeks before harvesting the tissue.

Notes

This protocol completely removes any sciatic nerve input to the hind limb. However, femoral nerve branches innervate cutaneous tissues of the leg and foot, as well as ligaments, joints and blood vessels and may overlap with territories supplied by the sciatic nerve. We therefore wished to ensure that complete denervation on hind limb tissues took place, and that femoral nerve supply does not substitute sciatic nerve requirements (electrically, chemically, or functionally) after sciatic denervation. To accomplish this, surgical denervation of both sciatic and femoral nerves was performed, and two weeks prior to digit tip amputations to allow full nerve degeneration to take place. Complete nerve degeneration can be verified by monitoring beta 3 tubulin immunoreactivity on histological sections of denervated hind limbs. While ablation of sciatic innervation alone denervates the distal limb, femoral ablation was performed to ensure that no nerve regeneration from the proximal limb would occur during the regeneration time interval. Additionally, an incision was made in the ventral skin of the thigh and the femoral fascial sheath. Femoral denervation was achieved by cutting a 5 mm section of the femoral nerve, leaving the femoral artery intact. Sham surgeries were performed by exposing the nerves without cutting them.

Acknowledgments

Y.R. was supported by the Machiah Foundation Fellowship, the Siebel Stem Cell Institute and the Thomas and Stacey Siebel Foundation (1119368-104-GHBJI), the Human Frontier Science Program (HFSP) Long Term Fellowship, and partly by the HFSP Career Development Award (CDA00017/2016) and from the German Research Foundation (DFG, RI 2787/1-1 AOBJ: 628819). C.D.M. is supported by the American College of Surgeons Resident Research Scholarship.

References

  1. Kumar, A., Godwin, J. W., Gates, P. B., Garza-Garcia, A. A. and Brockes, J. P. (2007). Molecular basis for the nerve dependence of limb regeneration in an adult vertebrate. Science 318(5851): 772-777.
  2. Rinkevich, Y., Montoro, D. T., Muhonen, E., Walmsley, G. G., Lo, D., Hasegawa, M., Januszyk, M., Connolly, A. J., Weissman, I. L. and Longaker, M. T. (2014). Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration. Proc Natl Acad Sci U S A 111(27): 9846-9851.
  3. Singer, M. (1947). The nervous system and regeneration of the forelimb of adult Triturus; the relation between number of nerve fibers and surface area of amputation. J Exp Zool 104(2): 251-265.
  4. Singer, M. (1952). The influence of the nerve in regeneration of the amphibian extremity. Q Rev Biol 27(2): 169-200.
  5. Stover, S. L., Hale, A. M. and Buell, A. B. (1994). Skin complications other than pressure ulcers following spinal cord injury. Arch Phys Med Rehabil 75(9): 987-993.
  6. Stover, S. L., Omura, E. F. and Buell, A. B. (1994). Clinical skin thickening following spinal cord injury studied by histopathology. J Am Paraplegia Soc 17(2): 44-49.

简介

周围神经系统对哺乳动物附属物中组织替换的需求和影响仍然很大程度上未定义。 来自附肢再生的蝾螈模型的报告(Singer,1952; Singer,1947; Kumar等人,2007),以及与脊髓损伤患者相关的人临床皮肤和指甲问题 et al。,1994)表明附肢再生可能具有重要的神经组分。 为了探索这个问题,我们生成没有神经供应的后肢组织。 该方案与多色"彩虹"记者小鼠品系组合允许在缺少神经供应的情况下的单细胞克隆分析和遗传谱系追踪研究(Rinkevich等人,2014),暴露神经需求 在组织生长,维持和再生期间的细胞替代和分化。

关键字:失神经支配, 外周神经系统, 组织维修, 肢体再生

材料和试剂

  1. 纱布垫
  2. 无菌罩
  3. 4-0平肠缝合,18",PS-4针(Ethicon)
  4. 手持烧灼:高温灼热细尖(Bovie Medical,型号:AA01)
  5. 野生型CD-1小鼠(8-12周龄)(Charles River Laboratories International)
  6. 异氟烷(异性)(Henry Schein Animal Health)
  7. 兽用软膏(Dechra,Puralube ?眼用软膏)
  8. 10%聚维酮碘制备溶液(Dynarex,目录号:1413)
  9. 0.3mg/ml丁丙诺啡(Buprenex)(Reckitt Benckiser)

设备

  1. 电动剪刀
  2. 剪刀
  3. CO 2室
  4. 远红外加热垫(Kent Scientific)

程序

  1. 程序说明
    1. 确保去神经治疗程序获得足够的机构动物护理批准。用2-3%吸入异氟烷诱导麻醉。眼睛软膏应用于双眼,以防止手术期间角膜的干燥。
    2. 使用脚趾捏趾操作,以确保麻醉深度足够。
    3. 在开始手术前,皮下施用0.1mg/kg丁丙诺啡
    4. 神经神经去神经
      1. 将鼠标放置在一个干净,吸湿的表面上的调节加热垫顶部的俯卧位。
      2. 用电动剪刀从大鼠后大腿和下背部摘除毛发(图1)。


        图1.坐骨神经暴露的后部手术部位。刮除足够大小的区域可防止手术部位的污染。

      3. 无菌制备剃光的皮肤与聚维酮碘溶液和悬垂无菌帷幕。确保在整个手术过程中保持无菌技术。只要手术区保持无菌,就不需要抗生素
      4. 使用无菌技术,切开平行的股骨在背大腿的皮肤。使用小的锋利的剪刀传播组织在皮肤下面,以动员皮肤远离下面的肌肉。使用自固定皮肤钩打开手术部位很有帮助。
      5. 股骨将在肌肉内可见(图2;箭头指示股骨)。使用锋利的剪刀,小心地划分肌肉平行于和刚好在股骨。这将揭示坐骨神经。 (图3;箭头表示坐骨神经)。皮肤钩子可以插入肌肉,以改善神经的暴露

        图2.股骨的暴露。坐骨神经最初不可见,但位于此处所见的股骨表面的下方和深处。


        图3.坐骨神经的暴露。暴露神经需要用细剪刀进行精细的解剖。

      6. 通过切除位于手术区域中心的坐骨神经的5mm截面获得去神经(图4; P,近端D,远端)。去除5毫米的神经,而不是简单的切割,可以防止神经的愈合和再生

        图4.坐骨神经的分割。去除5毫米的神经阻止再生。

      7. 用手持烧灼器灼烧近端神经的切断端1秒,以进一步防止再生。 (换句话说,烧灼仍附着在脊柱上的神经末端,而不是连接到远端肢体的末端)。
      8. 如果有任何出血,用纱布保持压力以获得止血。用几个中断的4-0可吸收缝线闭合皮肤切口(图5)。


        图5.后切口闭合。缝合线应足够靠近,切口中无间隙。

    5. 股神经去神经支配
      1. 将鼠标放在仰卧位置在清洁,吸收表面上的调节加热垫的顶部。
      2. 从下腹部,腹股沟和整个大腿的侧面剃刮头发进行手术(图6)。


        图6.用于股神经接近的前部手术部位。将头发刮过中线,并将其刮到前肢的中途。

      3. 无菌制备剃光的皮肤与聚维酮碘溶液和悬垂无菌帷幕。确保在整个手术过程中保持无菌技术。只要手术区保持无菌,就不需要抗生素
      4. 做一个切口平行于股骨几乎延伸到小鼠的中线。使用小锋利的剪刀,铺在皮下,以从下面的肌肉松开
      5. 股骨神经,动脉和静脉将立即可见,作为平行于股骨的束运行(图7;箭头表示束)。使用具有精细运动的小尖锐剪刀,从围绕它的组织释放股神经的近端部分(图8;箭头表示被夹在剪刀上的股神经的近端部分)。切除腹壁附近的5 mm神经节段,并使用手持烧灼器灼烧神经近端部分的切断端1秒钟。


        图7.股骨神经和动脉的暴露。从股动脉和静脉分离股骨近端神经需要使用细剪刀进行非常精细的解剖。

        图8.近侧股神经的暴露。看到被分开的神经的部分覆盖在剪刀上。小心避免剪切股骨血管。

      6. 小心不要打扰股动脉和静脉。如果偶然发生这些血管的出血,用纱布垫施加稳定的压力。如果出血不能在保持压力5分钟后停止,则应立即对动物实施安乐死。我们的典型安乐死方法是将静止麻醉的小鼠放入CO 2室中,直到呼吸停止,然后进行颈椎脱臼。然而,最好在开始手术前咨询机构兽医,以确定安乐死的首选方法。
      7. 用几个中断的4-0可吸收缝线闭合皮肤切口。

  2. 术后护理监护
    1. 手术后,允许小鼠在加热垫顶部的纸衬里笼中恢复。监测呼吸频率和模式,对刺激的反应,以及每15分钟恢复正常活动的能力,直到完全清醒(图9)。一旦小鼠完全从麻醉中恢复并可走动,将其放入一个干净的家笼。确保在鼠标清醒之前用温水清洁皮肤和毛皮上的大部分碘。


      图9.鼠标从手术中恢复。将鼠标放在干净的纸巾上,以避免吸入笼子。监视鼠标,直到它能够移动。

    2. 吸入麻醉停止后,小鼠的呼吸频率预计将恢复到80-200的正常速率,并应在5分钟内开始响应刺激,并在10分钟内恢复正常活动。呼吸频率持续低于每分钟60次呼吸,5分钟后不响应刺激或10分钟后恢复正常活动的小鼠可能无法恢复,应考虑进行安乐死。 

  3. 早期安乐死标准
    手术后一周,每天监测小鼠的一般外观,活动水平,体重减轻,感染迹象,以及该区域大型充满液体的囊肿的发展。如果这些迹象发展,咨询兽医或安乐死动物
  4. 程序端点
    基于实验的要求,在安乐死和收获肢体之前,对小鼠进行不同量的时间。在我们的组中,我们等待8-12周收获组织

笔记

此协议完全删除任何坐骨神经输入到后肢。然而,股神经分支支配腿和脚的皮肤组织以及韧带,关节和血管,并且可能与坐骨神经提供的区域重叠。因此,我们希望确保在后肢组织上进行完全去神经支配,并且在坐骨神经去神经支配后,股神经供应不能代替坐骨神经的需求(电学,化学或功能)。为了实现这一点,进行坐骨神经和股骨神经的手术去神经,并且在数字尖端截肢之前两周允许发生完全神经变性。完全神经变性可以通过监测β3微管蛋白在去神经后肢的组织切片上的免疫反应性来验证。虽然单独的坐骨神经支配消融使远端肢体不能神经支配,但是执行股骨消融以确保在再生时间间隔期间不会发生来自近端肢体的神经再生。此外,在大腿和股骨筋膜鞘的腹部皮肤中进行切口。通过切割股骨神经的5mm截面,留下股动脉完整来实现股神经去神经。假手术通过暴露神经而不切割它们来进行。

致谢

Y.R.由Machiah基金会奖学金,Siebel干细胞研究所和Thomas和Stacey Siebel基金会(1119368-104-GHBJI),人类前沿科学计划(HFSP)长期奖学金,以及部分由HFSP职业发展奖(CDA00017/2016)和来自德国研究基金会(DFG,RI 2787/1-1 AOBJ:628819)。 C.D.M.由美国外科医师学院驻地研究奖学金支持。

参考文献

  1. Kumar,A.,Godwin,JW,Gates,PB,Garza-Garcia,AA和Brockes,JP(2007)。  成年脊椎动物中肢体再生的神经依赖性的分子基础。 318(5851):772-777。 br />
  2. Rinkevich,Y.,Montoro,DT,Muhonen,E.,Walmsley,GG,Lo,D.,Hasegawa,M.,Januszyk,M.,Connolly,AJ,Weissman,IL和Longaker,MT(2014) 克隆分析揭示哺乳动物后肢组织的神经依赖性和独立作用维护和再生。 Proc Natl Acad Sci USA 111(27):9846-9851。
  3. Singer,M.(1947)。  神经系统和再生的成人Triturus的前肢;神经纤维数目和截肢表面积之间的关系。 104 104(2):251-265。
  4. Singer,M。(1952)。  神经再生的两栖肢。 27(2):169-200。
  5. Stover,SL,Hale,AM and Buell,AB(1994)。  除了脊髓损伤后的压力性溃疡以外的皮肤并发症。 Arch Phys Med Rehabil 75(9):987-993。
  6. Stover,SL,Omura,EF和Buell,AB(1994)。  通过组织病理学研究的脊髓损伤后的临床皮肤增厚。 J Am Paraplegia Soc 17(2):44-49。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rinkevich, Y., Montoro, D. T., Muhonen, E., Lo, D., Hasegawa, M., Marshall, C. D., Walmsley, G. G., Connolly, A., Weissman, I. L. and Longaker, M. T. (2016). Denervation of Mouse Lower Hind Limb by Sciatic and Femoral Nerve Transection. Bio-protocol 6(13): e1865. DOI: 10.21769/BioProtoc.1865.
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