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Aug 2021

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Simple Methods for Permanent or Transient Denervation in Mouse Sciatic Nerve Injury Models
小鼠坐骨神经损伤模型中永久性或暂时性去神经的简单方法   

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Abstract

Our ability to move and breathe requires an efficient communication between nerve and muscle that mainly takes place at the neuromuscular junctions (NMJs), a highly specialized synapse that links the axon of a motor neuron to a muscle fiber. When NMJs or axons are disrupted, the control of muscle fiber contraction is lost and muscle are paralyzed. Understanding the adaptation of the neuromuscular system to permanent or transient denervation is a challenge to understand the pathophysiology of many neuromuscular diseases. There is still a lack of in vitro models that fully recapitulate the in vivo situation, and in vivo denervation, carried out by transiently or permanently severing the nerve afferent to a muscle, remains a method of choice to evaluate reinnervation and/or the consequences of the loss of innervation. We describe here a simple surgical intervention performed at the hip zone to expose the sciatic nerve in order to obtain either permanent denervation (nerve-cut) or transient and reversible denervation (nerve-crush). These two methods provide a convenient in vivo model to study adaptation to denervation.


Graphical abstract:



Keywords: Muscle (肌肉), Denervation (去神经支配), Nerve crush (神经挤压), Neuromuscular junction (肌神经接点), Acetylcholine receptor (乙酰胆碱受体), synaptic vesicle glycoprotein 2B (突触小泡糖蛋白 2B)

Background

Neuromuscular disorders constitute a heterogeneous group of more than 200 diseases that present impaired motor function and are often debilitating and prematurely fatal. In vertebrates, the neuromuscular junction (NMJ) is a specialized cholinergic synapse with a complex molecular architecture that ensures reliable conversion of the nerve influx into muscle contraction. In various neuromuscular disorders, genetic alterations result in the failure of the neurotransmission caused by the loss of NMJs, characterized by skeletal muscle weakness and fatigue as observed in Congenital Myasthenic Syndromes, Amyotrophic Lateral Sclerosis, or Spinal Muscular Atrophy.


NMJ integrity requires both healthy presynaptic (motoneuronal) and healthy postsynaptic (muscular) compartments. The organization of the postsynaptic region relies on the heparan sulfate proteoglycan agrin secreted by motoneurons at the NMJ (Huzé et al., 2009 and references therein). Agrin binding to the LRP4/MusK complex induces Acetylcholine Receptors (AchR) clustering at the NMJs, as well as the recruitment of approximately 5 myonuclei that specialize in the expression of the genes coding for NMJs components (Simon et al., 1992; Vaittinen et al., 1999; Méjat et al., 2003; Ravel-Chapuis et al., 2007). Besides the control of postsynaptic gene expression, NMJ structural integrity require a meshwork of intermediate filaments (Mihailovska et al., 2014), linkers of the actin network such as the dystrophin glycoprotein complex (Belhasan and Akaaboune, 2020) and a precise patterning of the microtubule network (Osseni et al., 2020; Ghasemizadeh et al., 2021) that contribute to AChR addressing and clustering at the postsynaptic membrane of myofibers.


Here, we describe how to perform an efficient and rapid intervention on the sciatic nerve to create a permanent or a transient denervation. This method is adapted from standard protocols (Méjat et al., 2005; Bauder and Ferguson, 2012; Morano et al., 2018; Ghasemizadeh et al., 2021). We explain how to track efficiently the sciatic nerve allowing a simple surgical gesture involving a small incision through the skin and muscle. We propose two alternative methods for sciatic nerve injury: i) a permanent denervation (Nerve-cut), ii) a transient and reversible denervation (Nerve-crush). Each method provides specific advantages to evaluate the effects of denervation and/or monitor axon and NMJs regeneration (Morano et al., 2018; Ghasemizadeh et al., 2021).


These methods are very simple and permit the in vivo study of the neuromuscular integrity along with regeneration processes.

Materials and Reagents

  1. Metacam®, 1.5 mg/mL oral suspension for dogs (Boehringer Ingelheim)

  2. Ketamine, Imalgène® 1000 solution for injection (Boehringer Ingelheim)

  3. Buprenorphine, Buprecare® 0.3 mg/mL solution for dog and cat (Axience)

  4. Xylazine, Rompun® 2% (Bayer)

  5. Lurocaine, solution for injection/parenteral route (Vetoquinol®)

  6. Ocry-gel®, TVM-UK Company

  7. PBS (Sigma-Aldrich, catalog number: D1408-500mL)

Equipment

Surgical tools

  1. Tool box (Moria, catalog number: 11210)

  2. One pair of scissors straight pointed 10 cm long (Moria, catalog number: 4877)

  3. Two pairs of Dumont forceps N°5 Swiss model (Moria, catalog number: MC40)

  4. One pair of needle holder clamps (Securimed, catalog number: 100AAA100)

  5. One pair of Dauphin’s scissors (Securimed, catalog number: 2117)

  6. Suture thread (Ethicon, catalog number: JV390)

  7. Hemostatic clip 1,7 cm Glolink Tool (Vetolabo, catalog number: VT-004-413-13)

  8. Syringe 0.3 mL (BD Micro-Fine+)


Equipment

  1. Heating Mat (Acculux Thermolux, catalog number: 461265)

  2. Heating cabinet (ONO V.B., catalog number: RS5)

  3. Isoflurane anesthesia (Ref VI-1586) device (TEM SEGA, MiniTag V1/Evaporator Tec7)

  4. Fur trimmer (Moser, catalog number: VI-1586)

Procedure

  1. Turn on the heating cabinet to allow anesthetized mice to recover properly after the procedure.

  2. Anesthetize mouse with Isoflurane. For this purpose, place the mouse in a clean induction chamber connected to the anesthesia device (Figure 1). Adjust the isoflurane to 3–4% and the oxygen flow to 0.8–1.5 L/min. Once anesthetized the mouse loses all its tonus [For a more detailed procedure, please refer to previously described protocols (Davis, 2008)].



    Figure 1. Isoflurane anesthesia set-up.


  3. Cover Mouse cornea with a drop of Ocrygel® to prevent eyes from drying-up during the procedure.

  4. Anesthetize mouse using an intraperitoneal injection of Ketamine/xylazine mix (100 mg Ketamine/kg weight and 10 mg xylazine/ kg weight) previously diluted in a 10 mM phosphate buffered saline solution. Use a 25 G needle mounted on a 3 mL syringe.

  5. To prevent pain due to the surgical procedure, perform a subcutaneous injection of buprenorphine at the site of incision (0.1 mg/kg weight). Use a 25 G needle mounted on a 3 mL syringe.

  6. Place the mouse on the heating mat during the whole procedure to avoid any hypothermia.


  7. Shave the mouse at the hip (see Video 1 for the zone to shave).

    Video 1. Mouse Hair shaving process.


  8. Make a 3 mm incision in the skin over the trochanter (Figure 2A–2C) (see Video 2 to observe the approach to correctly identify the area to be incised).



    Figure 2. Sequential approaches to reach the sciatic nerve.


    Video 2. Localization of the skin incision to correctly reach the sciatic nerve.


  9. Incise the conjunctive tissue just on the right of the hip to open a small breach through the conjunctive tissue (Figure 2D) (see Video 3 to observe the size area incised).


    Video 3. Skin incision surgery.


  10. Push aside the muscular fascia in order to clearly visualize the nerve (see Video 4 to observe the extracted nerve) (Figure 2F–2E) (Caution: this must be done without any bleeding).


    Video 4. Sciatic nerve isolation.


  11. Apply drops (2–3) of lurocaïne directly on the incised zone to perform a local anesthesia of the nerve. Wait at least 2 min to continue the procedure.


For definitive denervation procedure

  1. Using a fine forceps (Moria N°5 or equivalent) in order to cut a small part of the nerve (1–3 mm) (Figure 3A) (see Video 5).


Video 5. Sciatic nerve section surgery.


For a transient denervation (nerve crush procedure)

  1. Pinch for 30 s the exposed sciatic nerve with a hemostatic clip (Figure 3B) (see Video 6).


    Video 6. Sciatic nerve crush surgery.



    Figure 3. Sequential approaches to reach the sciatic nerve.


  2. Stitch the skin with one or two sutures (Figure 3C–3D) (see Video 7).


    Video 7. Skin stitching surgery.


  3. Allow the mice to recover gently from anesthesia in the heating cabinet (25°C).

  4. Analysis of denervation effects can be appreciated by following NMJs structural changes on Tibialis anterior isolated muscle fibers. Using immunofluorescence approaches, 3 days post-surgery you will observe the absence of SV2B labeling on isolated fibers (Figure 4A–4B). Likewise, for the nerve crush procedure, you will observe the reinnervation after 15 days as SV2B staining’s begins to progressively reappear (Figure 4C–4D). Note that the overall cross section area (CSA) of muscle fibers will decreased rapidly in the course of denervation/regeneration (Ghasemizadeh et al., 2021). Alterations of the NMJs structure such as fragmentation of AchRs, disruption of NMJs area and/or decrease of synaptic myonuclei number above the NMJs can be tracked as previously described (Jones et al., 2016; Osseni et al., 2020; Ghasemizadeh et al., 2021). Analysis of transcriptional changes by monitoring gene expression along the regeneration processes also allow characterization of the time course of NMJs alterations. mRNA or proteins encoded by genes such as MyoD (Myoblast determination protein1), Myog (Myogenin), AchR-α,β,γ,δ,ϵ (Acetylcholine Receptor α,β,γ,δ,ϵ subunits), MITR (MEF2 interacting Transcription Repressor), NRG1-α,β (Neuregulin-α,β) and MusK (Muscle skeletal receptor tyrosine-protein kinase) will be key to determine the correct time course of denervation/reinnervation (Méjat et al., 2005; Thomas et al., 2015; Morano et al., 2018).



    Figure 4. Sciatic nerve denervation procedure effects.

    Isolated fibers of tibialis anterior muscle from 2-months-old WT double-stained with an antibody against synaptic vesicle glycoprotein 2B (SV2B, in green) to label the presynaptic domain of NMJs and with α-bungarotoxin–A594 (in red) to label the postsynaptic domain of NMJs. Representative immunofluorescence images of non-injured nerve (A), definitive denervation procedure (B) after 3 days and transient denervation procedure after 3 days (C) or 18 days (D). Scale bars, 10 µm.

Notes

  1. The main difficulty lay in the precision to locate the area for skin incision (Figure 2A). You must not be too much towards the spine or the knee or both too much above the hip or buttock at the same time; otherwise, it will be very difficult to find the nerve. In fact, the nerve comes to the surface close to the skin just behind the hip as it does for you. Outside of this location, the nerve goes deep into the muscle fascia and becomes very difficult to visualize and grasp.

  2. Note that the efficiency of the procedures is not easily visible as mice show a quasi-normal behavior after the procedure (see Video 8). Nevertheless, as a consequence of the completed procedure (Nerve-cut or Nerve-crush), after waking up, mice drag the denervated leg, as can be seen in Video 8. This situation is definitive for the Nerve-cut procedure whereas, in the case of nerve crush procedure, mice recover gradually the movement of the paw, which appears completely normal 3 weeks after the Nerve-crush procedure.


    Video 8. Behaviors of mice after the procedure.

Acknowledgments

This work was supported by grants from ATIP-Avenir and AFM via the MyoNeurAlp Alliance. We acknowledge contributions of the CELPHEDIA Infrastructure (http://www.celphedia.eu/), and especially the center AniRA of Lyon “Plateau de Biologie Expérimentale de la Souris”. We thank Tiphaine Dorel and members of CIQLE imaging center (Faculté de Médecine Rockefeller, Lyon-Est). All graphic abstracts were created with BioRender.com. These methods were derived from Méjat et al. (2005). This protocol was adapted from our recent work (Ghasemizadeh et al, 2021; DOI: 10.7554/eLife.70490).

Competing interests

The authors declare no competing financial interests.

Ethics

Animal experimentation: Experiments and procedures were performed in accordance with both the guidelines of the local animal ethics committee of the University Claude Bernard - Lyon 1 and French / European legislation on animal experimentation (Directive 2010/63 from European Union) approved by the ethics committee CECCAPP (agreement number D691230303 delivered by the French Ministry of Research).

References

  1. Bauder, A. R. and Ferguson, T. A. (2012). Reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis. J Vis Exp (60).
  2. Belhasan, D. C. and Akaaboune, M. (2020). The role of the dystrophin glycoprotein complex on the neuromuscular system. Neurosci Lett 722: 134833.
  3. Davis, J. A. (2008). Mouse and rat anesthesia and analgesia. Curr Protoc Neurosci Appendix 4: Appendix 4B.
  4. Ghasemizadeh, A., Christin, E., Guiraud, A., Couturier, N., Abitbol, M., Risson, V., Girard, E., Jagla, C., Soler, C., Laddada, L., et al. (2021). MACF1 controls skeletal muscle function through the microtubule-dependent localization of extra-synaptic myonuclei and mitochondria biogenesis. Elife 10: e70490.
  5. Huzé, C., Bauche, S., Richard, P., Chevessier, F., Goillot, E., Gaudon, K., Ben Ammar, A., Chaboud, A., Grosjean, I., Lecuyer, H. A., et al. (2009). Identification of an agrin mutation that causes congenital myasthenia and affects synapse function. Am J Hum Genet 85(2): 155-167.
  6. Jones, R. A., Reich, C. D., Dissanayake, K. N., Kristmundsdottir, F., Findlater, G. S., Ribchester, R. R., Simmen, M. W. and Gillingwater, T. H. (2016). NMJ-morph reveals principal components of synaptic morphology influencing structure-function relationships at the neuromuscular junction. Open Biol 6(12).
  7. Méjat, A., Ramond, F., Bassel-Duby, R., Khochbin, S., Olson, E. N. and Schaeffer, L. (2005). Histone deacetylase 9 couples neuronal activity to muscle chromatin acetylation and gene expression. Nat Neurosci 8(3): 313-321.
  8. Méjat, A., Ravel-Chapuis, A., Vandromme, M. and Schaeffer, L. (2003). Synapse-specific gene expression at the neuromuscular junction. Ann N Y Acad Sci 998: 53-65.
  9. Mihailovska, E., Raith, M., Valencia, R. G., Fischer, I., Al Banchaabouchi, M., Herbst, R. and Wiche, G. (2014). Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn-plectin 1f complexes. Mol Biol Cell 25(25): 4130-4149.
  10. Morano, M., Ronchi, G., Nicolò, V., Fornasari, B. E., Crosio, A., Perroteau, I., Geuna, S., Gambarotta, G. and Raimondo, S. (2018). Modulation of the Neuregulin 1/ErbB system after skeletal muscle denervation and reinnervation. Scientific Reports 8(1): 5047.
  11. Osseni, A., Ravel-Chapuis, A., Thomas, J. L., Gache, V., Schaeffer, L. and Jasmin, B. J. (2020). HDAC6 regulates microtubule stability and clustering of AChRs at neuromuscular junctions. J Cell Biol 219(8).
  12. Ravel-Chapuis, A., Vandromme, M., Thomas, J. L. and Schaeffer, L. (2007). Postsynaptic chromatin is under neural control at the neuromuscular junction. EMBO J 26(4): 1117-1128.
  13. Simon, A. M., Hoppe, P. and Burden, S. J. (1992). Spatial restriction of AChR gene expression to subsynaptic nuclei. Development 114(3): 545-553.
  14. Thomas, J. L., Moncollin, V., Ravel-Chapuis, A., Valente, C., Corda, D., Mejat, A. and Schaeffer, L. (2015). PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression. Mol Cell Biol 35(24): 4110-4120.
  15. Vaittinen, S., Lukka, R., Sahlgren, C., Rantanen, J., Hurme, T., Lendahl, U., Eriksson, J. E. and Kalimo, H. (1999). Specific and innervation-regulated expression of the intermediate filament protein nestin at neuromuscular and myotendinous junctions in skeletal muscle. Am J Pathol 154(2): 591-600.

简介

[摘要] 我们移动和呼吸的能力需要神经和肌肉之间的有效交流,这种交流主要发生在神经肌肉接头(NMJ)上,这是一种高度专业化的突触,将运动神经元的轴突与肌纤维连接起来。当 NMJ 或轴突被破坏时,肌肉纤维收缩的控制就会丧失,肌肉就会瘫痪。了解神经肌肉系统对永久性或暂时性去神经支配的适应性是了解许多神经肌肉疾病的病理生理学的挑战。这里仍然缺乏完全概括体内情况的体外模型,并且通过暂时或永久切断传入肌肉的神经进行的体内去神经仍然是评估再神经支配和/或后果的一种选择方法神经支配的丧失。我们在这里描述了一种在髋部进行的简单手术干预,以暴露坐骨神经,以获得永久性去神经(神经切断)或瞬时和可逆去神经(神经粉碎)。 这两种方法提供了一个方便的体内模型来研究去神经支配的适应。

图形概要:


[背景] 神经肌肉疾病是由 200 多种疾病组成的异质组,这些疾病表现出运动功能受损,并且常常使人衰弱和过早致命。在脊椎动物中,神经肌肉接头 (NMJ) 是一种特殊的胆碱能突触,具有复杂的分子结构,可确保神经流入可靠地转化为肌肉收缩。在各种神经肌肉疾病中,遗传改变导致由NMJ缺失引起的神经传递失败,其特征是骨骼肌无力和疲劳,如在先天性肌无力综合征、肌萎缩性侧索硬化症或脊髓性肌萎缩症中观察到的那样。
NMJ 完整性需要健康的突触前(运动神经元)和健康的突触后(肌肉)隔间。突触后区域的组织依赖于NMJ ( Huzé ) 的运动神经元分泌的硫酸乙酰肝素蛋白多糖集聚蛋白。 等。 , 2009 和其中的参考文献)。集聚蛋白与 LRP4/ MusK复合物的结合诱导乙酰胆碱受体 ( AchR ) 在 NMJs 处聚集,以及募集大约 5 个专门表达编码 NMJs 成分的基因的肌核 (Simon等人,1992 年; Vaittinen 等。 , 1999;梅亚特 等。 , 2003;拉威尔-查普伊 等。 , 2007) 。除了控制突触后基因表达外,NMJ 结构完整性还需要中间丝网 (米哈伊洛夫斯卡 等。 , 2014), 肌动蛋白网络的接头,如肌营养不良蛋白糖蛋白复合物 ( Belhasan和Akaaboune , 2020) 和微管网络的精确模式 ( Osseni 等。 , 2020;加塞米扎德 等。 , 2021) 有助于AChR在肌纤维的突触后膜处寻址和聚集。
在这里,我们描述了如何对坐骨神经进行有效和快速的干预,以产生永久性或暂时性的去神经支配。该方法改编自标准协议 (梅雅特 等人,2005;鲍德和弗格森,2012;莫拉诺 等人,2018 年;加塞米扎德 等人,2021)。我们解释了如何有效地跟踪坐骨神经,从而实现一个简单的手术姿势,包括穿过皮肤和肌肉的小切口。我们提出了两种用于坐骨神经损伤的替代方法: i )永久性去神经(神经切断),ii)瞬时和可逆去神经(神经粉碎)。每种方法都提供了特定的优势来评估去神经支配和/或监测轴突和 NMJs 再生的影响 (莫拉诺 等。 , 2018;加塞米扎德 等。 , 2021)。
这些方法非常简单,可以在体内研究神经肌肉完整性以及再生过程。

关键字:肌肉, 去神经支配, 神经挤压, 肌神经接点, 乙酰胆碱受体, 突触小泡糖蛋白 2B

材料和试剂
1. Metacam ® , 1.5 mg/mL 狗口服混悬剂 ( Boehringer Ingelheim)
2. 氯胺酮,Imalgène ® 1000 注射液(勃林格殷格翰)
3. 丁丙诺啡, Buprecare ® 0.3 mg/mL 狗和猫溶液( Axience )
4. 甲苯噻嗪,Rompun ® 2% (拜耳)
5. Lurocaine,注射/肠胃外途径溶液( Vetoquinol ® )
6. Ocry - gel ® , TVM-UK 公司
7. PBS ( Sigma-Aldrich,目录号:D1408-500mL)


设备


手术工具
1. 工具箱( Moria ,目录号:11210)
2. 一把剪刀直尖10厘米长( Moria ,目录号:4877)
3. 两对 Dumont 镊子 N°5 Swiss 型号( Moria ,目录号:MC40)
4. 一对持针器夹( Securimed ,目录号:100AAA100)
5. 一把 Dauphin 的剪刀( Securimed ,目录号:2117)
6. 缝合线(Ethicon,目录号:JV390)
7. 止血夹1,7 cm Glolink Tool( Vetolabo ,目录号:VT-004-413-13)
8. 注射器 0.3 mL(BD Micro-Fine+)


设备
1. 加热垫( Acculux Thermolux ,目录号:461265)
2. 加热柜(ONO VB,目录号:RS5)
3. 异氟醚麻醉 (Ref VI-1586) 装置 (TEM SEGA, MiniTag V1/Evaporator Tec7)
4. 毛皮修剪器(Moser,目录号:VI-1586)


程序


1. 打开加热柜,让麻醉小鼠在手术后正常恢复。
2. 用异氟醚麻醉小鼠。为此,将鼠标放在连接到麻醉装置的干净感应室中(图 1)。将异氟醚调整为3-4 %,将氧气流量调整为 0.8-1.5 L /min。一旦被麻醉,老鼠就会失去所有的紧张[有关更详细的程序,请参阅先前描述的协议 (Davis, 2008)]。


 
图 1.异氟醚麻醉设置。


3. 用一滴Ocrygel ®覆盖小鼠角膜,以防止在手术过程中眼睛干燥。
4. 麻醉 小鼠使用先前在 10 mM 磷酸盐缓冲盐溶液中稀释的氯胺酮/甲苯噻嗪混合物(100 毫克氯胺酮/公斤体重和 10 毫克甲苯噻嗪/公斤体重)腹腔注射。使用安装在 3 mL 注射器上的 25 G 针头。
5. 为防止因手术引起的疼痛,请在切口部位皮下注射丁丙诺啡(0.1 毫克/千克体重)。使用安装在 3 mL 注射器上的 25 G 针头。
6. 在整个过程中将鼠标放在加热垫上,以避免体温过低。
7. 在臀部剃须鼠标(请参阅视频 1了解要剃须的区域)。


 
视频 1. 鼠标剃毛过程。


8. 在转子上方的皮肤上做一个 3 mm 的切口(图 2A - 2C)(参见视频 2 ,观察正确识别要切开区域的方法)。


 
图 2.到达坐骨神经的顺序方法。


 
视频 2. 皮肤切口的定位以正确到达坐骨神经。


9. 切开臀部右侧的结膜组织,通过结膜组织打开一个小缺口(图 2D)(请参阅视频 3以观察切开的大小区域)。


 
视频 3.皮肤切口手术。


10. 推开肌肉筋膜,以便清楚地看到神经(参见视频 4观察提取的神经)(图 2F - 2E)(注意:必须在没有任何出血的情况下完成此操作)。


 
视频 4. 坐骨神经隔离。


11. 将卢卡因滴 (2 – 3)直接涂抹在切口区域,对神经进行局部麻醉。等待至少 2 分钟以继续该过程。


用于最终去神经手术
12. 使用细镊子( Moria N°5 或同等产品)以切割一小部分神经( 1-3毫米)(图 3A)(参见视频 5 )。


 
视频 5. 坐骨神经切片手术。


对于短暂的去神经支配(神经挤压手术)
13. 用止血夹夹住暴露的坐骨神经 30 秒(图 3B)(参见视频 6 )。


 
视频 6. 坐骨神经挤压手术。




 
图 3.到达坐骨神经的顺序方法。


14. 用一根或两根缝线缝合皮肤(图 3C - 3D)(参见视频 7) 。


 
视频 7. 皮肤缝合手术。


15. 让小鼠在加热柜 (25°C) 中从麻醉中轻轻恢复。
16. 胫骨前分离肌纤维的结构变化,可以了解去神经作用的分析。使用免疫荧光方法,术后 3 天,您将观察到孤立纤维上没有 SV2B 标记(图 4A - 4B)。同样,对于神经挤压过程,您将在 15 天后观察到神经再支配,因为 SV2B 染色开始逐渐重新出现(图 4C - 4D)。请注意,肌肉纤维的总横截面积 (CSA) 在去神经/再生过程中会迅速下降 (加塞米扎德 等。 , 2021)。如前所述,可以跟踪NMJ 结构的改变,例如 AchR 的碎片、 NMJ区域的破坏和/或 NMJ 上方的突触肌核数量减少 (琼斯等人,2016; Osseni 等。 , 2020;加塞米扎德 等。 , 2021)。通过监测再生过程中的基因表达来分析转录变化也可以表征NMJ 改变的时间过程。由MyoD等基因编码的 mRNA 或蛋白质 (成肌细胞测定蛋白 1)、 Myog (肌细胞生成素) 、 AchR - α 、 β 、 γ 、 δ 、 ε (乙酰胆碱受体α 、 β 、 γ 、 δ 、 ε亚基)、 MITR (MEF2 相互作用转录抑制因子)、 NRG1 - α 、 β (神经调节蛋白- α 、 β )和MusK (肌肉骨骼受体酪氨酸蛋白激酶)将是确定去神经/再神经支配正确时间过程的关键 (梅雅特 等。 , 2005;托马斯等人。 , 2015;莫拉诺 等。 , 2018)。


 
图 4. 坐骨神经去神经手术效果。
分离的胫骨前肌纤维,用针对突触小泡糖蛋白 2B(SV2B,绿色)的抗体进行双染色,以标记 NMJ 的突触前结构域,并用 α-银环蛇毒素-A594(红色)标记NMJ 的突触后结构域。未受伤神经 (A)、3 天后的最终去神经手术 (B) 和 3 天 (C) 或 18 天 (D) 后的瞬时去神经手术的代表性免疫荧光图像。比例尺,10 µm。


笔记


1. 主要困难在于精确定位皮肤切口区域(图 2A)。你不能太靠近脊椎或膝盖,或者同时太高过臀部或臀部;否则,将很难找到神经。事实上,就像它为您所做的那样,神经会靠近臀部后面的皮肤到达表面。在这个位置之外,神经深入肌肉筋膜,变得非常难以观察和掌握。
2. 请注意,程序的效率不容易看到,因为小鼠在程序后表现出准正常行为(参见视频 8 )。然而,作为完成程序(神经切断或神经粉碎)的结果,醒来后,老鼠拖着去神经的腿,如视频 8所示。这种情况对于神经切断手术是确定的,而在神经挤压手术的情况下,小鼠逐渐恢复爪子的运动,这在神经挤压手术后 3 周显得完全正常。


 
视频8. 小鼠手术后的行为。


致谢


这项工作得到了 ATIP-Avenir 和 AFM 通过MyoNeurAlp联盟的资助。我们感谢 CELPHEDIA 基础设施 ( http://www.celphedia.eu/ ) 的贡献,特别是里昂的AniRA中心“Plateau de Biologie ” 实验de la Souris”。我们感谢Tiphaine Dorel 和 CIQLE 成像中心 ( Faculté de Médecine Rockefeller, Lyon-Est) 的成员。所有图形摘要都是使用 BioRender.com 创建的。这些方法源自Méjat 等。 (2005 年)。该协议改编自我们最近的工作 (加塞米扎德 等, 2021; DOI:10.7554/eLife.70490)。


利益争夺


作者声明没有相互竞争的经济利益


伦理


动物实验:实验和程序均按照 Claude Bernard - Lyon 1 大学当地动物伦理委员会的指导方针和伦理委员会批准的法国/欧洲动物实验立法(欧盟指令 2010/63)进行CECCAPP(法国研究部提供的协议号 D691230303)。


参考


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Copyright Osseni et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Osseni, A., Thomas, J. L., Ghasemizadeh, A., Schaeffer, L. and Gache, V. (2022). Simple Methods for Permanent or Transient Denervation in Mouse Sciatic Nerve Injury Models. Bio-protocol 12(11): e4430. DOI: 10.21769/BioProtoc.4430.
  2. Ghasemizadeh, A., Christin, E., Guiraud, A., Couturier, N., Abitbol, M., Risson, V., Girard, E., Jagla, C., Soler, C., Laddada, L., et al. (2021). MACF1 controls skeletal muscle function through the microtubule-dependent localization of extra-synaptic myonuclei and mitochondria biogenesis. Elife 10: e70490.
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