Simple Methods for Permanent or Transient Denervation in Mouse Sciatic Nerve Injury Models

Abstract

This protocol was validated in: eLife (2021), DOI: 10.7554/eLife.70490Our 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 (nervecrush).These two methods provide a convenient in vivo model to study adaptation to denervation.

Graphical abstract: 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   15. Allow the mice to recover gently from anesthesia in the heating cabinet (25°C).16.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

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 Nervecrush), 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.

Figure 1 .Figure 2 .Video 2 .
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 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 12.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).Published: Jun 05, 2022 Video 5. Sciatic nerve section surgery.For a transient denervation (nerve crush procedure)13.Pinch for 30 s the exposed sciatic nerve with a hemostatic clip (Figure3B) (see Video 6).Video 6. Sciatic nerve crush surgery.

Figure 3 .
Figure 3. Sequential approaches to reach the sciatic nerve.

Figure 4 .
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 αbungarotoxin-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.