The overall goal of this procedure is to demonstrate a simple reproducible method of sciatic nerve crush and whole mount analysis of muscle reinnervation after sciatic nerve crush. This is accomplished by first exposing the sciatic nerve, including proper arrangement of surgical retractors to ensure good access to the nerve. Next, crush the nerve with hemostat forceps and mark the nerve with charcoal.
Then remove individual muscles innervated by sciatic nerve from the hind limb. The final step is to dissect individual muscles for whole mount analysis after immuno staining. Ultimately, the results demonstrate that reproducible sciatic nerve crush can be simply performed and muscle hole mounts can be used to analyze subsequent regeneration.
The main advantage of this procedure is that it provides a simple reproducible technique to crush sciatic nerve and subsequently score axonal regeneration. Begin this procedure by anesthetizing the mouse for surgery using a cocktail of 100 milligrams per kilogram of ketamine and 10 milligrams per kilogram of xylazine via intraperitoneal injection to minimize postoperative pain. The mouse also receives a subcutaneous injection of 10 milligrams per kilogram of meloxicam.
Next, shave both hindquarters carefully using a surgical clipper. Then apply nare hair removal cream for complete depletion. Clean the skin with a sterile cotton tipped applicator and Betadine surgical scrub.
After that, apply ophthalmic ointment to the eyes with a sterile cotton tipped applicator. Place the mouse on a clean stainless steel plate over a preheated homeo themic blanket, and keep the animal's temperature at 37 degrees Celsius. Then tape all the limbs down.
Carefully position the hind limb symmetrically so that the knee joint makes a right angle with the body. Subsequently, cover the surgical field with a sterile drape under the microscope. Make a semi-circular incision across the midline.
Separate the skin gently from the underlying musculature and fold it over from time to time. Apply about 0.1 milliliters sterile saline to keep the surgical site moist during the procedure. Next, open the fascial plane between the gluteus maximus and the anterior head of the biceps femoris.
To reveal the sciatic nerve for a surgical control. The contralateral sciatic nerve should be exposed and mobilized, but left intact. Then repose and suture the gluteal musculature.
Expose the experimental sciatic nerve in the same fashion with retractors. To ease visualization. Gently free the sciatic nerve from the surrounding connective tissue using a pair of iridectomy scissors.
Then use the fine 5 45 forceps to place the nerve at the bottom jaw of the super fine hemostatic forceps align the three fascial adjacently not on top of each other. The hemostatic forceps have been engraved with a mark at 1.5 millimeters from their tip. Place the outermost portion of the sciatic in line with this mark before crush.
This ensures a crush of uniform width and that the nerve does not extend beyond the jaws of the hemostatic forceps when flattened due to the crushing force. If the nerve extends beyond the forceps tips, the nerve will only be partially crushed. Use a second pair of hemostatic forceps that has been pre dipped and powdered carbon to mark the crush site.
Crush the nerve at the same crush site for 15 seconds at three clicks. No carbon marking should extend beyond the boundary of the initial crush. This is particularly important if precise marking of the crush site is required to pre dip the forceps in carbon and preclude widespread carbon at the surgical site.
Open the forceps in powdered carbon. Then gently close them. Use sterile gauze to wipe off the carbon on the outside of the hemostats.
Check the forceps under at least three x magnification to verify that the crushing surfaces are evenly coated in powdered carbon. If necessary, they are re dipped and wiped repose and suture the gluteal musculature the same way as the contralateral side. Finally, close the skin incision using the nine millimeter reflex clips.
If nine millimeter reflex clips are found to restrict movement, smaller reflex clips or six aut sutures can be used. Instead, after sacrificing the mouse, remove four muscles. The tibias anterior extensor digitorum longus, peroneus longus, and soleus in the hind limb carefully by pinning through their connective tissues to a black sard coated dish.
Then rinse them in PBS. Subsequently, fix them in 4%Paraform aldehyde for 30 minutes. To dissect each muscle on a black sard coated Petri dish, remove the tendons from it using the iridectomy scissors.
After that, thin the muscles by peeling away interior muscle fibers Carefully preserve the exterior surface of the muscle, including the endplate bands. Then mount the resulting muscles on plus slides with vector shield and 22 by 40 millimeter glass cover slips seal two sides with clear nail polish. When they are properly mounted on the slide, the nplate bands should be in contact with the cover slips.
Here is an example of a crush site in situ at the left hind limb. The crush site is indicated by black carbon. The asterisk marks a branch of the tibial nerve that innervates thigh musculature and serves as a useful landmark.
During crush surgery, the tibial division of the sciatic nerve is outlined in blue, the peroneal and green and the srel and yellow. This semi thin section demonstrates a complete crush performed with the hemostatic forceps. Arrowheads indicate crushed fibers here.
An incomplete crush performed with the angled forceps is indicated in another semi fin section. Arrowhead again indicate crushed fibers while arrows indicate preserved axons. These are the rendered images from the whole mount muscles.
After removal of connective tissue and subsequent thinning, the nplate bands are outlined on each muscle. Shown here is the sous whole mount muscle. 14 days after sciatic nerve crush.
The arrows indicate areas of the neuromuscular junction that have been re innervated by an axon. Here is the demonstration of denervated neuromuscular junction. The gap 43 positive schwan cell processes are indicated by the arrowheads.
No axons are seen in this denervated neuromuscular junction. After watching this video, you should have a good understanding of how to reproducibly crush static nerve and prepare muscle hormone mounts for the analysis of regeneration.
