The overall goal of this procedure is to examine the influence of nerves on normal and pathological skin. This method can be used to answer key questions in normal skin biology, such as whether cutaneous nerves affect homeostasis and disease. The main advantage of this technique is that you can compare intact and denervated skin samples from the same animal.
Visual demonstration of this technique is critical as nerves can be difficult to recognize and remove with minimal damage to the surrounding tissues. First, anesthetize the mouse and check that it has reached the proper plane of sedation using a toe pinch. Also, confirm that the mouse exhibits normal breathing and heartbeat.
Now set the animal on a warming pad in an aseptic surgical area. Using an electric clipper, carefully remove the hair from the dorsal side where the biopsy will be taken. Then wipe the shaved area using Betadyne and alcohol wipes.
Ensure that all the hair clippings are removed from the site. Now outline the biopsy site using a black marker. To obtain longitudinal sections of hair follicles, the longer edge of the biopsy should run in an anterior to posterior direction parasetidal to the dorsal midline.
Using a scalpel, make a full thickness excision without damaging the underlying muscular fascia. Bleeding is typically minimal. The biopsy sample should include the epidermis, dermis, subcutaneous fat, and panniculus carnosus.
Flatten the excised skin sample on a dry paper towel, dermis side down. Trim away the excess paper towel, and store the sample on cold TBS for up to an hour if other samples need to be collected. Returning to the mouse, suture the biopsy site using 60 nylon sutures about three millimeters apart.
Then monitor the mouse in a recovery cage until it regains consciousness. Use analgesics in accordance with designated institutional animal care, and follow their guidelines if the mouse appears distressed. Within seven to 10 days of surgery, remove the sutures.
Proceed with processing the biopsies for histology or whole-mount staining. Anesthetize the animal, shave the entire dorsal skin, and clean the area similar to the biopsy procedure. Now make an incision using a sterile scalpel along the dorsal midline from the base of the neck to roughly half a centimeter above the tail.
Using sterile blunt forceps, gently retract the skin on the left side away from the flank to visualize the underlying tissue from the scapular fat pads near the neck to just above the hind limb. Now identify the dorsal cutaneous nerve under a dissecting light microscope. These appear as white strands that travel caudally through the translucent fascia of the trunk wall before making sharp bends and entering the loose connective tissue underneath the skin.
Next, using ultrafine forceps, remove the nerve exclusively from the left side of the animal located at anatomical sites T3 to T12 by plucking them from where their segments bend at the trunk wall to their entry sites into the skin. Orient the forceps vertically and grasp the nerves about half a centimeter below their bend sites. Once grabbed, pull upwards to cause the nerves to stretch and separate from the surrounding tissue to remove them.
Avoid damaging the adjacent blood vessels. Be sure to keep the tissue moist throughout the procedure by periodically applying drops of 0.9%still saline solution. Continue removing all nerves extending from the trunk wall to the skin, but do not disrupt the nerves within the dense fascia of the trunk wall.
Next, remove any nerves from the exposed skin flap. These fibers comprise the distal branches of the dorsal cutaneous nerve and appear as white branching strands located sporadically within the connective tissue on the dermal side of the skin flap. To remove these fine branches, position the forceps roughly parallel to the dermal surface, grasp the nerve and pluck upwards.
Remove all the visible nerves in this fashion. Do not puncture the blood vessels and skin. During this step, it's important to avoid rupturing neighboring blood vessels.
If you find a nerve with an adjacent blood vessel, follow it eventually to an area where there is no adjacent vessel and remove it by plucking. Now, loosen the skin on the right side of the dorsal midline incision, but do not remove any nerves. This will serve as the contralateral sham operative control.
Finally, suture up the animal and monitor post-operatively as before. Later, to confirm the stable loss of nerves, weeks after surgery, gently prick the denervated area using a sterile hypodermic needle. Note whether the animal responds, typically by shuddering or turning its head.
If the skin area has been stably denervated, the animal will exhibit little or no response. Harvest skin biopsies from the area of no response and the contralateral sham side for matched experimental and control samples. From the biopsies, it is important to sample multiple histological sections to identify potential differences in touch dome abundance or morphology between the sham and denervated specimens.
The glebe one cree ERT2 mouse strain allows targeting of tamoxifen-induced genetic recombination to areas of the skin that display high hedgehog pathway activity such as touch dome epithelia. These mice were crossed to induce abolaxi reporter mice to visualize the touch dome epithelia. Nodes are crucial for maintaining both normal touch domes as well as their associated Merkel cells.
This is demonstrated experimentally by the gradual loss of normal touch dome morphology as well as loss of keratin eight deposited cells following denervation. Nerves are also crucial for promoting hedgehog signaling in the touch dome as monitored by Glebe one cree ERE2 expressions, and home-mount beta collective sided staining from both sham and denervated skin. After watching this video, you should have a good understanding of how to surgically ablate dorsal cutaneous nerves in order to test whether these nerves are involved in normal skin function or if they modulate disease.
Once mastered, this technique can be performed routinely and reliably with minimal stress to the animal. Following this procedure, methods such as amino fluorescent staining can be used to confirm whether nerves are fully ablated from the skin, and whether gene expression is being affected.
