Use of cholera toxin subunit B to label neural projections to lower urinary tract organs

Abstract

This protocol is used to visualise sensory and autonomic neurons innervating organs of the lower urinary tract in an experimental adult male or female rat. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation, including methods of anesthesia, surgical environment, and post-operative monitoring and care.

Before start

Steps

Preparation for surgery

Prepare cholera toxin subunit B solutions: low salt formulation with 0.05% Evans Blue.

Anesthetise animal (2.5% isoflurane in oxygen, or as required for maintenance)

Apply eye lubricant and place animal on heated pad.

Shave and clean the ventral abdomen.

Surgery

Perform a midline incision in the skin and then the muscle, then gently move organs to visualise the required injection site.

Microinject sterile tracer solution at the selected injection site using a Hamilton Neuros Syringe attached to a 33G needle. At each injection site, hold the needle in place for ~5 seconds after ejection of the dye, to enable the dye to spread to the underlying tissue. This also minimises leakage.

Note

Injections into the bladder body (dome) are made bilaterally on dorsal and ventral aspects; total volume ~ 5 µl. Single injections are made into the dorsal aspect of the proximal urethra (maximum 0.3 µl per injection).

Wash all injection sites with sterile saline.

Close the muscle and skin using approved procedures. Administer analgesics and monitor animal during postoperative period as per local approved procedure.

Tissue harvesting

To analyse tracer distribution in ganglia or the injection site, 4 days after surgery, fix animals by intra-cardiac perfusion, then remove tissues of interest for further study.