Neuromuscular nicotinic receptors mediate bladder contractions following bladder reinnervation with somatic to autonomic nerve transfer after decentralization by spinal root transection

Abstract

Purpose: We investigated whether the reinnervated neuronal pathway mediates contraction via the same neurotransmitter and receptor mechanisms as the original pathway.

Materials and methods: After decentralizing the bladder by transecting the sacral roots in dogs we performed peripheral nerve transfer, including bilateral genitofemoral to pelvic nerve transfer and unilateral left femoral nerve to bilateral pelvic nerve transfer. Reinnervation was assessed 7.5 months postoperatively by monitoring bladder pressure during electrical stimulation of the transferred nerves, spinal ventral roots and spinal cord.

Results: Of the 17 dogs with genitofemoral to pelvic nerve transfer 14 (82%) demonstrated functional bladder reinnervation as evidenced by increased bladder pressure during stimulation of the transferred genitofemoral nerve, or L3 or L4 spinal ventral roots. Lumbar spinal cord stimulation caused increased bladder pressure in 9 of 10 dogs (90%) with unilateral left femoral nerve to bilateral pelvic nerve transfer. Succinylcholine virtually eliminated the bladder pressure increases induced by electrical stimulation of the transferred somatic nerves or of the lumbar spinal segments that contribute axons to these donor nerves. In unoperated or sham operated controls succinylcholine had no effect on nerve evoked bladder pressure increases but it substantially decreased the urethral and anal sphincter pressure induced by stimulating the lumbosacral spinal cord or the S2-S3 spinal ventral roots. The reinnervated detrusor muscles of dogs with genitofemoral to pelvic nerve transfer and unilateral left femoral nerve to bilateral pelvic nerve transfer also showed increased α1 nicotinic receptor subunit immunoreactivity in punctate dots on detrusor muscle fascicles and in neuronal cell bodies. This staining was not observed in controls.

Conclusions: Succinylcholine sensitive nicotinic receptors, which normally mediate only skeletal muscle neuromuscular junction neurotransmission, appeared in the new neuronal pathway after genitofemoral to pelvic and unilateral femoral nerve to bilateral pelvic nerve transfer. This suggests end organ neuroplasticity after reinnervation by somatic motor axons.

Keywords: nerve regeneration; nerve transfer; nicotinic; receptors; spinal cord injuries; urinary bladder.

Figure 1

Top: Urodynamic traces before and after SCh. A&B: Control responses to stimulation of left (Lt) or right (Rt) anterior vesical branch of the pelvic nerve. C&D: Implanted RF micro-stimulator responses in a GFNT animal. E&F: Responses in a FNT animal to stimulation of the left transferred FN. ASphP = anal sphincter pressure; IVsP = intravesical pressure, UP = urethral pressure, RP = rectal pressure, DP = detrusor pressure (IVsP minus RP). Bottom: Rate of pressure generation in response to FES in control (CON), GFNT and FNT. *= Significantly different from control (p<0.01) Mann-Whitney U test.

Figure 2

FES induced (nerve evoked) detrusor pressure increases before and after SCh in GFNT, FNT and control animals and sphincter pressures in control urethra and anal sphincters. * = statistically significantly different from pre-SCh p<0.01 Student’s t test, # = Statistically significantly different from control detrusor pre-SCh p<0.01 Mann-Whitney U test.

Figure 3

Immunohistochemistry of α1 nicotinic receptor subunits (red) in detrusor muscle in sham GFNT and FNT. DAPI (blue) A-C: Arrowheads indicate axons (pgp9.5 immunopositive, green). D-F: Arrowheads-asterisks indicate axons, higher power in insets. Black arrows illustrate small punctate immunopositive profiles on muscle fascicles, enlarged in insets. G-I: Arrowheads and black arrows as above. White arrow-asterisk and insets show α1 subunit immunopositive non-neuronal structure on muscle fascicle surface. m=muscle fascicle; ct = connective tissue.

Figure 4

Photomicrographs of α1 nicotinic receptor subunits in intramural ganglia and muscularis. Colors are as indicated in Figure 3. A-C: Intramural ganglion neurons with low level of α1 nicotinic receptor subunit immunostaining. D-F: Black arrow indicates a small double-labeled neuronal cell body at edge of muscle fascicle. Insets show enlargement of asterisk indicated neuron. G-L: FNT dog. G-I: Intramural ganglion neurons with α1 nicotinic receptor subunit immunostaining. J-L: Black arrow indicates a small double-labeled neuronal cell body at edge of muscle fascicle. Insets show enlargement of asterisk indicated neuron.

Figure 5

Mean number of punctate profiles on muscles and nerve cell bodies co-immunostained with anti-α1 nicotinic receptor subunit and anti-pgp9.5 antibodies. * = Statistically significantly different from Sham/un-operated controls by ANOVA with post hoc Student-Newman-Keuls tests, p<0.05.