Nerve Transfer for Restoration of Lower Motor Neuron-Lesioned Bladder, Urethral, and Anal Sphincter Function in a Dog Model. Part 3. Nicotinic Receptor Characterization

Very little is known about the physiological role of nicotinic receptors in canine bladders, although functional nicotinic receptors have been reported in bladders of many species. Utilizing in vitro methods, we evaluated nicotinic receptors mediating bladder function in dogs: control (9 female and 11 male normal controls, 5 sham operated), Decentralized (9 females, decentralized 6–21 mo), and obturator-to-pelvic nerve transfer reinnervated (ObNT-Reinn; 9 females; decentralized 9–13 mo, then reinnervated with 8–12 mo recovery). Muscle strips were collected, mucosa-denuded, and mounted in muscle baths before incubation with neurotransmitter antagonists, and contractions to the nicotinic receptor agonist epibatidine were determined. Strip response to epibatidine, expressed as percent potassium chloride, was similar (∼35% in controls, 30% in Decentralized, and 24% in ObNT-Reinn). Differentially, epibatidine responses in Decentralized and ObNT-Reinn bladder strips were lower than controls after tetrodotoxin (TTX, a sodium channel blocker that inhibits axonal action potentials). Yet, in all groups, epibatidine-induced strip contractions were similarly inhibited by mecamylamine and hexamethonium (ganglionic nicotinic receptor antagonists), SR 16584 (α3β4 neuronal nicotinic receptor antagonist), atracurium and tubocurarine (neuromuscular nicotinic receptor antagonists), and atropine (muscarinic receptor antagonist), indicating that nicotinic receptors (particularly α3β4 subtypes), neuromuscular and muscarinic receptors play roles in bladder contractility. In control bladder strips, since tetrodotoxin did not inhibit epibatidine contractions, nicotinic receptors are likely located on nerve terminals. The tetrodotoxin inhibition of epibatidine-induced contractions in Decentralized and ObNT-Reinn suggests a relocation of nicotinic receptors from nerve terminals to more distant axonal sites, perhaps as a compensatory mechanism to recover bladder function

Nerve transfer for restoration of lower motor neuron-lesioned bladder function. Part 1: attenuation of purinergic bladder smooth muscle contractions.

This study determined the effect of pelvic organ decentralization and reinnervation 1 yr later on the contribution of muscarinic and purinergic receptors to ex vivo, nerve-evoked, bladder smooth muscle contractions. Nineteen canines underwent decentralization by bilateral transection of all coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7, and hypogastric nerves. After exclusions, 8 were reinnervated 12 mo postdecentralization with obturator-to-pelvic and sciatic-to-pudendal nerve transfers then euthanized 8-12 mo later. Four served as long-term decentralized only animals. Controls included six sham-operated and three unoperated animals. Detrusor muscle was assessed for contractile responses to potassium chloride (KCl) and electric field stimulation (EFS) before and after purinergic receptor desensitization with α, β-methylene adenosine triphosphate (α,β-mATP), muscarinic receptor antagonism with atropine, or sodium channel blockade with tetrodotoxin. Atropine inhibition of EFS-induced contractions increased in decentralized and reinnervated animals compared with controls. Maximal contractile responses to α,β-mATP did not differ between groups. In strips from decentralized and reinnervated animals, the contractile response to EFS was enhanced at lower frequencies compared with normal controls. The observation of increased blockade of nerve-evoked contractions by muscarinic antagonist with no change in responsiveness to purinergic agonist suggests either decreased ATP release or increased ecto-ATPase activity in detrusor muscle as a consequence of the long-term decentralization. The reduction in the frequency required to produce maximum contraction following decentralization may be due to enhanced nerve sensitivity to EFS or a change in the effectiveness of the neurotransmission

Nerve transfer for restoration of lower motor neuron-lesioned bladder, urethral and anal sphincter function. Part 4: Effectiveness of the motor reinnervation.

In pilot work we showed that somatic nerve transfers can restore motor function in long-term decentralized dogs. We continue to explore the effectiveness of motor reinnervation in 30 female dogs. After anesthesia, 12 underwent bilateral transection of coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7, and hypogastric nerves. Twelve months post-decentralization, 8 underwent transfer of obturator nerve branches to pelvic nerve vesical branches, and sciatic nerve branches to pudendal nerves, followed by 10 months recovery (ObNT-ScNT Reinn). The remaining 4 were euthanized 18 months post-decentralization (Decentralized). Results were compared to 18 Controls. Squat-and-void postures were tracked during awake cystometry. None showed squat-and-void postures during the decentralization phase. Seven of 8 ObNT-ScNT Reinn began showing such postures by 6 months post-reinnervation; one showed a return of defecation postures. Retrograde dyes were injected into the bladder and urethra 3 weeks prior to euthanasia, at which point, roots and transferred nerves were electrically stimulated to evaluate motor function. Upon L2-L6 root stimulation, 5 of 8 ObNT-ScNT Reinn showed elevated detrusor pressure and 4 showed elevated urethral pressure, compared to L7-S3 root stimulation. After stimulation of sciatic-to-pudendal transferred nerves, 3 of 8 ObNT-ScNT Reinn showed elevated urethral pressure; all showed elevated anal sphincter pressure. Retrogradely labeled neurons were observed in L2-L6 ventral horns (in laminae VI, VIII and IX) of ObNT-ScNT Reinn, versus Controls in which labeled neurons were observed in L7-S3 ventral horns (in lamina VII). This data supports the use of nerve transfer techniques for restoration of bladder function

Nerve transfer for restoration of lower motor neuron-lesioned bladder function. Part 2: correlation between histological changes and nerve evoked contractions.

We determined the effect of pelvic organ decentralization and reinnervation 1 yr later on urinary bladder histology and function. Nineteen canines underwent decentralization by bilateral transection of all coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7, and hypogastric nerves. After exclusions, eight were reinnervated 12 mo postdecentralization with obturator-to-pelvic and sciatic-to-pudendal nerve transfers, then euthanized 8-12 mo later. Four served as long-term decentralized only animals. Before euthanasia, pelvic or transferred nerves and L1-S3 spinal roots were stimulated and maximum detrusor pressure (MDP) recorded. Bladder specimens were collected for histological and ex vivo smooth muscle contractility studies. Both reinnervated and decentralized animals showed less or denuded urothelium, fewer intramural ganglia, and more inflammation and collagen, than controls, although percent muscle was maintained. In reinnervated animals, pgp9.5+ axon density was higher compared with decentralized animals. Ex vivo smooth muscle contractions in response to KCl correlated positively with submucosal inflammation, detrusor muscle thickness, and pgp9.5+ axon density. In vivo, reinnervated animals showed higher MDP after stimulation of L1-L6 roots compared with their transected L7-S3 roots, and reinnervated and decentralized animals showed lower MDP than controls after stimulation of nerves (due likely to fibrotic nerve encapsulation). MDP correlated negatively with detrusor collagen and inflammation, and positively with pgp9.5+ axon density and intramural ganglia numbers. These results demonstrate that bladder function can be improved by transfer of obturator nerves to pelvic nerves at 1 yr after decentralization, although the fibrosis and inflammation that developed were associated with decreased contractile function