Resiniferatoxin (RTX)- and tetrodotoxin (TTX)-induced changes in chemical coding of sensory neurons in lumbar and sacral/coccygeal dorsal root ganglia (DRG) supplying porcine urinary bladder
Article published in Urologia Polska 2008/61/Supl. 1.

authors

Agnieszka Bossowska, Andrzej Borkowski, Piotr Radziszewski, Mariusz Majewski

Katedra Fizjologii Człowieka, Wydział Nauk Medycznych, Uniwersytet Warmińsko-Mazurski w Olsztynie
Katedra i Klinika Urologii Ogólnej, Onkologicznej i Czynnościowej Uniwersytetu Medycznego w Warszawie

summary

Introduction.

At present time, RTX is used in experimental therapy as a drug, which can abolish the activity of afferent neurons involved in abnormal neural circuits causing micturition disorders during various neurogenic diseases like overactive bladder or interstitial cystitis. RTX and TTX have not only different binding points to the nerve terminals, but also dissimilar mechanisms of pharmacological action. Thus, it may be of great interest to compare the mode of action of both neurotoxins, what potentially could broaden our neurourological armamentarium. To reach this goal, changes in the chemical coding of sensory neurons supplying

porcine urinary bladder, an animal, which can be used as a very good model for human lower urinary tract, have to be elucidated in detail after exposure of the organ to studied neurotoxins.

Objectives.

The aim of that study was to compare changes in the chemical coding of lumbar and sacral/coccygeal sensory neurons after bladder instillation with RTX or TTX.

Materials and methods.

The study was performed on eighteen juvenile female pigs. The retrograde tracer Fast Blue (FB) was injected into the urinary bladder wall in all animals. Three weeks later, bladder instillation of RTX (500 nmol per animal) was carried out in six animals and another group of six pigs was treated by TTX instillation (12 μg per animal), while remaining 6 animals (control group) were instilled with the vehiculum only. After a week, all animals were sacrificed using transcardial perfusion method and DRGs of interest were then collected. The neurochemical characteristic of FB+ neurons was performed using routine single- and double-immunofluorescence labelling techniques on 10-μm-thick cryostat sections.

Results.

In control group, FB+ neurons, immunoreactive (-IR) to substance P (SP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (NOS), galanin (GAL), calbindin (CB) and somatostatin (SOM) constituted 40%, 35%, 26%, 6%, 6% and 2% of all retrogradelly traced DRG

neurons, respectively. The bladder instillation with RTX lead to a significant decrease in the number of neurons containing CGRP, SOM and NOS (9%, 1.5% and 0%, respectively). After TTX treatment, a distinct increase in the number of neurons containing PACAP (37%), and a significant decrease in the number of NOS-, CB- and SOM-IR traced neurons (4%, 0.2% and 0.2%, respectively) was observed. Single retrogradelly labeled cells contained also Leu5-enkefalin (0.2%), but they were not observed in the control DRGs. There were also significant differences in the number of neurons containing particular neurotransmitters between lumbar and sacral/coccygeal DRGs. In the control group, retrogradelly labeled lumbar DRG neurons contained SP – 44%, CGRP – 47%, PACAP – 23%, NOS and GAL – 8% (each population), SOM – 4.5% and CB – 2.5%, while these substances constituted 49%, 23%, 31%, 1.5%, 2%, 0.2 and 3% in the sacral/coccygeal DRGs neurons, respectively. The instillation of bladder with RTX

caused changes in the chemical coding of lumbar FB+ neurons, leading to significant increase in number of PACAP-, GAL- and NOS-IR cells (60%, 60% and 20% respectively) and the lack of neurons containing CB. On the other hand, a decrease in number of cells containing CGRP, NOS, SOM and CB (16%, 0.5%, 0.5% and 0.5%, respectively), paralleled with an increase in the number of PACAP- or GAL-IR sensory neurons (50% and 35%, respectively) was observed in the population of sacral/coccygeal DRG cells

after RTX treatment. Furthermore, significant increase in number of SP-, CGRP- and SOM-IR cells (80%, 80% and 18%, respectively) and the lack of neurons containing GAL and CB was observed in lumbar FB+ neurons after TTX instillation, while this procedure induced an increase in the number of cells containing GAL and NOS (5% and 4%) in sacral/coccygeal DRGs.

Conclusions.

These preliminary data show that both neurotoxin influences the chemical coding of DRG cells supplying porcine urinary bladder, but the effects of their action are distinctly different. Although it needs further studies, it appears that TTX, in contrast to RTX, can probably be used in the case of afferent hypoactivity rather, than as a drug controlling the afferent limb of the micturition reflexes in a way similar to RTX.