An exploration of the control of micturition using a novel in situ arterially perfused rat preparation

Abstract

Our goal was to develop and refine a decerebrate arterially perfused rat (DAPR) preparation that allows the complete bladder filling and voiding cycle to be investigated without some of the restrictions inherent with in vivo experimentation [e.g., ease and speed of set up (30 min), control over the extracellular milieu and free of anesthetic agents]. Both spontaneous (naturalistic bladder filling from ureters) and evoked (in response to intravesical infusion) voids were routinely and reproducibly observed which had similar pressure characteristics. The DAPR allows the simultaneous measurement of bladder intra-luminal pressure, external urinary sphincter-electromyogram (EUS-EMG), pelvic afferent nerve activity, pudendal motor activity, and permits excellent visualization of the entire lower urinary tract, during typical rat filling and voiding responses. The voiding responses were modulated or eliminated by interventions at a number of levels including at the afferent terminal fields (intravesical capsaicin sensitization-desensitization), autonomic (ganglion blockade with hexamethonium), and somatic motor (vecuronium block of the EUS) outflow and required intact brainstem/hindbrain-spinal coordination (as demonstrated by sequential hindbrain transections). Both innocuous (e.g., perineal stimulation) and nociceptive (tail/paw pinch) somatic stimuli elicited an increase in EUS-EMG indicating intact sensory feedback loops. Spontaneous non-micturition contractions were observed between fluid infusions at a frequency and amplitude of 1.4 ± 0.9 per minute and 1.4 ± 0.3 mmHg, respectively and their amplitude increased when autonomic control was compromised. In conclusion, the DAPR is a tractable and useful model for the study of neural bladder control showing intact afferent signaling, spinal and hindbrain co-ordination and efferent control over the lower urinary tract end organs and can be extended to study bladder pathologies and trial novel treatments.

Keywords: bladder afferent activity; bladder external urinary sphincter; brainstem; capsaicin; decerebrate; hexamethonium; incontinence; voiding.

Figure 1

Schematic of decerebrate arterially perfused rat (DAPR) in situ preparation for bladder studies. Shows the preparation with a double lumen cannula inserted via the left ventricle into the ascending aorta, allowing perfusate to be pumped into the arterial tree, as well as allowing continuous monitoring of perfusion pressure. Recording of phrenic nerve activity was used as a physiological indicator of brainstem viability. A needle (30G) inserted into the bladder dome allowed infusion of fluid and monitoring of intravesical pressure (see inset photograph of filled bladder). Simultaneous recordings of EUS–EMG activity and bladder afferent nerve were possible. Naturalistic stimuli could also be applied to the perineum, tail, or hindlimbs to evoke somatic and autonomic responses.

Figure 2

Typical evoked micturition response. (A) As the bladder is filled there is a gradual rise in pressure and a tonic increase in activity of the EUS–EMG. This pressure rise triggers a void with a generalized bladder contraction, a series of bursts on the EUS–EMG trace (mirrored by small oscillations in intravesical pressure) and the ejection of urine. Note at the end of the void as the tonic sphincter activity returns to baseline and the sphincter closes, the still contracting bladder generates a spike of pressure as it contracts iso-volumetrically (also refer to Videos S1 and S2 in Supplementary Material). (B) Expanded time scale showing increase in EUS activity during filling, followed by discrete bursting activity during voiding (inset: three individual bursts), in time with bladder pressure oscillations, where each burst is followed by a mini pressure rise (superimposed on inset), characteristic of the rat voiding pattern. Note in (A), the preparation also shows a eupnoeic pattern of phrenic nerve discharge with respiratory sinus arrhythmia seen in the heart rate trace consistent with intact brainstem–autonomic coupling yet none of these variables (nor perfusion pressure) are altered during the micturition reflex.

Figure 3

Comparison of filling evoked and natural voiding responses. Natural voids occurred in most preparations as fluid from the kidneys filled the bladder. These were of qualitatively similar to filling evoked voids, with no significant differences in pressure trajectory or voiding threshold. Note also the presence of small, spontaneous non-micturition contractions (NMCs; arrowed).

Figure 4

Rate of fluid infusion and bladder pressure at void. (A) Example voids showing that at slower infusion rates (e.g., 20 μl/min) it took longer for a void to be triggered and a larger number of NMCs were observed before the void. Note also the EMG activity that accompanied each NMC. At higher infusion rates, fewer NMCs occurred before the void. (B) The rate of fluid infusion did not significantly alter the bladder pressure at which the void occurred (C) A statistically significant linear relationship (P < 0.0001; R² = 0.83; n = 4) was observed between infusion rate and the volume infused into the bladder before voiding was triggered.

Figure 5

Non-micturition contractions (NMCs). (A) Low amplitude NMCs occurred under basal conditions, when bladder volume was low. Each NMC was accompanied by tonic firing of the EUS. (B) During fluid infusion in the same preparation, NMCs became larger in amplitude with bladder distension until voiding was triggered. (C) When brainstem control had deteriorated (as indicated by a loss of phrenic activity and voiding), the NMCs became biphasic and their amplitude significantly increased. In the first contractile phase of each NMC, tonic EUS firing was observed. The subsequent single burst of the EUS was followed by a second pressure oscillation (dotted line) and they could now be associated with leakage of fluid.

Figure 6

Effect of nicotinic receptor blockade on micturition. (A) Topical vecuronium bromide (a competitive antagonist of neuromuscular transmission) applied to the EUS (10 μl; 2 μg/μl; (ii), caused a decrease in EUS–EMG activity within 2 min but voiding still occurred, albeit at a lower pressure [compared to control; (i)]. In particular, during the void, fewer and lower amplitude EUS-EMG bursts were observed. (iii) A second application of vecuronium completely abolished EUS–EMG activity and further infusion caused passive leakage of urine. In order to test the EUS activity, the distal urethra was clamped to allow bladder pressure to increase. No EUS activity was evoked. This block was maintained for the following 30 min without sign of recovery. (B) Application of the ganglion blocker hexamethonium [330μM; (ii)] to the perfusate blocked the cardiovascular responses to peripheral chemoreflex activation indicating that the sympathetic and parasympathetic outflows were blocked. There was an initial decrease in the voiding pressure, although the characteristic pressure trajectory and EUS bursting activity remained. (iii) After a further 2 min, NMCs became larger (arrowed) in amplitude, with associated EUS activity to maintain continence. Voiding was present, but altered, with low voiding pressure and high baseline intravesical pressures.

Figure 7

Capsaicin sensitization–desensitization of bladder responses. (A,B) Control void and spontaneous NMCs. (C) Filling with capsaicin solution caused an initial voiding response of similar pressure characteristics as control. However, an increased level of tonic EUS activity particularly post-void was observed and voiding was incomplete. (D) At this time there was a marked increase in the amplitude of NMCs and they were accompanied by larger bursts of EMG activity. (E) Some 3 min after the application of capsaicin, intravesical infusion at the same rate continued for a longer period of time until almost twice the volume had been administered and intravesical pressure had increased twofold above that seen during the control void. This infusion triggered a striking increase in the level of EUS–EMG activity as bladder pressure rose above 20 mmHg, previously the pressure at which voiding occurred. However, small incomplete voiding episodes were seen to occur at the highest pressures accompanied by irregular EUS bursting. Normal voiding responses were non recoverable. (F) The subsequent NMCs resulted in synchronous low level tonic firing, as the EUS was markedly desensitized.

Figure 8

Bladder pelvic nerve afferent activity during micturition cycle. (A) Bladder afferent nerve activity increased during filling, followed by a drop in activity before the void (n = 4). The ramping discharge of the afferent nerve was progressive and followed the increase in bladder pressure. At void, afferent nerve bursting was observed to be in anti-phase with the EUS bursting activity [expanded in (B)]. (C) Changes in bladder pressure during NMCs were associated with increase in both, EUS–EMG and afferent nerve discharge. To ascertain that this was a nerve response, muscle relaxant was applied (vecuronium dose 2 μg/ml, systemically) (D), which resulted in complete abolition of EUS activity, while afferent activity remained on the pelvic nerve in synchrony with the NMCs.

Figure 9

Acute sequential hindbrain transections. (A) Histological parasagittal section (0.18 mm lateral) through hindbrain showing the mid-collicular line of transection (Neutral red stained section) and (B) annotated schematic (Paxinos and Watson, 2007) of the two acute brainstem transections during active filling and voiding. The initial transection dissociated the rostral tissues from the brainstem (e.g., hypothalamic nuclei) leaving the brainstem intact. Voiding remained unaffected by this intervention. The second transection (shown) effectively disrupted the rostral periaqueductal gray (PAG), and resulted in loss of coordinated voiding, although filling responses remained. DRn, dorsal raphe nucleus; IC, inferior colliculus, Pn, pontine nucleus; tz, trapezoid body, VMHDM, ventromedial hypothalamic nucleus dorsomedial.