Dopamine loss in the midbrain substantia nigra and the ventral tegmental area lead to dysfunction in the urinary bladder contraction in the 6-OHDA rat model of Parkinson´s disease

Murillo P1, Winder M1, Aronsson P1, Carlsson T1

Research Type

Basic Science / Translational

Abstract Category

Neurourology

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Abstract 273
Best Basic Science
Scientific Podium Session 16
Thursday 5th September 2019
09:45 - 10:00
Hall G3
Animal Study Basic Science Pathophysiology Detrusor Overactivity Pharmacology
1.Department of Pharmacology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
Presenter
P

Pilar Murillo

Links

Abstract

Hypothesis / aims of study
Patients suffering from Parkinson´s disease (PD) not only show characteristic motor symptoms, but also develop several troublesome non-motor symptoms including gastrointestinal and urinary dysfunction. The latter, in particular, has shown to strongly correlate with the patient’s quality of life, where symptoms are observed as overactive bladder, including nocturia, urgency and increased micturition frequency. In fact, as much as three out of four patients report lower urinary tract symptoms (LUTS), and up to 39% report severe symptoms connected to the urinary tract [1]. Moreover, standard dopaminergic treatments are also unpredictable where the effects may vary from significant improvement of the symptoms to no effect, or to even worsening in some patients. This indicates that the mechanisms behind these problematic symptoms are complex and need to be further pre-clinically investigated. Limited studies have looked into the urinary dysfunction in animal models of the disease, which display that dopaminergic degeneration in the nigrostriatal pathway in the 6-OHDA lesioned rats lead to similar symptoms as seen in PD patients [2]. It has further been shown that the central, brain, noradrenergic system is important for the hyperactivity of the urinary bladder in rodents. However, local changes in the urinary bladder smooth muscle have been sparsely investigated. Previously, we have shown that the detrusor muscle contractility is significantly altered following central dopaminergic lesion in the same 6-OHDA MFB (medial forebrain bundle) lesion rat model [3]. In particular, we saw that the response to cholinergic stimulation was significantly increased when observing detrusor muscle contraction as compared to normal healthy animals. The MFB model, however, lead to significant dopaminergic cell loss in the substantia nigra (SN) as well as the ventral tegmental area (VTA), both of which are suggested to be important for the micturition control by their direct and indirect innervations of the pontine micturition centre in the brainstem. This leads to the question if one or both of these regions are involved in the local changes in the detrusor muscle contractility observed in the 6-OHDA rat model. The aim of the current study was therefore for the first time to investigate which region, SN or VTA, is involved or more prominent in the local urinary bladder dysfunction in rats.
Study design, materials and methods
All experimental procedures were approved by the local ethical committee according to permit #145-15. For this study, we have in the current experiment used Sprague-Dawley rats (Charles River, Germany). One group of animals received intracerebral injections of 6-OHDA in the MFB (“6-OHDA MFB”, n=4) to substantially degenerate the SN and the VTA, while a second group received 6-OHDA into the striatum, which degenerates the SN, while sparing the VTA (“6-OHDA intrastriatal”, n=6). These groups where further compared with normal healthy rats [for the electrical field stimulation (EFS), as sham-operated animals were missing in this test, n=6)] or controls (n=11) where healthy rats (n=6) were pooled together with sham-operated animals (n=5), in which the latter received saline only in the MFB. At four weeks following the 6-OHDA lesions the animals were sacrificed by an overdose of pentobarbital and the urinary bladders were excised and two bladder strips (approx. 2 x 6 mm) from each bladder were prepared and mounted in an organ bath setup filled with Krebs solution. Following pre-stretching (5-8 mN) and an equilibrium period, the tissue was challenged with EFS (1-40 Hz), the muscarinic agonist methacholine (10-8 – 10-3 M) and the purinergic agonist ATP 10-6 – 5*10-3 M) and the muscle tissue contractions were measured using Biopac Acknowledge software (Biopac Systems inc., Goleta, CA). The data were finally presented as dose-response curves, using GraphPad Prism for Mac OSX (GraphPad Software Inc., San Diego, USA). Appropriate one- or two-way ANOVAs followed by Tukey’s or Sidak’s post hoc tests were used for group comparisons.
Results
Following stimulations with EFS the 6-OHDA groups (6-OHDA MFB and 6-OHDA intrastriatal) showed an increased contraction, with a trend evident already at 5 Hz, and at the highest frequency tested (40 Hz) the normal animals contracted 13.7±2.1 mN as compared to 19.8±2.7 mN and 20.2±2.2 mN in the 6-OHDA MFB and 6-OHDA intrastriatal groups respectively (two-way ANOVA, interaction p=0.0035). Further evaluating the response of the muscarinic agonist, methacholine (10-8 M - 10-3 M), the 6-OHDA MFB group showed a trend to increase (28.0±3.3mN at 10-3 M), while the 6-OHDA intrastriatal was less affected (24.5.0±2.6 mN) as compared to control animals (23.4.0±2.6 mN; two-way ANOVA, group and interaction p=0.12 and 0.095 respectively). In fact, a significant increased contraction in the 6-OHDA MFB group could be observed at the concentration of 10-5 M as compared to controls (one-way ANOVA, p=0.010). Further, the ATP-induced contractions in the 6-OHDA MFB group, although not significant (two-way ANOVA, group p=0.18), showed, similarly as to the methacholine response, a trend to increase (9.7±2.1 mN at 10-3 M) as compared to control (7.2±0.6 mN at 10-3 M). However, the ATP response in the 6-OHDA intrastriatal group was not at all affected (7.2±1.7 mN at 10-3 M).
Interpretation of results
The current preliminary data show that the SN, and not the VTA, is mainly responsible for the local changes seen following EFS in the urinary bladder tissue. This is in line with that PD patients often have a dopaminergic degeneration mainly in the SN, while the VTA is, at least in early-to-mid disease, intact. However, as altered responses to methacholine, but also to some extent ATP, are mainly evident in the 6-OHDA MFB group this may indicates that damages in the VTA may further aggravate the urinary bladder dysfunction, at least in this PD rat model. This could possibly explain why the urinary dysfunction significantly worsens in later stages of the disorder in some PD patients, as the disease progresses it may start involve dopaminergic degeneration in the VTA.
Concluding message
The current data show that both the SN and VTA may be involved in the urinary bladder dysfunction seen in patients with PD. However, dopamine lesions restricted to SN may not involve additional muscarinic or purinergic (ATP) receptor response alterations in the urinary bladder tissue. These data could explain why the urinary bladder dysfunction is worsening in PD patients as the disease progresses, as well as why the effect of standard dopaminergic treatment varies from patient to patient. Finally, we believe this is a step forward to understand the underlying mechanisms of urinary bladder problems in patients with PD.
References
  1. Winge K, Nielsen KK. Bladder dysfunction in advanced Parkinson's disease. Neurourol Urodyn. 2012 Nov;31(8):1279-83. doi: 10.1002/nau.22237. Epub 2012 Apr 6.
  2. Soler R, Füllhase C, Santos C, Andersson KE. Development of bladder dysfunction in a rat model of dopaminergic brain lesion. Neurourol Urodyn. 2011 Jan;30(1):188-93. doi: 10.1002/nau.20917. Epub 2010 Jun 29.
  3. Mitra R, Aronsson P, Winder M, Tobin G, Bergquist F, Carlsson T. Local Change in Urinary Bladder Contractility Following CNS Dopamine Denervation in the 6-OHDA Rat Model of Parkinson's Disease. J Parkinsons Dis. 2015;5(2):301-11. doi: 10.3233/JPD-140509.
Disclosures
<span class="text-strong">Funding</span> ParkinsonFonden, Wilhelm och Martina Lundgrens Vetenskapsfond, Parkinson Research Foundation, Kungl. vetenskaps-. och Vitterhets-Samhället (KVVS) <span class="text-strong">Clinical Trial</span> No <span class="text-strong">Subjects</span> Animal <span class="text-strong">Species</span> Spraley-Dawley rats <span class="text-strong">Ethics Committee</span> Local ethical committee, permit #145-15