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NEUROSCIENCE |
1 INSERM E358, Institute François Magendie; University Bordeaux 2, 146 rue Léo Saignat, 33077 Bordeaux Cedex, France
2 University Bordeaux 2, EA2966, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France
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Abstract |
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(Received 17 July 2006;
accepted after revision 15 October 2006;
first published online 26 October 2006)
Corresponding author F. Nagy: INSERM E358, Institut François Magendie, Université Bordeaux 2, 146 rue Léo Saignat, 33077 Bordeaux Cedex, France. Email: frederic.nagy{at}bordeaux.inserm.fr
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Introduction |
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To gain a greater understanding of the neuronal mechanisms of urinary bladder dysfunction in MS, we first characterized the phenotypes of normal, hyperactive and areflexic activities of the detrusor that occur during the different stages of EAE. We then tested the hypothesis that these functional alterations were caused by modifications in the balance between inhibitions and excitations within the spinal centres controlling the micturition reflex. For that purpose, we considered the role of inhibition by activating inhibitory glycine and GABA receptors in EAE rats presenting detrusor hyperactivity and by blocking these receptors in EAE rats presenting detrusor areflexia. In the next step, we addressed the question of whether the alterations occur preferentially at the segmental level or depend on descending controls from supraspinal centres. Finally, given the importance of alterations in inhibition, we tested the mechanisms of an inhibitory effect induced by electrical stimulation of sacral roots as this treatment is applied to suppress detrusor hyperactivity in clinical MS.
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Methods |
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Acute EAE was induced in 115 female 6- to 7-week-old Lewis rats, (160 ± 10 g, Charles River, France), by intradermal inoculation of an emulsion of 50 µg guinea-pig CNS extract, 100 µl Complete Freund's adjuvant (CFA, Difco, France) and 2 mg attenuated Mycobacterium tuberculosis H37Ra strain (Deloire et al. 2004). Control rats were immunized with CFA–M. tuberculosis (n = 10). Weight and clinical scores of motor function of the rats were determined daily: 0, no clinical sign; 1, flaccid tail; 2, flaccid tail and hindlimb weakness; 3, complete paralysis of one hindlimb; 4, paraplegia. Animals were kept in cages (five animals per cage) with standard conditions of light and free access to water and food.
Animal handling and experimentation conformed to guidelines of the European Union (permissions No. 6305 and 33/00055 of local Animal Experimentation Commission). All animals were anaesthetized with 1.25 g kg–1 urethane I.P. injection, supplemented if required. Absence of spontaneous body or vibrissae movement, absence of heart rate modification (monitored with LE 5002 Storage Pressure Meter, Bioseb, France), absence of nociceptive reflex (no movement after hindlimb pinching) and absence of behavioural reflex (no eye blinking when approaching an object) were ensured throughout the experiment. Finally, under anaesthesia, animals were decapitated.
Micturition reflex experiments
A lubricated polyethylene transurethral catheter (o.d., 0.96 mm; i.d., 0.58 mm, A. Systems Inc., USA) was passed into the bladder. Catheter placement, absence of detrusor abnormality and complete bladder voiding after each cystometrogram were controlled visually by a 1.5 cm midline incision in the lower abdominal wall (Jaggar et al. 1998). Saline-soaked swabs over the wounds and a heating pad protected rats from hypothermia during the experiments. To evaluate bladder motility, two infusion protocols were used, either ramp stimulation at constant infusion rate (50 µl min–1 during 14 min; maximum of 0.7 ml intravesical volume) (McMahon & Abel, 1987), or sustained stimulation at constant volume (0.5 ml). A pressure transducer (SensoNor SP 8444, Norway) coupled to the infusion system amplified cystometrogram signals of continuously monitored intravesical pressure, which were processed by Spike 2 software (CED, UK). Bladder activity was quantified by several cystometrogram parameters, the area under the pressure–volume curve (AUC; cmH2O ml), the duration and the amplitude of contractions. AUC was calculated from the first contraction to the last recorded contraction. In addition, a micturition threshold (Vmic; ml) was determined during ramp stimulations.
Bladder inflammation
Intravesical instillation of 0.5 ml 50% turpentine dissolved in olive oil was performed for a period of 45 min (McMahon & Abel, 1987) causing an inflammatory response lasting for at least 48 h (Jaggar et al. 1999). This stimulation activates afferent C-fibres of the bladder (Habler et al. 1990) which are necessary (Jasmin et al. 1998) in segmental spinal reflexes (Yoshimura, 1999).
Peripheral nerve histology
Six EAE rats presenting either functional detrusor alteration at clinical scores 2–4 and two control rats were perfused intracardially with 0.9% NaCl followed by 0.5% paraformaldehyde–2.5% glutaraldehyde in 0.1 M cacodylate buffer. Bilaterally close to the spine, the individual roots of sacral nerves from L6 and S1, before they form the sciatic nerve, were removed, immersed in 2% osmium tetroxide for postfixation, dehydrated and embedded in Epon resin. On semi-thin sections (1 µm), myelin was stained by alkaline toluidine blue and examined by light microscopy.
Intrathecal drug characterization
A laminectomy allowed insertion of a polyethylene catheter at spinal cord L6–S1 junction. Skin was sutured to prevent desiccation. The composition of vehicle solution was (mM): NaCl 101, KCl 3.8, MgCl2 18.7, MgSO4 1.3, KH2PO4 1.2, Hepes 10, CaCl2 1 and glucose 25; pH was adjusted to 7.4. Solutions were dissolved from frozen stock, just before intrathecal (I.T.) injection. Drugs administered I.T. (15 µl) were the agonists glycine (Q-Biogene, France), GABA, muscimol (Sigma, France) and baclofen (RBI, Germany), and the non-selective glycine receptor antagonist strychnine (Sigma, France) and GABAA receptor antagonist bicuculline methiodide (Sigma, Germany).
Spinalization
After laminectomy at the upper thoracic level, using microsurgical techniques, the dura was opened and the spinal cord was exposed. I.T. administration of 50 µl 2% lidocaine hydrochloride (Astra-Zeneca, France) caused reversible chemical spinalization (Ness & Castroman, 2001). Alternatively, mechanical spinalization was made by sectioning the spinal cord (Jasmin et al. 1998).
Electrical stimulation of sacral dorsal roots in rats presenting detrusor hyperactivity
In order to test whether sacral root stimulation inhibited the micturition reflex, a monopolar stimulation electrode (Interstim° 3057, Medtronic, USA) was positioned in sacral foramen S1. Electrical pulses (500 µs, 25 Hz) were delivered using a stimulator (Master 8, Ampi, Israel). Electrode positioning was controlled by stimulation-induced movements of the tail or ipsilateral hindlimb (Zvara et al. 1998). For experimentation, electrical stimulation intensity (from 0.4 to 2 mA) was kept below the threshold for movement induction and tested for various durations (800–3200 s) for adequate suppression of detrusor hyperactivity.
Statistical analyses
Comparisons of proportions (detrusor areflexia versus detrusor hyperactivity) in the different clinical stages as established on clinical scores were tested using 
2 test. Unpaired, non-parametric data comparisons were statistically analysed by Mann–Whitney U test (InStat 2.03, GraphPad software, USA) and reported as medians and range (minimum–maximum). In case of multiple comparisons of non-parametric data of functional and pharmacological characteristics of detrusor hyperactivity and detrusor areflexia, we used the Kruskall-Wallis test. For parametric comparison of detrusor hyperactivity treatment by electrical stimulation of sacral roots, we used paired Student's t test, and data were summarized as means and S.E.M. Probability values of 
0.05 were considered statistically significant. Throughout the text n indicates the number of animals in each experiment.
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Results |
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Mean onset of symptomatic EAE (score 1) occurred between 9 and 11 days after immunization with a peak of disease severity (score 3 or 4) between 10 and 14 days. Cystometrogram recordings of EAE rats distinguished three micturition reflex patterns (Fig. 1). Ten rats with normal bladder function (8.7%), 56 rats with detrusor hyperactivity (48.7%) and 49 rats with detrusor areflexia (42.6%) were observed. Rats injected with vehicle presented no clinical EAE and normal bladder function. The relative proportion of detrusor areflexia and hyperactivity varied with EAE development. At the pre-symptomatic phase (n = 17; score 0 at 7 or 8 days after immunization), we observed a predominance of areflexia (n = 9; 53%) versus hyperactivity (n = 4; 23.5%). Conversely, at symptomatic EAE onset (n = 19; score 1), rats presented predominantly with detrusor hyperactivity (n = 14; 73.7%) versus areflexia (n = 5; 26.3%). The frequency of detrusor areflexia versus detrusor hyperactivity was significantly different between these two early disease stages (P = 0.046) suggesting that areflexia precedes hyperactivity in EAE. During the progression of EAE, areflexia and hyperactivity frequency were almost equally distributed at clinical stages of established disease (n = 20; score 2) with detrusor areflexia (n = 11; 55%) versus detrusor hyperactivity (n = 9; 45%), peak of disease (n = 42; score 3 and 4) with detrusor areflexia (n = 20; 47.6%) versus detrusor hyperactivity (n = 22; 52.4%) and during spontaneous clinical recovery phase (n = 17) with detrusor areflexia (n = 4; 23.5%) versus detrusor hyperactivity (n = 7; 41.2%). Additionally, a normal micturition reflex was observed in EAE-induced rats at the pre-symptomatic phase (score 0, n = 4; 23.5%) and during spontaneous recovery (score 0 or 1, n = 6; 35.3%). However, normal micturition reflex was absent during EAE progression with symptomatic scores 1–4. In rats studied at the peak of symptomatic EAE (scores 3 and 4) or during spontaneous recovery, no significant correlation was found between the frequency of either detrusor areflexia or hyperactivity and the delay of disease onset or the duration of EAE. Clinical and micturition reflex alterations during EAE are summarized in Table 1.
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Functional and pharmacological characteristics of detrusor hyperactivity
Among the 115 EAE-induced rats, the cystometrograms for 10 rats showed a profile that was comparable to those of vehicle-treated animals. Therefore, we first determined whether this profile could be considered as a normal micturition reflex. Quantitative comparison of cystometrogram parameters between the 10 immunized EAE rats (four rats in preclinical phase and six rats in spontaneous recovery phase) considered as presenting normal bladder function and the 10 vehicle-injected rats was performed. It revealed no significant differences for Vmic (0.57 ml (0.54–0.6 ml) (median (range), for immunized EAE rats versus 0.54 (0.52–0.56) ml for vehicle-injected rats), for increase of bladder activity as indicated by the AUC (1752 (1342–2030) cmH2O ml versus 2658 (2310–3001) cmH2O ml), and for duration (46 (43–52) s versus 53.5 (45–66) s) and amplitude of contractions (29 (27–31) cmH2O versus 25.5 (22–30) cmH2O;), confirming the normal micturition reflex pattern in the 10 immunized EAE animals.
In the following experiments, animals from the control group injected with vehicle representing normal bladder function served as controls for comparison with functional alterations of detrusor hyperactivity. Three cystometrograms for rats with detrusor hyperactivity (n = 24) were recorded. Vmic measured in hyperactivity (0.15 (0.07–0.19) ml) was significantly lower compared to vehicle-injected control (0.54 (0.52–0.56) ml; P < 0.001). AUC was significantly increased in hyperactivity (11961 (9816–15552) cmH2O ml) compared to control (2658 (2310–3001) cmH2O ml; P < 0.001). Detrusor hyperactivity presented significantly shorter duration of contractions (32.6 (24.1–38.7) s) than control (53.5 (45–66) s; P < 0.001); however, the amplitude of contraction in hyperactivity (36.9 (24.2–38.7) cmH2O) was significantly higher than in animals with normal bladder function (25.5 (22–30) cmH2O; P < 0.05).
We explored the possibility of restoring bladder function by inhibiting detrusor hyperactivity with agonists of glycine receptors (glycine), of ionotropic GABAergic receptors (GABA and muscimol), and metabotropic GABAergic receptors (baclofen). I.T. administration of 100 µM glycine (Fig. 2A) suppressed bladder activity for up to 30 min and abolished micturition in the defined limits of maximum bladder filling (P < 0.001). I.T. administration of 10 µM GABA significantly increased Vmic compared to untreated detrusor hyperactivity (P < 0.001). Muscimol also significantly modified Vmic (P < 0.05; Fig. 2C). As GABAB receptors are known to modulate spinal neurones, we also applied the GABAB agonist baclofen at 10 µM (not shown) and 20 µM (Fig. 2B), which caused similar but smaller inhibition to glycine (both P < 0.01). Regarding the increased Vmic, I.T. administration of glycinergic and GABAergic agonists significantly inhibited bladder activity compared to untreated detrusor hyperactivity as determined by AUC measurements (Fig. 2D). In EAE rats, we observed that suppression of hyperactivity always resulted in areflexia. There was never a transition from detrusor hyperactivity to normal micturition reflex. Detrusor hyperactivity seems to be highly sensitive to activation of inhibitory receptors and can be completely suppressed by glycine. We therefore investigated whether areflexia in EAE rats was caused by an over-inhibition in the dorsal horn.
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In EAE rats presenting detrusor areflexia, I.T. administration of strychnine (specific glycine receptor antagonist) at 50–100 µM caused a dose-dependent partial recovery of bladder activity indicating that areflexia depends on glycinergic inhibition (Fig. 3A). Higher strychnine doses induced convulsions in EAE rats. The concentration of strychnine resulting in 50% recovery (half-maximal inhibition; IC50) from detrusor areflexia was determined to be 65 µM (Fig. 3B). Regarding the regained bladder activity induced by I.T. administration of strychnine, AUC increased significantly in a dose-dependent manner (Fig. 3C). Again, the shift from areflexia to hyperactivity in EAE rats by modulating glycinergic inhibition did not involve normal bladder function, confirming that once the micturition status is altered, the inhibitory and excitatory control only allows either detrusor areflexia or hyperactivity status in EAE.
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Descending excitatory pathway
In rats showing detrusor areflexia, reactivation of detrusor function by suppression of a local inhibition with inhibitory receptor antagonists suggests that areflexia is associated with a strong inhibitory control in which glycine receptors predominate. To elucidate the origin of this powerful inhibition, we investigated whether local interneurones and/or an inhibitory descending pathway are involved. We observed that in EAE rats with detrusor areflexia, neither chemical lidocaine spinalization (n = 3) nor mechanical transection (n = 3) of upper spinal cord induced reactivation of bladder activity (not shown). At least two possibilities could explain the persistence of areflexia after spinalization. Either the inhibition originates locally in the segmental dorsal horn, or expression of bladder activity requires an excitatory descending pathway, which was also suppressed by spinalization.
To determine the potential control of the bladder by a descending excitatory pathway, bladder activity was first reinitiated in EAE rats with detrusor areflexia by lumbosacral I.T. administration of 100 µM strychnine to block the glycine receptors (Fig. 4Aa and Ab). We then performed mechanical spinalization causing immediate suppression of hyperactivity in these rats (n = 6; Fig. 4Ac). Under these conditions (n = 3) or in rats spinalized chemically with lidocaine (n = 3), the subsequent I.T. administration of 100 µM strychnine did not induce bladder reactivation (not shown). Moreover, in EAE rats with detrusor hyperactivity (n = 10), both chemical and mechanical spinalization immediately and completely suppressed bladder activity (Fig. 4B). Taken together, these data demonstrate the existence of a descending excitatory control, which is obligatory for the expression of the micturition reflex in EAE rats presenting hyperactivity or areflexia of the detrusor. This control determines the bladder hyperactivity in EAE rats presenting detrusor hyperactivity. On the other hand, the descending excitatory control is dominated by a strong segmental inhibition in rats presenting detrusor areflexia.
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Given that in the rat model of spinal cord injury the restoration of bladder hyperactivity could depend on the activation of a short segmental reflex loop, we investigated whether such reorganization of the micturition reflex circuit occurs in EAE rats. In EAE rats presenting areflexia, instillation of 0.4 ml turpentine into the urinary bladder did not cause bladder reactivation (n = 7; not shown). Moreover, in EAE rats presenting areflexia that develop detrusor hyperactivity after I.T. administration of 100 µM strychnine, and in which the regained micturition reflex was suppressed by spinalization (Fig. 4Aa–c), further instillation of turpentine into the urinary bladder (n = 3) did not reactivate bladder function (Fig. 4Ad). Similarly, in EAE rats presenting detrusor hyperactivity, in which mechanical or chemical (Fig. 4B) spinalization abolished contractions, intravesical instillation of turpentine did not reinduce bladder activity (n = 10; not shown). Taken together, our data demonstrate that a short micturition reflex is not present in EAE rats.
Detrusor hyperactivity treatment by electrical stimulation of sacral roots
Electrical stimulation is a potential treatment to suppress hyperactivity of detrusor in MS patients. To better characterize the inhibitory receptors involved in inhibition of detrusor hyperactivity, EAE rats (n = 30) received electrical stimulation of sacral S1 roots. All the treated rats presenting detrusor hyperactivity responded independently of clinical scores to this electrical stimulation by complete (n = 15; Fig. 5Aa) or incomplete inhibition (n = 15; Fig. 5Ab) of which some presented intermitting inhibition (Fig. 5Ac) of bladder contractions. Complete suppression of hyperactivity in EAE rats induced by electrical stimulation for 1200 s exceeded the time of stimulation with a significant delay of reactivation of 130 ± 46 s for the first contraction (n = 9; Fig. 5Aa). Electrical stimulation for longer periods of 1500 and 1800 s further retarded the reappearance of the first contraction for 182 ± 32 (n = 4) and 199 ± 16 s (n = 4), respectively. We used stimulations of 1200 s for the following experiments. Efficacy of detrusor hyperactivity suppression by electrical stimulation varied during EAE (Fig. 5B) being strongest at the preclinical stage (score 0) in which four out of five rats with detrusor hyperactivity responded with complete inhibition. The effect was reversed at clinical onset (score 1) of EAE (Fig. 5B). With EAE disease progression, about half of the rats with detrusor hyperactivity at each defined disease score (see Methods) responded to electrical stimulation with complete inhibition of detrusor hyperactivity. Bladder contraction reappeared and increased progressively within 1200 s after stimulation. In animals presenting complete inhibition of detrusor hyperactivity during electrical stimulation, the AUC measured at reappearance of bladder activity was significantly reduced compared to AUC before stimulation (n = 9; Fig. 5C). Conversely, when inhibition of detrusor hyperactivity was incomplete during electrical stimulation (n = 9; Fig. 5Ab), detrusor activity started with an AUC of 90.9 ± 12.3% (12021 ± 912 cmH2O ml), which is not significantly different from the AUC before stimulation.
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Discussion |
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Clinical evaluation and cystometry reveal neurogenic bladder dysfunction with detrusor areflexia and hyperactivity in more than 90% of immunized EAE rats. Urethane used for anaesthesia may interfere with the micturition reflex (Yoshiyama et al. 1994). However, this effect acts essentially at the supraspinal sites (Yoshiyama & de Groat, 2005). Moreover, our study compared cystometric parameters recorded in the same conditions in normal and EAE animals.
During development of symptomatic disease (scores 1–4), all immunized EAE rats suffered from detrusor reflex abnormalities. The animals presenting normal bladder activity were monitored at pre-symptomatic disease stage (score 0) or in spontaneous recovery phase after severe clinical EAE (scores 1 or 0). These controls, however, are different from those recorded in unanaesthetized animals presenting shorter duration and more variable periods of bladder contraction (Yoshiyama et al. 1999). Pharmacological characterization of these pathological detrusor reflex alterations revealed the involvement of inhibitory mechanisms in the lumbosacral spinal cord. Detrusor hyperactivity could be suppressed by activation of glycinergic and GABA receptors, whereas blockade of these receptors resulted in transformation of detrusor areflexia into detrusor hyperactivity. Histopathological observations showing that the peripheral nerves in EAE rats did not present any pathological signs, confirm that the pathological detrusor abnormalities are caused by spinal cord alterations. We also showed that electrical stimulation of sacral roots could efficiently reduce detrusor hyperactivity in EAE rats. The inhibitory effect of electrical stimulation is mostly based on activation of glycine receptors in the lumbosacral spinal cord.
The present study reveals the high frequency of neurogenic bladder dysfunction in EAE rats, as in patients suffering from MS, even in the early pre-symptomatic disease phase when clinical signs were not yet visible. During MS disease progression, the vesicosphincter status changes in many patients (Litwiller et al. 1999; Ciancio et al. 2001). Among them, 30% presenting detrusor hyperactivity change to areflexia, and 50% with areflexia change to detrusor hyperactivity (Ciancio et al. 2001). Detrusor hyperactivity is most frequent being observed in about two-thirds of patients, especially with prolonged disease evolution. Based on our experimental data, we propose a model illustrating the transition from normal to pathological alterations of bladder activity in EAE (Fig. 7). Inhibition predominates at the segmental level in the dorsal horn during the pre-symptomatic disease, as evidenced by the prevalence of detrusor areflexia and by the fact that detrusor hyperactivity, when present, can be easily suppressed by electrical stimulation of the sacral root. A direct transition from normal to detrusor hyperactivity is rare (Mizusawa et al. 2000), confirming the predominance of inhibition at these early clinical stages. At onset of symptomatic disease, it is most likely that a compensatory mechanism strongly favours excitation in the dorsal horn. This may account for the predominant occurrence of detrusor hyperactivity and for only partial inhibition of hyperactivity by electrical stimulation. In established and advanced symptomatic EAE, equal occurrence of both phenotypes of the detrusor dysfunction, areflexia and hyperactivity, suggests the installation of a dynamic balance between inhibition and excitation. Pharmacological blockade of glycine receptors in the lumbosacral spinal cord pushes the balance towards excitation and presentation of the hyperactivity phenotype. Conversely, I.T. administration of glycine favours expression of the areflexia phenotype. As in MS, in EAE the areflexia phenotype directly switches to hyperactivity without expressing the normal phenotype of bladder activity.
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GABAA receptors also regulate micturition reflexes in the spinal cord (Igawa et al. 1993; Miyazato et al. 2003). Our data show that GABAergic inhibition participates in lumbosacral over-inhibition in bladder dysfunction of EAE. In detrusor areflexia, I.T. administration of bicuculline induced a partial recovery of detrusor activity. However, the GABAA antagonist was much less efficient than strychnine in restoring a reflex. This partial effect cannot be explained by an experimental bias, as the maximum bicuculline concentration administered (160 µM) was significantly higher than bicuculline concentrations (100 µM) in other studies (Kanie et al. 2000; Sugaya & de Groat, 2002; Lin, 2003). GABAA receptor-mediated inhibition rather acts at the supraspinal level, in lateral and medial pons centres, where it inhibits descending excitatory pathways to the lumbar spinal cord (Kanie et al. 2000). Inhibitory descending pathways were also proposed in rats (Baez et al. 2005) and in cats, as glutamate injection in the raphe serotoninergic nucleus caused inhibition of micturition (Chen et al. 1993). Such a descending pathway might also contribute to spinal over-inhibition in EAE rats presenting areflexia.
As a mirror image of detrusor areflexia, the balance between excitation and inhibition was most probably shifted towards excitation in detrusor hyperactivity, as the latter was efficiently blocked by I.T. administration of inhibitors such as glycine or GABAA receptor agonists as shown for muscimol. Baclofen-mediated activation of GABAB receptors was also effective in reducing detrusor hyperactivity in EAE, although in human bladder dysfunction caused by spinal lesion, baclofen has only a limited effect (Stempien & Tsai, 2000). A descending excitatory pathway is most probably involved in over-excitation in detrusor hyperactivity, because in our EAE rats with detrusor hyperactivity, mechanical or chemical spinalization abolished bladder contraction. The participation of a descending pathway from the pons in the generation of detrusor hyperactivity has also been shown in animal models of cerebral ischaemia caused by permanent middle cerebral artery occlusion (Kanie et al. 2000).
In physiological conditions, the micturition reflex relies on a spinobulbospinal loop (de Groat et al. 1990) characterized, in cats, by a latency of 65–100 ms between pelvic afferent stimulation and sacral parasympathetic neuronal discharge (Mallory et al. 1989). In spinalized cats showing detrusor hyperactivity, however, the reflex latency is much shorter (15–40 ms) and similar to that in spinalized rats (Mallory et al. 1989). These alterations argue for a reorganization of the micturition reflex pathway from a long bulbospinal loop to a short spinal loop after spinalization (de Groat et al. 1981). Furthermore, afferent fibres involved in this short reflex loop seem to be different. Subcutaneous injections of capsaicin, a specific activator of C-fibres, had no effect in intact cats, whereas it suppressed the micturition reflex for 3–6 weeks after spinalization in animals presenting detrusor hyperactivity, indicating involvement of non-myelinated afferent C-fibres (de Groat et al. 1990). Similarly, in human with complete suprasacral spinal cord injury, detrusor hyperactivity can be reduced by intravesical instillation of capsaicin (Kim et al. 2003) also suggesting a reorganization in a short reflex loop activated by C-fibres (Dinis et al. 2004).
Such reorganization in EAE is therefore possible. Previous studies using chemical inflammation of the bladder indicated recruitment of C-fibres, inducing bladder hyperactivity (Yoshimura & de Groat, 1999). However, intravesical instillation of turpentine did not induce bladder contraction in our EAE rats with detrusor areflexia. This indicates that the micturition reflex does not depend on C-fibre activation in EAE rats arguing against the development of a short reflex loop. We confirmed this in EAE rats showing detrusor hyperactivity, which turned into areflexia by spinalization. In these spinalized rats, stimulation of bladder nociceptive afferents with turpentine was also unable to trigger bladder contraction.
In humans, several types of bladder hyperactivity (Tanagho & Schmidt, 1989; Weil et al. 2000) including those occurring in MS patients (Rund Bosch & Groen, 1996), can be suppressed by low frequency chronic stimulation of sacral roots (i.e. by sacral neuromodulation). This approach is applied in functional surgery to partially restore bladder function. We show here, in EAE rats presenting detrusor hyperactivity, that electrical stimulation of sacral nerves suppressed bladder activity. Inhibition of bladder contractions was maintained during long periods of stimulation (up to 1 h). When reappearing, contractions presented with a reduced frequency and lower amplitude. However, the restored activity, although reduced, never regained a normal phenotype indicating that even when inhibited, the micturition centres remained in a sensitized state. Various types of fibres involved in sacral neuromodulation have been characterized. Stimulation of somatic afferent fibres of the pelvis decreased bladder contraction in cats (de Groat & Ryall, 1969) and rats (Sato et al. 1992; Morrison et al. 1995). Whereas A-fibre stimulation decreases contractions in rats, C-fibre activation promotes bladder contractions (de Groat et al. 1990). A-fibres contained in bladder afferents are probably involved in inhibition of small fibres. Involvement of small C-fibre inhibition in sacral neuromodulation is supported by the observed decrease of neuropeptide contents (substance P and neurokinine A) in primary afferents of the L6 dorsal root (containing fibres originating from the bladder), but not L5, during stimulation of bladder afferents in spinalized animals (Shaker et al. 2000). In EAE rats, we showed that the inhibition of bladder contraction occurs immediately, in opposition to the situation of complete spinal cord lesion that requires long periods of electrical stimulation to elicit an inhibitory effect (Shaker et al. 2000). Again, this indicates that bladder hyperactivity is based on different mechanisms in spinal cord injury and inflammatory neurodegenerative diseases.
In conclusion, our data reveal the existence of an exaggerated descending excitatory control in EAE rats developing either areflexia or hyperactivity of the detrusor. This over-excitation at the spinal segmental level determines bladder hyperactivity. In EAE rats presenting detrusor areflexia, however, this excitatory control is dominated by a strong segmental inhibition mostly mediated by glycine receptor activation. In rats presenting with hyperactivity of the detrusor muscle, electrical stimulation of afferent fibres in the sacral dorsal root favours inhibition via the activation of local glycinergic interneurones and suppresses bladder activity. The present study in EAE rats substantially improves understanding of the mechanisms of spinal cord affection causing bladder dysfunction in MS, and sheds new light on pharmacological mechanisms involved in suppression of detrusor hyperactivity by electrical stimulation of lumbosacral roots in MS patients.
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Footnotes |
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, first quartile; 
, minimum. *P < 0.05, **P < 0.01, ***P < 0.001.
) and incomplete (
