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Keywords:

  • electrical stimulation;
  • bladder;
  • spinal cord injuries;
  • synaptic transmission

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

OBJECTIVE

To investigate the effects of intravesical electrical stimulation (IVES) on bladder function and synaptic neurotransmission in the lumbosacral spinal cord in the spinalized rat, as the clinical benefits of IVES in patients with increased residual urine or reduced bladder capacity have been reported but studies on the mechanism of IVES have mainly focused on bladder Aδ afferents in central nervous system-intact rats.

MATERIALS AND METHODS

In all, 30 female Sprague-Dawley rats were divided equally into three groups: normal control rats, sham-stimulated spinalized rats and IVES-treated spinalized rats. IVES was started 5 weeks after spinal cord injury (SCI) and was performed 20 min a day for 5 consecutive days. At 7 days after IVES, conscious filling cystometry was performed. Sections from the L6 and S1 spinal cord segments were examined for n-methyl-d-aspartic acid receptor 1 (NMDAR1) subunit and γ-aminobutyric acid (GABA) immunoactivity.

RESULTS

In IVES-treated spinalized rats, the number and maximal pressure of nonvoiding detrusor contractions were significantly less than in sham-stimulated spinalized rats. The mean maximal voiding pressure was also lower in IVES-treated than in sham-stimulated spinalized rats. IVES significantly reduced the interval between voiding contractions compared with the untreated spinalized rats. There was an overall increase in NMDAR1 immunoactivity after SCI, which was significantly lower in IVES-treated spinalized rats. Immunoactivity of GABA after SCI was significantly lower than in the control group and was significantly higher in IVES-treated spinalized rats.

CONCLUSION

Our results suggest that IVES might affect voiding contractions in addition to inhibiting C-fibre activity and that IVES seems to have a more complex effect on the bladder control pathway. For synaptic neurotransmission in the spinal cord, IVES could possibly shift the balance between excitation and inhibition towards inhibition.


Abbreviations
GABA

γ-aminobutyric acid

IVES

intravesical electrical stimulation

NMDA(R)

n-methyl-d-aspartic (receptor)

SCI

spinal cord injury.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

The first trials with intravesical electrical stimulation (IVES) can be traced back to 1878 when Saxtorph treated patients with urinary retention by transurethral bladder stimulation. This technique was re-introduced by Katona [1] as a treatment for neurogenic voiding disorders. Several investigators worldwide have been using IVES to treat bladder and bowel dysfunction secondary to various conditions including spinal cord injury (SCI), myelodysplasia, hypocontractile or acontractile bladders and lazy bladder syndrome [2–6].

Several studies have investigated the anatomical and physiological aspects of the mechanism of action of IVES. In those experimental studies, it was shown that IVES involves the direct artificial activation of the Aδ afferents from low-threshold bladder mechanoreceptors, which comprise the sensory system responsible for both initiating and maintaining the micturition reflex [7]. Recently, it was suggested that IVES-induced modulation of the micturition reflex was due to enhanced excitatory synaptic transmission in the central micturition reflex pathway [8]. However, while IVES is clinically applied to pathological bladders, previous experimental studies have been limited to the myelinated Aδ afferents in spinal-intact rats.

Nervous control of normal bladder function relies on a spinobulbospinal pathway involving the coordination of sympathetic, parasympathetic and somatic controls. This complex control depends crucially on the activation of excitatory and inhibitory sacral spinal neurones. In the CNS, various amino acids are known to be important neurotransmitters in the micturition reflex pathway [9], e.g. glutamate is a major excitatory neurotransmitter and facilitates the micturition reflex [10]. Conversely, γ-aminobutyric acid (GABA) is a major inhibitory neurotransmitter and inhibits the micturition reflex at the level of the lumbosacral cord [11]. SCI rostral to the lumbosacral level causes urinary bladder dysfunction. There is a considerable reorganization of the reflex connection in the spinal cord after the interruption of both ascending and descending pathways. SCI produces an initial period of urinary bladder areflexia that persists for several weeks to months. This period is followed by the emergence of a micturition reflex at the spinal level and detrusor hyperreflexia has been recorded in chronic SCI rats. This reorganization of the micturition reflex is related to an alteration of the ratio of inhibitory to excitatory amino acid concentrations in the spinal cord. In the acute phase after SCI, levels of inhibitory amino acids in the spinal cord are higher and maintained for several days, and this increase is associated with detrusor areflexia [12]. Conversely, the micturition reflex is characterized by detrusor hyperreflexia in the chronic phase of SCI. The inhibitory amino acid concentration is reduced while there is no change in the excitatory amino acid concentration [13]. Thus, the equilibrium between excitatory and inhibitory amino acids is shifted towards excitation in chronic SCI rats.

To gain a greater understanding of the mechanisms of action of IVES, we examined the effects of IVES on bladder function in a chronic SCI rat model. In the chronic phase of SCI, a spinal micturition reflex subsequently becomes active and evokes detrusor hyperreflexia. Thus, the synaptic neurotransmission in the lumbosacral spinal cord in the spinalized rat was also investigated. Glutamate acts on spinal neurones through various glutamatergic receptor subtypes including the n-methyl-d-aspartic acid (NMDA) subtype. NMDA receptors (NMDARs) control urogenital and digestive reflex pathways at the lumbosacral level [14]. GABA and glycine are major inhibitory neurotransmitters. It is known that GABAergic mechanisms in the spinal cord inhibit the micturition reflex. However, very little is known about changes in GABA levels in the lumbosacral spinal cord after SCI. We also investigated the change in expression levels of the NMDAR 1 (NMDAR1) subunit and GABA in the lumbosacral spinal cord after IVES.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

In all, 30 female Sprague-Dawley rats weighing 250–300 g were divided equally into three groups: ‘normal’ control rats that did not undergo any procedure, sham-stimulated spinalized rats (spinalized rats with stimulation catheter insertion, but no stimulation) and IVES-treated spinalized rats. The animal procedures were approved by the Institutional Animal Care and Use Committee of the Yonsei University College of Medicine.

The SCI procedure was performed under general anaesthesia with a combination of an s.c. injection of acepromazine (0.05 mg/kg), ketamine (50 mg/kg) and xylazine (5 mg/kg). A 2-cm dorsal midline incision was made and the vertebral body was exposed. The paraspinal muscles were dissected from the spinal process and retracted laterally. After laminectomy of the T9–T10 vertebrae the spinal cord was completely sectioned with iris scissors. The space between the retracted ends of the spinal cord was packed with Gelfoam and the incision was sutured. Body temperature was maintained at 37 °C during the first 24 h after SCI using a heating plate and i.m. ampicillin (150 mg/kg) was used for prophylaxis for the next 7 days. During the first 2 weeks, urine was evacuated by digital compression twice daily until reflex micturition was restored.

IVES was started 5 weeks after the SCI. Each rat in the stimulation group had IVES for 20 min a day for 5 days. For the experimental IVES procedure [7] previously described, a specially designed catheter with a monopolar electrode inside the tip was inserted into the bladder through the urethra. The intravesical electrode was used as the cathode. A brass plate wrapped in a saline-soaked gauze was placed on the abdominal skin at the level of the bladder and it served as the anode. IVES was administered with an MS-310 stimulator (Vitacon, Trondheim, Norway) giving unipolar square waves of 0.2 ms duration, with an intensity of up to ≈10 mA and frequencies of 20 Hz. These parameters are identical to those of a previous study, which evoked the best reflex bladder contraction in spinal-intact rats [7].

At 7 days after IVES, we performed conscious filling cystometry. The rats were anaesthetized with isoflurane and the bladder was exposed through a midline incision. A polyethylene-50 tube was placed through a small incision in the dome of the bladder. The bladder was closed in a purse-string manner. After surgery, the rats were placed in a restraining cage and were allowed to recover from anaesthesia for 1–2 h. The intravesical catheter was connected via a three-way stopcock to a pressure transducer and a syringe pump for recording intravesical bladder pressure and infusing saline into the bladder, respectively. Saline at room temperature was infused at 0.04 mL/min. Cystometric variables were then measured during saline infusion for 2 h to evaluate bladder function. Bladder contractions were designated as voiding or nonvoiding depending on whether urine was collected after the contraction. The interval between voiding contractions and nonvoiding contractions, the frequency of nonvoiding contractions and the amplitude of bladder contractions were calculated using the Power-lab system (AD instruments, Australia).

After bladder infusion for 2 h, the rats underwent intracardial perfusion with 0.1 m phosphate buffer pH 7.4 followed by 4% paraformaldehyde fixative in phosphate buffer (0.1 m, pH 7.4). The spinal cord sections (L6, S1) were removed and post-fixed for 12 h in the same fixative at 4 °C before being cryoprotected in 0.1 m, pH 7.4, phosphate-buffered 30% sucrose solution overnight. They were then sectioned (40 µm) on a freezing microtome (Microm, Walldorf, Germany). The free-floating tissue sections were processed for immunohistochemistry using rabbit anti-NMDAR1 polyclonal antibodies (1:1000, Novus Biologicals Inc.) and rabbit anti-GABA (1:1000, Abcam, Cambridge, MA, USA). Sections were incubated overnight at 4 °C with the primary antibodies. After 14 h, the sections were reacted for 2 h with a biotinylated goat anti-rabbit IgG (1:200, Vectastain Inc.) and ABC solutions (Vectastain Inc.), respectively. The tissue sections were washed, mounted on the gelatine-coated slides and coverslipped with polymount. The sections were examined in an image analyser (Olympus, Albertslund, Denmark). Immunodensity was quantified by measuring and averaging the intensity of staining [15]. The density of white matter was also used as a reference point to ensure equal staining between sections. The immunodensities (arbitrary units) of NMDAR1 and GABA were expressed as the mean (sem). Nonspecific binding was examined by omitting the corresponding primary antibody during the incubation process.

All data are expressed as the mean (sem). For statistical analyses anova and Student’s t-test (unpaired) were used; with P < 0.05 considered to indicate statistical significance.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

During conscious filling cystometry, all spinalized rats had nonvoiding detrusor contractions before a large amplitude voiding bladder contraction occurred (Fig. 1). This phenomenon was not detected in normal control rats. The amplitude of the nonvoiding detrusor contractions increased as the bladder was filled, reaching a mean (sem) maximum of 29.2 (10.7) cmH2O. There was a mean of 12.2 (5.8) nonvoiding detrusor contractions per voiding cycle. However, in IVES-treated rats, the mean number of nonvoiding detrusor contractions per voiding was 6.9 (3.7) (P < 0.01) and mean maximal nonvoiding detrusor contraction pressure was 20.3 (9.1) cmH2O (P < 0.05), which were significantly less than in the sham-stimulated spinalized rats (Table 1). The mean maximal voiding pressure was also less in IVES-treated than in sham-stimulated spinalized rats at 33.9 (5.4) cmH2O vs 57.3 (12.3) cmH2O (P < 0.05). The interval between voiding contractions was significantly larger after spinalization, at 642 (180) s compared with normal control rats at 373 (153) s. However, IVES significantly reduced the interval between voiding contractions to 275 (88) s compared with the sham-stimulated spinalized rats (P < 0.05).

image

Figure 1. Cystometrogram in the normal control group (A), the sham-stimulated spinalized group (B) and IVES treated spinalized group (C).

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Table 1.  Effects of IVES on cystometric variables and synaptic transmission in the lumbosacral spinal cord
Variable, mean (sem)Groups
Normal controlSCISCI + IVES
  • *

    P < 0.05;

  • †arbitrary units.

Cystometric measurement
Voiding contraction:
 Interval between voiding, s 373 (153) 642 (180)* 275 (88)*
 Maximal pressure, cmH2O27.5 (6.2) 57.3 (12.3)*33.9 (5.4)*
Nonvoiding contraction:
 Number per voiding cycle, n  12.2 (5.8) 6.9 (3.7)*
 Maximal pressure, cmH2O  29.2 (10.7)20.3 (9.1)*
Synaptic transmission
Mean density:
 NMDAR168.6 (1.5) 117.2 (2.1)*68.7 (3.2)*
 GABA84.3 (3.1) 66.4 (1.0)*83.1 (2.1)*

There were NMDAR1-positive cells in the superficial layers of the dorsal horn. In the ventral horn, the large motor neurones were not immunoreactive compared with the nearby smaller cells. After SCI, there was an overall higher immunodensity of NMDAR1 in L6–S1 spinal cord compared with the normal control group, at 117.19 (2.12) vs 68.62 (1.53), and this was significantly lower in the IVES-treated spinalized group at 68.73 (3.21). GABA-positive cells were localized as a dense band within lamina II of the dorsal horn and sacral parasympathetic nucleus region. The immunodensity of GABA was 84.39 (3.02) in the normal control group, 66.38 (1.01) in the spinalized group and 83.17 (2.18) in the IVES-treated spinalized group (Fig. 2, Table 1).

image

Figure 2. Photomicrographs showing the density of immunohistochemical staining for NMDAR1 and GABA.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

IVES markedly reduced the number and maximal detrusor pressure of the nonvoiding contractions in spinalized rats. It is well known that two types of afferent fibres, namely Aδ and C-fibres, carry sensory information from the bladder to the spinal cord [16]. Recent experiments have shown that there is reorganization of the reflex connections in the spinal cord after SCI [17]. A major breakthrough has been the recognition that C-fibre bladder afferents can trigger bladder hyperactivity after SCI. In CNS-intact rats, C-fibre bladder afferents are usually silent or inactive. After SCI, these fibres become sensitized and saline distension of the bladder induces multiple nonvoiding detrusor contractions. In the present study, IVES suppressed the nonvoiding detrusor contractions that occurred before voiding, indicating that IVES might inhibit C-fibre activity emerging after SCI. We have further data to support that IVES can inhibit C-fibre activity. A c-fos gene expression study is a tool to evaluate the spinal processing of afferent C-fibre input; IVES reduced c-fos gene expression in the L6–S1 spinal cord segment in spinalized rats [18]. IVES also significantly reduced the relative density of calcitonin gene-related peptide and substance P at the lumbosacral dorsal root ganglia in the spinalized rat [19]. Inhibition of afferent C-fibre activity in the spinal reflex pathway may be one of the underlying mechanisms of IVES.

Previous studies on the effects of IVES on the micturition reflex showed that IVES induced a prolonged decrease in the micturition threshold volume [20] and enhanced excitatory synaptic transmission in the central micturition reflex pathway [8]. The conclusion from those studies was that the IVES-induced threshold change was due to activation of bladder Aδ afferents and bladder C-afferents were not involved in the modulation of the micturition reflex. In those studies, they attempted to avoid co-activation of bladder C-afferents by keeping the stimulation intensity below threshold for activation of unmyelinated bladder fibres (10 mA, 20 Hz). They suggested that the stimulation intensity used was well below threshold for electrical activation of C-afferents or their receptors. So, Ebner et al.[7] offered a theoretical rationale for the use of IVES as treatment for weak detrusor contractility. In previous studies, IVES in CNS-intact rats consisted of continuous stimulation at 20 Hz, and the intensity was adjusted to give maximal reflex bladder contraction (7–11 mA). The same stimulus of IVES was applied to neuropathic bladder in SCI rat. We expected different findings in the neuropathic bladder after IVES because there is reorganization of the reflex connections in the spinal cord after SCI. There is evidence in the rat that C-fibre afferents increase their excitability to induce bladder hyperreflexia after SCI. Bladder afferent neurones exhibit various types of functional (more excitable due to increased expression of tetrodotoxin-sensitive Na+ channels) and morphological changes (larger in size and more likely to exhibit neurofilamment immunoreactivity) after SCI [21]. C-fibre bladder afferents are usually silent or inactive in the spinal-intact rat, but become more excitable after SCI. The IVES effect on C-fibres is based on silent C-fibres with low excitability according to previous studies. In the present study, IVES suppressed nonvoiding bladder contractions that occur prematurely before voiding bladder contractions. These data suggest that it is possible for IVES to inhibit C-fibre activity, which becomes more excitable after SCI.

The cystometric outcome of IVES is different from that of capsaicin. Pretreatment of chronic SCI rats with capsaicin eliminated the nonvoiding contractions but the amplitude of voiding contractions and bladder capacity were not altered in unanaesthetized rats [22]. These effects of capsaicin were consistent in chronic SCI rats on the second day after urethane anaesthesia, as the rats were recovering from deep urethane anaesthesia [23]. Those results show that capsaicin treatment has an inhibitory effect on C-fibres that mediate nonvoiding contractions. A blockade of capsaicin-sensitive afferents by capsaicin, suppressed nonvoiding bladder contractions, but not voiding contractions in SCI rats. In the present study, the mean maximal voiding pressure was lower in IVES-treated rats than in untreated spinalized rats. IVES significantly reduced the interval between voiding contractions compared with untreated spinalized rats. Voiding contractions are still triggered by Aδ fibre afferent in SCI rats [22]. It is possible that IVES might not only inhibit C-fibre activity of the pelvic nerve but involve other lower urinary tract reflex mechanisms.

How does IVES affect voiding contraction in addition to inhibition of C-fibre activity? Numerous studies have shown that the hypogastric nerve conveys sympathetic efferent fibres to provide inhibitory input to the bladder and excitatory input to the bladder neck and proximal urethra [24,25]. Previous studies [26] have shown that bilateral transection of the hypogastric nerve in conscious SCI rats prominently reduced maximal voiding pressure and threshold volume and improved voiding efficiency. Those results indicate that a sympathetic pathway is involved in the abnormal voiding of SCI rats. This abnormal sympathetic activity might occur from the elimination of the modulatory influence of the pontine micturition centre on the spinal sympathetic reflex pathway after SCI. Therefore, enhancement of the inhibitory input to the bladder would increase the intervals between voiding contractions and enhancement of the excitatory input to the bladder neck would maintain a high outlet resistance during voiding in spinalized rats. IVES significantly reduced the maximal voiding pressure and the interval between voiding contractions. Cystometric results of IVES are similar to those of bilateral transection of the hypogastric nerve. Thus, it is possible that IVES may influence the spinal sympathetic reflex pathway controlling the detrusor and bladder neck/proximal urethra. The effect of IVES on sympathetic reflex pathway may occur by suppressing the inhibitory input to the bladder enhanced by SCI. Although the effect of IVES on voiding contractions was similar to that of bilateral transection of the hypogastric nerve, further study to evaluate its influence on the sympathetic reflex pathway of IVES is warranted.

We attempted to investigate the effects of IVES on the synaptic neurotransmission that controls the micturition reflex pathway in the lumbosacral spinal cord. A complex family of ionotropic and metabotropic glutamate receptors mediate the various responses of the excitatory signal in the CNS [27,28]. The ionotropic receptors are further classified into NMDARs (NMDAR1 and NMDAR2A-D subunits) and non-NMDARs. The NMDAR1 glutamatergic receptor subunit is widespread throughout the L6 and S1 segments of the rat spinal cord. NMDAR1 labelling is present in the dorsal root ganglia, the dorsal horn, the sacral parasympathetic nucleus, the dorsal grey commissure, and groups of motoneurones in the ventral horn of CNS-intact rat [29]. It is well known that NMDAR1 receptors can be activated by glutamate released by afferents from peripheral and supraspinal origins to elicit bladder contractions. Intravesical injection of MK-801, a noncompetitive NMDA receptor antagonist, decreased voiding pressure and increased bladder capacity in SCI rats [30]. Grossman et al.[31] quantified the protein levels of the NMDAR1 in the lumbar spinal cord at both 24 h and 1 month after SCI. The levels of NMDAR1 protein in acute SCI were lower than in the uninjured control. In chronic SCI, lumbar regions showed a trend toward up-regulation, but this was not significant. In the present study there was a significant up-regulation in the NMDAR1 labelling in chronic SCI. Excitatory amino acids acting on the NMDA receptor may play a role in the control of micturition.

In chronic SCI, the change in the expression level of inhibitory amino acids in the spinal cord is more obvious. Glycine is an important inhibitory neurotransmitter as well as GABA. In the spinal shock phase after SCI, the glycine level in the lumbosacral cord is increased and in chronic SCI the glycine level is lower compared with the CNS-intact rats [13]. Detrusor areflexia may be caused by an increase in inhibitory amino acids in the spinal cord and detrusor hyperreflexia during the chronic phase of SCI may be related to a decrease of inhibitory amino acids in the spinal cord. The present study also showed that the immunoactivity of GABA in chronic SCI was lower than that in the controls. IVES in the chronic SCI rat significantly restored the balance between levels of excitatory and inhibitory amino acids in the lumbosacral spinal cord. The immunoactivity of NMDR1 was lower and that of GABA was higher in the lumbosacral spinal cord. The balance between excitation and inhibition was most probably shifted toward inhibition in detrusor hyperreflexia after IVES.

In conclusion, IVES significantly reduced the number and maximal pressure of nonvoiding detrusor contractions in spinalized rats. Inhibition of afferent C-fibre activity in the spinal reflex pathway may be one of the underlying mechanisms of IVES. IVES also reduced the interval between voiding contractions. This suggests that the effect of IVES is not restricted to inhibition of afferent C-fibre activity. It seems reasonable to attribute the effect of IVES to other lower urinary tract reflex mechanisms that decrease the interval between voiding contractions. In present study, there were no data concerning the optimal parameters for inhibition of afferent C-fibre activity. A challenge for future studies will be to identify such optimal parameters. For synaptic neurotransmission in the spinal cord, the effect of IVES on micturition reflex was related to an alteration in the balance between excitatory and inhibitory amino acid levels in the lumbosacral spinal cord.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

This work was supported by Korean Research Foundation Grant (KRF-2004-042-E00093)

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES