Background
Overactive bladder syndrome (OAB) is characterized by urinary urgency accompanied by frequent urination and nocturia, with or without urinary urge incontinence [
1]. OAB severely affects the patients’ quality of life, since frequent urination can cause severe inconvenience, whereas long-term incontinence can lead to urinary tract infections, possibly leading to severe clinical complications affecting the patient’s physical and mental well-being [
2]. Therefore, the micturition reflex which is subject to complex control from both the central and the peripheral nervous system, is attracting more attention. Anatomic studies have uncovered that the relevant afferent nerves mainly pass through the pelvic posterior, mainly from the L6 and S1 dorsal root ganglia (DRG) to the spinal cord, to trigger the micturition reflex [
3]. According to the diameter of the neurons, the afferent DRG nerve fibers are classified as large, medium and small, which are further categorized into A
α/β, A
δ and C-type fibers [
4,
5]. The afferent nerves controlling of the urinary bladder include the myelinated A
δ and the unmyelinated C-type fibers [
6]. The hyperpolarization-activated cyclic nucleotide-gated cation (HCN) currents (
Ih) have been reported to be expressed by many cell types, such as cardiac pacemaker cells and retinal photoreceptor cells, as well as central and peripheral nerve cells, including DRG cells.
It has been discovered that small- and medium-size nerve cells of the DRG which can control urinary bladder all possess
Ih currents, and the
Ih current of the medium-sized neurons is significantly higher than that of the small neurons [
7]. This indicates that the
Ih current is predominantly present in the medium-size DRG neurons. Even though the afferent neurons that control the bladder include C and A
δ type fibers, in normal physiological conditions, the A
δ fibers are solely responsible for the mechanical nociceptive reflex when the bladder expands to threshold pressure. This indicates that the medium-size neurons of the DRG play an important role in the micturition reflex. The
Ih current activated by hyperpolarization of the HCN-gated channels has been shown to be involved in many important physiological functions, including the pacemaker function of the heart and brain as well as maintaining the resting membrane potential and cell excitability [
8,
9].
Masuda and coworkers have discovered that ZD7288, a specific inhibitor of HCN-gated channels, can inhibit the
Ih current in the medium-size cells that control the bladder, markedly increasing the duration of the afterhyperpolarization potential of an action potential [
7]. This in turn implies that the
Ih current influences the excitability of the medium-size DRG neurons that innervate the bladder. In order to find out the relationship between OAB and the
Ih current in medium-size DRG neurons, this study was performed to characterize the electrophysiological properties of the
Ih current in the medium-size cells of the L6 dorsal root ganglion in a rat model of overactive bladder, and to investigate its influence on the model animals’ urodynamics.
Discussion
The urinary bladder weight of the overactive bladder syndrome (OAB) model rats, as well as the excitability and Ih current density of the animals’ medium-size DRG neurons were significantly increased. Intrathecal injection of the specific Ih current inhibitor ZD7288 was able to alleviate the observed changes in the OAB rats, including a prolonged micturition time and shortened micturition interval. At the same time, ZD7288 completely blocked the Ih current density, and lowered the excitability of the medium-size DRG cells. Taken together, these results strongly suggest that the Ih current may be a factor in the development of OAB.
The partial bladder outlet obstruction rat model is a good approximation of the condition seen in clinical patients with urinary bladder overactivity and urethral blockage syndromes, including a high degree of agreement in the observed pathological changes [
19]. Therefore, the partial bladder outlet obstruction rat model was used in this study, and the urinary bladder weight of the successfully induced OAB rats was significantly increased, in agreement with the literature [
13]. Recordings of the micturition process have uncovered that the micturition interval of the OAB rats was significantly shortened, their micturition time was significantly increased, and micturition volume was significantly decreased, indicating that the rats with the partially-blocked urethra indeed had an abnormally enhanced micturition reflex.
According to anatomical studies, the neural fibers that innervate the bladder include the myelinated A
δ and the myelin-free C-type fibers [
6,
19,
20]. One part of these afferent fibers transverses the pelvis, arriving at the L6 and S1 DRG of the spine, and takes part in the initial stage of the micturition signal, while another part transverses the lower abdomen, arriving at the L1 and L2 level of the spine [
3,
21,
22]. The DRG neurons are classified into small, medium and large types according to their cross-sectional diameter, and further separated according to the A
α/β, A
δ and C typology [
4,
5]. A subset of the nerve fibers that control the micturition process terminate at the L6 DRG, making the question pertinent whether the excitability of the OAB rats’ L6 DRG has exhibited any changes. The results of present study indicated that the AP amplitude and number of APs elicited by a 500-pA stimulus were significantly increased, whereas the half-width, threshold strength, threshold value and the afterhyperpolarization potential, as well as the input resistance were significantly decreased. The membrane potential also had an obvious depolarization tendency. All these results suggested that the medium-size L6 DRG cells of the OAB rats had a significantly increased excitability. The membrane potential of the L6 DRG neurons of the OAB rats also displayed obvious depolarization, with an increase in the number of APs elicited by a 500-pA stimulus, as well as a decrease of the input resistance and τ value. Thus, the hyperpolarization-induced cation current (
Ih) might play an important role in the observed increased excitability of the medium-size neurons in the L6 DRG of the OAB rats. The
Ih current is present in excitable cells in both the center and the periphery of the body, including the photoreceptor cells of the retina and the cardiac pace-maker cells, as well as cells of the central and peripheral nervous system [
23,
24]. The
Ih current channel is activated when the AHP is rapidly increasing, and the influx of Na
+ causes a slow depolarization of the cell membrane. In peripheral nerves,
Ih is found in the sensory DRG neurons [
25]. Within these sensory neurons, it participates in the AHP process, and induces the cell membrane to recover to the resting potential after depolarization, shortening the AHP amplitude and duration [
26,
17,
27]. Studies have also reported that the
Ih current plays a role in the maintenance of membrane potential, influencing the cells’ spontaneous and repeated discharge, as well as related phenomena [
28,
29]. In our experiments, it was shown that the specific
Ih current inhibitor ZD7288 is able to clearly inhibit the excitability of the medium-size DRG cells in the OAB rats, including a reduction of the number of APs elicited by a 500-pA stimulus and an increase of the τ value, indicating that the
Ih current within the L6 DRG neurons plays an important role in the etiology of OAB. After administration of ZD7288, DRG in sham+ZD7288 group also showed elevated τ value and reduction in current density, suggested that
Ih current inhibitor could reduce the cell membrane excitability in sham rats. However, the reduction range of current density of DRG neurons after ZD7288 administration were obviously wider in OAB + ZD7288 group than sham+ZD7288 group. These results suggested that ZD7288 could inhibit cell excitability of DRG cells relatively more strongly in OAB rats than in sham rats.
A study by Masuda and coworkers has found that the
Ih current is primarily present in the medium-size DRG neurons that control the urinary bladder, and that inhibition of the current significantly increases the time from the highest AHP point until the return to the resting potential, and reduces the excitability of the medium-size DRG neurons [
7]. These results imply that the excitability of the medium-size mechanical nociceptor cells in the DRG that control the urinary bladder may be influenced by the
Ih current. This raises the question whether the
Ih current in the medium-size DRG cells of the OAB mice experienced changes. Our results show that the
Ih current density was significantly increased, and functional experiments have shown that intrathecal injection of ZD7288 significantly improved the micturition parameters of the OAB rats. The experimental results indicate that an increase in the strength of the
Ih current in the medium-size cells of the L6 DRG might be an important component of the OAB development process. Furthermore, the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN) mediates the
Ih current, and an investigation of HCN expression in the neurons that control the bladder, which are found in the L6-S1 DRG, has uncovered rich expression of HCN2 in the average medium-size DRG cells.
It is thus theoretically possible that the significant increase of the
Ih current in the medium-size neurons of the L6 DRG of OAB rats is related to the HCN2 channel, and this notion merits further research. Furthermore, these experimental results demonstrate that the half-width, amplitude, and afterhyperpolarization potential of the action potential are not influenced by ZD7288, which is in agreement with other studies, which have also found that ZD2788 cannot influence the shape of the AP or the AHP amplitude [
28,
29]. This indicates that the observed changes of the
Ih current may be part of the electrophysiological mechanism responsible for the increase of excitability in the medium-size DRG cells of the OAB rats. The limitations of current study is that the precise functional activity of the
Ih current in the medium-size DRG neurons of OAB rats has not been fully unveiled, and we do not observe the small-size DRG neurons. Further studies using structurally dissimilar HCN channel blockers such as ivabradine will be needed to strengthen our present behavioral and electrophysiological findings. Further electrophysiological mechanisms influencing the cells’ excitability still remain to be resolved. Since the etiology of OAB is complicated and multifactorial, additional studies are needed in different animal models and different OAB related cells to clarify the real role of
Ih current in bladder dysfunction.
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