Therapeutic effects of imidazoline 2 receptor activation on lower urinary tract dysfunction in a mouse model of spinal cord injury

Abstract

This study evaluated the effects of the imidazoline I2 receptor (I2R) agonist 2-(2-benzofuranyl)-2-imidazoline (2BFI) on lower urinary tract dysfunction (LUTD) in a mouse spinal cord injury (SCI) model. Female mice were divided into three groups: spinal intact (SI), SCI with vehicle, and SCI with 2BFI beginning 2 wk postinjury. SCI was induced by complete Th8–9 spinal transection. Four weeks after SCI, conscious cystometrograms (CMG) and external urethral sphincter (EUS) electromyography (EMG) were recorded. L6-S1 dorsal root ganglia (DRG) were collected for qPCR analysis of TRPV1, tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS). In a separate group of animals, intrathecal (i.t.) 2BFI was tested during CMG/EMG in SCI. Organ bath studies assessed 2BFI-induced relaxation in bladder strips precontracted with KCl or carbachol. SCI mice showed increased nonvoiding contractions (NVCs) and reduced voiding efficiency versus SI. 2BFI significantly decreased NVCs, improved voiding efficiency, and prolonged EUS relaxation, evident as reduced EMG activity compared with vehicle. SCI-induced upregulation of TRPV1, TNF-α, and iNOS in DRG was significantly attenuated by 2BFI. I.t. 2BFI reduced residual urine and NVCs and increased EUS relaxation in SCI mice. In organ baths, 2BFI produced significant relaxation of precontracted bladder strips from both SI and SCI mice. I2R activation in the lumbosacral spinal cord and bladder may provide an effective therapeutic strategy for SCI-induced LUTD.

NEW & NOTEWORTHY

This study shows that selective activation of the imidazoline-2 receptor improves both detrusor overactivity and detrusor-sphincter dyssynergia after spinal cord injury. Systemic and intrathecal administration of 2BFI produced coordinated actions in the spinal cord and lower urinary tract, including enhanced relaxation of the external urethral sphincter, reduced inflammation mediated by C-fiber afferents, and direct relaxation of bladder smooth muscle, indicating a promising therapeutic target.

INTRODUCTION

Lower urinary tract dysfunction (LUTD) significantly reduces quality of life by exacerbating storage and voiding problems (1). Chronic spinal cord injury (SCI) rostral to the lumbosacral spinal cord region results in detrusor overactivity (DO) during the storage phase, a phenomenon mediated by spinal reflexes triggered through hyperexcitable C-fiber afferent pathways (2–5). Moreover, during the voiding phase, SCI individuals often experience ineffective voiding due to detrusor-sphincter dyssynergia (DSD). Although several studies have proposed potential pharmacological targets for treating LUTD following SCI (3, 4), drug therapies often fail to sufficiently alleviate SCI-induced LUTD (6).

Imidazoline receptors were initially identified as nonadrenergic-binding sites for clonidine, which induces hypotensive effects through activation of α2-adrenoceptors in the brainstem (7, 8). Subsequent studies clarified that the hypotensive action of clonidine can be achieved by selective activation of imidazoline receptors, while adverse effects such as sedation are mainly associated with α2-adrenoceptor activation (9). In addition, based on biochemical, pharmacological, and functional characterizations, imidazoline receptors have been classified into three subtypes: imidazoline 1, 2, and 3 receptors. Among them, imidazoline 2 receptors (I2Rs) have been shown to be expressed in multiple organs, including the central nervous system, vascular smooth muscles, and urinary tract (7, 8). It has also been reported that clonidine ameliorated detrusor overactivity and DSD conditions in patients with SCI (10, 11); however, it remains unclear whether I2R activation is involved in the therapeutic effects of clonidine on SCI-induced LUTD. Furthermore, the previous basic and clinical studies have demonstrated that the drugs acting on I2Rs are effective for the treatment of chronic pain without severe adverse events (12, 13).

Therefore, we investigated the effects of selective I2R stimulation on storage and voiding dysfunctions using 2-(2-benzofuranyl)-2-imidazoline (2BFI), a selective I2R agonist, in a mouse model of SCI. Our aim was to determine whether the imidazoline receptor subtype, I2R, represents a novel target for the treatment of LUTD following SCI.

MATERIALS AND METHODS

Animal Preparation

We utilized female C57BL/6N mice aged 9–10 wk weighing between 18 and 22 g (Envigo, Frederick, MD). Female mice were chosen because we previously reported that, in both spinal intact (SI) and SCI conditions, they exhibit external urethral sphincter (EUS) relaxation during voiding without apparent EUS bursting activity, behavior that mimics human EUS function, whereas male mice show EUS bursting during voiding (2). A complete transection of the Th8–9 spinal cord was performed to produce SCI under 1.5%–2.0% isoflurane anesthesia according to the methods reported in previous studies (14). The bladders of animals with SCI were manually emptied by bladder compression and perineal stimulation once daily for a duration of 4 wk following SCI. Spinal intact (SI) mice underwent a sham procedure that did not involve spinal cord transection. Postsurgery, the animals received daily subcutaneous injections of ampicillin (100 mg/kg) for 7 days and ketoprofen (5 mg/kg) twice daily for 3 days. The subjects were split into three subgroups: 1) SI with vehicle treatment (20 μL normal saline, administered twice daily via intraperitoneal injection), 2) SCI with vehicle treatment (20 μL normal saline, administered twice daily via intraperitoneal injection), and 3) SCI with 2BFI treatment (20 mg/kg, diluted in 20 μL normal saline, administered twice daily via intraperitoneal injection) (2-BFI hydrochloride; Tocris Bioscience). 2BFI or vehicle treatment commenced at 2 wk post-SCI or sham surgery. The dosage and treatment period of 2BFI are based on the previous study (13). At 4 wk following the SCI, cystometrograms (CMGs) and electromyography (EMG) of the external urethral sphincter (EUS) were conducted.

The mice were housed in groups of three to four per cage, in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines for humane treatment, in an air-conditioned environment maintained at 22°C to 24°C, with a 12-h light-dark cycle, lights on at 7:00 AM, and access to food and water ad libitum.

Cystometric Analysis

At 4 wk post-SCI, we performed cystometrograms (CMGs) under a conscious condition. First, a midline abdominal incision was made, and a PE-50 tube (Clay-Adams, Parsippany, NJ) was inserted into the bladder from the dome under isoflurane anesthesia. After closing the incision, the animals were gently secured in a cage (Economy holder 15–30 g; Torrington, CT) to allow them to recover from anesthesia, and the CMG catheter was connected through a three-way stopcock to a pressure transducer and a syringe pump. To alleviate surgical pain during awake CMG recordings, a local anesthetic, lidocaine hydrochloride, was applied to the abdominal skin wound to reduce surgical pain. Saline was infused through the catheter at a rate of 0.01 mL/min to record CMGs for a minimum of 180 min until consistent voiding contractions were observed. CMG parameters were then measured during individual CMGs, with the bladder being emptied after each voiding contraction. Nonvoiding contractions (NVCs) were identified as increases in bladder pressure amplitude exceeding 8 cmH₂O. The number of NVCs was counted during the filling phase of at least two voiding cycles of single CMGs. We also assessed maximal micturition pressure and post-void residual (PVR) volume by draining the remaining intravesical fluid through the bladder catheter by gravity after voiding. Bladder capacity was determined by multiplying the infusion rate (0.01 mL/min) by the duration (in minutes) from the start of saline infusion to the onset of voiding contractions. Voided volume (VV) was calculated by subtracting PVR from the bladder capacity. In addition, we calculated the percent voiding efficiency (VV/bladder capacity × 100). These parameters were analyzed in two consecutive CMGs by measuring PVR after each micturition event.

EUS-Electromyography Measurement

Following the CMG evaluation, a pair of epoxy-coated stainless-steel wire EMG electrodes (50.8 μm diameter, stainless steel wire; A-M Systems, WA) was implanted in the external urethral sphincter (EUS) under isoflurane anesthesia. Thirty-gauge needles attached to the EUS electrodes were inserted into the EUS through the perineum and then retracted to leave the EMG wires in position. After conducting urodynamic tests, the positions of the EMG wire tips were verified by dissecting each animal, and when EMG wires were located more than 1.0 mm apart from the urethra, the data were excluded from analyses. After verification of electrode placement, the data from 3 mice were excluded from the analysis because the EMG wire tips were located more than 1.0 mm away from the urethra. EUS-EMG activity was assessed using data-acquisition software (sampling rate 2,000 Hz; Chart, AD Instruments, Colorado Springs, CO). The mice were placed in restraining cages, and EUS-EMG activity was recorded after they recovered from anesthesia. After a 60-min stabilization period for the EMG, EUS-EMG activities were recorded. In this study, we utilized EUS-EMG activity as a functional surrogate for EUS function and measured the total duration of decreased EUS-EMG activity, which was periodically observed between periods of tonic EMG activity, which we defined as the EUS relaxation time during voiding.

Intrathecal Administration of 2BFI

We conducted intrathecal (i.t.) application of 2BFI during CMG and EMG using another set of mice. After CMG and EUS-EMG measurements were completed according to the methods described earlier, we performed a laminectomy of the L1-L2 vertebra, where the L6 spinal cord was located, with special caution not to injure the spinal cord. We prepared a PE10 tube, whose catheter tip was flared and tapered, and inserted it into the intrathecal space through the window of laminectomy at the level of L6 spinal cord. The catheter was secured tightly with surrounding tissues. We slowly administered artificial cerebrospinal fluid (ACSF, Tocris Bioscience) (10 μL per administration) or 2BFI (20 μg diluted in 10 μL ACSF per administration) through the catheter and observed the changes in CMG and EUS-EMG activity. Any mice whose voiding reflexes disappeared after i.t. catheter implantation likely due to additional spinal cord damage, were excluded from data analyses. The data from 4 mice were excluded from the analysis due to loss of the voiding reflex after intrathecal catheter implantation.

Muscle Strips Study

In the organ bath study, we cut the harvested bladder of SI and SCI mice into two and four longitudinal strips, respectively. Bladder muscle strips were mounted in a vertical double-jacketed organ bath with 15 mL of 37°C oxygenated Krebs solution (composition in mM: NaCl 118, KCl 4.7, CaCl₂ 1.9, MgSO₄ 1.2, NaHCO₃ 24.9, KH₂PO₄ 1.2, dextrose 11.7; pH 7.4, bubbled with 95% O₂ and 5% CO₂). Tissue was stretched by applying 10 mN (1 g) baseline tension and was equilibrated for 1–2 h before drug testing.

To induce precontraction of muscle strips, we added KCl (80 mM) or carbachol (10 μM) to each bath. We waited for 30–40 min after inducing precontraction with KCl or carbachol until test drugs were added. Thereafter, 100 μM of BU224 (I2R antagonist) (BU 224 hydrochloride, Tocris Bioscience) or vehicle (Krebs solution) was applied to each bath, and we waited for 15 min for equilibration before cumulative 2BFI application (1, 10, and 100 μM) (I2R agonist). The treatment interval of cumulative 2BFI application was 15 min each. We calculated the relaxation effects of 2BFI as the percentage of relaxation from the maximal contraction elicited by KCl or carbachol according to the previous research (15).

Real-Time RT-PCR

Following completing CMG and EMG recordings, bilateral L6-S1 dorsal root ganglia (DRG) were harvested to measure mRNA expression levels of the transient receptor potential cation channel subfamily V member 1 (TRPV1), tumor necrosis factor α (TNFα), and inducible nitric oxide synthase (iNOS) using real-time RT-PCR. The tissues were immediately frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA), and 2 μg of RNA was reverse transcribed into cDNA with the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). The cDNA was analyzed using the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, CA) in a 12-μL reaction volume with SYBR Green PCR Master Mix (Qiagen, Valencia, CA). Primers were sourced from Qiagen, including β-actin (Rn_Actb_1_SG, a housekeeping gene), TRPV1 (Mm_Trpv1_1_SG), TNF-alpha (Mm_Tnf_1_SG), and iNOS (Mm_Nos2_1_SG) using qPCR. The ΔΔ CT method was used, and the results were reported as fold changes in gene expression (2 ^−ΔΔCt) to quantify mRNA levels.

Ethical Considerations

All animal experiments were carried out in compliance with the ARRIVE and NIH guidelines and received approval from the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh (Approval No. 22061313). After completion of each experimental procedure, animals were euthanized by CO₂ asphyxiation followed by cervical dislocation.

Statistics

For statistical analysis, EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) (16), was utilized. We used the one-way ANOVA and post hoc unpaired t test to analyze statistical significance between groups. Paired t test and Mann–Whitney U test were taken for detecting statistical significance on the same individuals and between groups, respectively. Results are presented as the mean ± standard deviation, with statistical significance defined as P < 0.05. In addition, no formal adjustment for multiple comparisons across all endpoints was applied. Therefore, primary endpoints were prespecified (NVCs, voiding efficiency, and EUS relaxation time), whereas qPCR and organ bath outcomes were considered exploratory and interpreted accordingly.

RESULTS

CMG Analysis and EUS-EMG Measurement

Typical CMG and EUS-EMG traces are shown in Figs. 1 and 2, and the parameters are summarized in Table 1. Vehicle-treated SCI mice (n = 5) showed a significantly higher number of NVCs and lower voiding efficiency than SI mice (n = 5) (NVCs: 0.55 ± 0.16 vs. 0.1 ± 0.08, P < 0.001; voiding efficiency: 0.17 ± 0.03 vs. 0.96 ± 0.08, P < 0.001). However, 2BFI-treated SCI mice (n = 5) exhibited a lower number of NVCs and improved voiding efficiency along with increased EMG relaxation time during voiding compared with vehicle-treated SCI mice (n = 5) (NVCs: 0.26 ± 0.16 vs. 0.55 ± 0.16, P = 0.03; voiding efficiency: 0.31 ± 0.06 vs. 0.17 ± 0.03, P = 0.002; EMG relaxation time: 1.11 ± 0.60 vs. 0.36 ± 0.21, P = 0.03).

Figure 1.

Representative traces of single CMG recorded after the start of normal saline infusion until a voiding contraction in SI mice (A, n = 5 mice), vehicle-treated SCI mice (B, n = 5 mice), and 2BFI-treated SCI mice (C, n = 5 mice). A black arrow shows the voiding event. The right-side trace shows the expanded view of the voiding phase. CMG, cystometrogram; SCI, spinal cord injury; SI, spinal intact.

Figure 2.

Representative traces of CMG and EUS-EMG recordings during the voiding phase in SI mice (A, n = 5 mice), vehicle-treated SCI mice (B, n = 5 mice), and 2BFI-treated SCI mice (C, n = 5 mice). Black arrows indicate voiding events and corresponding EUS relaxation periods, highlighting differences in voiding coordination among SI mice, vehicle-treated SCI mice, and 2BFI-treated SCI mice. CMG, cystometrogram; EUS-EMG, external urethral sphincter electromyography; SCI, spinal cord injury; SI, spinal intact.

Table 1.

Cystometric and EUS-EMG parameters in spinal intact, vehicle-treated SCI, and 2BFI-treated SCI mice

	SI (n = 5)	SCI with Vehicle (n = 5)	SCI with 2BFI (n = 5)
CMGs parameters
Micturition intervals, min	7.47 ± 4.5	36.8 ± 11.5†	22.3 ± 6.8‡*
Micturition pressure, cmH2O	29.4 ± 5.2	57.4 ± 15.2†	59.5 ± 8.4‡
Nonvoiding contraction, min−1	0.1 ± 0.08	0.55 ± 0.16†	0.26 ± 0.16*
Bladder capacity, mL	0.07 ± 0.04	0.37 ± 0.11†	0.22 ± 0.07‡*
Voided volume, mL	0.07 ± 0.04	0.06 ± 0.01	0.07 ± 0.03
Residual urine, mL	0.002 ± 0.004	0.31 ± 0.1†	0.15 ± 0.04‡*
Voiding efficiency	0.96 ± 0.08	0.17 ± 0.03†	0.31 ± 0.06‡*
EMG parameters during voiding bladder contraction
EMG relaxation time, s	4.6 ± 2.2	0.36 ± 0.21†	1.11 ± 0.60‡*
Number of notch-like reductions	N/A	1.3 ± 0.44	2.23 ± 0.32*

Data are presented as means ± SD. Statistical analyses were performed using one-way ANOVA followed by post hoc tests. n represents the number of animals per group. CMGs, cystometrograms; EMG, electromyography; EUS, external urethral sphincter; SCI, spinal cord injury; SI, spinal intact.

^*P < 0.05 SCI with Vehicle vs. SCI with 2BFI.

^†P < 0.05 SCI with Vehicle vs. SI.

^‡P < 0.05 SCI with 2BFI vs. SI.

Intrathecal Administration of 2BFI

Typical CMG and EUS-EMG traces are shown in Figs. 3 and 4, and the parameters are summarized in Table 2. Soon after i.t. application of 2BFI in SCI mice (n = 7), significant increases of NVCs, micturition pressure, and maximum EMG amplitudes were observed compared with pre-drug values (NVCs: 0.59 ± 0.18 vs. 1.42 ± 0.82, P = 0.04; micturition pressure: 49.8 ± 10.4 vs. 55.5 ± 11.5, P = 0.03; maximum EMG amplitude: 5.91 ± 3.7 vs. 8.40 ± 4.5, P = 0.01). However, significant reductions in NVCs, and an increase of EUS relaxation time, along with reductions of PVR, were seen after around 10 min following i.t. application of 2BFI (NVCs: 0.59 ± 0.18 vs. 0.33 ± 0.22, P = 0.049; EUS relaxation time: 0.082 ± 0.12 vs. 0.104 ± 0.13, P = 0.029; PVR: 0.33 ± 0.23 vs. 0.27 ± 0.22, P = 0.036). In SCI mice with i.t. application of ACSF (n = 5), no significant changes were observed before and after the treatment.

Figure 3.

Representative CMG recordings before and after intrathecal (i.t.) administration of 2BFI (20 μg) in SCI mice (n = 7 mice). Filled arrows indicate voiding events, and an arrow with a horizontal bar indicates the timing of i.t. application of 2BFI. CMG, cystometrogram; SCI, spinal cord injury; i.t., intrathecal.

Figure 4.

Representative traces of CMG and EUS-EMG recordings during the voiding phase before intrathecal (i.t.) administration of 2BFI (A) and approximately 10 min after i.t. 2BFI administration (B) in SCI mice (n = 7 mice). CMG, cystometrogram; EUS-EMG, external urethral sphincter electromyography; SCI, spinal cord injury.

Table 2.

Effects of intrathecal 2BFI or ACSF on cystometric and EUS-EMG parameters in SCI mice

SCI with i.t. 2BFI (n = 7)	Before 2BFI	Soon After 2BFI	Around 10 min After 2BFI	SCI with i.t. ACSF (n = 5)	Before ACSF	Soon After ACSF	Around 10 min After ACSF
ICI, min	4.26 ± 1.5	3.94 ± 1.9	6.58 ± 4.5	ICI, min	6.25 ± 3.4	4.79 ± 1.9	6.55 ± 3.1
NVC, min−1	0.59 ± 0.18	1.42 ± 0.82*	0.33 ± 0.22†	NVC, min−1	0.70 ± 0.51	0.58 ± 0.41	0.68 ± 0.44
Micturition pressure, cmH2O	49.8 ± 10.4	55.5 ± 11.5*	47.7 ± 7.3	Micturition pressure, cmH2O	58.3 ± 10.1	62.4 ± 9.12	64.3 ± 11.8
Maximum EMG amplitude, mV	5.91 ± 3.7	8.40 ± 4.5*	5.28 ± 3.2	Maximum EMG amplitude, mV	6.15 ± 1.13	7.2 ± 1.62	6.74 ± 1.99
sum of EUS relaxation time, s	0.082 ± 0.12	0.064 ± 0.03	0.104 ± 0.13†	sum of EUS relaxation time, s	0.062 ± 0.02	0.051 ± 0.01	0.061 ± 0.01
PVR, mL	0.33 ± 0.23		0.27 ± 0.22†	PVR, mL	0.375 ± 0.18		0.39 ± 0.21

Data are presented as means ± SD. n represents the number of animals per group. Paired t test,

^*P < 0.05 Before vs. soon after 2BFI or ACSF.

^†P < 0.05 Before vs. around 10 min after 2BFI or ACSF. ACSF, artificial cerebrospinal fluid; EMG, electromyography; EUS, external urethral sphincter; ICI, intercontraction interval; i.t., intrathecal; NVC, nonvoiding contraction; PVR, post-void residual; SCI, spinal cord injury.

Muscle Strips Study

Representative traces of the organ bath study are shown in Fig. 5. The traces showing the drug effects in muscle strips are shown in Fig. 6, and the data are summarized in Table 3.

Figure 5.

Representative recordings from bladder muscle strip experiments pretreated with carbachol (A) or KCl (B) in SCI mice. 2BFI was applied cumulatively (1, 10, and 100 μM) at 15-min intervals. The number of bladder strips used ranged from 5 to 8 depending on the experimental condition. SCI, spinal cord injury.

Figure 6.

Concentration-response curves showing 2BFI-induced relaxation of bladder muscle strips. SI pretreated with carbachol (10 μM) (A), SCI pretreated with carbachol (10 μM) (B), SI pretreated with KCl (80 mM) (C), and SCI pretreated with KCl (80 mM) (D). Relaxation is expressed as a percentage of the maximal precontraction induced by carbachol or KCl. 2BFI was applied cumulatively (1, 10, and 100 μM). Data are presented as means ± SD. Sample sizes were as follows: SI + vehicle, n = 6; SI + BU224, n = 5 (A); SCI + vehicle, n = 8; SCI + BU224, n = 8 (B); SI + vehicle, n = 6; SI + BU224, n = 7 (C); and SCI + vehicle, n = 8; SCI + BU224, n = 5 (D). n represents the number of bladder strips. *P < 0.05 vs. 2BFI 1 μM; ^†P < 0.05 vs. vehicle. SCI, spinal cord injury; SI, spinal intact.

Table 3.

Effects of 2BFI on precontracted bladder strips from SI and SCI mice

Precontracted with Carbachol	2BFI 1 μM/L	2BFI 10 μM/L	2BFI 100 μM/L	Precontracted with KCL	2BFI 1 μM/L	2BFI 10 μM/L	2BFI 100 μM/L
+ Vehicle				+ Vehicle
SI (n = 6)	5.28 ± 3.0	10.4 ± 4.8*	22.8 ± 5.7*	SI (n = 6)	3.75 ± 2.7	5.14 ± 3.8	19.0 ± 4.7*
SCI (n = 8)	3.05 ± 1.9	7.62 ± 4.3*	23.4 ± 9.1*	SCI (n = 8)	−0.79 ± 8.6	−0.55 ± 15.8	43.8 ± 17.2*
+ BU224 100 μM/L				+ BU224 100 μM/L
SI (n = 5)	0.76 ± 0.77	2.31 ± 0.99*†	8.09 ± 1.71*†	SI (n = 7)	−0.16 ± 3.9	1.12 ± 6.7	10.3 ± 6.6*†
SCI (n = 8)	2.34 ± 0.83	5.04 ± 1.58*	13.6 ± 2.4*‡	SCI (n = 5)	−2.01 ± 2.85	−1.06 ± 4.88	10.7 ± 3.7*‡

Data are presented as means ± SD. n represents the number of bladder strips. Paired t test,

^*P < 0.05 2BFI 1 μM/L vs. 10 μM/L or 100 μM/L. Mann–Whitney U test,

^†P < 0.05 SI with vehicle vs. SI with BU224.

^‡P < 0.05 SCI with vehicle vs. SCI with BU224. SCI, spinal cord injury; SI, spinal intact.

In SI and SCI mice muscle strips precontracted with carbachol, 2BFI induced dose-dependent relaxation (SI, n = 6; SCI, n = 8), and BU224 significantly reduced the 2BFI-induced relaxation effects (SI, n = 5; SCI, n = 8) (Fig. 6 and Table 3). On the other hand, 100 μM of 2BFI, but not at other concentrations, significantly relaxed the muscle strips precontracted with KCl (SI, n = 6; SCI, n = 8), and BU224 significantly reduced the 100 μM 2BFI-induced relaxation effects (SI, n = 7; SCI, n = 5) (Fig. 6 and Table 3).

Real-Time PCR

One-way ANOVA analysis followed by post hoc unpaired t test revealed that mRNA expression levels of TRPV1, TNF-α, and iNOS in L6-S1 DRG were significantly higher in vehicle-treated SCI mice (n = 5) than in SI mice (n = 5) (TRPV1: 8.1-fold; P = 0.002; TNF-α: 8.9-fold, P = 0.003; iNOS: 10.1-fold, P = 0.03). However, the expression levels of these molecular markers were significantly reduced in 2BFI-treated SCI mice (n = 5) compared with vehicle-treated SCI mice (n = 5) (TRPV1: 3.0 vs. 8.1-fold; P = 0.027; TNF-α: 3.8 vs. 8.9-fold, P = 0.041; iNOS: 2.1 vs. 10.1-fold, P = 0.049; Fig. 7).

Figure 7.

RT-PCR analyses of mRNA expression levels of TRPV1, TNF-α and iNOS in L6-S1 dorsal root ganglia. Data are presented as means ± SD. Sample sizes were as follows: SI mice, n = 5; vehicle-treated SCI mice, n = 5; and 2BFI-treated SCI mice, n = 5. Statistical analyses were performed using one-way ANOVA followed by post hoc tests. *P < 0.05 vs. SI mice; †P < 0.05 vs. vehicle-treated SCI mice. iNOS, inducible nitric oxide synthase; SCI, spinal cord injury; SI, spinal intact; TNF-α, tumor necrosis factor α.

DISCUSSION

In this study, we demonstrated that 1) 2BFI (administered by daily intraperitoneal injection or by i.t. application) improved DO, evident as a reduction in NVC during bladder filling, and DSD, evident as an increase of EUS relaxation time during voiding, in SCI mice, and 2) the therapeutic effects of I2R activation by 2BFI were induced at least in part through its central action, which enhanced the coordination between the spinal cord and the bladder, thereby ameliorating SCI-induced DO and DSD, the latter being associated with inefficient voiding. Furthermore, 2BFI-mediated effects on DO may also involve peripheral actions on bladder smooth muscles, evident as 2BFI-induced significant relaxation of precontracted bladder muscle strips in the organ bath study.

Although imidazoline receptors were first discovered over a decade ago, their molecular identities, including I2R, have remained elusive due to various factors such as receptor heterogeneity, lack of cloning, and complex pharmacology (8). However, radioligand pharmacological studies using [³H]-idazoxan and [³H]-2-BFI have demonstrated that I2R are abundantly expressed in the central nervous system and, to a lesser extent, in other organs including the urinary bladder (7, 17). Previous studies also reported that clonidine, an agonist of α2-adrenoceptors and imidazoline receptors, can alleviate SCI-induced LUTD through its action in the spinal cord (10, 11), although it is not known whether I2R are involved in these clonidine-induced positive effects. Thus, the present study demonstrated for the first time that I2R activation directly contributes to the improvement of SCI-induced LUTD, including both DO and DSD, through central and peripheral mechanisms.

Although clonidine has been shown to improve detrusor overactivity and detrusor-sphincter dyssynergia after spinal cord injury, its pharmacological profile is complex because clonidine activates both α2-adrenoceptors and imidazoline receptors. Importantly, these receptor systems are pharmacologically and mechanistically distinct. Previous studies have demonstrated that many of the clinically limiting adverse effects of clonidine, such as sedation and hypotension, are predominantly mediated by α2-adrenoceptor activation, whereas imidazoline receptor activation can occur independently and engage different intracellular signaling pathways.

In this context, the present study provides novel evidence that selective activation of I2 receptors alone is sufficient to improve both storage and voiding dysfunction following spinal cord injury. By using 2BFI, a selective I2R agonist, we were able to dissociate therapeutic effects on lower urinary tract function from classical α2-adrenergic actions, which are known to be associated with clinically relevant adverse effects such as hypotension and sedation (9). Importantly, I2R ligands have shown generally acceptable safety profiles in clinical trials for chronic pain (12), suggesting that selective I2R activation represents a mechanistically distinct and potentially safer therapeutic strategy that may preserve efficacy while reducing α2-adrenoceptor-mediated side effects compared with nonselective agents such as clonidine. From a translational perspective, these properties support the feasibility of I2R-targeting regimens using either systemic administration for chronic management or intrathecal delivery for refractory cases, although optimization of dosage and administration routes will be needed in future studies (18).

Moreover, I2R activation reportedly mitigated the spinal cord damage by inhibiting oxidative stress and neuronal apoptosis (19). Previous studies have shown that clonidine inhibited central but not peripheral pudendal nerve-evoked urethral constriction in cats (20) and that intrathecal application of clonidine improved bladder overactivity and spinal spasticity in a clinical study with patients with SCI (18). In this study, i.t. application of 2BFI at the L6 spinal cord level significantly relaxed the EUS, improving inefficient voiding after SCI. It has been suggested that targeting therapy to inhibit Onuf’s nucleus can alleviate DSD caused by SCI (21, 22); therefore, we speculate that I2R activation could suppress EUS overactivity (i.e., DSD) through inhibition of Onuf’s nucleus located at the L6-S1 spinal cord, resulting in improved coordination between the bladder and EUS. In addition, as I2R allosteric binding sites are reported to exist on monoamine oxidase (MAO) A and B (23), I2R may act as an inhibitor of MAO-A activities, which causes elevated levels of neurotransmitters including serotonin (5-HT) (24). It is known that i.t. administration of 5-HT into the lumbosacral spinal cord improves voiding efficiency through EUS relaxation in SCI rats (25). Therefore, the improvement of DSD after systemic treatment with 2BFI in SCI mice in this study could be mediated by 2BFI-induced 5HT upregulation in the spinal cord. Further studies are needed to clarify these points regarding the underlying mechanisms in I2R-induced therapeutic effects on DSD after SCI.

Our study also demonstrated that therapeutic effects of 2BFI on DO are associated with decreased expression of TRPV1, a C-fiber afferent marker, along with reductions in inflammatory markers such as TNF-α and iNOS in L6-S1 DRG. It has been well documented that C-fiber afferent hyperexcitability contributes to the emergence of DO after SCI (2–5). 2BFI is also known to have neuroprotective effects on the damaged spinal cord by inhibiting oxidative stress in SCI (19). Neuroprotection after SCI is closely associated with reduced levels of oxidative stress and inflammation (16). Thus, it is reasonable to assume that 2BFI-induced improvement of DO in SCI mice observed in this study is mediated by anti-inflammatory effects of the drug in conjunction with reduced C-fiber afferent hyperexcitability, evident as a reduction of TRPV1 in L6-S1 DRG following 2BFI treatment, after SCI.

In the present study, 2BFI significantly reduced bladder contractility through peripheral action, as evidenced by the relaxation effects on bladder muscle strips of SCI mice. In a previous study, relaxation effects of I2R activation on bladder muscles from a rat model of diabetes mellitus were mediated by activation of K_ATP channels (15). Potassium channels play a crucial role in modulating the membrane potential of smooth muscle cells, which in turn influences calcium influx via voltage-dependent calcium channels. This process ultimately affects the contractility of the muscle by altering intracellular calcium levels (26). Because it has been reported that clonidine induces dose-dependent hyperpolarization responses in precontracted smooth muscle cells (27), we hypothesize that 2BFI may act as a K_ATP channel opener in precontracted urinary bladder strips, resulting in significant relaxation of bladder muscle strips. Further studies are needed to test this hypothesis using K_ATP channel modulators to examine whether K_ATP channels are involved in 2BFI-induced bladder smooth muscle relaxation in SCI.

2BFI was systemically administered at 20 mg/kg twice daily (intraperitoneal) based on prior in vivo studies demonstrating biological efficacy of repeated 2BFI treatment in chronic disease models (13). To directly assess spinal mechanisms, we additionally used intrathecal administration of 2BFI (20 μg in 10 μL ACSF) to demonstrate that lumbosacral spinal delivery of 2BFI can modulate SCI-induced LUTD. Because rapid dilution and clearance of drugs in the intrathecal space make it difficult to estimate actual spinal tissue concentrations, these findings should not be interpreted as a direct guide to clinical dosing. In ex vivo organ bath experiments, 2BFI was applied at 1–100 μM to establish concentration-response relationships under diffusion-limited conditions typical of isolated tissue preparations; therefore, these concentrations are not directly comparable with intrathecal injectate concentrations. Although the present study was not designed to define a therapeutic dose in humans, spinal drug delivery for refractory LUTD is clinically feasible, as intrathecal clonidine has been successfully administered in SCI patients with detrusor hyperreflexia (11, 18), and the clinical development of selective I2R ligands would support the feasibility of systemic I2R-targeting regimens (12).

This study has some limitations. First, in the acute experiment setup, we examine the effects of i.t. application of 2BFI, but not systemic application (e.g., intravenous injection) of the drug. Thus, additional experiments comparing spinal versus systemic routes of administration are warranted. Second, we did not investigate the mechanism underlying the transient excitatory effects observed shortly after i.t. administration of 2BFI. In the present experiments, intrathecal 2BFI induced a transient increase in bladder and EUS activity within the first ~10 min after administration, which was followed by subsequent improvement of LUTD, including DO and DSD in SCI mice. Such biphasic responses may be related to heterogeneous expression of I2 receptors in the spinal cord, where early activation of excitatory neurons transiently facilitates spinal reflex activity, whereas later engagement of inhibitory spinal circuits may contribute to functional improvement in bladder and urethral coordination. However, the initial excitatory phase was short-lived and consistently resolved within ~10 min after i.t. application. No sustained worsening of voiding function or overt adverse neurological signs were observed, suggesting that these transient effects are unlikely to raise immediate safety concerns in the present experimental setting. Third, the relatively small sample sizes may limit statistical power, particularly for detecting modest effect sizes; therefore, the results should be interpreted with caution and validated in future studies with larger cohorts. In addition, our molecular analyses were limited to transcript-level measurements, and corresponding protein expression and localization were not examined in the present study.

Conclusions

Activation of imidazoline I2 receptors, which exerts central actions at the lumbosacral spinal cord and peripheral effects in the bladder, may represent a promising and mechanistically distinct therapeutic strategy for the treatment of SCI-induced lower urinary tract dysfunction.

DATA AVAILABILITY

Data will be made available upon reasonable request.