Upregulated 5-HT 1A Receptors Regulate Lower Urinary Tract Function in Rats after Complete Spinal Cord Injury

Animal groups and experimental design

All animals were housed in standard laboratory cages under 12:12-h light–dark cycle conditions with standard rodent chow and water available ad libitum. All experiments were performed during the light cycle. All animal procedures were approved by the Cleveland Clinic Institutional Animal Care and Use Committee. Forty-two adult female Sprague-Dawley rats (220–250 g; Harlan Laboratories, Madison, WI, USA) were randomly divided into two groups: (1) spinally intact group (n = 12) and (2) T8 complete transection group (n = 30). In the spinally intact group total of 12 animals, all 12 were used to pharmacologically test LUT function and then 6 animals were used for Western blot study and the other 6 were used for histological study. In the T8 complete transection group, there were four subgroups at four different time points, including 1 (n = 6), 2 (n = 6), 4 (n = 6), and 8 weeks (n = 18) post-injury. All animals in the 1, 2, and 4 week post-injury groups were used for Western blot study. In the 8 weeks post-injury group, all 18 animals were used to pharmacologically test LUT function by intravenous (n = 12) or intrathecal (n = 6) drug delivery. After assessments of LUT function, 6 of the 12 animals with intravenous delivery were used for Western blot study and the other 6 animals were used for histological study. The spinal cord surgery was performed using ketamine/xylene anesthesia, and LUT functional assessments were prepared using isoflurane surgery and then recovery for 1 h prior to recordings when the animals were awake. All efforts were made to minimize suffering. All procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

T8 spinal cord laminectomy and complete transection surgery

Animals had operations performed under general anesthesia using intraperitoneal administration of ketamine (60 mg/kg) and xylazine (10 mg/kg). The body temperature was monitored by a rectal probe and maintained using a heating pad underneath the animal, which automatically adjusted the temperature based on feedback from the rectal probe. A sterile eye lubricant (Paralube) was applied to prevent dehydration. The musculature was cut from T7-T9 following the skin incision and the dorsal surface of the T8 segment was exposed by laminectomy. The spinally intact group did not receive the following injury procedures. For the complete transection group, a complete cut was made by a scissor at T8, resulting a separated gap (2–3 mm) between two stumps. A high-magnification surgical microscope was used to confirm that there was no remaining neural tissue between the two stumps of cord. The overlying musculature was sutured closed and the skin was closed using 4-0 black monofilament suture.

Western blot analyses of spinal cord tissues

The spinally intact animals and animals at four different time points (1, 2, 4, and 8 weeks) post-SCI were subjected to transcardial perfusion with ice-cold phosphate-buffered saline (PBS). The spinal cord rostral to the injury (T5/6), and sympathetic (T10∼L2) and parasympathetic (L6/S1) segments of spinal cord were homogenized in a radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). All samples were centrifuged, and the supernatant was collected and stored at −80°C until use. Protein concentration was estimated using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Protein lysates were prepared in 4 × Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and dithiothreitol (DTT) and then incubated at 95°C for 5 min. Equal amounts of protein were loaded into each well (50 μg) of stain-free Novex 4–12% Tris-Glycine Mini Gels, WedgeWell format (Thermo Fisher Scientific, Waltham, MA, USA). After gel electrophoresis, protein samples were transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). Blots were blocked in 5% dry milk in tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) for 1 h at room temperature (RT). Primary antibody (5-HT_1A receptor, MAB11041, 1/1000 in TBST containing 5% bovine serum albumen (BSA); Millipore Sigma, Temecula, CA, USA) was incubated overnight at 4°C. After three washes in TBST for 5 min, blots were incubated in secondary antibodies (HRP-conjugated anti-mice IgM, Abcam, Waltham, MA, USA) at RT for 2 h. Chemiluminescence was detected using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA), imaged using a Bio-Rad ChemiDoc MP, and analyzed using Imagelab software version 6.1 (Bio-Rad, Hercules, CA, USA) for volumetric analysis of protein expression. 5-HT_1A receptor levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (a loading control, 2118, Cell Signaling Technology, Danvers, MA, USA) as a loading control. All results were normalized by taking the value of the spinally intact group as 100% to obtain a comparative value. The significance was assessed using one-way analysis of variance (ANOVA) followed by the Tukey's post hoc test with Graphpad Prism 8.0 (GraphPad). The 95% confidence level (p < 0.05) was considered statistically significant.

Immunohistochemistry for 5-HT_1A receptor and analyses of immunoreactivity intensity

At 8 weeks post-SCI, animals were transcardially perfused with 4% paraformaldehyde (PFA). The spinal cords were removed and stored in 4% PFA overnight, and then transferred to 30% sucrose solution. The L6/S1 segment of spinal cords were sectioned by cryostat in 30 m pieces for an immunohistochemistry process, and the 15 sections (120 m apart) in each animal were selected. The sections were blocked by blocking solution (PBS containing 0.5% Triton X-100 and 3% BSA) and then incubated with antibodies against 5-HT_1A receptor (MAB11041, Millipore Sigma) and NeuN (Abcam) overnight at room temperature. All sections were rinsed three times with PBS for 10 min and then incubated at RT for 1 h with secondary antibodies (Abcam) including Alexa Fluor 488-conjugated secondary antibodies and Alexa Fluor 594-conjugated secondary antibodies. The sections were rinsed three times with PBS, and mounting media (Vector Laboratory, Newark, CA, USA) was added before they were cover-slipped. The sections were photographed by a Zeiss confocal microscope for further analysis.

Standardized areas for sampling in sections from each animal in each group were selected by Photoshop and ImageJ. The mean number of pixels containing immunoreactive product in the sampled area was measured and multiplied by the average intensity. This value was subtracted from background immunolabeled intensity, as measured in a separate adjacent section. Graphs were plotted and statistics were assessed using Graphpad Prism 8.0 (GraphPad). The intensities are shown as mean ± standard error of the mean (SEM) in units of the percentage of the mean intensities from spinally intact rats, which are presented as 100%. Significant differences between groups are indicated by a signifier (p < 0.05; p < 0.001) using an unpaired t test. Light intensity and threshold values were maintained at constant levels for all analyses.

Awake urodynamics, EUS electromyography (EMG) recording, and pharmacological interventions

Animals were anesthetized under 2.5% isoflurane with oxygen to perform surgeries and then cystometry and EUS EMG recording were performed when the animals were awake. The urinary bladder was exposed by an incision of the midline abdomen. A catheter (polyethylene 50) was inserted into the bladder through a small incision at the apex of the bladder dome. The bladder end of a pulmonary embolism (PE) catheter was heated to form a collar and was secured with a purse-string suture on the opening of the detrusor. The end of bladder catheter was connected to a pressure transducer (model P23XL-1; Gould Ohmeda, Valley View, OH, USA), an amplifier (Astro-Med, Inc., West Warwick, RI, USA), and an Aladdin single-syringe infusion pump (World Precision Instruments, Sarasota, FL, USA) for signal recording and saline infusion. A normal saline-filled femoral vein catheter was inserted into the femoral vein for drug delivery. The bladder catheter exited from the rostral edge of the abdominal wound, which was closed in layers. The skin above the vascular catheters was also closed. Fine (50μm diameter) Teflon-insulated platinum wire electrodes (A-M Systems, Carlsborg, WA, USA) were inserted percutaneously on both sides of the mid-urethra using a 27-gauge needle for subsequent measurements of EUS EMG activity. Next, rats were placed in a universal restrainer (Braintree Scientific, Inc., MA, USA) to perform physiological tests. Following a 1 h recovery period from anesthesia, continuous cystometrograms (CMGs) were collected using constant infusion (0.1 mL/min) of saline by an infusion pump (World Precision Instruments) through the catheter into the bladder to elicit repetitive voids. The electrodes were connected to an AC amplifier (Astro-Med, Inc.) with high- and low-pass frequency filters at 10 Hz and 1 kHz and a recording system (DASH 8X, Astro-Med, Inc.) at a sample frequency of 10 kHz. Continuous cystometry at a flow rate of 6 mL/h for 1 h prior allowed the LUT to adjust to the flow rate. After 1 h the pump was stopped and the bladder was emptied by aspiration through the bladder catheter via a stopcock port. The pump was restarted and after at least three reproducible filling CMGs were obtained, WAY100635 from Sigma (0.02 mg/kg, 0.2 mg/kg, 1 mg/kg, 2 mg/kg) for spinally intact or WAY100635 (0.2 mg/kg, 1 mg/kg, 2 mg/kg) and propranolol (2 mg/kg) for complete SCI animals were intravenously administered through the right site of femoral vein, and then another filling CMG was performed, from which measurements were taken for three to five filling cycles. Drug solutions were administered in a volume of 0.1 mL followed by a 0.1 mL flush of saline. In addition to the systemic delivery of WAY100635, the intrathecal injection of WAY100635 (0.002 mg/kg, 0.02 mg/kg) with the tip at lumbar segments was used to determine site of action in the SCI animals. For intrathecal catheter preparation, the laminectomy at T10 spine was made to expose the upper lumbosacral segment of spinal cord after midline incision on the back of the skin. A 27 G needle was used to make a small hole in the dura mater and a 32 G intrathecal catheter (CS-32, Braintree Scientific, Inc.) was inserted into the intrathecal space advancing 0.3 cm caudally to approximately reach the L1/L2 spinal segment. The catheter was secured with tissue glue (3M) and the other end of catheter was used for the drug injection during the awake urodynamics and EUS EMG recordings.

Quantification of CMG and EUS EMG recordings

During each of the individual interventions in the CMG and EUS EMG recordings, three to five consecutive and stabilized micturition events were segmented for analysis as described in our previous studies. ^21,22 Segmented data included the full duration between the completions of two consecutive voiding contractions. The defined parameters used to analyze both CMG and EUS EMG recoding data are shown in Figure 1. CMG voiding parameters include: the voiding efficacy (%), peak voiding pressure (cmH₂O), bladder volume capacity (mL), voiding duration (sec), voiding interval (min), and high-frequency oscillation (HFO) (sec). The period of the voiding cycle from the empty bladder with saline infusion until the first micturition occurred was used to determine voiding efficacy. It was calculated by the percentage of voiding volume in the amount of total saline infused. Non-voiding contractions (NVCs) were determined as contractions with amplitudes >5 cm H₂O that were not observed with any successful micturition. The CMG non-voiding parameters include: amplitude (peak pressure – baseline pressure) (cmH₂O), single non-voids duration (sec), number of non-voids, and percentage of time with non-void activity during the bladder-filling phase (%). The EMG parameters included (during the bursting phase): the number of EMG bursts, EMG bursting duration (sec), EMG burst interval (sec), and EMG burst amplitude (mV). EMG bursts were qualified as coordinated when they were timed in sync with the CMG HFO. The amplitude of tonic EUS EMG activity during the bladder filling phase also was analyzed. All data collection and data analysis were performed in a blinded fashion. Data are presented as group mean ± SEM, and were analyzed using a one-way ANOVA with a Fisher's post hoc test. Significance level was set to 0.05 for all comparisons.

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FIG. 1.

The illustration of parameters of voiding, non-voiding, and electromyography (EMG) in the analyses of cystometrograms (CMG) and external uretheral sphincter (EUS) EMG recordings. (A) In the voiding analyses: a, bladder capacity; b, peak pressure; c, voiding interval; d, duration. Asterisks indicate the voiding contraction. (B) In the non-voiding analyses: a, non-voiding amplitude; b, non-voiding duration. (C) In CMG/EMG analyses, a, CMG high-frequency oscillation period; b, EMG bursting duration, c, EMG bursting interval; d, EMG bursting amplitude. Color image is available online.

Upregulation of spinal 5-HT_1A receptor after chronic SCI

The expression levels of spinal 5-HT_1A receptor at 1, 2, 4, and 8 weeks after T8 SCI in the spinal cord segments both rostral and caudal to the injured site were determined by Western blot analyses. The caudal segments were divided into sympathetic segments (T10-L2) and parasympathetic segments (L6/S1). There were no significant differences in the levels of 5-HT_1A receptor rostral to the injured site in SCI animals when compared with the spinally intact animals (Fig. 2A, B). In contrast, there was significant upregulation of spinal 5-HT_1A receptor below the injured site over a period of 8 weeks after T8 complete lesion. In the sympathetic spinal cord segment, there was significant upregulation of 5-HT_1A receptor starting at 1 week and continuously increasing, even at 4 and 8 weeks post-SCI. These increases were ∼35% (1 week), 42% (2 weeks), 50% (4 weeks), and 58% (8 weeks) following SCI when compared with the spinally intact animals (Fig. 2C, D). Similar to the sympathetic spinal cord segment, the parasympathetic spinal cord segment also showed robust upregulation of 5-HT_1A receptor starting at 1 week and continuing through 4 or 8 weeks post-SCI. These increases were ∼30% (1 weeks), 50% (2 weeks), 80% (4 weeks), and 82% (8 weeks) following SCI when compared with the spinally intact animals (Fig. 2E, F).

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FIG. 2.

The spatiotemporal expression of the 5-HT_1A receptor in the spinal cord after T8 complete spinal cord injury (SCI). At the designated time points after SCI, spinal cords were harvested and total tissue lysate was extracted for Western blot analyses of 5-HT_1A receptors. The expression levels of 5-HT_1A receptor in both the sympathetic (T10∼L2 spinal segments, C) and parasympathetic (L6/S1 spinal segments, E) spinal cord were gradually increased over time after T8 complete SCI, which was not seen in the rostral spinal cord (T5/6 spinal segments, A). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Statistical analyses of 5-HT_1A receptor immunoreactivity are shown in rostral (B), sympathetic (D), and parasympathetic (F) spinal cord in T8 complete SCI rats. **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared with individual spinal fragments taken from spinally intact rats; ⁺ p < 0.05, ⁺⁺ p < 0.01 when compared with individual spinal fragments taken from rats with T8 complete SCI for 1 week. (mean ± standard error of the mean [SEM] % to individual spinal fragment taken from spinally intact rats; n = 6 per group). Color image is available online.

In addition, we further studied the anatomical distribution of upregulated spinal 5-HT_1A receptor. The L6/S1 segment of spinal cords from spinally intact animals and SCI animals at 8 weeks post-injury were collected for this study. We analyzed the immunoreactivity of 5-HT_1A receptor with double-staining of 5-HT_1A receptor and a neuronal marker (NeuN) from laminae I to X of the gray matter (Fig. 3A–L). There was a significant increase in the immunoreactivity of 5-HT_1A receptor from laminae I to V, and a trend toward increased immunoreactivity of 5-HT_1A receptor in lamina VI, VII, and X. These increases were ∼250% (lamina I), 70% (lamina II and III), and 30% (lamina IV and V) following SCI when compared with the spinally intact animals (Fig. 3M, N). The overall increase in 5-HT_1A receptor immunoreactivity is consistent with the outcomes of the Western blot analyses, and supports the upregulation of spinal 5-HT_1A receptor in the chronic phase of SCI. These results indicated an upregulation of 5-HT_1A receptor in the areas caudal to the injured site, including both sympathetic and parasympathetic segments, which are involved in the regulation of micturition reflex pathways.

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FIG. 3.

Immunoreactivity of 5-HT_1A receptor in L6/S1 segments. Representative confocal images of double staining with antibodies for 5-HT_1A receptor and NeuN from a spinally intact animal (A–C) and a complete spinal cord injured (SCI) animal (D–F) show the upregulation of 5-HT_1A receptor after SCI. The higher magnification images from boxed areas (A–F) in the center portion of laminae IV and V were shown in (G–L). (M, N) Statistical analyses showed that 5-HT_1A receptor immunoreactivity (ir) was significantly increased in laminae I–V of the gray matter in the complete SCI animals compared with the spinally intact animals. Lamina VI, VII, and X showed a trend toward increased 5-HT_1A receptor immunoreactivity, but no statistically significant difference. *p < 0.05, ***p < 0.001 when compared with individual spinal sections taken from spinally intact rats. n = 6 per group. Color image is available online.

Pharmacological blockade of spinal 5-HT_1A receptor regulates detrusor and EUS activities in awake spinally intact animals

Previous studies showed the inhibitory effects of the 5-HT_1A receptor antagonist (WAY100635) on detrusor activity in anesthetized spinally intact rats. ^15,16 We next investigated the dose-response effect of WAY100635 on both detrusor and EUS activity in awake animals. The CMG data showed continuous infusion of saline into the bladder without generating non-voiding activity prior to the micturition event (Fig. 4A). The application of WAY100635 demonstrated significant inhibition of detrusor activity in a dose-response manner in different CMG parameters (Figs. 4A and 5A). Trends toward a decrease in voiding efficacy (Fig. 5A, left graph of first row) and voiding interval (Fig. 5A, graph column of second row) were observed following the higher doses of WAY100635, including the highest dose (2 mg/kg) in voiding efficacy and the two higher doses (1 mg/kg and 2 mg/kg) in voiding interval shown the significant difference among them while compared with the baseline (Fig. 5A).

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FIG. 4.

Dose-dependent effects of an intravenously injected 5-HT_1A receptor antagonist (WAY100635) on the lower urinary tract (LUT) function of spinally intact animals. (A) Representative cystometrogram (CMG) and external uretheral sphincter (EUS) electromyographic (EMG) recordings under awake conditions (three to four voiding cycles) show that bladder contractions only occurred in the voiding events (asterisks) during baseline. Application of WAY100635 resulted in inhibitory effects on detrusor activity and on tonic EUS EMG activity in the bladder filling phase in a dose-dependent fashion. (B) Tracings of both CMG and EUS EMG in the voiding period show high-frequency oscillation (HFO) in the detrusor contraction and phasic activity of EUS. Note that increases in HFO and the reduction in amplitude of EUS EMG activity were observed with increasing doses of WAY100635. (C) Highlights of EUS EMG tracing during the bladder filling phase, as indicated by the significant reduction of the amplitude of tonic EUS activity. Note that higher doses of WAY100635 caused more extensive reductions over several micturition cycles.

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FIG. 5.

The effects of an intravenously injected 5-HT_1A receptor antagonist (WAY100635) on the micturition reflex of spinally intact animals. Statistical analyses of parameters of voiding (A) and external uretheral sphincter (EUS) electromyographic (EMG) (B) activity in a WAY100635 pharmacological dose-response study in spinally intact rats. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared with baseline; ⁺ p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, ⁺⁺⁺⁺ p < 0.0001 when compared with 0.02 mg/kg WAY100635 treatment; ^{^} p < 0.05; ^{^^} p < 0.01; ^{^^^^} p < 0.0001 when compared with 0.2 mg/kg WAY100635 treatment. n = 12 per group. Color image is available online.

The measurement of peak voiding pressure (Fig. 5A, right graph of first row) demonstrated a significant dose-dependent decrease following WAY10065 (from 0.2 mg/kg, 1 mg/kg to 2 mg/kg) compared with baseline and the smaller dose of 0.02 mg/kg WAY100635. Measurements of bladder capacity (Fig. 5A, middle graph of first row) and HFO during the voiding period (Fig. 5A, right graph of second row) showed significant increases following the higher doses of WAY100635 (from 0.2 mg/kg, 1 mg/kg to 2 mg/kg) compared with the baseline and the smaller dose of 0.02 mg/kg WAY100635. Voiding duration (Fig. 5A, left graph of second row) showed a significant increase following 2 mg/kg WAY100635 compared with baseline and 0.2 mg/kg WAY100635.

In addition to the inhibitory effect on detrusor activity, we also found alterations in EUS recording following WAY100635 (Fig. 4B and 5B). There were significant increases in both EUS burst number and duration (Fig. 5B, left and middle graphs of first row) with the higher doses of WAY100635. The significant difference can be observed from 0.2 mg/kg, 1 mg/kg to 2 mg/kg. The EUS burst interval (Fig. 5B, left graph of second row) did not show any significant difference with or without WAY100635. The amplitude of EUS burst activity (Fig. 5B, right graph of first row) showed a significant decrease with the higher dose of WAY100635 (1 mg/kg and 2 mg/kg) among them. We further analyzed the tonic activity of EUS EMG during the filling phase (Fig. 5B, right graph of second row) and found a significant reduction (65–85%) following all doses of WAY100635 compared with baseline (Fig. 4C, 5B). We did not observe any incontinence or leakage of urine during the filling phase, even with such alterations in EUS activity.

Pharmacological blockade of spinal 5-HT_1A receptor regulates detrusor and EUS activities in awake animals with T8 complete lesion

We next investigated the effects of WAY100635 on LUT function after complete SCI. In the CMG recordings, SCI animals showed typical neurogenic bladders, with many NVCs prior to the occurrence of a micturition event (Fig. 6A). In the voiding parameters of CMG analysis (Fig. 7A), the bladder-capacity analyses (Fig. 7A, second graph) demonstrated a significant increase with the higher dose of WAY100635 (1 mg/kg and 2 mg/kg) when compared with the baseline. The analyses of voiding duration (Fig. 8A, fourth graph) showed a significant increase at the highest dose of WAY100635 (2mg/kg) compared with the other lower doses and baseline. The HFO-duration analyses (Fig. 7A, sixth graph) showed a significant decrease following WAY100635 (from 0.2 mg/kg, 1 mg/kg to 2 mg/kg) compared with the baseline. There were no significant differences in either peak voiding pressure (Fig. 7A, third graph) or voiding efficacy (Fig. 7A, first graph) for any dose of WAY100635. For the non-voiding parameters (Fig. 7B), including the number of NVCs, the percentage of NVCs during the filling phase, and NVC amplitude, there were significant differences in the number of NVCs (Fig. 7B, first graph) and in the amplitude (Fig. 7B, third graph) of NVCs for the three highest doses of WAY100635 compared with baseline. There were significant differences in the percentage of NVCs during filling phase (Fig. 7B, second graph) at the two highest doses of WAY100635 when compared with the lower dose and baseline. For single NVC duration (Fig. 7B, fourth graph), both 1 mg/kg and 2 mg/kg WAY100635 caused a significantly longer duration than 0.2 mg/kg WAY100635 and baseline.

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FIG. 6.

Dose-dependent response of an intravenously injected 5-HT_1A receptor antagonist (WAY100635) on lower urinary tract (LUT) function in T8 complete spinal cord injury (SCI) animals. (A) Representative cystometrogram (CMG) and external uretheral sphincter (EUS) electromyographic (EMG) recordings under awake conditions (three to four voiding cycles) showed many non-voiding contractions of the detrusor prior to the voiding events (asterisks) after SCI. The application of WAY100635 clearly reduced non-voiding contractions (number and amplitude) in a dose-dependent fashion. (B) Highlights of representative high-frequency oscillation (HFO) and EMG activity during the voiding event. Phasic EUS activity was observed at baseline. Note that increased EUS phasic duration and a decreased HFO period were found after 2 mg/kg WAY100635.

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FIG. 7.

Effects of an intravenously injected 5-HT_1A receptor antagonist (WAY100635) on micturition reflexes of T8 complete spinal cord injury (SCI) animals. Statistical analyses of parameters of voiding (A), non-voiding (B), and external uretheral sphincter (EUS) electromyographic (EMG) (C) activity in a WAY100635 pharmacological dose-response study in T8 complete SCI animals. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared with baseline; ⁺ p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, ⁺⁺⁺⁺ p < 0.0001 when compared with 0.2 mg/kg WAY100635 treatment; ^{^} p < 0.05; ^{^^^} p < 0.001 when compared with 1 mg/kg WAY100635 treatment. n = 12 per group. Color image is available online.

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FIG. 8.

Dose-dependent response of an intrathecally delivered 5-HT_1A receptor antagonist (WAY100635) on lower urinary tract (LUT) function in T8 complete spinal cord injury (SCI) animals. (A) Representative cystometrogram (CMG) and external uretheral sphincter (EUS) electromyographic (EMG) recordings under awake conditions (three voiding cycles) indicated many non-voiding contractions of the detrusor prior to the voiding events (asterisks) after SCI. The application of WAY100635 clearly reduced non-voiding activity in a dose-dependent fashion. (B) Highlights of representative high-frequency oscillation (HFO) and EMG activity during the voiding event. Phasic EUS activity was observed at baseline. The increased EUS phasic duration and a decreased HFO period were found after 0.02 mg/kg WAY100635 application.

In addition to detrusor activity, we also investigated EUS activity during the voiding period (Fig. 7C). There was EUS burst activity observed in awake baseline recordings after T8 complete SCI (Fig. 6B). In the EUS EMG analysis (Fig. 7C), both the number of EUS bursts (Fig. 7C, first graph) and EUS burst duration (Fig. 7C, second graph) showed significant increases following 2 mg/kg WAY100635 when compared with the other doses and baseline. There were no significant differences found in either EUS burst amplitude (Fig. 7C, third graph) or EUS burst interval (Fig. 7C, fourth graph) for any dose of WAY100635.

To further test the findings of systemic delivered WAY100635 on LUT function, we performed the intrathecal injection of WAY100635 in two different doses (0.002 mg/kg and 0.02 mg/kg) with CMG and EUS recordings in the SCI animals (Fig. 8). In general, we have observed the similar results in parameters of voiding, non-voiding, and EUS EMG activity in a dose-dependent manner as what was found by systemic delivery. The significant increase in bladder capacity and the significant decrease in HFO duration were observed in the higher dose of WAY100635 treatment (Fig. 9A). The number, percentage, and amplitude of NVC were significantly decreased, but the single NVC duration was significantly increased with the higher dose of WAY100635 (Fig. 9B). The number and duration of EUS bursts were also significantly increased with the increase of the dose of WAY100635 (Fig. 9C). These data indicated that the acting sites of WAY100635 were mainly on the spinal cord, wherein WAY100635 regulated LUT function in SCI animals.

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FIG. 9.

Effects of an intrathecally delivered 5-HT_1A receptor antagonist (WAY100635) on micturition reflexes of T8 complete spinal cord injury (SCI) animals. Statistical analyses of parameters of voiding (A), non-voiding (B), and external uretheral sphincter (EUS) electromyographic (EMG) (C) activity in a WAY100635 pharmacological dose-response study in T8 complete SCI animals. *p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001 when compared with baseline; ⁺ p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, ⁺⁺⁺⁺ p < 0.0001 when compared with 0.002 mg/kg WAY100635 treatment; n = 6 per group. Color image is available online.

Application of propranolol diminishes WAY100635 regulation of LUT activity after T8 complete SCI

We next investigated whether the reduction in NVC activity produced by WAY100635 after complete SCI is caused bt activation of the sympathetic pathway, which relaxes the detrusor during the filling phase of micturition. The application of propranolol (a β1/β2-adrenergic receptor antagonist with weaker affinity for β3-adrenergic receptors; 2 mg/kg) diminished the effects of WAY100635 on both detrusor and EUS activities during the urodynamic and EUS EMG recordings (Fig. 10A, B). For the voiding parameters (Fig. 11A), the voiding interval (Fig. 8A, fifth graph) and HFO duration (Fig. 11A, sixth graph) were significantly decreased, and the peak voiding pressure (Fig. 11A, third graph) was significantly increased, as compared with 2 mg/kg WAY100635 alone or baseline. The propranolol did not alter the effects of WAY100635 on volume capacity (Fig. 11A, second graph) or voiding efficiency (Fig. 11A, first graph). For the non-voiding parameters (Fig. 11B), propranolol diminished the effects of WAY100635 on the number of NVCs (Fig. 11B, first graph), the amplitude of NVCs (Fig. 11B, third graph), and the percentage of NVCs during the filling phase (Fig. 11B, second graph). For the EUS EMG parameters (Fig. 11C), the increased number (Fig. 11C, first graph) and burst duration (Fig. 11C, second graph) of EUS produced by WAY100635 were diminished by propranolol. In contrast, there were no significant differences in EUS burst amplitude (Fig. 11C, third graph) or interval (Fig. 11C, fourth graph) after propranolol treatment.

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FIG. 10.

Propranolol diminished the effects of WAY100635 on lower urinary tract (LUT) function after T8 complete SCI. (A) Representative cystometrogram (CMG) and external uretheral sphincter (EUS) electromyographic (EMG) recordings under awake conditions (three voiding cycles) showed that propranolol diminished the effects of WAY100635 on the LUT, including voiding and non-voiding contractions. (B) Highlights of representative high-frequency oscillation (HFO) and EMG activity during the voiding event over the periods of baseline and after application of WAY100635 and propranolol.

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FIG. 11.

Effects of a 5-HT_1A receptor antagonist (WAY100635) and a beta-adrenoceptor antagonist (propranolol) on micturition reflexes of T8 complete spinal cord injury (SCI) animals. Statistical analyses of parameters of voiding (A), non-voiding (B), and external uretheral sphincter (EUS) electromyographic (EMG) (C) activity in T8 complete SCI rats. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared with baseline; ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, ⁺⁺⁺⁺ p < 0.0001 when compared with 2 mg/kg WAY100635 treatment. n = 12 per group. Color image is available online.