Chronic, Twice-Daily Dosing of an NK2 Receptor Agonist [Lys⁵,MeLeu⁹,Nle¹⁰]-NKA(4-10), Produces Consistent Drug-Induced Micturition and Defecation in Chronic Spinal Rats

Abstract

Acute administration of [Lys5,Me,Leu9,Nle10]-NKA(4-10) (LMN-NKA) produces contractions of the detrusor and rectum with voiding in intact and acutely spinal cord injured (SCI) rats. In the current study, the ability of LMN-NKA (10 μg/kg or 100 μg/kg, subcutaneous [SC], twice a day [bid]) or vehicle to induce voiding and defecation in chronic SCI rats was examined across 30 days. After the last day of administration, voiding response rates and bladder pressure (BP) responses to LMN-NKA (intravenous [IV] and SC) were evaluated under anesthesia.

In conscious rats, LMN-NKA (100 μg/kg) produced dose-dependent micturition within 5 min, with response rates >90%, and voiding efficiency >80% in males and >60% in females, which remained stable across the 1-month test period. Similarly, LMN-NKA administration rapidly induced defecation, which also remained stable. Under anesthesia, LMN-NKA increased BP, voiding efficiency, and voiding response rates, which reached 100% at 3 and 10 μg/kg IV in males and females, respectively. SC administration produced 100% response rates in males (30 μg/kg) but only 71% in females (100 μg/kg). Efficacy in rats chronically treated with LMN-NKA was similar to naïve and vehicle-treated rats, except for reduced voiding efficiency in chronically dosed female rats (100 μg/kg). No differences in bladder weights or collagen-to-smooth muscle ratios in histological sections were seen between the groups. Thus neither tolerance, nor sensitization, to LMN-NKA-induced micturition and defecation occurs with chronic administration in rats with chronic SCI. Efficacy was higher in male than in female rats.

Introduction

Spinal cord injury (SCI) disrupts the ascending and descending neural pathways that are responsible for efficient, voluntary micturition and defecation. In addition, SCI triggers inflammatory responses, both locally at the tissue level and within the central nervous system (CNS), that alter bladder and gastrointestinal (GI) function, including alterations in vagal nerve afferent sensitivity and enteric nervous system remodeling, all of which contribute to changes in bladder and GI function after SCI.^1,2,3 Thus, most individuals suffering from SCI cannot initiate voiding and require catheters to void urine and digital bowel programs to void feces.^4,5 Catheters promote urinary tract infections, and digital bowel programs are time-consuming and stigmatizing. An “on-demand, rapid-onset, short duration, drug-induced, voiding therapy” that is safe and effective^6,7 would provide a monumental improvement in the daily routines of those with SCI.

Neurokinin 2 receptor (NK2R) agonists are known bladder and colorectal, prokinetic agents that produce smooth muscle contractions by stimulation of NK2Rs located on the smooth muscle cells.^8–11 Urodynamic and manometric studies in anesthetized animals of various species show that NK2R agonists can produce rapid-onset (<5 min) and short-duration (<10 min), dose-dependent elevations in bladder pressure (BP) and GI activity after various routes of administration.^7,12,13,14 In conscious, spinal intact dogs, NK2R agonists produce efficient voiding of urine and feces within minutes of intravenous (IV) and subcutaneous (SC), administration, and repeat administration for up to 7 days was without any attenuation of the voiding and defecation responses.¹⁴ This drug-induced micturition and defecation is also observed in anesthetized acute SCI female rats.^6,7

Because drug-induced micturition would require chronic, multiple-daily dosing to be therapeutically useful for individuals with SCI, the present study was conducted to determine if [Lys5,Me,Leu9,Nle10]-NKA(4-10) (LMN-NKA)-induced micturition and defecation could be evoked in the chronic SCI rat. We examined if tolerance to, or sensitization of, response to an NK2R agonist (LMN-NKA) was observed with twice-daily dosing across 1 month. In addition, we examined if chronic, drug-induced micturition produces histological changes in the bladder.

Methods

Animals

Female and male Sprague-Dawley rats (n = 103; 230–500 g, Charles River Laboratories, Raleigh, NC) were maintained under standard conditions of laboratory housing with free access to food and water. Experiments conformed with National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and were approved by the Integrated Laboratory Systems Animal Care and Use Committee.

Spinal cord transection and post-surgical procedures

Rats were anesthetized with a mixture of ketamine and xylazine (Henry Schein; 50–100 mg/kg and 5–10 mg/kg intraperitoneal, respectively). Using aseptic procedures the skin and muscle over the middle thoracic vertebrae were incised, the spinal cord was exposed by a laminectomy, and the cord was transected at T9.¹⁵ After the transection, a piece of Gelfoam^R was placed between the cut ends of the spinal cord, the surrounding muscle was closed with absorbable sterile sutures, and the skin was closed with wound clips (removed 10 days post-surgery). Prior to the surgery, all rats received an SC injection of dual penicillin (3000 U/mL, penicillin G coupled with procaine; PenJect, Henry Schein). Immediately following surgery, rats were given buprenorphine (0.05 mg/kg SC; Henry Schein) and 5 mL of sterile saline SC. Buprenorphine was continued for 2 days (0.03 mg/kg SC, twice a day [bid]), and gentamicin (5 mg/kg SC; Gentafuse, Henry Schein) was administered once a day, for 5–7 days to prevent bladder infections. Bladders were manually expressed 2–3 times a day until spontaneous voiding was observed (7–14 days).

Behavioral monitoring and dosing

At 4–5 weeks post-SCI, rats were habituated to metabolism cages and randomly assigned to one of three groups to receive twice daily (i.e., bid, at 8–10 AM and 1–4 PM) SC injections of saline vehicle (10 female, 8 male), 10 μg/kg LMN-NKA (8 female, 7 male), or 100 μg/kg LMN-NKA (10 female, 8 male) for 1 month. The doses of LMN-NKA for the daily dosing were based on approximately effective dose 50 (ED50) and ED80 that produced voiding in naïve, anesthetized, chronic SCI rats. Twice each week (i.e., Monday and Thursday or Tuesday and Friday) for 5 weeks, animals were observed for 30 min after the morning dose, and voiding parameters were recorded (see below). A pilot study in females rats verified that drug-induced voiding was similar during the morning and afternoon dosing, and only morning behavioral observations were recorded for efficiency.

Behavioral parameters were measured by persons blinded to the treatment. The time and volume of each void, the number and wet weight of fecal pellets, and the presence/absence of diarrhea were recorded. At the end of the 30-min observation period, urine was expressed by Credé maneuver and recorded as residual volume. The voiding efficiency (VE) was calculated at 10 min post-dose (voided volume/[voided volume + residual volume] × 100%). The responder rate was determined as the number of responders (within 10 min of dosing)/total number of observations × 100%. Any unexpected or unusual changes in behavior, the presence of flushing of the extremities, and any atypical muscle contractions were recorded, if present.

Urodynamic and cardiovascular study

The day after the last dose of the 30-day regimen, rats from each of the three treatment groups were anesthetized with urethane (1.2–1.4 g/kg SC), and voiding cystometry was conducted as previously described.^3,4 A separate group of naïve (i.e., untreated) chronic SCI rats (17 female, 22 male) was also studied.

Arterial blood pressure was continuously recorded by a catheter (PE 50) inserted into the carotid artery. Systolic, diastolic, and mean blood pressure and heart rate values were calculated off-line (LabChart 8, AD Instruments). Baseline and post-dose parameters were recorded and used to calculate the change in blood pressure or heart rate. Values were expressed as a change from baseline values collected from a 20- to 60-sec period before and 5 min after each dose. Salivation was measured by placing a weighed piece of absorbent paper under each rat's tongue before each dose. At 5–10 min post-dosing the paper was removed and re-weighed and the difference in weight recorded. We assumed that 1 g weight change = 1 mL fluid production. Rats did not salivate in response to vehicle injections.

For urodynamic studies, a flared-tipped catheter (PE 50) was inserted into the bladder through a small incision at the dome and secured in place with suture. The catheter was then connected via a three-way stopcock to an infusion pump (Perfusion Pump PhD2000, Harvard Apparatus) and a pressure transducer (DelTran II, Utah Medical Products) coupled to a bridge amplifier (Transbridge 4M, World Precision Instruments) to allow for bladder filling (0.04–0.1 mL/min with 0.9% saline) and pressure measurement, respectively. Bladder capacity was determined by filling the bladder until release of fluid through the urethra was observed. Subsequently, the bladder was filled to 70% of capacity and the effect of vehicle or ascending doses of LMN-NKA on BP and voided volumes were recorded. The bladder was then emptied to measure the residual volume. When voiding occurred, bladder voiding pressure (BVP) was recorded, and the VE was calculated. Subsequently, continuous infusion cystometry was performed until the bladder returned to pre-dose physiological bladder capacity, and stable baseline characteristics. The next-higher dose of LMN-NKA was then administered.

Statistical analysis

BP was recorded via Chart software through the PowerLab/8SP data acquisition system (version 7 and 8, AD Instruments). Measurements included: baseline BP; maximal BP; time to onset of voiding; and VE and responder rate (number of animals voiding within 5 min of dosing/total number of animals observed × 100%). Data were analyzed using Excel (Microsoft, Redmond, WA) and Prism 6 (GraphPad Software, Inc., San Diego, CA). Statistical analysis was performed using a one-way, or a two-way analysis of variance (ANOVA), with Dunnett's or Turkey's multiple comparisons tests, as appropriate. Response rates were analyzed by conditional logistic regression using vehicle as the reference group and stratifying for dosing week. The regression was performed using R (R Foundation for Statistical Computing, Vienna, Austria). A p-value <0.05 was considered statistically different. All values are expressed as the mean + standard deviation (SD), except the cardiovascular data, which are presented as mean + standard error (SE).

Bladder histology and tissue analysis

A separate group of rats were treated bid for 30 days with vehicle (n = 3/sex), 10 μg/kg LMN-NKA (n = 2 females), or 100 μg/kg LMN-NKA (n = 3/sex) and were euthanized with an overdose of urethane; the bladders were carefully dissected from the abdominal cavity, weighed, and placed into 10% formalin for 2–5 days before being embedded in paraffin wax. Sections were cut (5-μm thickness) at 200-μm intervals through the bladder (including the mid-bladder and the bladder base). Two consecutive sections from each level were stained with hematoxylin and eosin or Verhoeff/Masson Trichrome and examined for evidence of general tissue damage, by an observer who was blinded to the sex and treatment group.

Analysis of female bladder tissue was performed using Definiens Architect Client and Tissue Studio Portal by the University of North Carolina (UNC) Translational Pathology Laboratory (TPL) group. High-resolution images (digital slides) were made by scans on an Aperio ScanScope XT (Leica Biosystems) with a 20 × power objective and a camera resolution of 0.4942 microns per pixel. Images were then uploaded to eSlide Manager as JPEG2000-compressed Aperio SVS files and visualized with ImageScope 12.3 (Leica Biosystems). Images were subsequently imported to Definiens Architect XD 2.7 for analysis with Tissue Studio version 4.4.2. Each tissue section was detected separately. The Definiens Composer algorithm was used to segment the tissue into five regions of interest (ROIs): collagen, elastin, urothelium, smooth muscle, and glass or adipose. This algorithm was trained on representative input regions to classify all the tissue within the ROIs in the final analysis. The program then calculated the total tissue area and the area percentages for each of the ROIs.

Results

Behavioral assessment of chronic, bid treatment with LMN-NKA in conscious, chronic SCI rats

S/c administration of LMN-NKA rapidly induced urination in a dose-dependent manner, in both female and male rats, with 100 μg/kg of LMN-NKA producing urination in <4 min on virtually every dose (Fig. 1A,B). In addition, in males, 10 μg/kg LMN-NKA also significantly reduced the time to void at weeks 2, 4, and 5 compared with vehicle-treated male rats (Fig. 1B). The response rate was significantly higher after 100 μg/kg LMN-NKA in both females and males by conditional logistic regression (likelihood ratio 65.6, p < 0.0001 for females, 51.5, p < 0.0001 for males) (Fig. 1C,D). In addition, the males (but not females) had a significantly greater responder rate to 10 μg/kg of LMN-NKA compared with vehicle (Fig. 1C,D).

FIG. 1.

LMN-NKA induced rapid-onset voiding that was maintained over the 30-day period. The time to urination (A, B), the responder rate (C, D; number of animals voiding within 10 min of dosing/total number of animals observed × 100), and the VE (E, F) after each dose is shown. If no voiding occurred within the 30-min observation period the animal was assigned a 30-min time and a VE of 0. Number of animals/group: vehicle (n = 8 male, 10 female), 10 μg/kg (n = 7 male, 8 female), or 100 μg/kg (n = 8 male, 10 female) LMN-NKA. Group differences in time to voiding was determined using a two-way ANOVA followed by Sidak's or Tukey's multiple comparisons test comparing each treatment group within sex. There was no difference in the time to voiding within treatment groups over the observation weeks. Differences in the time to void was found between treatments groups as follows: females, F(2, 119) = 51.52, p < 0.0001; males, F(2, 99) = 47.43, p < 0.0001. *100 μg/kg dose different to both vehicle and 10 μg/kg; #100 μg/kg group different to vehicle; $10 μg/kg group different to vehicle. ANOVA, analysis of variance; LMN-NKA, [Lys5,Me,Leu9,Nle10]-NKA(4-10); VE, voiding efficiency.

Bladder voiding efficiency was also significantly increased by LMN-NKA in a dose-dependent manner reaching 80 and 86% in females and males, respectively, after 5 week's treatment (Fig 1D,E) at the 100 μg/kg dose. The voided volumes increased dose-dependently and were maintained throughout the dosing period. On the last week of dosing females voided on average 0.75 + 0.58 and 1.81 + 0.96 mL (mean + SD) in the 10 and 100 μg/kg group, respectively; males voided on average 1.03 + 0.79 and 1.94 + 0.70 mL, in the 10 and 100 μg/kg group, respectively, within 10 min of dosing. These volumes were significantly higher than the vehicle controls (females 0.36 + 0.48 mL; males 0.05 + 0.14 mL; one-way ANOVA (females F = 10.96, p = 0.0004 and males F = 19.55, p < 0.0001). For LMN-NKA-induced urination, males appear to be more sensitive than females as males demonstrated a faster time to void, higher responder rate, and higher VE to 10 μg/kg LMN-NKA compared with females (F = 18.4, p < 0.0001). Importantly, the time to void, responder rate, and VE were consistent from the first through the last day of drug administration.

LMN-NKA also induced rapid-onset, dose-dependent defecation in female (Fig. 2A,C,E) and male (Fig. 2B,D,F) rats that was consistent across the 5-week period. Defecation onset time at the 100 μg/kg dose of LMN-NKA was similar to the time to induce urination (i.e., <4 min post-dosing) in both females and males. Both the females and males defecated more quickly after 10 μg/kg and 100 μg/kg LMN-NKA compared with vehicle (Fig. 2A,B). The time to onset of defecation was similar to that of urination in males, but females dosed with 10 μg/kg LMN-NKA defecated before urinating (F[1, 70] = 10.81, p = 0.0016). The responder rates in female and males at both doses of LMN-NKA were significantly higher compared with vehicle (likelihood ratio 77.9, p < 0.0001 for males, and 71.7, p < 0.0001 for females), with the females being more likely to respond compared with the males at the 10 μg/kg dose (Fig. 2C,D). Defecation (number of fecal pellets expelled over time in epochs of 5 min) continued throughout the 30-min observation period after 100 μg/kg LMN-NKA, and was significantly increased (p < 0.001) compared with vehicle and 10 μg/kg LMN-NKA in both females (F[2, 100] = 57.59) and males (F[2, 80] = 101.4). Over the 30-min observation period, defecation also increased after 10 μg/kg LMN-NKA in females and males compared with vehicle with the largest increase occurring in the first 5 min.

FIG. 2.

LMN-NKA induced rapid-onset defecation that was maintained over the 30-day period. The time to defecation (A, B), the responder rate (C, D; number of animals defecating within 10 min of dosing/total number of animals observed × 100), and fecal weight (E, F) after each dose is shown. If no defecation occurred within the 30-min observation period the animal was assigned a 30-min time and 0 g fecal weight. Number of animals/group: vehicle (n = 8 male, 10 female), 10 μg/kg (n = 7 male, 8 female), or 100 μg/kg (n = 8 male, 10 female) LMN-NKA. Group differences in time to defecation was determined using a two-way ANOVA followed by Sidak's or Tukey's multiple comparisons test comparing each treatment group within sex. There was no difference in the time to defecation within treatment groups over the observation weeks. Differences in the time to defecate was found between treatments groups as follows: females, F(2, 120) = 67.94, p < 0.0001; males, F(2, 99) = 69.76, p < 0.0001. *100 μg/kg dose different to both vehicle and 10 μg/kg; #100 μg/kg group different to vehicle; $10 μg/kg group different to vehicle. ANOVA, analysis of variance; LMN-NKA, [Lys5,Me,Leu9,Nle10]-NKA(4-10).

Total fecal weight over the 30-min observation period was significantly higher with 100 μg/kg LMN-NKA compared with 10 μg/kg LMN-NKA and vehicle groups (Fig. 2E,F). A two-way ANOVA was also conducted between the vehicle and the 10 μg/kg group only, and it demonstrated a significant increase (approximately twofold over vehicle) with 10 μg/kg compared with vehicle (female F[1, 80] = 19.08 and male F[1, 64] = 17.68; p < 0.0001). Therefore, females were more sensitive than males in demonstrating a significantly higher responder rate at 10 μg/kg LMN-NKA; however, males had a higher fecal output at 100 μg/kg. Soft stools or diarrhea was observed in males with a 50% incidence rate after the 100 μg/kg dose but was rarely observed (10% incidence rate) in females. Soft stools or diarrhea was not observed with the 10 μg/kg dose in either sex.

In addition to observing LMN-NKA-induced micturition and defecation, flushing of the ears and paws occurred within 30 sec after the 100 μg/kg dose. Contractions of the abdominal muscles was also observed, but its rare occurrence and transient nature made it impractical to quantify using observation alone.

Urodynamic assessment in anesthetized chronic SCI rats after chronic, b.i.d. treatment with LMN-NKA

During constant saline infusion (0.04–0.1 mL/min) BP slowly rose as the bladder filled to capacity and low amplitude (3–12 mm Hg) non-voiding contractions occurred before the bladder started to leak. Once leak point pressure was obtained rhythmic contractions either subsided or continued as the bladder leaked and was continuously refilled. During this “physiological” voiding, maximum BPs were significantly higher in females compared with males (all groups combined n = 19–20: male = 32.42 + 6.21, females = 39.12 + 7.21, mean + SD, unpaired t test, p = 0.00182) and voiding was incomplete. When the bladder was filled to 70% capacity, IV administration of LMN-NKA produced dose-dependent increases in voiding responses, BP, and voiding efficiency to a similar extent in all four groups except for VE in female rats treated with 100 μg/kg for 30 days, which was significantly lower than the other groups (Table 1, Figs. 3 and 4). Voiding occurred within 30 sec of IV dosing, and VE was >40% in both females and males at ≥10 μg/kg LMN-NKA. SC administration of LMN-NKA also produced a dose-related increase in voiding, which occurred within 3 min of dosing, with the males being more likely to void at each dose level compared with females (Table 1). Similar to what was seen in the conscious studies, bladder voiding responder rates (Table 1) and VE were greater (and voiding BP lower) in males than females (Fig. 4).

Table 1.

Voiding Responder Rates in Drug-Naïve, Anesthetized, Chronic SCI Male and Female Rats following IV or SC LMN-NKA

	Female		Male
Dose (μg/kg)	SC	IV	SC	IV
1	ND	9 ( 1/11)	ND	60 (6/10)
3	50 (1/2)	89 (8/9)	25 (3/12)	100 (10/10)
10	33 (2/6)	100 (9/9)	58 (8/12)	100 (9/9)
30	57 (4/7)	100 (10/10)	100 (12/12)	100 (6/6)
100	71 (5/7)	100 (9/9)	100 (11/11)	100 (6/6)
300	71 (5/7)	100 (9/9)	100 (10/10)	100 (7/7)
1000	100 (3/3)	ND	100 (3/3)	ND

Values are the % of responders/dose group (number of responders/total number of rats tested/dose group). A void was determined as >0.03 mL of fluid expelled within 5 min of dosing.

IV, intravenous; LMN-NKA, [Lys5,Me,Leu9,Nle10]-NKA(4-10); ND, not determined; SC, subcutaneous; SCI, spinal cord injury.

FIG. 3.

Physiograph tracing of arterial (upper panels) and bladder pressure (lower panels) in an anesthetized female and male rat dosed with 100 μg/kg IV LMN-NKA. Arrows show the time of each injection; *shows the onset of the void. The rats had previously been dosed with 100 μg/kg SC, bid for 1 month, before the terminal cystometry experiment. Note that the bladder voiding pressure was higher in the female compared with the male. bid, twice daily; IV, intravenous; SC, subcutaneous.

FIG. 4.

VE and bladder pressure response from anesthetized, drug-naïve (non-treated) (no preTx), 30-day vehicle, and LMN-NKA treated groups. Drug-induced voiding was examined at 70% of the bladder capacity. Data are mean + SD. A two-way ANOVA and Tukey's multiple comparison test was used to identify differences between the treatment groups. No differences were observed in males. The VE in females rats treated for ∼30 days with 100 μg/kg SC was significantly different (F[1, 81] = 27.45, p < 0.0001) from the vehicle group at 10–100 μg/kg IV doses (#) and compared with the naïve group at 100–300 μg/kg doses ($). Bladder pressure increased dose-dependently in males and females to IV LMN-NKA (females: F[5, 147] = 41.95, and males: F[5, 147] = 41.82; p < 0.0001). ANOVA, analysis of variance; IV, intravenous; LMN-NKA, [Lys5,Me,Leu9,Nle10]-NKA(4-10); SD, standard deviation; VE, voiding efficiency.

LMN-NKA produced hypotension in both females and males after IV administration of ≥10 μg/kg (Figs. 3 and 5). No change in heart rate was observed. These cardiovascular responses were not different between naïve (no pre-treatment), vehicle-, and LMN-NKA-treated groups. IV administration of LMN-NKA induced salivation at doses ≥30 μg/kg, (18.4 + 16.1 μL with 30 μg/kg; 41.3 + 20.8 μL with 100 μg/kg and 100.7 + 36.7 μL with 300 μg/kg; n = 18–22/dose group). No difference in volume of salivation was seen between females and males.

FIG. 5.

Peak changes in heart rate and mean arterial blood pressure in response to IV administration of LMN-NKA in anesthetized, drug-naïve, female and male rats and following 30-day treatment with saline vehicle or LMN-NKA (10 or 100 μg/kg). Dose-related decrease in blood pressure were observed at doses starting at 10 μg/kg; the peak hypotension occurred between 0.5 and 2 min post-dose. No significant change in heart rate was observed. Values are mean + SE, n = 4–7/group/timepoint. For mean blood pressure two-way ANOVA for males: F(5, 96) = 12.38; and for females: F(5, 117) = 33.39; both p < 0.0001). *All doses were compared with the 1 μg/kg dose using a one-way or two-way ANOVA followed by Dunnett's or Sidak's multiple comparison test, respectively. ANOVA, analysis of variance; IV, intravenous; SE, standard error.

Histological assessment of the bladder after 30 days of repeated chronic, bid administration of LMN-NKA

Bladders from chronic SCI rats were larger and thicker than non-spinalized naïve animals, as previously reported.¹⁶ Figure 6 shows an example of coronal sections of bladder tissue from a saline vehicle- and 100 μg/kg LMN-NKA-dosed female rat (Fig. 6A.1, A.2), and an example of how the tissue was digitized to quantify the smooth muscle and collagen (Fig. 6B.1, 6B.2). No qualitative differences in the urothelium, elastin, smooth muscle, or collagen were observed between female and males or between treatment groups, but the small number of animals examined did not provide sufficient power to conclude that quantitative differences might not be found in a sufficiently powered study. Similarly, there were no differences in the muscle:collagen ratios or bladder weights between the treatment groups (Fig. 7A,B). Finally, LMN-NKA produced no differences in body weight gain between the three groups across the treatment period (Fig. 7C,D).

FIG. 6.

Bladder tissue collected from chronic SCI rats following SC doses of vehicle (saline) or 100 μg/kg LMN-NKA, twice daily for 30 days. The urothelium of all the bladders was intact and the urothelial thickness appeared similar between treatment groups (Fig 6A.1,A.2). No differences were found in tissue morphology between vehicle and LMN-NKA dosed rats. The digitized image (Fig. 6B.2) shows smooth muscle (orange) and collagen (green) content and the urothelium (red). SC, subcutaneous; SCI, spinal cord injury. Color image is available online.

FIG. 7.

Bladder morphology and body weights of chronic SCI rats following SC doses of vehicle (saline), 10 or 100 μg/kg LMN-NKA, bid for 30 days. All data are mean + SD. (A) Bladder smooth muscle and collagen content. Data from two sections/level. N of rats = 3 for V and 100 μg/kg and n = 2 for 10 μg/kg. No differences were observed in the area of smooth muscle or collagen between the treatment groups. (B) Bladder weights. Data are from two sections/level. N of rats = 3 for V and 100 μg/kg and n = 2 for 10 μg/kg. Bladder weights were similar between groups. (C, D) Body weights of females and males. Body weight on the first (day 1) and the last day (day 30) of female (C) and male (D) rats dosed with saline vehicle, 10 or 100 μg/kg SC LMN-NKA. Body weights were compared using a two-way ANOVA and no difference between day 1 and day 30 was observed. Females, F(2, 52) = 1.914, p = 0.1577 and males, F(2, 40) = 0.3007, p = 0.7420. ANOVA, analysis of variance; bid, twice daily; LMN-NKA, [Lys5,Me,Leu9,Nle10]-NKA(4-10); SC, subcutaneous; SCI, spinal cord injury; SD, standard deviation; V, vehicle.

Discussion

The present study demonstrated that dosing bid with LMN-NKA, an NK2R agonist, maintains very consistent induction of micturition and defecation in conscious rats with chronic SCI throughout a 1-month study, indicating that neither tolerance nor sensitization of the NK2 receptor occurred. Similarly, chronic dosing had no effect on urodynamic or cardiovascular dose-response response curves to acute administration of LMN-NKA in anesthetized rats, except for a reduction in VE in female rats treated with 100 μg/kg (SC) for 1 month. This is puzzling, because there was no decline in VE in conscious female rats at this dose. The cardiovascular effects of LMN-NKA after IV administration, which are mediated by off-target stimulation of NK1 receptors,^13,17 were also not affected by chronic dosing.

This study also addressed the possibility that repeated drug-induced contractions of the bladder, sufficient to produce highly efficient voiding, might induce remodeling of bladder detrusor smooth muscle or collagen content or signs of pathology. However, bladder weights and the ratio of smooth muscle to connective tissue and collagen to tissue area content remained the same in vehicle- and LMN-NKA-treated rats. After SCI the bladder capacity increases, the bladder wall thickens, and compliance changes along with hypertrophy of the smooth muscle.^16,18–20 Similar changes have been reported in the colon of SCI rats.²⁰ Despite these SCI-induced morphological changes, LMN-NKA induced a bladder contraction that produced efficient voiding and defecation. In the current anesthetized cystometry study, the LMN-NKA-induced VE in chronic SCI females was 50–60% (dose range 10–300 μg/kg IV), which was similar to reports in spinal intact female rats (60–75%) and better than the acute SCI model (25–40%).⁷ Similarly, LMN-NKA produced increases in bladder voiding pressure in chronic spinal cord injury (cSCI) rats that was similar to that of intact rats, which are both substantially greater than those produced in acute SCI rats.⁴ NK2 receptors are expressed at several sites along the micturition and defecation pathways, including bladder, urethral, and GI smooth muscle and are expressed in afferent neurons (see article by Kullmann and collegues⁷). These data suggest that LMN-NKA-induced bladder contractions result from both direct stimulation of the bladder smooth muscle (acute SCI) and stimulation of afferent (or efferent) NK2R and that augments voiding, resulting in near normal VE at normal BPs, in chronic SCI rats.

In conscious SCI rats, SC injection of LMN-NKA induced voiding within 4 min, with a VE of 60–80%, and a 90–100% responder rate, at the highest dose administered in both females and males. LMN-NKA was well tolerated and effective with no attenuation throughout the 5-week dosing period. Males demonstrated a faster time to urinate, higher responder rate, and higher VE to 10 μg/kg LMN-NKA compared with females (Table 1). A significant sex difference was also noted in regards to responder rates, VE, and BP in the anesthetized studies, with males showing higher response rates and VE with lower bladder voiding pressures. These data suggest that males have lower urethral resistance during drug-induced voiding, presumably due to the “peristaltic” action provided by the larger, sexually dimorphic, urethral rhabdosphincter, ischiocavernosus, and bulbocavernosus muscles in the male.^21,22 Indeed, preliminary studies in both intact and chronic spinal animals (Marson and Thor, unpublished) indicate that LMN-NKA-induced micturition is accompanied by high-frequency bursting of the perineal musculature, similar to natural voiding in spinal intact rats.

LMN-NKA also induced defecation that occurred within 5 min at a dose of 100 μg/kg SC with a 90–100% responder rate that was maintained across the 5-week bid dosing period. Whereas both females and males dosed with 10 μg /kg of LMN-NKA also defecated within 10 min, the responder rate was higher in females compared with males; however, males had a higher fecal output at 100 μg /kg, (ratio of fecal weight/100 g body weight in females = 0.83 and in males = 1.13 g). Soft stools or diarrhea was observed in males only, which may be related to the increased fecal output.

No concerning side effects were seen during the dosing period. Transient flushing of the ears and paws occurred with the highest dose of LMN-NKA (100 μg/kg), but no flushing was seen with the low (10 μg/kg) dose. It is thought that flushing is an NK1R-mediated effect, because flushing in humans given IV injection of neurokinin A, the endogenous NK2R agonist, was not blocked by selective NK2R antagonists,²³ and flushing in conscious spinal intact rats was blocked by the NK1R antagonist CP99,994, but not the NK2R selective antagonist GR1599897.²⁰ In anesthetized chronic SCI rats, IV administration of LMN-NKA doses ≥10 μg/kg produced a transient hypotension (Figs. 3 and 5). The hypotension observed in the current study was similar to that observed after IV administration of LMN-NKA in anesthetized acutely spinalized rats and in spinal intact rats.⁶ The LMN-NKA-induced hypotension was significantly less with SC administration in both chronic (data not shown) and acute SCI rats¹² suggesting that the large plasma concentrations (Cmax) resulting from IV administration may have contributed to the hypotension. It is possible that the hypotension seen under urethane anesthesia in the current study would be reduced in the awake animal, because anesthetics can lower the threshold for hypotensive effects of NK2 agonists.¹⁷ The LMN-NKA-induced hypotension is mediated by NK1 receptors, because it is blocked by pre-administration of NK1 receptor antagonist CP99,994,¹⁷ and IV administration of the more selective NK2R agonist GR64349 did not produce a hypotension in anesthetized acute SCI rats.¹² No change in heart rate was observed, suggesting that the drop in BP was insufficient to trigger the baroreceptor reflex.

Although LMN-NKA induced defecation in the awake chronic SCI rats, the lack of colorectal pressure monitoring and histological analysis of the rectum and colon are limitations of this study. We have previously shown both in acute SCI anesthetized rats and awake spinal intact rats that LMN-NKA produces a dose-related increase in colorectal pressure and defecation, respectively, which is mediated via NK2 receptors.^6,24 However, future studies of changes in colorectal and anal sphincter function associated with chronic SCI (i.e., neurogenic bowel) should examine colorectal pressures and histology to determine if high colorectal pressures and histopathological changes occur in chronic SCI animals during chronic induction of defecation with LMN-NKA. Future studies should also address the potential mechanisms (e.g., GI smooth muscle vs. enteric nervous system) and further examine possible sex differences of NK2 agonist-induced defecation.

The current study, demonstrating that chronic, bid, SC administration of LMN-NKA produced consistent and efficient voiding and defecation in SCI rats, supports the possibility that an NK2 agonist may be a useful therapy to induce “on demand” voiding in people with SCI. Previous studies in rats⁶ demonstrated that intranasal and sublingual dosing of LMN-NKA also produced similar increases in bladder and colorectal pressure, which might be preferred for clinical use.

Funding Information

This work was supported by the National Institutes of Health through an National Institute of Neurological Disorders and Stroke grant, NS089880. The University of North Carolina Translational Pathology Laboratory is supported in part by grants from the NCI (2-P30-CA016086-40), National Institute of Environmental Health Sciences (2-P30ES010126-15A1), University of Cancer Research Fund, and National Carolina Biotechnology Center (2015-IDG-1007).

Author Disclosure Statement

No competing financial interest exist