Dog and human bladders have different neurogenic and nicotinic responses in inner versus outer detrusor muscle layers

Abstract

The aim of this study was to investigate layer and species variations in detrusor muscle strip responses to myogenic, neurogenic, and nicotinic, and muscarinic receptor stimulations. Strips from bladders of 9 dogs and 6 human organ transplant donors were dissected from inner and outer longitudinal muscle layers, at least 1 cm above urethral orifices. Strips were mounted in muscle baths and maximal responses to neurogenic stimulation using electrical field stimulation (EFS) and myogenic stimulation using potassium chloride (KCl, 120 mM) determined. After washing and re-equilibration was completed, responses to nicotinic receptor agonist epibatidine (10 μM) were determined followed by responses to EFS and muscarinic receptor agonist bethanechol (30 μM) in continued presence of epibatidine. Thereafter, strips and full-thickness bladder sections from four additional dogs and three human donors were examined for axonal density and intramural ganglia. In dog bladders, contractions to KCl, epibatidine, and bethanechol were 1.5- to 2-fold higher in the inner longitudinal muscle layer, whereas contractions to EFS were 1.5-fold higher in the outer (both pre- and post-epibatidine). Human bladders showed 1.2-fold greater contractions to epibatidine in the inner layer and to EFS in the outer, yet no layer differences to KCl or bethanechol were noted. In both species, axonal density was 2- to 2.5-fold greater in the outer layer. Dogs had more intramural ganglia in the adventitia/serosa layer, compared with more internal layers and to humans. These findings indicate several layer-dependent differences in receptor expression or distribution, and neurogenic responses in dog and human detrusor muscles, and myogenic/muscarinic differences between dog versus humans.

INTRODUCTION

The urinary bladder can be divided into three anatomical regions (from cranial to caudal): the apex (dome), body (central middle region), and base that includes the trigone. The bladder wall is structured into four layers (from the inside out): mucosa, submucosa, muscle (detrusor), and adventitia/serosa. The smooth muscle fibers of the detrusor are arranged in inner and outer longitudinal layers, and a middle circular layer (1). The function of smooth muscle fibers is to provide relaxation during the filling phase and contraction during emptying phase (2). These processes of detrusor muscle contraction and relaxation are controlled by the central nervous system through the coordination between the activity of sympathetic and parasympathetic nerve fibers, and activity in sensory fibers, and includes involvement of a variety of nerves, neurotransmitters, and receptors (3). Pathological alterations can lead to bladder dysfunction and loss in quality of life. Understanding contractile properties of the healthy bladder is key to better understanding the treatment of bladder disorders and diseases.

Stimulation of receptors on bladder smooth muscle constitutes a major component in the physiology of bladder contractions. Treatment with a variety of agents targeting the contractile machinery of the bladder has been used to treat patients with insufficient bladder contractility. Although the physiological responses to pharmacological agents are well studied in whole bladders in vivo and in full-thickness bladder strips in vitro (1), little is known about whether the smooth muscle across regions or layers of the bladder respond differently to different contractile agents. Previous investigations have focused mainly on studying the biomechanical and microstructural properties of muscle tissues in different bladder regions or layers (4–8). No studies have examined for layer-dependent differences or dog versus human bladder differences in detrusor smooth muscle contractile properties.

The primary aim of this study was to explore in vitro contractile responsiveness of smooth muscle strips dissected from inner versus outer longitudinal muscle layers of normal dog and human bladders and to compare their contractile responses under various stimulating conditions. Specifically, we tested their responses to depolarization induced by myogenic stimulation with high potassium, the nicotinic receptor agonist epibatidine, neurogenic stimulation with electrical field stimulation both pre- and post-epibatidine, and the muscarinic receptor agonist bethanechol in the continued presence of epibatidine. Our secondary goal was to compare between dog and human bladders in their responses to the different stimulations within a muscle layer.

MATERIALS AND METHODS

Experiments on the dog tissues were approved by the Institutional Animal Care and Use Committee according to guidelines of the National Institute of Health for the Care and Use Laboratory Animals, the United States Department of Agriculture and the Association for Assessment and Accreditation of Laboratory Animal Care (ACUP no. 5043). A total of 13 dog bladders (7 males and 6 females) were used in this study. The seven males and four of six females were mixed-breed hound dogs, 6–8 mo old, weighing 18–25 kg, and were procured from Marshall BioResources, North Rose, NY. The other two female dogs were adult beagles, 8 mo old, and were procured from Envigo Global Services, Inc., Denver, PA. Dogs were group housed using standard husbandry and exposed to a 12-h light/dark cycle. Four of six females and all the seven males were sham operated or unoperated control animals derived from other larger studies focusing on nerve transfer for pelvic organ reinnervation or heart failure.

A total of nine human bladders (4 male and 5 female) were procured as whole human bladders from human organ transplant donors from the National Disease Research Institute (Philadelphia, PA). Donor age ranged between 22 and 63 yr, with an average age of 49.2 ± 15.3 yr. Of the six human donors, five were White and one was Black. These bladders were transferred to us through the procurement agency as deidentified tissues. Since these human tissues samples were deidentified and were not used in clinical investigations, their use did not require Institutional Review Board approval under the common Rule of Protection of Human Subjects Regulation. That said, their use was approved by the Temple University Institutional Biosafety Committee (no. 10799) and met Biosafety in Microbial and Biosafety Laboratory and OSHSA Standards.

Collection and Testing of Bladder Muscle Strips

Whole bladders collected from nine dogs (7 male sham operated mixed-breed hound dogs and 2 adult female beagles) were used for the in vitro muscle strip contractility studies. Bladders were washed in Tyrode buffer (125 mM NaCl, 27 mM KCl, 4.2 mM NaH₂PO₄, 1.8 mM CaCl₂, 5 mM MgCl₂, 23.8 mM NaHCO₃, and 5.6 mM dextrose), immersed in Custodiol HTK organ transport media (5 mM NaCl, 9 mM KCl, 1 mM potassium hydrogen 2-ketoglutarate, 4 mM MgCl₂, 18 mM histidine HCl, 180 mM histidine, 2 mM tryptophan, 30 mM mannitol, and 0.015 mM CaCl₂), and saved on ice at 4°C for contraction studies performed the following day.

Whole human bladders collected from six organ transplant donors (3 males and 3 females) were also used for the in vitro muscle strip contractility studies. The specimens were harvested by the National Disease Research Institute (Philadelphia, PA) within 30 min after cross-clamping the aorta and transported to the laboratory within 40 h immersed in Belzer’s Viaspan University of Wisconsin organ transport Solution or Custodiol HTK on wet ice. Differences in the organ transport media used for dog and human bladders were tested in a dog bladder; no differences were found in the responses of muscle strips dissected from half of the bladder stored in Custodiol HTK and of strips dissected from the other half stored in Belzer’s Viaspan University of Wisconsin (data not shown).

All specimens were dissected in a cold room (0°C–5°C). Dog bladders were first marked with sutures to demarcate the ventral surface, as were human bladders when possible (Fig. 1A). Bladder muscle strips were dissected from the dorsal and the ventral aspects of the dome and the central middle part, at least 1 cm above the ureteral orifices. These dissections were performed using sharp micro scissors and ×5 magnifying loops. Although fascicles of myofibrils were arranged in random directions in the dome region, strips were dissected along visible fascicles from tissue adjacent to the mucosa, and separate strips from visible fascicles adjacent to the adventitia/serosa (Fig. 1B). For the inner longitudinal muscle layer strips, the mucosa was separated from the underlying layers by sharp dissection, and inner longitudinal muscle strips were obtained with the long axis parallel to the direction of the muscle fibers and separated from the circular and the outermost muscle layer (which were visible using the ×5 loops). Thus, the middle circular muscle layer was not included in this study. For the outer longitudinal muscle layer strips, peritoneal fat was removed (specifically for human bladders), and sharp dissection was performed to remove the outermost muscle fibers. Strips were clamped between force transducers and positioners and mounted in muscle baths containing 10 mL of Tyrode solution aerated with 95% O₂ and 5% CO₂ at 37°C. Strips from both dog and human bladders were initially stretched slowly to 20 mN of isometric tension and allowed to relax to ∼10 mN of basal tension (9, 10).

Figure 1.

A: representative photograph showing the different regions of the dog bladder. B: hematoxylin and eosin (H&E)-stained section of full bladder thickness to show different layers and areas from where muscle strips were dissected. Ganglia are indicated by G and arrows.

For each bladder, both inner and outer longitudinal muscle layers were collected, and a range of 6–32 strips were tested per layer, per bladder (8–9 per dog bladder and 5–6 per human bladder), and per experimental condition. Contractile responses were monitored with isometric force transducers, as described previously (9). EFS of 12 V, 1-ms pulse duration, and 30-Hz frequency was delivered to each strip using a Grass S88 stimulator (Natus Neurology, Inc., Warwich, RI) interfaced with a Stimu-Splitter II (Med-Lab Instruments, Loveland, CO) power amplifier and LabChart software (ADInstruments). After a 30-min equilibration, strips were exposed sequentially to an isotonic buffer containing 120 mM potassium chloride (KCl), which was washed out after maximal responses were determined. After re-equilibration for ∼1 h, 10 μM of the nicotinic receptor agonist epibatidine (Cat. No. 0684, Tocris Bioscience, R&D Systems, Minneapolis, MN) was added to the same subsets of strips, followed by stimulation with EFS and then 30 μM of the muscarinic receptor agonist bethanechol (Cat. No. 1071009, Sigma-Aldrich, St. Louis, MO) in the continued presence of epibatidine. Thus, in all experiments, each strip (dissected either from the inner or the outer longitudinal muscle) once suspended in its respective muscle bath, was tested sequentially for its response to all contractile agents: EFS, KCl, epibatidine, and bethanechol. All responses are presented in millinewtons (mN). In both muscle layers of dog and human bladders, some strips contracted to epibatidine, and some did not (raw data provided in Tables 1–4). We indicated strips that responded to epibatidine with tension ≥2 mN as “responders” and strips did not contract to epibatidine or contracted with tension <2 mN as “nonresponders.”

Table 1.

Strip response to epibatidine plus number and percentage of responders in dog inner longitudinal muscle

Dog No.	Gender	Response Means ± 95% CI	Number of Total Strips	Number of Responders	Percentage of Responders	Responsiveness Means ± 95% CI
	Male	6.5 ± 2.4	16	14	88	7.3 ± 2.4
2	Male	9.0 ± 3.0	32	26	81	10.8 ± 3.1
3	Male	5.7 ± 2.7	24	19	79	7.0 ± 3.2
4	Male	7.7 ± 1.6	30	26	87	8.7 ± 1.5
5	Male	6.7 ± 2.2	30	24	80	8.1 ± 2.4
6	Male	9.4 ± 5.7	20	15	75	12.2 ± 7.1
7	Male	9.5 ± 3.6	20	17	85	11.0 ± 3.7
8	Female	6.1 ± 2.0	24	18	75	8.0 ± 2.0

Strip responses to epibatidine are measured in millinewtons and shown as means ± 95% confidence interval (CI).

Table 4.

Strip response to epibatidine plus number and percentage of responders in human outer longitudinal muscle

Bladder No.	Gender	Response Means ± 95% CI	Number of Total Strips		Number of Responders	Percentage of Responders	Responsiveness Means ± 95% CI
1	Male	38.3 ± 7.0	32		32	100	38.3 ± 7.0
2	Male	10.3 ± 3.4	31		23	74	13.6 ± 3.7
3	Female	9.6 ± 4.3	31		17	55	16.8 ± 6.0
4	Female	7.0 ± 3.4	24		15	63	11.0 ± 4.5
5	Female	53.6 ± 13.0	8		8	100	53.6 ± 13.0

Strip responses to epibatidine are measured in millinewtons and shown as means ± 95% confidence interval (CI).

Epibatidine was selected because it has been shown to be a very potent nicotinic acetylcholine receptor agonist (11). At a concentration below or equal to 10 μM, epibatidine induces its effect by stimulating nicotinic receptors, without binding to any other receptors (including muscarinic receptors) (12). Bethanechol was selected because it has been shown to be a potent muscarinic receptor agonist due to its resistant to degradation by acetylcholinesterase (its potency in inducing muscle contractions may be higher than acetylcholine itself) (13). Concentrations of drugs used in this study were as previously established in our laboratory (9, 14), as well as by others (13, 15–17).

In these studies, a strip’s force of contractions was not normalized to strip size because the setup used for such studies required mounting each strip along its length between two spring wire clips (Cat. No. 158802, Radnoti, LLC, Covina, CA), so, the amount of muscle involved in generating the force is very similar for all strips, matching previously reported methods from our laboratory (9, 18, 19). Responses to KCl are presented in millinewtons and were not normalized to strip size because the length and weight of muscle strips were comparable in strips from the inner versus outer longitudinal muscle layers of dog bladders [9.4 ± 2.5 (means ± 95% confidence interval) vs. 9.0 ± 1.6, P = 0.06, and 9.3 ± 2.1 vs. 10.3 ± 2.6, P = 0.06, respectively; data from 5 dog bladders and a total of 100 muscle strips], and in strips from the inner versus outer longitudinal muscle layers of human bladders (9.5 ± 1.0 vs. 9.9 ± 1.7, P = 0.07, and 16.7 ± 2.6 vs. 15.7 ± 3.2, P = 0.08, respectively; data from 6 human bladders and a total of 148–153 muscle strips). Responses to drug treatments were not normalized by KCl responses, specifically in dog bladders because KCl responses were different between the two muscle layers, nor in human bladders for consistency purposes (20).

No differences in contractile properties were found between strips from ventral and dorsal aspects or from dome and middle locations of the bladder, as previously reported (5); therefore, data from these muscle strips were grouped together. Sex differences could not be examined in dog tissues because samples of convenience were used. Specifically, only one to two female dogs were included versus seven males. No sex differences were found in male versus female humans; therefore, data from these muscle strips were grouped together, although different symbols are used to indicate male versus female in the graphs of contractile responses. Comparable contractile responses between male and female bladder samples have also been reported in human (21) or rat (22), which are in line with our findings.

Confirmation of Urothelium Removal

After completion of the muscle experiments, muscle strips were fixed in 4% paraformaldehyde in phosphate buffer for 4 h at 4°C and equilibrated in 10% phosphate-buffered sucrose overnight, following by 30% phosphate-buffered sucrose for approximately the same length of time. Tissues were embedded in OCT Compound (Scigen, Gardena, CA) and stored at −80°C until processed. They were then cryosectioned into 14-µm sections, mounted immediately onto coated slides (Fisher Plus), and kept frozen at −80°C until use. A subset of slides from each muscle strip was stained with hematoxylin and eosin and examined to confirm removal of the mucosa.

Immunostaining and Quantification of Axon Density

Muscle strips of two male dog and two female human bladders were collected at the end of the muscle contraction experiment and were fixed and cyrosectioned, as described in the previous section. Also, full-thickness bladder specimens from the midbladder regions of three additional female dogs and one female and two male human bladders were collected, fixed, and cyrosectioned as described in the previous section. This brought the total specimen number for axon density analysis to five dogs and five humans. Subsets of these slide sections were permeabilized with 0.5% pepsin, blocked using 10% goat serum, and stained using a monoclonal primary antibody against pgp9.5 (a pan neuronal marker, Abcam, Cat. No. ab8189, RRID:AB_306343, Cambridge, MA) at 1:100 dilution in phosphate-buffered saline (PBS) overnight at room temperature. The next day after being washed, the sections on slides were incubated with a secondary antibody Cy3 Goat anti-Mouse tagged with AF488 (red; Jackson ImmunoResearch Labs, Cat. No. 115–545-166, RRID:AB_2338852, West Grove, PA) at 1:100 dilution in PBS, for 2 h at room temperature. Slides were then stained with 0.1 µg/mL 4′,6- diamidino-2-phenylindole, dihydrochloride (DAPI) in PBS for 15 min before being cover slipped with 80% glycerol in PBS. Specificity of the anti-pgp9.5 antibody was determined using no primary antibody controls in which serum was substituted for the primary antibody, followed by the secondary antibody; no labeling was observed in the sections as a result of incubation of tissues with serum and then the secondary antibody alone. This antibody has also been shown to detect specifically neuronal cell bodies and axons in dog nerves (23, 24).

Slides were imaged using a Nikon Eclipse E800 upright microscope (Nikon, Melville, NY) equipped with a Retina cooled imaging camera (QImaging, Surrey, BC, Canada) and imaging software (Bioquant Life Science, Bioquant Image Analysis, Corp., Nashville, TN). In the muscle strips, at least 4 randomly chosen fields were quantified per dog or human muscle strip section, in a blinded fashion by M.F.B. and N.F. In sections from full-thickness bladders, 3–4 random fields located within the regions of interest (inner longitudinal layer and outer longitudinal layer) were quantified, from at least two sections per dog or human specimen. The immunopositive axons were counted using a ×20 objective (×300 magnification in the digital image) in a 280,287-µm² field size. The same size field was quantified per strip and per full-thickness bladder layer. Axon density was assayed using a previously described stereological grid count method (25), with data normalized to the area of the region of interest and presented as the number of pgp9.5 immunopositive axons per micrometer².

Histology and Quantification of Intramural Ganglia

Histological studies were performed on full-thickness bladder sections obtained from a total of 13 dogs and 8 humans, including full-thickness midbladder specimens reserved from the nine dog and five human bladder specimens used in the earlier mentioned muscle strip experiments. Histological data from 4 of 13 dogs were used in prior studies (9, 24). Tissues were fixed, prepared, and sectioned as described in the Confirmation of Urothelium Removal section. Sections were stained with hematoxylin and eosin. Slides were then examined for the location and number of intramural ganglia per bladder layer, and for the number of neuronal cell bodies per ganglia, by an individual naive to group assignment (M.F.B.). This assessment was performed using a Retina cooled imaging camera (QImaging, Surrey, BC, Canada), imaging software (Bioquant Life Science, Bioquant Image Analysis Corp., Nashville, TN), and previously published methods.

Statistical Analyses

Data from replicates of individual bladders are indicated as symbols with error bars within each bar graph (Figs. 2, 3, and 4). The numbers of specimens per group and muscle strips per treatment are indicated in the figure legends. Statistical analyses were performed using GraphPad Prism version 8.4.2 or 9 (La Jolla, CA). Data are presented as means ± 95% confidence intervals. P values were adjusted for multiple comparisons whenever applicable and values of 0.05 or less were considered as statistically significant for all analyses. Repeated-measures mixed-effects restricted maximum likelihood (REML) models were used to compare each treatment’s muscle strip contractile results, using the factors individual bladder specimens (and their replicate muscle strips) and muscle layer (26), and to compare axonal density using the same factors. A mixed-effects model analysis was used to compare the responses to different stimulations per layer, in dogs versus humans, using the factors species and muscle layer. For each mixed-effects model, Sidak’s multiple comparison post hoc tests were used to determine differences between layers, regions, or species. Wilcoxon matched-pairs signed-rank test was used to compare the number of responders to epibatidine (Tables 1, 2, 3 and 4) between the two muscle layers. One-way ANOVAs were used to examine differences in number of ganglia per bladder wall layer and number of neurons per ganglia per bladder wall layer separately for dogs and humans, followed by Tukey’s multiple comparison post hoc tests to determine differences between the layers. A mixed-effects model analysis was used to compare the number of ganglia per bladder wall layer between dogs and humans, using the factors species and bladder layer, followed by Sidak’s multiple comparison post hoc tests. Because this study did not test a prespecified statistical null hypothesis, the study is exploratory; therefore, the calculated P values are interpreted as descriptive, not hypothesis testing (27).

Figure 2.

Maximal contractile responses to myogenic, muscarinic- and nicotinic-receptor, and neurogenic stimulations in strips from the inner and outer longitudinal bladder muscle. Responses to 120 mM KCl in dog (A) and human (B) bladders. Responses to 10 µM of the nicotinic receptor agonist epibatidine in dog (C) and human (D) bladders. Responses to EFS at intensity of 12 V, 30 Hz, and 1-ms pulse duration that was delivered to each strip before and after epibatidine treatment in dog (E) and human (F) bladders. Responses to 30 µM of the muscarinic receptor agonist bethanechol in dog (G) and human (H) bladders. The maximal responses are expressed in millinewtons (mN). For dog data, N = 8 or 9 bladders per muscle layer per treatment and n = 196–228 strips per muscle layer per treatment. For human data, N = 5 or 6 bladders per muscle layer per treatment and n = 159–126 strips per muscle layer per treatment. Data are presented as means ± 95% CI. Data were analyzed using mixed-effects model analyses of variance with Sidak’s multiple comparison post hoc tests to determine differences between inner and outer muscle layers within each species. *P < 0.05 and **P < 0.01, respectively, compared between muscle layers of the same treatment group, as shown. #P < 0.05 and ##P < 0.01, respectively, comparing pre- versus post-responses to EFS, as shown. CI, confidence interval; EFS, electrical field stimulation; Hz, hertz; ms, millisecond; N, number of bladders; n, number of strips per group; V, volt.

Figure 3.

Density of pgp9.5 immuno-sensitive axons in dog and human bladder muscle strips. A and B: representative images of pgp9.5-immunostained axons (red immunofluorescence) in sections of muscle strips from dog (A) and human (B) bladders. C and D: quantification of axon density of the nerve fibers with pgp9.5 immunopositive stain (arrows) in strips from the inner and outer longitudinal muscle layer, as well as sections of full thickness of dog (C) and human (D) bladders. For dog data, N = 5 bladders, and n = 37–40 strips per muscle layer. For human data, N = 5 bladders, and n = 85–87 strips per muscle layer. Scale bar = 100 μm. Data are presented as means ± 95% CI. Data were analyzed using mixed-effects model analyses of variance with Sidak’s multiple comparison post hoc tests. *P < 0.05 and **P < 0.01, respectively. CI, confidence interval; N, number of bladders; n, number of strips per group.

Figure 4.

Intramural ganglia in the layers of dog and human bladders. Number of ganglia (A) and the number of neurons per ganglia (C) in dog bladders. Number of ganglia (B) and the number of neurons per ganglia (D) in human bladders. E and F: representative images of H&E-stained bladder sections for intramural ganglia in the adventitia/serosa layer in dog and human bladders, respectively. N = number of bladders. For dog data, N = 13 bladders per count per layer, and for human data, N = 6–8 bladders per count per layer. Scale bar = 100 μm. Data are presented as means ± 95% CI. Data were analyzed using mixed-effects model analyses of variance with Tukey’s multiple comparison post hoc tests to determine differences between layers within each species or with Sidak’s multiple comparison post hoc tests to compare the number of ganglia per bladder wall layer between dogs and humans. **P < 0.01 compared between layers as shown. &P < 0.05 compared with adventitia/serosa layer in dog bladders. CI, confidence interval; H&E, hematoxylin and eosin.

Table 2.

Strip response to epibatidine plus number and percentage of responders in dog outer longitudinal muscle

Dog No.	Gender	Response Means ± 95% CI	Number of Total Strips	Number of Responders	Percentage of Responders	Responsiveness Means ± 95% CI
1	Male	2.5 ± 1.4	16	6	38	5.4 ± 2.4
2	Male	4.2 ± 1.4	32	21	66	6.0 ± 1.7
3	Male	3.3 ± 1.7	24	10	42	7.1 ± 2.7
4	Male	6.3 ± 3.5	30	19	63	9.5 ± 5.1
5	Male	3.3 ± 1.3	30	16	53	5.5 ± 1.8
6	Male	3.7 ± 1.8	20	11	55	6.2 ± 2.4
7	Male	4.1 ± 2.2	20	11	55	6.8 ± 3.3
8	Female	5.3 ± 2.2	24	16	67	7.6 ± 2.6

Strip responses to epibatidine are measured in millinewtons and shown as means ± 95% confidence interval (CI).

Table 3.

Strip response to epibatidine plus number and percentage of responders in human inner longitudinal muscle

Bladder No.	Gender	Response Means ± 95% CI	Number of Total Strips	Number of Responders	Percentage of Responders	Responsiveness Means ± 95% CI
1	Male	51.8 ± 11.0	32	32	100	51.8 ± 11.0
2	Male	12.9 ± 2.4	31	30	97	13.4 ± 2.4
3	Female	14.0 ± 5.4	32	23	72	19.3 ± 6.3
4	Female	15.4 ± 8.6	24	19	19	19.1 ± 10.2
5	Female	49.6 ± 26.5	8	8	100	49.6 ± 26.5

Strip responses to epibatidine are measured in millinewtons and shown as means ± 95% confidence interval (CI).

RESULTS

Responses to Myogenic, Nicotinic-Receptor, Neurogenic, and Muscarinic-Receptor Stimulations

Responses to myogenic stimulation with KCl.

Strips obtained from the inner longitudinal muscle layer of dog bladders showed greater responses to 120 mM KCl than those from the outer longitudinal muscle layer (28.5 ± 7.7 vs. 20.4 ± 4.1, P < 0.0001; Fig. 2A). In human bladders, no differences were found in KCl-induced force between inner and outer longitudinal muscle layer strips (47.2 ± 25.4 vs. 49.7 ± 16.8, P = 0.40; Fig. 2B). Male and female human bladders had similar responses to KCl. Human bladders showed greater responses overall to KCl than dog bladders (P = 0.003), yet no muscle layer or muscle layer × species interaction differences (P = 0.43 and P = 0.13, respectively).

Responses to nicotinic receptor agonist epibatidine.

In dog bladders, strips from the inner longitudinal muscle layer showed greater responses to 10 µM epibatidine than strips from the outer longitudinal muscle layer (7.6 ± 1.3 vs. 4.1 ± 1.0, P < 0.0001; Fig. 2C). In both layers, some strips contracted in response to epibatidine, and some did not (raw data provided in Tables 1 and 2). The proportion of strips that responded to epibatidine with tension ≥ 2 mN (responders) was greater in strips from the inner longitudinal versus the outer longitudinal muscle layer for each dog (Table 1 vs. Table 2, P = 0.008). Also, the average strip responsiveness to epibatidine (data that included the responders only and measured in mN of tension) was greater in strips from the inner longitudinal versus the outer longitudinal muscle layer (9.1 ± 1.6 vs. 6.8 ± 1.1, P = 0.005; data in Table 1 vs. Table 2).

In human bladders, strips from the inner longitudinal muscle had greater responses to epibatidine than strips from the outer longitudinal muscle (28.8 ± 25.0 vs. 23.8 ± 26.1, P = 0.008; Fig. 2D). In both layers from the human bladders, some strips contracted in response to epibatidine, and some did not (raw data provided in Tables 3 and 4). In humans, the proportion of strips that responded to epibatidine with tension ≥ 2 mN (responders) showed no statistical differences between strips from inner versus outer longitudinal muscle layers (Table 3 vs. Table 4, P = 0.25). Yet the average strip responsiveness to epibatidine (again, data that included responders only; measured in mN of tension) was greater in strips from the inner versus outer longitudinal muscle layer (30.6 ± 23.0 vs. 26.7 ± 23.0, P = 0.046; Table 3 vs. Table 4). Male and female human bladders had similar responses to epibatidine (Fig. 2D).

In the mixed-effects model analysis comparing human to dog results, although the human bladders showed greater responses to epibatidine than dog bladders (P = 0.01), both species showed similar greater responses in the inner longitudinal muscle layer than in the outer layer (P = 0.005).

Responses to neurogenic stimulation using EFS before and after epibatidine treatment.

Strips obtained from the two muscle layers of dog and human bladders were tested for their responses to EFS, which was delivered to each strip before and after the epibatidine treatment described earlier. In dog bladders, the repeated-measures mixed-effects analysis showed a difference between the inner versus outer longitudinal muscle layers in their responses to EFS, both before and after treatment with epibatidine (P < 0.0001; Fig. 2E). Responses to EFS pre-epibatidine treatment were greater in strips obtained from the outer longitudinal muscle layer, versus the inner layer (41.1 ± 7.8 vs. 29.8 ± 5.0, P < 0.0001; Fig. 2E). Similarly, responses to EFS post-epibatidine treatment were greater in strips obtained from the outer longitudinal muscle layer versus the inner layer (25.0 ± 3.7 vs. 20.6 ± 5.6, P = 0.04; Fig. 2E). In addition, post hoc tests showed that responses to EFS post-epibatidine were lower than pre-epibatidine, in each layer (P < 0.0001 each; Fig. 2E).

In human bladders, the repeated-measures mixed-effects statistical analysis showed that the primary effect of EFS pre- and post-epibatidine were different between the inner and outer muscle layers (P < 0.0001; Fig. 2F). Although post hoc tests showed no statistical differences in the effect of EFS pre-epibatidine in inner versus outer muscle layers (44.5 ± 18.6 vs. 37.4 ± 18.3, P = 0.09; Fig. 2F), they showed that the effect of EFS post-epibatidine was greater in strips from the outer layer (55.0 ± 28.5 vs. 45.7 ± 22.1, P = 0.002; Fig. 2F). Yet, in contrast to the dog bladders, in human bladders, post hoc tests showed that whereas epibatidine increased the responses to EFS in muscle strips from the outer longitudinal muscle layer (i.e., EFS results pre- vs. post-epibatidine, P = 0.01; Fig. 2F), it had no effect on EFS responses in strips from the inner layer (i.e., EFS results pre- vs. post-epibatidine, P = 0.40). Male and female human bladders had similar responses EFS, pre- and post-epibatidine.

Bladders of both species responded similarly to EFS pre-epibatidine treatment (P = 0.38), with both showing greater responses in the outer longitudinal muscle layer that in the inner (P = 0.009). In response to EFS post-epibatidine treatment, human bladders showed greater responses than dog bladders in both layers (P = 0.003), although maintaining similar greater responses in the outer longitudinal muscle layer that in the inner (i.e., the muscle layer × species interaction was not statistically different, P = 0.43).

Responses to muscarinic receptor agonist bethanechol.

Responsiveness to bethanechol was then tested in the continued presence of epibatidine. In dog bladders, strips from the inner longitudinal muscle had higher responses to 30 µM of the muscarinic agonist bethanechol than strips from the outer longitudinal muscle (27.6 ± 7.7 vs. 18.3 ± 3.8, P < 0.0001; Fig. 2G). In human bladders, there was no statistically significant difference in the responses to bethanechol in the inner versus outer layers (57.8 ± 30.0 vs. 61.8 ± 29.2, P = 0.30; Fig. 2H). Male and female human bladders had similar responses to bethanechol. In the mixed-effects model analysis comparing human to dog results, human bladders had a greater response to bethanechol than dog bladders (P = 0.003). They also showed a muscle layer × species interaction difference (P = 0.04) because the dog bladders showed a statistically significant difference between the inner versus outer longitudinal muscle layers that the human bladders lacked.

Density of pgp9.5 Immuno-Sensitive Nerve Fibers

Bladders from five dog and five human bladders (muscle strips from two dog and two human bladders, and sections of midbladder full thickness of an additional three dog and three human bladders) were fixed, cryosectioned, and immunostained for pgp9.5, a pan neuronal marker that detects axons. Axon density, calculated as a percentage of pgp9.5 positive nerve fibers per area, was greater in the outer longitudinal muscle compared with the inner longitudinal muscle layer in five dog bladders (0.70 ± 0.3 vs. 0.35 ± 0.1, P = 0.01; Fig. 3, A and C). Similarly, axonal density was greater in the outer versus inner longitudinal muscle layers in 5 human bladders (1.0 ± 0.2 vs. 0.4 ± 0.2, P < 0.0001, Fig. 3, B and D). A species difference was observed (P = 0.02) due to a higher axonal density in the outer longitudinal muscle layer of human bladders than in dog bladders (P = 0.01). The inner longitudinal muscle layer showed a similar axonal density in both species (P = 0.89). That said, in both species, axonal density was higher in the outer longitudinal muscle layer than in the inner layer (P < 0.0001).

Location of Intramural Ganglia

Full-thickness of bladder sections obtained from 13 dogs and 8 humans were examined for the location and number of intramural ganglia and for the number of neuronal cell bodies in these ganglia (Fig. 4). Within dog bladder walls, more ganglia were observed in the adventitial/serosal layer compared with the lamina propria and inner longitudinal muscle layer (Fig. 4A and Fig. 1B). Within human bladder walls, ganglia were more evenly distributed across the various layers, with no significant differences between the layers (Fig. 4C). A species difference was observed with human bladders containing fewer intramural ganglia overall within their bladder walls than in dog bladders (P = 0.002) and a muscle layer × species interaction difference (P = 0.02). The latter was due to the presence of fewer intramural ganglia in the adventitial/serosal layer in human bladders compared with dog bladders (P = 0.04; Fig. 4C).

In dog bladders, numbers of neuronal cell bodies per ganglion were higher in the adventitial/serosal layer compared with the lamina propria and inner longitudinal muscle layers (Fig. 4B). The numbers of neuronal cell bodies per ganglion were similar across the layers in human bladders (Fig. 4D). In the mixed-effects model analysis comparing human to dog results, no statistically significant differences were observed.

DISCUSSION

To our knowledge, this is the first study investigating variations in contractile responses to various stimulation and neuroanatomical features across different layers of dog and human bladder tissues. In dog bladders, responses induced by the myogenic stimulator KCl, the nicotinic receptor agonist epibatidine, and the muscarinic receptor agonist bethanechol were greater in strips dissected from the inner longitudinal muscle, whereas responses induced by EFS (a neurogenic stimulator) were greater in strips dissected from the outer longitudinal muscle layer. Similarly, in human bladders, responses to epibatidine were greater in strips dissected from the inner longitudinal muscle, and responses to EFS post-epibatidine were greater in strips dissected from the outer longitudinal muscle layer. In contrast to the dog bladders, human bladders showed similar responses to KCl and bethanechol in the two muscle layers examined. In both dog and human bladders, axonal densities in the outer longitudinal muscle layer were greater than in the inner longitudinal muscle layer. In addition, there were more intramural ganglia (and neurons within those ganglia) in the outer adventitia/serosa layer of dog bladders than more internal layers. The number of intramural ganglia (and neurons within those ganglia) was similar across all layers in human bladders.

High potassium (KCl, 120 mM) has previously been shown to be effective in inducing maximal contractions in mucosa-denuded muscle strips collected from dog bladders (9, 19) and human bladders (16) in which the inner and outer muscle layers were not separated. Here, strips from the inner and outer longitudinal muscle layers of the dog and human bladders were separated and individual responses to KCl were examined. In dog bladders, the enhanced KCl-induced contractions in the inner versus outer longitudinal muscle layer (Fig. 2A) are suggestive of enhanced muscle tone or possibly increasing Ca²⁺ sensitivity in the inner layer. Although one could speculate that the outer muscle layer may have a lower density of smooth muscle fibers than the inner to accommodate different mechanical strains occurring within each during physiological filling, we found that the muscle strips’ weights were comparable in the two muscle layers, matching results from Morales-Orcajo et al. (7) showing that the inner and outer longitudinal layers are of almost similar thicknesses. In contrast to the dog bladders, in human bladders, the similar contractions in response to KCl in both the inner and outer longitudinal muscle layers (Fig. 2B) demonstrate comparable nonspecific functional and receptor-independent contractile properties in both bladder layers (i.e., similar myogenic responses in each layer).

The expression of functional nicotinic receptor subunits in dog bladder tissue has previously been described by our group (28). In this study, the higher epibatidine-induced responses in the inner layer in both species, despite the increased axonal density in the outer layer (Fig. 2, C and D, Tables 1, 2, 3 and 4, Fig. 3, C and D), suggest that the direct excitatory effects of this nicotinic agonist are due to stimulation of intramural nerve terminals and not activation of action potentials along the axons, as was previously reported in rat (29). The different epibatidine responses in inner versus outer layers (Fig. 2, C and D) could also be due to differing numbers of nicotinic receptors on the nerve terminals in these layers.

EFS at 30 Hz has previously been shown to evoke maximal contractions in dog and human bladder strips (9, 30). Our observed increased contractile force in response to EFS in dog and human bladder strips from the outer longitudinal muscle layers versus inner layers (Fig. 2, E and F) might indicate that there are more autonomic receptors on the smooth muscle cells in this layer that become stimulated by the release of neurohumoral transmitters from nerve endings. Although our axon counts were derived from a pan neuronal marker (pgp9.5+) that does not differentiate between axon subtypes, we did observe higher axonal density in the outer longitudinal muscle layer than in the inner, in both species, in support of this hypothesis. In the pig urinary bladder, it has also been shown that the outer muscle layer dissected from beneath the serosa produces a higher contractile force in response to EFS than the inner muscle from the mucosal side (31). The variability that we observed in the contractile responses of different human bladder specimens could be because of the natural variation in normal human bladder contractile responses, or other unknown factors. Further explanation is not possible because of the limited information available from the organ procurement agency.

The decrease in EFS post-epibatidine responses compared with EFS pre-epibatidine in dog bladders (Fig. 2E) suggests that a proportion of epibatidine-induced acetylcholine release through nicotinic receptors might involve the generation of action potentials, as was previously reported in the vas deferens (32). In contrast, in human bladders, the data in Fig. 2F suggest that intramural nerve terminal depolarization via EFS and the release of neurotransmitters onto the smooth muscle occurs without stimulation of nicotinic receptors (33). We speculate that the layer-dependent effects of epibatidine on EFS-evoked contractions in dog and human bladders could be due to an interaction of epibatidine with other neurotransmitters that induce either inhibitory or excitatory effects, as was previously reported (34, 35).

In dog bladders, 30 μM of bethanechol was previously shown as an effective concentration for inducing muscle strip contractions (9). The enhanced responses to bethanechol in the inner versus outer longitudinal strips from dog bladders (Fig. 2G) but not in human bladders (Fig. 2H) could be due to species differences. Species differences in the distribution of adrenoceptor subtypes in bladder muscle have been previously reported (36). It is possible that in dogs, the lower axonal density in the inner muscle layer (Fig. 3C) and, consequently, a lower release of acetylcholine into bladder muscle, enhances muscarinic receptor sensitivity to this agonist, as previously reported (29). In humans, the similar effect of bethanechol on both muscle layers could be because, in both men and women, cholinergic nerves are similarly distributed in different parts of the bladder and run across the bundles of smooth muscle cells after originating from larger trunks located deep in the bladder layers (37).

Previous studies have reported that epibatidine can interact with muscarinic receptors to induce the release of acetylcholine (38). However, based on the similar results in Fig. 2A vs. Fig. 2G, and in Fig. 2, B vs. Fig. 2H, we suggest that epibatidine does not alter muscarinic receptor function since the responses to bethanechol and KCl were almost identical in both species’ bladders. In both dogs and humans, some strips did not contract when stimulated with epibatidine (nonresponders) and some contracted (responders). Interestingly, strips’ characteristic of being “nonresponders” was only in their response to nicotinic receptor stimulation because they still responded to other stimulations, i.e., to KCl, EFS, and bethanechol, with similar response levels as in “responders.”

The enhanced axonal density in the outer muscle layers (Fig. 3, A–D) is in accordance with reports that nerve fibers and associated macroscopic intramural ganglia ramify in the adventitia (39, 40). Then, while penetrating the muscle and the submucosa layers, the nerve fibers repeatedly branch into smaller fibers in association with small, microscopic intramural ganglia (39, 40). In both species, the enhanced axonal density in the outer muscle layer (Fig. 3) matched the EFS-induced contractility presented in Fig. 2, E and F.

In full-thickness dog bladder walls, although the submucosa and the internal longitudinal muscle layers contained no ganglia, ganglia were found in connective tissues of the middle circular muscle, outer longitudinal muscle, and adventitia/serosa layers (Fig. 4A), matching findings from another group (41). In full-thickness human bladder walls, intramural ganglia were detected in all layers (lamina propria, in connective tissues of different muscle layers, and adventitia/serosa layers; Fig. 4C). Others have also reported intramural ganglia in the lamina propria and between bundles of detrusor muscles in human bladders (42, 43).

Perspectives and Significance

The findings in this study are suggestive of layer-dependent and interspecies variations in the expression or distribution of receptors and the effect of these receptors on neurotransmitters responsible for bladder muscle contractions. Specifically, in dog bladders, the inner longitudinal muscle layer showed greater muscle layer-specific myogenic and muscarinic responses, as well as nicotinic responses than the outer layer. In contrast, the outer longitudinal muscle layer of dog bladders showed more neurogenic responses concomitant with higher axonal density, ganglia numbers, and numbers of neurons/ganglia in this same outer layer. Human bladders had similar nicotinic responses (more in the inner layer) and neurogenic (more in the outer layer) as dog bladders, the latter concomitant with higher axonal density in the outer layer. In contrast to dogs, the human bladder specimens did not show muscle layer-dependent myogenic and muscarinic responses. This potential species difference in myogenic responses should be further explored since limitations of this study include the low number of human subjects with a broad range in age and incomplete medical health information.

We prefer not to speculate about the possible physiological and pathophysiological relevance of these findings until more definitive evidence emerges. Yet, these findings expand the knowledge regarding layer-dependent differences in bladder detrusor muscle. To understand and treat bladder disease in both dogs and humans, characterizing fundamental mechanisms of physiological contractile properties in normal bladders at the tissue level is a prerequisite for the identification of causes of disease and the advancement of medical treatments.

DATA AVAILABILITY

Data are provided in the tables and are also available on request from the authors.

GRANTS

Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award R01NS070267 (to M.R.R. and M.F.B.) and R01AG049321 (to M.R.R. and M.F.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

N.F., M.F.B., and M.R.R. conceived and designed research; N.F., M.F.B., D.G., and M.A. performed experiments; N.F., M.F.B., D.G., A.S.B., and D.Y. analyzed data; N.F. and M.F.B. interpreted results of experiments; N.F. and M.F.B. prepared figures; N.F. and M.F.B. drafted manuscript; N.F., M.F.B., and M.R.R. edited and revised manuscript; N.F., M.F.B., and M.R.R. approved final version of manuscript.