This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology, and pathology.
We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
The bladder smooth muscle relaxes to allow urine storage, and contracts to elicit urine elimination. Relaxation is mediated by intrinsic smooth muscle properties and by tonic release of norepinephrine (NE) from the sympathetic nerves, which activates beta adrenergic receptors (β3AR in human) in the detrusor. Voiding is achieved by inhibiting the sympathetic input and activating the parasympathetic nerves that release ACh/ATP to contract the bladder smooth muscle1. Numerous pathological conditions, including brain and/or spinal cord injury, neurodegenerative diseases, diabetes, bladder outlet obstruction or interstitial cystitis, can profoundly alter bladder function, with severe impact on the patient’s quality of life2. These conditions alter the contractility of the smooth muscle by affecting one or more components of the bladder: the smooth muscle, the afferent or efferent nerves and/or the mucosa.
Several in vivo and in vitro methods to study bladder function have been developed. In vivo, cystometry is the primary measurement of bladder function. Though this is an intact preparation that allows collection of information under close to physiological conditions, there are a number of circumstances in which the use of smooth muscle strips is preferred. These include situations when surgical and/or pharmacological manipulations would affect the survival and stability of the in vivo preparation, or when the studies require the use of the human tissue or expensive chemicals. This method also facilitates an examination of the effects of drugs, age and pathology on each component of the bladder, i.e. smooth muscle, mucosa, afferent and efferent nerves.
Bladder strips have been employed over the years by many groups to answer a number of scientific questions. They were used to evaluate changes in myogenic spontaneous activity induced by pathology. This activity is believed to contribute to the urgency and frequency symptoms of overactive bladder (OAB), and is therefore a target for drugs being developed for OAB3-9. Bladder strips were also used to investigate myogenic and neuronal factors that modulate smooth muscle tone with the aim of discovering ion channels and/or receptors and/or intracellular pathways that could be targeted to induce either relaxation or contraction of the smooth muscle3,10-13. Other studies have focused on the nature of neurotransmission, including transmitters and receptors involved and changes induced by pathology14,15. In addition, the method has been used for comparisons between tissues from different species16-18, between organs19-21, and evaluation of drug structure-activity relationships22-24. An extension of this method has been used to measure the effect of drugs on transmitter release from efferent nerves25. Furthermore, a variety of tissues (bladder, urethra, gastrointestinal tract, GI) harvested from animals or humans (from surgeries or organ donor tissue approved for research) and from a variety of animal models including spinal cord injury (SCI), bladder outlet obstruction (BOO), or interstitial cystitis (IC) can be investigated using this technique.
In this paper we illustrate the use of this method along with necessary experimental protocols, to address several scientific questions mentioned above.
All procedures described here are approved by the IACUC committee at University of Pittsburgh.
1. Solutions
2. Experimental Set-up (Schematic Figure 1A)
3. Tissue (Figure 1B)
Remove the bladder from an adult naïve female Sprague Dawley rat (200-250 g; ~10-12 weeks old) following these steps:
4. Stimulation Protocols
5. Data Analysis
Analyze data using adequate software (e.g., Windaq, LabChart).
Spontaneous Myogenic Activity
Spontaneous myogenic activity is an important smooth muscle characteristic that undergoes changes with postnatal development6-9 and pathology (e.g., SCI, BOO)3-5. Because this activity is believed to contribute to the symptoms of overactive bladder (OAB)2, an evaluation of receptors, intracellular pathways and pharmacological agents that modulate it, is of high interest for developing effective treatments for OAB and other smooth muscle dysfunctions. The method presented here can easily investigate these questions. Figure 2 illustrates different patterns of myogenic spontaneous activity during development in neonatal (i), juvenile (ii) adult (iii) and spinal cord injured rats (SCI; iv). Strips from neonatal rats exhibit large amplitude, low frequency rhythmic contractions (Figure 2Ai), while strips from adult rats exhibit small amplitude, high frequency activity (Figure 2Aii, iii). After SCI the neonatal pattern re-emerges (Figure 2Aiv). In addition to using strips from animal models, various pharmacological agents can be used to induce spontaneous contractions in strips from naive animals, with the aim of understanding the mechanisms underlying the spontaneous contractions. Examples of suitable pharmacological agents include muscarinic receptor agonists (carbachol; CCh), compounds that increase ACh levels (such as acetylcholine esterase inhibitors), low concentrations of KCl (e.g., 20 mM) or other experimental drugs. Figures 3A-B, illustrate modulation of spontaneous activity by pharmacological agents that act on KCNQ channels located on the smooth muscle. The KCNQ channel opener, flupirtine, decreases the amplitude and frequency of spontaneous activity in a concentration-dependent manner (Figure 3Ai-iii), while the KCNQ channel blocker, XE991, decreases the amplitude but increases the frequency of spontaneous activity (Figure 3Bi-iii).
Smooth Muscle Tone
Smooth muscle tone and contractility properties are important factors for proper function of the bladder during storage and voiding. This method can easily screen the effects of pharmacological agents on smooth muscle tone. Figures 3Aiv and 3Biv show that flupirtine decreases basal tone, consistent with smooth muscle relaxation, while the XE991 increases smooth muscle tone. Figure 4 illustrates concentration dependent increases in smooth muscle tone by activating bombesin receptors with neuromedin B (NMB; Figure 4A, B) or muscarinic receptors with carbachol (CCh; Figures 4C, D). Furthermore, intracellular pathways mediating these smooth muscle responses can be investigated using specific modulators (data not shown).
Neurally-mediated Responses and Modulation of Neurotransmission
Bladder contraction is achieved by the release of ACh/ATP from the parasympathetic efferent nerves. The contribution of muscarinic and purinergic systems to bladder contraction varies among species and pathological conditions, with predominant increase in purinergic contribution in pathologies such as interstitial cystitis, partial outlet obstruction, and overactive bladder26. Figure 5C demonstrates the use of this method to determine the contribution of muscarinic and purinergic components to neurotransmission in bladder strips from the mouse. The contribution of the cholinergic component was assessed using the muscarinic receptor antagonist, atropine. The contribution of the purinergic system was assessed using the purinergic receptor activator and desensitizer, alpha,beta-methylene ATP (ABMA). Additionally, the frequency dependent contribution of each component was assessed by varying the stimulation frequency from low to high frequencies (2-50 Hz).
The strength of the bladder contraction plays a significant role in voiding efficiently. Using this method, receptors and pathways that modulate neural transmission can be investigated as drug targets for voiding dysfunctions. The 5HT4 receptors are expressed pre-junctionally in parasympathetic neurons and their activation increases ACh levels27. Figure 6 illustrates the excitatory effect of the 5HT4 receptor agonist, cisapride, in human bladder and ileum strips.
Various experimental protocols can be employed to determine the site of action of a test compound. Diagram in Figure 7A illustrates a protocol used to assess pre- vs. post-junctional sites. If drug X reduces (or increases) the EFS response but has no effect on the CCh response, the most likely site of action is pre-junctional. If drug X alters both EFS and CCh response, then it may act on receptors located post-junctionally or both pre- and post-junctionally.
Role of Each Component: Smooth Muscle, Mucosa, and Neuronal
Different pathological conditions may affect various components of the bladder. For example interstitial cystitis (IC) affects primarily the urothelium, while OAB may result in altered smooth muscle contractility. Also, different receptors may be expressed in each bladder component and thus could be specifically targeted in a certain pathology. As opposed to in vivo methods, which measure a net effect of all bladder components, this in vitro method allows the investigation of particular components by using a combination of surgical and pharmacological procedures. To test smooth muscle contraction/relaxation in the absence of neuronal transmission, TTX (0.5-1 µM) can be added to the bath. In Figure 4, NMB and CCh were tested in the presence of TTX. To test the contribution of the mucosa (urothelium and lamina propria) to the smooth muscle contractility, strips with and without the mucosal layer are compared. Figure 7B shows that responses to CCh are reduced in the presence of the mucosa in the pig28. Similar results were reported in human bladder strips29. To test the role of nerve fibers, several approaches can be taken. One is to activate or inhibit specific fibers using pharmacological agents. For example, capsaicin activates a specific population of afferent nerves and causes species dependent smooth muscle contraction or relaxation17,18. Guanethidine inhibits the release of norepinephrine from sympathetic fibers, thus eliminating the contribution of these fibers. Another approach is to desensitize/eliminate specific fibers in vivo prior to the experiment. For example, systemic treatment of the animal with capsaicin desensitizes capsaicin sensitive afferent nerves. Other bladder components that can be studied in this preparation are interstitial cells or gap junctions by activating or blocking them with specific agents.
Species Differences
While most drug development is intended for the treatment of human disorders, basic research is primarily performed in animal tissue. Species differences exist in a number of receptors. For example, 5HT4 receptor agonists enhance neurally-evoked contractions in the human bladder but not in the rat bladder19,30, EFS-induced contractions are almost exclusively atropine-sensitive in human and old-world monkey detrusor from stable bladders31 but become partially atropine-resistant in human detrusor from patients with unstable bladder conditions (e.g., neurogenic, obstructed bladders)15,32,33, capsaicin elicits an excitatory response in rat and human bladder strips, no response in pig bladder strips and inhibitory response in guinea pig bladder strips17,18. Figure 4 shows that bombesin receptor agonists have excitatory effects on rat bladder and no effects on mouse and pig bladder strips16. This information is critical for selecting the appropriate animal model for studying a specific receptor.
Comparison of Sensitivity across Organs
Drugs intended for the treatment of bladder disorders may also affect smooth muscle from other organs, such as the gastrointestinal tract, urethra, gallbladder, etc. This method allows estimation of organ selectivity and sensitivity to a pharmacological agent by comparing different tissues side by side. As illustrated in Figure 6, the 5HT4 receptor agonist, cisapride, has different efficacy and potency in human bladder vs. ileum tissue.
Figure 1. Experimental set-up and bladder strip preparation. A) Schematic of the experimental set-up. Bladder strips are submersed in tissue chambers filled with aerated Krebs solution kept at 37 °C via a circulating water pump. One end of the strip is attached to an isometric force transducer to measure tissue contractility, the other to a fixed rod. The force transducer is connected to an amplifier and computer for data recording. Electric field stimulation electrodes connected to a stimulator are placed in the chamber and used for evoking neurally-mediated bladder contractions. B) Preparation of tissue strips. The bladder is pinned down in a dish and the following procedures are performed: #1 vertical cut though ventral half of bladder from urethra to dome to open the bladder into a flat sheet. #2 horizontal cuts removing the dome and base of the bladder/proximal urethra. #3 vertical cuts dividing the mid bladder into equal strips (4 strips from a rat bladder). C) Schematic of strip components: smooth muscle and mucosa, both containing afferent (blue) and efferent (green) nerves. Mucosa consists of the urothelium and lamina propria. Lamina propria contains blood vessels [1], interstitial cells [2], and muscularis mucosae [3]. Dotted line labeled #2b indicates the procedure for removing mucosa layer. Please click here to view a larger version of this figure.
Figure 2. Myogenic spontaneous activity during development and after pathology. A) Examples of spontaneous activity in neonatal (i), juvenile (ii), spinal intact adult (iii) and spinal cord injured (SCI) adult (iv) rat bladder strips. The SCI rat was used at 4 weeks after surgery. B, C) Summary of amplitude (B) and frequency (C) of spontaneous contractions in the four groups investigated. (Reproduced with permission from Artim DE, Kullmann FA, Daugherty SL, Bupp E, Edwards CL, de Groat WC. Neurourol Urodyn. 2011 Nov;30(8):1666-74.) Please click here to view a larger version of this figure.
Figure 3. Modulation of myogenic spontaneous activity and smooth muscle tone. A) The effect of the KCNQ channel opener, flupirtine, on spontaneous activity and baseline tone in adult rat bladder strips. (i) Flupirtine was added in increasing concentrations (cumulative) at the times indicated by arrows. The enlargements under the trace show 4 min of strip activity during control and after application of 10 µM and 50 µM flupirtine. (ii-iv) Summary of effects of flupirtine (7 strips from 4 rats) on the amplitude (ii) and frequency (iii) of spontaneous activity and baseline tone (iv), expressed as % change from control (pre-drug) values, which were set to 100%. B) The effect of the KCNQ channel blocker, XE991, on spontaneous activity and baseline tone in adult rat bladder strips. (i) XE991 was added in increasing concentrations (cumulative) at the times indicated by arrows. The enlargements under the trace show 2 min of strip activity during control and after application of 10 µM and 50 µM XE991. (ii-iv) Summary of effects of XE991 (9 strips from 4 rats) on the amplitude (ii) and frequency (iii) of spontaneous activity and baseline tone (iv), expressed as % change from control (pre-drug) values, which were set to 100%. Please click here to view a larger version of this figure.
Figure 4. Species differences. A) Concentration dependent smooth muscle contractions in response to the bombesin receptor agonist, neuromedin B (NMB), in rat bladder strips. B) Summary of effects of NMB on smooth muscle contraction in the rat bladder strips. Data are normalized to the KCl (80 mM) response. C, D) Absence of responses to NMB in mouse (C) and pig (D) bladder strips. Carbachol (CCh) elicits strong concentration dependent contractions in both mouse and pig strips, indicating that the strips can respond to stimuli. TTX (0.5 µM) was present in the bath in all strips. (Reproduced with permission from Kullmann FA, McKenna D, Wells GI, Thor KB. Neuropeptides 2013 Oct;47(5):305-13.) Please click here to view a larger version of this figure.
Figure 5. Electric field stimulation. A) Schematic of single pulse stimulation parameters. Abbreviations: d = duration of pulse, i = intensity of pulse, ipi = inter pulse interval. B) Schematic of train stimulation parameters. Abbreviations: td = train duration, i = intensity of pulse, iti = inter train interval. Inset shows the number of pulses in a train and the interval between them, which together with train duration determine the frequency of train stimulation. C) Contribution of purinegic and cholinergic components to neurally-evoked bladder contractions. EFS-FR represent stimulation frequencies, 2, 5, 10, 20, 50 Hz. Three stimuli delivered every 90 sec were tested for each frequency and each frequency series was repeated twice in control and twice after adding each compound. Alpha,beta-methylene ATP, abbreviated ABMA (strip 1), was used to desensitize purinergic receptors and atropine (strip 2) was used to block muscarinic receptors. Strip 3 served as control and was treated with the vehicle, water. Arrows indicate the time when each compound was added to each strip. Note that EFS-evoked contractions are strongly reduced by ABMA and atropine, while not affected by the vehicle. TTX was added at the end of the experiment while the EFS was delivered at 20 Hz. Note that remaining contractions observed in the control strip 3 were abolished by TTX, demonstrating their neural nature (i.e. initiated by transmitter release from the intramural nerves). # indicates smooth muscle responses to ABMA in the absence of EFS. Scale bars are 5 min for x axis and 2 g for y axis. Please click here to view a larger version of this figure.
Figure 6. Modulation of neurally-evoked bladder contractions. A, B) Examples of the enhancement of the neurally-evoked contractions by the 5HT4 receptor agonist, cisapride in human bladder (A) and ileum strips (B). Cisapride (black records) or DMSO (grey records) was added in a concentration dependent manner at the times indicated by arrows. Black bars below the records in each panel represent EFS, which consisted of 10 sec trains delivered at 20 Hz every 120 sec. Vertical scale bars are 1 g for all examples. TTX concentration was 0.5 µM. C-F) Summary of the area under the curve (AUC) of EFS-evoked contractions in response to cisapride (black bars) or DMSO (grey bars) in bladder strips (C, D) and ileum strips (E, F). In C-F, SB stands for SB-203186, representing a summary of data obtained after the addition of the 5HT4 receptor antagonist. Dotted lines are set to 100% and represent control. (Reproduced with permission from Kullmann FA, Kurihara R, Ye L, Wells GI, McKenna DG, Burgard EC, Thor KB. Auton Neurosci. 2013 Jun;176(1-2):70-7.) Please click here to view a larger version of this figure.
Figure 7. Sites of action of drugs and role of different components of bladder. A) Schematic of protocol for identifying the site of action of a drug. Strips are stimulated with ESF and carbachol (CCh). In i drug X reduces the EFS response but not the CCh response, indicating a pre-junctional site of action. In ii, drug X alters both responses, indicating an action on post-junctional or both pre- and post-junctional receptors. B) Influence of mucosa on smooth muscle contraction. Effects of carbachol are diminished in strips with the mucosa present (intact) compared to strips with the mucosa removed (denuded). (B is reproduced with permission from Hawthorn MH, Chapple CR, Cock M, Chess-Williams R. Br J Pharmacol. 2000 Feb;129(3):416-9). Please click here to view a larger version of this figure.
In this paper we described a simple in vitro smooth muscle contractility method that can be used to address a number of important scientific questions related to bladder physiology and pathology, as well as aiding the discovery of new drugs to treat bladder dysfunctions. We have illustrated the use of this method for assessing developmental, pathological and pharmacological properties of bladder smooth muscle contractility (Figures 2-4), neurotransmission modulation (Figures 5-7A), species differences (Figure 4), organ differences (Figure 6) and relevance of specific bladder components (e.g., mucosa, Figure 7B). Additional applications not illustrated here include evaluation of intracellular pathways using pharmacological agents3,10,11, structure-activity relationships of various drugs22-24, or evaluation/quantification of transmitter release after neural stimulation25.
While bladder function may ultimately be assessed in vivo, this in vitro method overcomes many situations that are problematic in vivo. These include situations when surgical and pharmacological manipulations would reduce the viability and/or survival of the organ or the animal, the use of human tissue, the need to identify and characterize responses from specific components (e.g., smooth muscle vs. epithelium vs. nerves) or the use of expensive chemicals. The method allows systematic investigation of the effects of various pharmacological agents as well as pathology on contractile activity of the smooth muscle and in a well-controlled fashion and environment.
The method provides a plethora of information; however, care should be taken when interpreting and extrapolating this information. This is an in vitro method of a reduced preparation, disconnected from its normal environment and neural control. The experimental conditions are not physiologic, thus the data may not entirely reflect in vivo physiological situations. For example, the method cannot account for changes in blood flow, hormones, humoral substances, external mechanical forces, or extrinsic neural control. Tissue is acutely decentralized, thus injury and ischemia related responses need to be evaluated and taken into account. Pathological changes occurring in the brain or spinal cord cannot be tested using this method unless they have already altered afferent, smooth muscle, mucosa or intramural nerve function (i.e. cellular plasticity). Electric field stimulation (EFS) allows the evaluation of neurally mediated responses. However, it excites indiscriminately all nerves in the strip (e.g., sympathetic, parasympathetic, afferents), as opposed to in vivo situation where the micturition reflex activates only particular pathways. One way to overcome this situation is to combine EFS with specific antagonists that selectively block different pathways. For example, guanethidine could be used to block norepinephrine release when studying contraction properties, or atropine could be used to block muscarinic receptors to prevent bladder contractions when studying relaxation properties. Finally, viability of the tissue is limited to a certain number of hours. In general, most components of bladder tissue are viable and stable (i.e. responding to EFS without deteriorating responses) over a period of 6-8h or longer. However, other tissues may be more sensitive (e.g., ileum lasts ~6 hr or less; author’s personal experience).
Although the method is technically feasible and with good reproducibility, there are several critical steps necessary to ensure its success. First, tissue preparation should be performed carefully to ensure viability by making necessary changes to the dissection procedure (avoid stretching the tissue while preparing the strips) and/or media if needed for different tissue types or species. Another critical step is setting-up neuronal stimulation parameters, such that ceiling effects are avoided. As described in the method section, this depends on the type of the experiment performed and expected mechanism of action of the test compound. For example, for testing effects of cisapride, a 5HT4 receptor agonist, on bladder strips (Figure 6), we set the amplitude of EFS-evoked contraction to ~50% of the maximal. This was based on the known mechanism of action of 5HT4 receptor agonists, namely enhancing ACh release from the pre-junctional parasympathetic nerves27, which in turn is expected to increase EFS-evoked contractions. Stimulation of muscle vs. nerves should be tested using TTX, which inhibits neural transmission and thus should inhibit EFS-evoked contractions. Adequate controls for vehicle and time must be performed during the drug testing to account for deterioration of the tissue with time and for any possible effects of the vehicle. For example, many drugs are dissolved in DMSO or ethanol. Our data (Figure 6) show that DMSO (0.1% and higher) can increase neurally-evoked contractions, an effect which needs to be subtracted from the effect of the test drug. Similarly, ethanol (up to 1%) reduces the spontaneous smooth muscle contractions but has no effect on neurally-evoked contractions34,35. If using genetically engineered animals or surgical models (e.g., spinal cord injury or ovariectomy), controls should include tissue from the appropriate background mouse strain or sham operated animals, respectively. In addition, some tissues, such as human, mouse and guinea pig bladders contain intramural ganglia. When working with these tissues, protocol selection and data interpretation must take into account effects of drugs or EFS on intramural neurons that further stimulate the smooth muscle.
Designing the experimental protocol, choosing the right parameters (for EFS, for drug stimulation) and concentrations to be tested are critical to ensure meaningful data. While parameters should be adjusted for individual tissues and drugs, general principles/guidelines outlined below are applicable. Cumulative concentration response curves are desirable, however this is not possible for all compounds. Drugs targeting receptors that desensitize, such as the purineric ionotropic receptors (P2X), or drugs that are metabolized quickly in the tissue (example ACh), cannot be reliably tested using cumulative concentration response curves in the same tissue. In these cases, single concentrations are tested in different groups of tissue. To evaluate desensitization, it is recommended to compare the magnitude of response elicited by a single highest concentration to that achieved at the end of a cumulative concentration response curve.
For accurate fitting of the data obtained from concentration response curves, it is desirable to test half log concentrations (example CCh in Figure 6). However, log concentrations (example NMB in Figure 4A) are acceptable when tissue viability may be limited or other constraints may be in place.
To select a concentration range for a novel compound, in preliminary experiments, it is useful to consider the binding affinity of the compound and test two power of 10 above and below that concentration. In subsequent experiments, the protocol is refined to determine a starting point where no effect of the drug is observed and an end point where either the response is maximal or the concentration tested is no longer specific for the intended target.
The time interval for applying a drug should be chosen taking into consideration several factors: a) Time for a drug to have an effect. In general drugs targeting membrane receptors have a relatively fast response (seconds to minutes), whereas drugs for intracellular targets (e.g., forskolin and other enzyme inhibitors36, botulinum toxin37) require additional incubation time (30 min – 3 hr). Additionally, tissue thickness may play a role. b) Duration and mechanism of action of drug. For cases when the drug effect reaches a plateau that is sustained, such as NMB in Figure 4A and cisapride in Figure 6, time intervals of 5-15 min between drug applications are adequate for collecting sufficient data. This is not possible with drugs having a much shorter duration of action or different mechanism of action (ATP, CCh). For example the effect of CCh in Figure 4C or 4D, reaches a plateau rapidly but the tissue tension tends to return to baseline. In this case, the time intervals need to be adjusted accordingly, usually adding the next concentration when the first response reaches a maximum.
Data analysis, particularly normalization of data to allow comparisons between strips is a very important step. Different studies use different parameters for normalization, including strip weight38, strip cross sectional area39, KCl response12, % of maximal response28 or % of the maximal response to another contractile agent (e.g., CCh38). The normalization parameter should be chosen depending on the purpose of the experiment, such that the parameter is not influenced by the test compounds, pathology or experimental design. For example, normalization to KCl response eliminates the weight and other dimensions of the strips, and thus could be used to compare responses in tissues where pathological condition may increase the weight of the strips (e.g., diabetes increases bladder mass). In addition, the response to KCl is not influenced by the removal of the mucosa/urothelium29, thus could be used in experiments evaluating different components of the bladder (e.g., mucosa vs. smooth muscles).
In summary, this contractility method provides a fast, easy and very powerful approach to assess bladder (and other organ) physiology and pharmacology. When used properly, it provides the ability to manipulate tissue in a reduced and well controlled environment. In the study of the urinary bladder function, this method was instrumental in the discovery and testing of compounds currently used for OAB management, such as the antimuscarinics and newly developed β3AR agonists.
The authors have nothing to disclose.
This study was supported by NIH R37 DK54824 and R01 DK57284 grants to LB.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Equipment | |||
Tissue Bath System with Reservoir | Radnoti, LLC | 159920 | isolated tissue baths |
Warm water recirculator pump | Kent Scientific Corporation | TPZ-749 | to keep tissue baths to 37 C |
Computer | |||
Data Acquisiton System | DataQ Instruments | DI-710-UH | To view, record and analyze data |
Transbridge Transducer Amplifier | World Precision Instruments | SYS-TBM4M | Transducer amplifier |
Grass stimulator | Grass Technologies | Model S88 | Stimulator |
Anesthesia System | Kent Scientific Corporation | ACV-1205S | To anesthetesize the animal |
Anesthetizing Box | Harvard Apparatus | 500116 | To anesthetesize the animal |
Anesthesia Masks | Kent Scientific Corporation | AC-09508 | To anesthetesize the animal |
Materials and surgical instruments | |||
sylgard | Dow Corning Corp | 184 SIL ELAST KIT | To pin, dissect & cut tissue |
Petri Dish | Corning | 3160-152 | To dissect/cut tissue |
Insect Pins | ENTOMORAVIA Austerlitz Insect Pins | Size 5 | To pin tissue |
Bench Pad | VWR International | 56617-014 | Absorbent bench underpads |
Rat surgical Kit | Kent Scientific Corporation | INSRATKIT | To remove and dissect tissue |
2 Dumont #3 Forceps | Kent Scientific Corporation | INS500064 | To remove and dissect tissue |
Tissue Forceps | Kent Scientific Corporation | INS500092 | To remove and dissect tissue |
Scalpel | Kent Scientific Corporation | INS500236 | To remove and dissect tissue |
Scalpel blade | Kent Scientific Corporation | INS500239 | To remove and dissect tissue |
Professional Clipper | Braintree Scientific, Inc. | CLP-223 45 | To remove fur |
Suture Thread | Fine Science Tools | 18020-50 | Tie tissue |
Tissue Clips | Radnoti, LLC | 158802 | Attach tissue to rod/transducer |
1g weight | Mettler Toledo | 11119525 | For transducer calibration |
Chemicals | |||
Krebs Solution: Sodium Chloride Potassium Chloride Monobasic Potassium Phosphate Magnesium Sulfate Dextrose Sodium Bicarbonate Calcium Chloride Magnesium Chloride |
Sigma Fisher Fisher Fisher Fisher Sigma EMD Baker |
S7653 P217-500 P285-3 M65-500 D16-500 S5761 CX0130-2 2444 |
To prepare Krebs solution |
Isoflurane | Henry Schein | 029405 | To anesthetesize the animal |
Oxygen tank | Matheson Tri Gas | ox251 | To use with anesthesia system |
Carbogen Tank (95% Oxygen; 5% Carbon Dioxide) | Matheson Tri Gas | Moxn00hn36D | To aerate Krebs solutions |