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Optimization of a pain model: effects of body temperature and anesthesia on bladder nociception in mice.
PUBLISHED: 01-01-2013
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a debilitating urological condition that is resistant to treatment and poorly understood. To determine novel molecular treatment targets and to elucidate the contribution of the nervous system to IC/BPS, many rodent bladder pain models have been developed. In this study we evaluated the effects of anesthesia induction and temperature variation in a mouse model of bladder pain known as urinary bladder distension (UBD). In this model compressed air is used to distend the bladder to distinct pressures while electrodes record the reflexive visceromotor response (VMR) from the overlying abdominal muscle. Two isoflurane induction models are commonly used before UBD: a short method lasting approximately 30 minutes and a long method lasting approximately 90 minutes. Animals were anesthetized with one of the methods then put through three sets of graded bladder distensions. Distensions performed following the short anesthesia protocol were significantly different from one another despite identical testing parameters; this same effect was not observed when the long anesthesia protocol was used. In order to determine the effect of temperature on VMRs, animals were put through three graded distension sets at 37.5 (normal mouse body temperature), 35.5, and 33.5°C. Distensions performed at 33.5 and 35.5°C were significantly lower than those performed at 37.5°C. Additionally, Western blot analysis revealed significantly smaller increases in spinal levels of phosphorylated extracellular-signal regulated kinase 2 (pERK2) following bladder distension in animals whose body temperature was maintained at 33.5°C as opposed to 37.5°C. These results highlight the significance of the dynamic effects of anesthesia on pain-like changes and the importance of close monitoring of temperature while performing UBD. For successful interpretation of VMRs and translation to human disease, body temperature should be maintained at 37.5°C and isoflurane induction should gradually decrease over the course of 90 minutes.
Authors: Katelyn E. Sadler, Jarred M. Stratton, Benedict J. Kolber.
Published: 04-27-2014
Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.
19 Related JoVE Articles!
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Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models
Authors: Duong D. Tu, Abhishek Seth, Eun Seok Gil, David L. Kaplan, Joshua R. Mauney, Carlos R. Estrada Jr..
Institutions: Harvard Medical School, Tufts University.
Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5. Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed. In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.
Bioengineering, Issue 66, Medicine, Biomedical Engineering, Physiology, Silk, bladder tissue engineering, biomaterial, scaffold, matrix, augmentation, cystometry
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The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation
Authors: Pieter Uvin, Wouter Everaerts, Silvia Pinto, Yeranddy A. Alpízar, Mathieu Boudes, Thomas Gevaert, Thomas Voets, Bernd Nilius, Karel Talavera, Dirk De Ridder.
Institutions: KU Leuven, Belgium, KU Leuven, Belgium, KU Leuven, Belgium.
The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion. After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3. Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.
Medicine, Issue 66, Physiology, Chemistry, cystometry, urodynamics, bladder function, bladder chemosensation, animal model, urinary tract
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An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment
Authors: Moo Rim Kang, Glen Yang, Klaus Charisse, Hila Epstein-Barash, Muthiah Manoharan, Long-Cheng Li.
Institutions: University of California, San Francisco , Alnylam Pharmaceuticals, Inc..
We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.
Cancer Biology, Issue 65, Medicine, Physiology, bladder tumor, orthotopic, bioluminescent, ultrasound, small RNA
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Murine Renal Transplantation Procedure
Authors: Jiao-Jing Wang, Sara Hockenheimer, Alice A. Bickerstaff, Gregg A. Hadley.
Institutions: The Ohio State University, The Ohio State University.
Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with > 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses. Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.
immunology, Issue 29, mouse, kidney, renal, transplantation, procedure
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A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts
Authors: Shaoping Hou, Armin Blesch, Paul Lu.
Institutions: Drexel University College of Medicine, Heidelberg University Hospital, Veterans Administration Medical Center, San Diego, CA, University of California, San Diego.
High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.
Medicine, Issue 92, spinal cord injury, telemetric recording, blood pressure, heart rate, autonomic dysreflexia, embryonic neural stem cell
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Transurethral Induction of Mouse Urinary Tract Infection
Authors: Kim H. Thai, Anuradha Thathireddy, Michael H. Hsieh.
Institutions: Stanford University , Stanford University School of Medicine.
Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37°C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.
Microbiology, Issue 42, UTI, urinary tract infection, urethra, mice, bacterial, cystitis, pyelonephritis, mouse, bacteria, urethral
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Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents
Authors: Phillip R. Kramer, Larry L. Bellinger.
Institutions: Texas A&M University Baylor College of Dentistry.
A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of humans with orofacial pain. To measure meal duration unrestrained rats are continuously kept in sound attenuated, computerized feeding modules for days to weeks to record feeding behavior. These sound-attenuated chambers are equipped with chow pellet dispensers. The dispenser has a pellet trough with a photobeam placed at the bottom of the trough and when a rodent removes a pellet from the feeder trough this beam is no longer blocked, signaling the computer to drop another pellet. The computer records the date and time when the pellets were taken from the trough and from this data the experimenter can calculate the meal parameters. When calculating meal parameters a meal was defined based on previous work and was set at 10 min (in other words when the animal does not eat for 10 min that would be the end of the animal's meal) also the minimum meal size was set at 3 pellets. The meal duration, meal number, food intake, meal size and inter-meal interval can then be calculated by the software for any time period that the operator desires. Of the feeding parameters that can be calculated meal duration has been shown to be a continuous noninvasive biological marker of orofacial nociception in male rats and mice and female rats. Meal duration measurements are quantitative, require no training or animal manipulation, require cortical participation, and do not compete with other experimentally induced behaviors. These factors distinguish this assay from other operant or reflex methods for recording orofacial nociception.
Behavior, Issue 83, Pain, rat, nociception, myofacial, orofacial, tooth, temporomandibular joint (TMJ)
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Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior
Authors: Ethan M. Anderson, Richard Mills, Todd A. Nolan, Alan C. Jenkins, Golam Mustafa, Chris Lloyd, Robert M. Caudle, John K. Neubert.
Institutions: University of Florida College of Dentistry, University of Florida College of Medicine , Stoelting Co., University of Florida .
We present an operant system for the detection of pain in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) assesses pain behaviors in a more clinically relevant way by not relying on reflex-based measures of nociception. Food fasted, hairless (or shaved) rodents are placed into a Plexiglas chamber which has two Peltier-based thermodes that can be programmed to any temperature between 7 °C and 60 °C. The rodent is trained to make contact with these in order to access a reward bottle. During a session, a number of behavioral pain outcomes are automatically recorded and saved. These measures include the number of reward bottle activations (licks) and facial contact stimuli (face contacts), but custom measures like the lick/face ratio (total number of licks per session/total number of contacts) can also be created. The stimulus temperature can be set to a single temperature or multiple temperatures within a session. The OPAD is a high-throughput, easy to use operant assay which will lead to better translation of pain research in the future as it includes cortical input instead of relying on spinal reflex-based nociceptive assays.
Behavior, Issue 76, Neuroscience, Neurobiology, Anatomy, Physiology, Medicine, Biomedical Engineering, Surgery, Neurologic Manifestations, Pain, Chronic Pain, Nociceptive Pain, Acute Pain, Pain Perception, Operant, mouse, rat, analgesia, nociception, thermal, hyperalgesia, animal model
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Mouse Bladder Wall Injection
Authors: Chi-Ling Fu, Charity A. Apelo, Baldemar Torres, Kim H. Thai, Michael H. Hsieh.
Institutions: Stanford University School of Medicine.
Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.
Medicine, Issue 53, stem cell, bladder cancer, intramural injection, bladder wall injection, bladder
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An Orthotopic Model of Murine Bladder Cancer
Authors: Georgina L. Dobek, W. T. Godbey.
Institutions: Tulane University, Tulane University.
In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.
Medicine, Issue 48, Bladder tumor, orthotopic, mouse, ultrasound
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A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation
Authors: Erik J. Zmuda, Catherine A. Powell, Tsonwin Hai.
Institutions: The Ohio State University, The Ohio State University, The Ohio State University.
Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation. Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.
Medicine, Issue 50, islet isolation, islet transplantation, diabetes, murine, pancreas
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An Orthotopic Murine Model of Human Prostate Cancer Metastasis
Authors: Janet Pavese, Irene M. Ogden, Raymond C. Bergan.
Institutions: Northwestern University, Northwestern University, Northwestern University.
Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.
Medicine, Issue 79, Urogenital System, Male Urogenital Diseases, Surgical Procedures, Operative, Life Sciences (General), Prostate Cancer, Metastasis, Mouse Model, Drug Discovery, Molecular Biology
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Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development
Authors: Daniel P. Vang, Gregory T. Wurz, Stephen M. Griffey, Chiao-Jung Kao, Audrey M. Gutierrez, Gregory K. Hanson, Michael Wolf, Michael W. DeGregorio.
Institutions: University of California, Davis, University of California, Davis, Merck KGaA, Darmstadt, Germany.
A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.
Medicine, Issue 80, Urinary Bladder, Animals, Genetically Modified, Cancer Vaccines, Immunotherapy, Animal Experimentation, Models, Neoplasms Bladder Cancer, C57BL/6 Mouse, MUC1, Immunotherapy, Preclinical Model
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Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia
Authors: Tristan M. Nicholson, Kristen S. Uchtmann, Conrad D. Valdez, Ashleigh B. Theberge, Tihomir Miralem, William A. Ricke.
Institutions: University of Wisconsin-Madison, University of Rochester School of Medicine & Dentistry, University of Wisconsin-Madison.
New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.
Medicine, Issue 78, Cancer Biology, Prostatic Hyperplasia, Prostatic Neoplasms, Neoplastic Processes, Estradiol, Testosterone, Transplantation, Heterologous, Growth, Xenotransplantation, Heterologous Transplantation, Hormones, Prostate, Testosterone, 17beta-Estradiol, Benign prostatic hyperplasia, Prostate Cancer, animal model
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An Orthotopic Bladder Cancer Model for Gene Delivery Studies
Authors: Laura Kasman, Christina Voelkel-Johnson.
Institutions: Medical University of South Carolina.
Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.
Medicine, Issue 82, Bladder cancer, gene delivery, adenovirus, orthotopic model, catheterization
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Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Authors: Khadija Elhabazi, Safia Ayachi, Brigitte Ilien, Frédéric Simonin.
Institutions: Université de Strasbourg.
Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.
Neuroscience, Issue 89, mice, nociception, tail immersion test, tail pressure test, morphine, analgesia, opioid-induced hyperalgesia, tolerance
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Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Authors: F. Aura Kullmann, Stephanie L. Daugherty, William C. de Groat, Lori A. Birder.
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
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.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
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Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats
Authors: Saeed Alshahrani, Filipe Fernandez-Conti, Amanda Araujo, Mauricio DiFulvio.
Institutions: Wright State University, Universidade São Judas Tadeu.
Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by Bölcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 °C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of Bölcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Almási et al 5 and Bölcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.
Neuroscience, Issue 63, Diabetes, painful diabetic neuropathy, nociception, thermal nociceptive threshold, nocifensive behavior
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Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain
Authors: John Jarrell.
Institutions: University of Calgary.
Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2). The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis. The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.
Medicine, Issue 28, Chronic pelvic pain, cutaneous allodynia, trigger points, dysmenorrhea, endometriosis, dyspareunia
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