This work was supported by grants from the Belgian Federal Government (IUAP P6/28), the Research Foundation-Flanders (F.W.O.) (G.0565.07 and G.0686.09), the Astellas European Foundation Award 2009 and the Research Council of the KU Leuven (GOA 2009/07, EF/95/010 and PFV/10/006). P.U. and W.E. are doctoral fellows of the Research Foundation-Flanders (FWO). M.B. is a Marie Curie fellow. D.D.R. a fundamental-clinical fellow of the FWO.

1. Laboratory Animals
<ol>
 <li>Animals (mice, rats) are housed in a specialized animal facility with a 12-hr light-dark cycle and ad libitum access to water and standard food pellets. Both age and sex of the animals are important parameters that should be standardized according to the needs. We typically perform cystometry in 10 - 12 week old female animals5,6.</li>
 <li>All animal experiments are carried out in accordance with the European Union Community Council guidelines and approved by the local ethics committee.</li>
</ol>
2. Anesthesia
<ol>
 <li>Isoflurane anesthesia is used for performing small surgical procedures, since it is easy to dose due to its short half-life. Appropriate waste gas scavenging is necessary when using isoflurane.
 <ol class="lalpha">
 <li>Animals are placed in a closed box, gassed with isoflurane 5% in pure oxygen (1 l/min).</li>
 <li>After induction, anesthesia at the appropriate level is maintained by a small mask (a cut 10 ml syringe) by 0.8 - 1 l/min oxygen with 1 - 1.5% of isoflurane.</li>
 </ol>
 </li>
 <li>For mice, urethane anesthesia is used during functional recordings (cfr. Infra). Urethane anesthesia is used during functional recordings because, in contrast to most other anesthetics, the micturition reflex is not suppressed by this anesthetic agent4. Urethane 1.2 g/kg body weight is administered subcutaneously (s.c.) 60 min before the start of the recordings. If necessary, the dose of urethane needed to obtain proper anesthesia can be titrated by administering additional doses (0.1 g/kg) subcutaneously. It should be noted, however, that additional anesthesia may alter the voiding efficiency and consequently the micturition frequency7,8. Thus, the cystometric recordings should be closely monitored and discarded whenever significant deviations from the control behavior are observed. Anesthetics should not be administered while test compounds are applied to avoid misinterpretation of the data.</li>
</ol>
3. Surgical Procedure - Bladder Catheter Implantation
<ol>
 <li>Surgical instruments are sterilized in an autoclave prior to use. Shave the abdomen of the animal, disinfect with ethanol 70% and perform a low midline laparotomy to expose the bladder.</li>
 <li>Under a surgical microscope, place a purse-string suture in the bladder dome using a non-absorbable, monofilament suture (size 6-0).</li>
 <li>Perform a small cystostomy (we preferentially use a 18 G needle) inside the purse string and insert a PE 50 polyethylene catheter, with a small cuff at the end (obtained by carefully heating the tubing) through this hole. In our hands, thinner tubes (PE 10) have a higher and more variable resistance. The tubing can be disinfected by putting it in an ethanol 70% solution or sterilized in an autoclave. Before implantation, the tubing should be flushed with saline.</li>
 <li>Secure the purse string around the tube with a surgeon's knot. Pull the catheter gently outwards until the bell-shaped tip is positioned right under the suture. Push a piece of 18 G catheter towards the suture to prevent leaking.</li>
 <li>Infuse a small volume of saline (100 - 200 &mu;l) gently to check for leaks. If leaks are present, an additional suture should be placed.</li>
 <li>Close the abdominal muscles using monofilament sutures, leaving a passage for the catheter in the upper part of the incision.</li>
 <li>Tunnel the tube to the interscapular region using a hollow metal rod, thereby preventing the animals to bite the tube during the experiment.</li>
 <li>The skin is closed using non-absorbable, monofilament sutures. The implanted catheter is flushed with saline to check its permeability.</li>
 <li>Before the animals awaken, analgesics are given subcutaneously (buprenorphine, 0.05 mg/kg).</li>
</ol>
4. Setup and Cystometry
<ol>
 <li>Depending on the experimental question (and the species used) cystometry is performed in awake (restraint or unrestraint) or urethane-anesthetized animals. Whereas cystometry is perfectly feasible in conscious rats, which tend to remain calm in a restrained environment, we usually perform cystometry under urethane anesthesia (1.2 mg/kg, subcutaneously) when using mice3,5,6,9.</li>
 <li>In anesthetized mice, body temperature is constantly monitored and kept at 36.5 &deg;C &plusmn; 0.2 &deg;C by means of a temperature probe and a heating lamp.</li>
 <li>The set-up is calibrated before every experiment according to the manufacturers' instructions.</li>
 <li>The implanted catheter is connected by a luer stub to a 3-way tap, which is connected to the pressure transducer (Edwards Lifesciences) on one side and an infusion pump on the other (Harvard Apparatus). The pressure transducer is connected via an amplifier (78534c monitor, Hewlett Packard) to the data acquisition system (DI-730-USB, Dataq instruments) and a computer. Windaq/Lite software can be used for recording.</li>
 <li>The infusion pump is started, enabling the infusion of saline or PBS at, for example, 100 &mu;l/min (in rats) or 20 &mu;l/min (in mice). As such the bladder will be filled at a constant rate, while the intravesical pressure is recorded.</li>
 <li>Figure 5 depicts a typical pressure trace: during fluid infusion, there is a slow pressure buildup in the bladder, until a certain volume/pressure is reached, the micturition threshold. Once this threshold is reached, the bladder will contract and subsequently the urinary sphincter will open, allowing the passage of urine through the urethra. As such, the bladder is emptied, the contraction will stop and the pressure will drop again to the "basal" level.</li>
 <li>As the rat is placed in a metabolic cage and above a digital balance, the voided volume can be measured simultaneously, giving information about the voided volume. Due to the small voided volumes in mice, it can be very challenging using this system. Therefore, voided volume is determined using small filter papers weighed before and after voiding.</li>
 <li>Typically, we let the system equilibrate for 30 min, followed by a recording period of 30 min. Then, drugs can be administered systemically or intravesically and another 30 min are recorded.</li>
 <li>Recorded data can be exported as .csv files that can be imported into a specialized software for quantitative analysis. We typically use Origin (OriginLab Corporation, MA, USA).</li>
 <li>After experiments, animals are sacrificed by cervical dislocation (mice) or CO2 intoxication (rats).</li>
</ol>
5. Representative Results
<img alt="Figure 1" src="/files/ftp_upload/3869/3869fig1.jpg" /> 
Figure 1. Laparotomy overview. A) Place the rat in the supine position. B) Shave and antiseptically prepare the surgical site. C) Incision of the skin. D) Incision of the abdominal muscles, and bladder exposure.
<img alt="Figure 2" src="/files/ftp_upload/3869/3869fig2.jpg" /> 
Figure 2. A purse-string suture.
<img alt="Figure 3" src="/files/ftp_upload/3869/3869fig3.jpg" /> 
Figure 3. Catheter implantation.
<img alt="Figure 4" src="/files/ftp_upload/3869/3869fig4.jpg" /> 
Figure 4. Tunnelling of the catheter.
Examples of pressure measurements obtained during intravesical perfusion of saline in a conscious rat and an anesthetized mouse are shown in Figure 5. Multiple parameters can be extracted from the pressure signal (e.g. the intercontractile interval, the baseline pressure and the threshold pressure). For comprehensive descriptions of these parameters, please see Andersson et al. (ref 4) and Yoshiyama et al. (ref 10).
We have recently used cystometry to identify the molecular targets of mustard oil (MO), a highly reactive compound that has been long used in experimental models of inflammation and hyperalgesia of visceral organs such as the urinary bladder11,12. Intravesical infusion of 10 mM MO induced a strong increase in the voiding frequency (decrease in the intercontractile interval) in wild type mice (Figure 6A, B) and a decrease of the voided volume6. Interestingly, MO induced similar changes in mice deficient of the MO receptor TRPA1. In contrast, MO induced much weaker changes in cystometric parameters in Trpv1 KO mice than in WT mice and was without any effect in Trpa1/Trpv1 KO mice. Together with measurements of the release of the Calcitonin Gene Related Peptide (CGRP)6, these data indicate demonstrate that TRPV1 may play a key role in visceral irritation induced by MO.
<img alt="Figure 5" src="/files/ftp_upload/3869/3869fig5.jpg" /> 
Figure 5. Representative traces of intravesical pressure recorded in a conscious female rat (A) and in an anesthetized female mouse (B). The lowest pressure is defined as the "baseline pressure" (red arrows). The pressure at the end of the filling phase is marked with blue arrows. The volume of fluid infused between these points, divided by the pressure difference, allows calculation of the compliance of the bladder wall (compliance = infused volume/(threshold pressure - baseline pressure). The "intercontractile interval" (ICI) is the time between two voiding contractions.
 
<img alt="Figure 6" src="/files/ftp_upload/3869/3869fig6.jpg" /> 
Figure 6. Effects of intravesical application of mustard oil on the cystometry pattern in wild type and Trpa1, Trpv1 and Trpa1/Trpv1 knockout mice. (A) Representative examples of intravesical pressure changes recorded in WT, Trpa1 KO, Trpv1 KO and Trpa1/Trpv1 KO mice in response to infusion of saline and 10 mM MO. (B) Time course of the average instantaneous voiding frequency before and during intravesical infusion of MO. For all mice, the data were normalized to the average frequency obtained during saline infusion. These data is adapted from Everaerts et al. (ref 6), with permission from Elsevier.

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Urodyn. 29, 603-608 (2010).</li><li>Gevaert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+transient+receptor+potential+cation+channel+TRPV4+impairs+murine+bladder+voiding.">Deletion of the transient receptor potential cation channel TRPV4 impairs murine bladder voiding.</a> J. Clin. Invest. 117, 3453-3462 (2007).</li><li>Andersson, K. E., Soler, R., Fullhase, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+urodynamic+investigation.">Rodent models for urodynamic investigation.</a> Neurourol. Urodyn. 30, 636-646 (2011).</li><li>Everaerts, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+cation+channel+TRPV4+improves+bladder+function+in+mice+and+rats+with+cyclophosphamide-induced+cystitis.">Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 19084-19089 (2010).</li><li>Everaerts, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+capsaicin+receptor+TRPV1+is+a+crucial+mediator+of+the+noxious+effects+of+mustard+oil.">The capsaicin receptor TRPV1 is a crucial mediator of the noxious effects of mustard oil.</a> Curr. Biol. 21, 316-321 (2011).</li><li>Yoshiyama, M., Roppolo, J. R., Thor, K. B., de Groat, W. 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Ther. 265, 844-850 (1993).</li><li>Boudes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Characterization+of+a+Chronic+Cyclophosphamide-Induced+Overactive+Bladder+Model+in+mice.">Functional Characterization of a Chronic Cyclophosphamide-Induced Overactive Bladder Model in mice.</a> Neurourol. Urodyn. , (2011).</li><li>Yoshiyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-related+differences+in+activity+of+lower+urinary+tract+in+response+to+intravesical+acid+irritation+in+decerebrate+unanesthetized+mice.">Sex-related differences in activity of lower urinary tract in response to intravesical acid irritation in decerebrate unanesthetized mice.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 295, R954-R960 (2008).</li><li>McMahon, S. B., Abel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+the+study+of+visceral+pain+states:+chronic+inflammation+of+the+chronic+decerebrate+rat+urinary+bladder+by+irritant+chemicals.">A model for the study of visceral pain states: chronic inflammation of the chronic decerebrate rat urinary bladder by irritant chemicals.</a> Pain. 28, 109-127 (1987).</li><li>Du, S., Araki, I., Yoshiyama, M., Nomura, T., Takeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+receptor+potential+channel+A1+involved+in+sensory+transduction+of+rat+urinary+bladder+through+C-fiber+pathway.">Transient receptor potential channel A1 involved in sensory transduction of rat urinary bladder through C-fiber pathway.</a> Urology. 70, 826-831 (2007).</li><li>Streng, T., Santti, R., Talo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+and+differences+in+female+and+male+rat+voiding.">Similarities and differences in female and male rat voiding.</a> Neurourol. 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Urol. 172, 1166-1170 (2004).</li><li>Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+lower+urinary+tract+in+mice+lacking+alpha1d-adrenoceptor.">Function of the lower urinary tract in mice lacking alpha1d-adrenoceptor.</a> J. Urol. 174, 370-374 (2005).</li><li>May, V., Vizzard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+dysfunction+and+altered+somatic+sensitivity+in+PACAP-/-+mice.">Bladder dysfunction and altered somatic sensitivity in PACAP-/- mice.</a> J. Urol. 183, 772-779 (2010).</li><li>Thorneloe, K. S., Meredith, A. L., Knorn, A. M., Aldrich, R. W., Nelson, M. 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No conflicts of interest declared.

The cystometry technique presented here allows performing in vivo measurements of bladder function in animal models. Rats are probably the most used animal model. Mice are more difficult to handle, but offer the advantage of using genetically manipulated animals. Because of the technical difficulty of using conscious mice, which tend to be very active resulting in loosening of the implanted catheter and changes in the intra-abdominal pressures that may influence the intravesical pressure, we advise to keep them anesthetized with urethane during the full cystometry protocol. Of course, the benefits of having sedated mice, should be weighed against the effects of the anesthetics. As such, urethane can have a significant influence on post-voiding residual volume7,8. We therefore wait until we have a stable degree of anesthesia, before we start our recordings and interventions.
	Researchers should consider the differences between male and female rodent micturition13. Also, the age of the animals is of great importance. We perform cystometry in animals that are 10 - 12 weeks old. All the experiments should be performed in a calm environment, especially when using conscious animals. A number of infusion rates have been described for our animal models. We typically use infusion rates of 20 &mu;l/min for mice and 100 &mu;l/min for rats.
	As mentioned above, micturition in humans and rodents differ significantly. For example, ATP is a major contributor to the detrusor contraction in mice and rats, whereas in humans, the bladder contraction, under physiological conditions is mainly mediated by acetylcholine4. However, the possibilities to perform invasive measurements that are generally prohibitive in the clinical context and the use of genetically modified animals, allow exploring the current boundaries of bladder pathophysiology and pharmacology. In this respect, significant advances have been recently obtained in elucidating the role of muscarinic14, prostaglandin15 and adrenergic receptors16, neuropeptides17, Ca2+-activated K+ channels18 and TRP channels3,5,6.

<table border="1">
	<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Commercial name, company,</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>urethane</td>
 <td>Urethane, Sigma-Aldrich</td>
 <td>315419</td>
 <td>group 2B carcinogen</td>
 </tr>
 <tr>
 <td>isoflurane</td>
 <td>Isoba, Schering-Plough Animal Health</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>polyethylene catheter</td>
 <td>Intramedic Polyethylene tubing PE50, Becton Dickinson </td>
 <td>427411</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>surgical microscope</td>
 <td>Op-Mi 6, Carl Zeiss</td>
 <td>Op-Mi 6</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>purse-string suture</td>
 <td>Prolene 6/0, Ethicon</td>
 <td>8610H</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>fascia and skin suture</td>
 <td>Ethilon 4/0 or 5/0, Ethicon</td>
 <td>662G or 661G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>postoperative analgesics</td>
 <td>Temgesic, Schering-Plough Animal Health</td>
 <td>&nbsp;</td>
 <td>dosage for rats: 0.05 mg/kg</td>
 </tr>
 <tr>
 <td>amplifier</td>
 <td>78534c monitor, Hewlett Packard</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>analytical balances and balance data acquisition software</td>
 <td>FZ 300i, A&amp;D</td>
 <td>FZ-300i</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>infusion pumps</td>
 <td>pump 33, Harvard apparatus</td>
 <td>HA33</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>cystometry recording system</td>
 <td>Dataq instruments, DI-730 series and Windaq/Lite</td>
 <td>DI-730-USB Windaq/Lite</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>temperature registration</td>
 <td>Fluke 52 KJ thermometer</td>
 <td>52 KJ</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pressure transducers</td>
 <td>Edwards Lifesciences, pressure monitoring set</td>
 <td>T322247A</td>
 <td>&nbsp;</td>
 </tr>
	 </tbody>
 </table>

the use of cystometry in small rodents: a study of bladder chemosensation

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.

Watch this Scientific Journal Video about The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation at JoVE.com

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

	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 ...

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22.3K Views.  KU Leuven, Belgium. Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

Video: The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

Mouse Bladder Wall Injection

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.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

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.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children. Recent investigations in animals have raised concerns that anesthesia exposure may lead to neuronal apoptosis, known as anesthesia-induced developmental neurotoxicity (AIDN). Furthermore, some clinical studies in children have suggested that anesthesia exposure may lead to neurodevelopmental deficits later in life. Nonetheless, an ideal animal model for preclinical study has yet to be developed. The neonatal piglet represents a valuable model for preclinical study, as they share a striking number of developmental similarities with humans.
The anatomy and physiology of piglets allow for implementation of rigorous human perioperative conditions in both survival and non-survival procedures. Femoral artery catheterization allows for close monitoring, thus enabling prompt correction of any deviation of the piglet's vital signs and chemistries. In addition, there are multiple developmental similarities between piglets and human neonates. The techniques required to use piglets for experimentation will require experience to master. A pediatric anesthesiologist is a critical member of the investigative team. We describe, in a general sense, the appropriate use of a piglet model for neurodevelopmental study.

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity.

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children.  Recent investigations in animals have raised concerns that ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...