Tutti i ratti sono stati utilizzati secondo i protocolli approvati dal Comitato sperimentale sugli animali del Beijing Friendship Hospital, Capital Medical University.
1. Preparazione chirurgica, anestetizzazione e tecniche chirurgiche
NOTA: Un totale di 40 femmine di ratto Wistar, del peso di 200-220 g, sono stati ottenuti commercialmente per il presente studio. Dei 40 ratti, 10 sono stati selezionati casualmente come gruppo di controllo e il resto è stato trattato come gruppo di test. Tutti gli animali sono stati ospitati in un ambiente sterile nelle strutture per animali del Beijing Friendship Hospital, Capital Medical University.
<ol><li>Eseguire l'anestesia generale somministrando pentobarbital di sodio per via intraperitoneale (40 mg/kg). In alternativa, indurre l'anestesia usando isoflurano al 3% -4% e mantenerla all'1% -3% (inalato). Applicare unguento oftalmico sugli occhi per prevenire la secchezza. Quindi, posizionare il ratto sulla piattaforma chirurgica e fornire supporto termico. NOTA: Somministrare analgesici come buprenorfina, 0,05 mg/kg, SC, 0,1-0,2 mL all'inizio della procedura.</li>
	<li>Controllare la profondità dell'anestesia dalla mancanza di risposta al pizzico della punta. Rasare la pelliccia da tutta la zona posteriore con un rasoio.</li>
	<li>Sterilizzare il sito chirurgico con almeno 3 cicli di uno scrub a due stadi come clorexidina o iodio povidone seguito da alcool isopropilico. Fissare gli arti con nastro chirurgico e fare un'incisione mediana di circa 3 cm sulla schiena con forbici chirurgiche.</li>
	<li>Approfondisci l'incisione attraverso i tessuti sottocutanei usando le forbici chirurgiche e taglia i muscoli attaccati alla colonna vertebrale.</li>
	<li>Identificare ed esporre visivamente la 13a costola (lo spazio intervertebrale collegato aquella costola è l'intervallo T13\u2012L1). Segna la 13acostola usando una sutura.</li>
	<li>Dopo l'identificazione, resecare attentamente i muscoli attaccati alla colonna vertebrale ed esporre la colonna vertebrale. Resecare il legamento sopraspinoso e il legamento interspinoso per un'accurata identificazione della colonna vertebrale. Esporre il livello di L4\u2012L5 con forbici chirurgiche e pinze. NOTA: Il legamento sopraspinoso può essere identificato facilmente a causa della presenza di tessuto sottocutaneo sottile. Dopo la resezione del legamento sopraspinoso, il legamento tra il processo spinoso è il legamento interspinoso.</li>
	<li>Sezionare attentamente il processo spinoso vertebrale L4\u2012L5 e parti del processo trasversale usando una pinza Kelly per esporre il midollo spinale (Figura 1).</li>
	<li>Esporre completamente il cono midollare al livello L4\u2012L5 e transectare il midollare del cono totalmente con le forbici per iridectomia. Inserire un imballaggio di tessuto per bloccare il recupero del midollo spinale.</li>
	<li>Chiudere il muscolo e la pelle sovrastanti sullo strato esterno della pelle utilizzando una sutura non assorbibile 4-0.</li>
	<li>Per il gruppo di controllo, eseguire i passaggi 1.1\u20121.7 e lasciare intatto il cono midollare. Chiudere l'incisione secondo il punto 1.9.</li>
</ol>2. Recupero degli animali
<ol><li>Tenere i ratti in un'incubatrice a temperatura controllata (37 °C) durante la prima ora dopo l'operazione e monitorarli fino a quando non sono sternali o in movimento attivo. NOTA: Ci vuole circa mezz'ora per il recupero totale.</li>
	<li>Trasferire l'animale in una gabbia pulita con cibo e acqua sufficienti. Tieni i ratti in gabbie separate. NOTA: Il successo della transezione è indicato quando i ratti nel gruppo di prova si muovono solo con l'aiuto delle zampe anteriori, mentre i ratti nel gruppo di controllo potrebbero camminare normalmente.</li>
</ol>3. Gestione post-operatoria
<ol><li>Iniettare penicillina G, un antibiotico (50.000 U/ml per animale) per via intraperitoneale. Somministrare analgesici come buprenorfina, 0,05 mg/kg, SC, 0,1-0,2 ml ogni 6-12 ore per 48 ore dopo l'intervento.</li>
	<li>Comprimere la vescica urinaria nell'ipogastrio per aiutare con lo svuotamento. Eseguilo due volte al giorno alla stessa ora (8 am e 8 pm) per sei settimane. NOTA: La perdita della normale costrizione del detrusore è il simbolo dell'uranio impoverito.</li>
	<li>Ospitare tutti i ratti in gabbie metaboliche, ciascuna contenente un imbuto di raccolta delle urine posto su una carta assorbente precedentemente pesata per monitorare la minzione e l'incontinenza.</li>
	<li>Raccogliere e annotare separatamente la variazione di peso della carta assorbente, che indica separatamente il volume vuoto (VV) e il volume residuo di urina.</li>
</ol>4. Test urodinamici
<ol><li>A sei settimane dopo l'operazione, eseguire un cistometrogramma, utilizzando apparecchiature di misurazione urodinamica come segue.<ol><li>Anestetizzare i ratti iniettando il 10% di cloralio idrato nella cavità peritoneale (3 ml/kg).</li>
		<li>Comprimere la vescica per lo svuotamento, quindi fissare il ratto alla piattaforma chirurgica usando un nastro.</li>
		<li>Inserire il catetere epidurale (3F) nella vescica e collegare l'apparecchiatura di misurazione urodinamica, il catetere epidurale e la pompa per infusione tramite il tubo a tre arti.</li>
		<li>Pompare soluzione fisiologica salina ad una velocità di 0,2 ml/min per la misurazione urodinamica (vedi Tabella dei materiali). Registrare l'MCC e il DOP e la conformità della vescica (calcolata dividendo δ volume della vescica con δ pressione del detrusore).</li>
	</ol></li>
</ol>5. Analisi statistica
<ol><li>Eseguire analisi statistiche utilizzando software disponibili in commercio.</li>
	<li>Utilizzare il test di Kolmogorov-Smirnov per testare la normalità dei dati.</li>
	<li>Esprimere le variabili normalmente distribuite come valori medi con deviazioni standard. Utilizzare i test t di Student accoppiati a due code per confrontare i parametri del cistometrogramma in entrambi i gruppi. NOTA: p < 0,05 indica che la differenza aveva rilevanza statistica.</li>
</ol>

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L'uranio impoverito è una causa comune di sintomi del tratto urinario inferiore sia negli uomini che nelle donne. È una costellazione complessa di sintomi con poche opzioni di trattamento che possono ridurre significativamente la qualità della vita (Qol) delle persone colpite18. Sebbene si ritenga che l'uranio impoverito sia multifattoriale, la comprensione della sua patogenesi rimane rudimentale. Gli studi hanno dimostrato che la patogenesi dell'uranio impoverito potrebbe essere correlata a fa...

L'underactivity del detrusore (DU) è una tipica disfunzione del tratto urinario inferiore che è rimasta sotto studiata. Anche se l'uranio impoverito è stato definito dall'International Continence Society (ICS)1, vengono utilizzate numerose terminologie diverse per riferirsi a questa malattia, ad esempio "insufficienza del detrusore", "vescica acontrattile", "areflessia del detrusore"2. L'uranio impoverito, come definito dall'International Continence Society (ICS) nel 2002, è una contrazione di forza e durata ridotte, che si traduce in un aumento prolungato del tempo per lo svuotamento della vescica, con conseguente incapacità di raggiungere lo svuotamento completo della vescica entro un periodo normale.
L'uranio impoverito può colpire il 48% degli uomini e il 12% delle donne (di età compresa >70 anni)3 con sintomi del tratto urinario inferiore. Sembra essere multifattoriale e non esiste un trattamento efficace. È stato riportato che l'uranio impoverito è onnipresente nei pazienti con disfunzione neurogena della vescica, come la sclerosi multipla4, il diabete mellito5, il morbo di Parkinson6 o l'ictus cerebrale7. L'uranio impoverito può anche essere causato da danni ai nervi iatrogeni, come l'isterectomia laparoscopica, la prostatectomia o altri interventi chirurgici nella piccola pelvi8. I cambiamenti fisiopatologici e i trattamenti disponibili dell'uranio impoverito sono ancora confusi a causa della mancanza di un modello animale appropriato per lo studio.
Il riflesso della minzione è controllato da vie spino-bulbospinali che combinano il centro di minzione pontina, il nucleo parasimpatico sacrale e i centri della corteccia più anziani9. L'attivazione e il mantenimento del riflesso della minzione dipendono principalmente dal trasporto regolare dei segnali sensoriali dalla vescica ai centri della corteccia più anziani. Si può postulare che la disfunzione sensoriale contribuisca all'uranio impoverito.
La maggior parte degli studi sperimentali sugli animali relativi alle disfunzioni del tratto urinario inferiore si sono concentrati su modelli di vescica iperattiva (OAB)10. Questi modelli forniscono una ragionevole comprensione della fisiopatologia e della prognosi dell'OAB. Tuttavia, sono stati riportati solo pochi modelli di uranio impoverito, ad esempio lesioni sopraspinali (lesioni locali, decerebrazione e occlusione dell'arteria cerebrale media), trassezione del midollo spinale o lesione da contusione, somministrazione sistemica (ad es. ciclofosfamide) o intravescicale di agenti irritanti o infiammatori (ad es. acido, acroleina e lipopolisaccaride)11,12,13,14 . Tra questi metodi, solo il metodo di transezione del midollo spinale o lesione da contusione può essere utilizzato per stabilire un modello animale di DU13. I tentativi di lesione del centro di minzione pontino e dei centri della corteccia superiore sono stati abbandonati a causa del grave trauma. Quindi, viene prestata maggiore attenzione per trovare una posizione accurata nel centro del riflesso della minzione per indurre l'uranio impoverito con effetti collaterali minimi.
Come accennato in precedenza, uno dei meccanismi di induzione dell'uranio impoverito è quello di danneggiare il midollo spinale per danneggiare la via di segnalazione del riflesso della minzione. Il metodo di caduta del peso di Allen è stato sviluppato per stabilire animali da laboratorio con midollo spinale ferito15. Tuttavia, non sono disponibili ulteriori dati sperimentali su questo metodo. Inoltre, poiché parti degli animali hanno recuperato la funzione spinale dopo l'ictus senza DU, non può essere considerato un metodo perfetto per generare un modello animale di uranio impoverito16.
Nel 1987, Bregman ha escogitato un processo di transetto del midollo spinale per generare il modello animale all'uranio impoverito e ha acquisito dati sperimentali17. Tuttavia, questo metodo non è stato applicato per stabilire il modello animale all'uranio impoverito. A quel tempo, i ricercatori erano ancora confusi sulla patogenesi dell'uranio impoverito. Poiché le posizioni nel midollo spinale associate all'induzione di OAB o DU sono adiacenti l'una all'altra, non sono state in grado di trovare il sito preciso del danno al midollo spinale per indurre DU17. OAB e DU sono stati introdotti insieme o separatamente con questo metodo. Quindi, sebbene questo metodo abbia introdotto l'uranio impoverito, era impreciso e non poteva essere utilizzato per la comprensione dell'occorrenza e dell'elaborazione dell'uranio impoverito.
Come detto sopra, la mancanza di un modello animale adatto di uranio impoverito è uno dei principali ostacoli per lo studio dell'uranio impoverito. I ricercatori sono continuamente alla ricerca di un modello accurato e gestibile in grado di simulare la patologia dell'uranio impoverito. Anche le opzioni di trattamento per l'uranio impoverito non sono migliorate significativamente negli ultimi 20 anni. Collettivamente, c'è un grande bisogno di descrivere un protocollo standard per stabilire un modello animale di uranio impoverito.
Quindi, in questo articolo, descriviamo un metodo per stabilire con successo un modello di ratto di uranio impoverito mediante transezione del cono midollare. La transezione è stata eseguita a livello L4\u2012L5 per separare il cono midollare. La capacità cistometrica massima (MCC), la pressione di apertura del detrusore (DOP) e la conformità della vescica sono state registrate e analizzate per convalidare il protocollo. Il protocollo indicato di seguito combina fattibilità e affidabilità in modo standardizzato per stabilire il modello animale di uranio impoverito, simulando la presenza e l'elaborazione dell'uranio impoverito. Il protocollo può essere utilizzato come tecnica per ulteriori studi sull'uranio impoverito.

<table><tbody><tr><td>0.9% saline</td><td>Wuhan Prosai Company</td><td>EY-C1178</td><td>pump for urodynamic measurement</td></tr><tr><td>10% chloral hydrate</td><td>Shandong Yulong Co., Ltd</td><td>H37022673</td><td>3mL/kg, administered intraperitoneally</td></tr><tr><td>Buprenorphine Hydrochloride Injection</td><td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co. LTD</td><td>H12020275</td><td>0.05mg/kg subcutaneously 24h and 48h postoperation</td></tr><tr><td>Epidural Catheter</td><td>Shandong Xinghua Co, Ltd</td><td>VABR3L</td><td>for urodynamic measurement</td></tr><tr><td>Penicillin G</td><td>Alta Technology Co., Ltd</td><td>1ST5637</td><td>50,000 unit/ml per animal</td></tr><tr><td>pentobarbital</td><td>Beijing solabo Technology Co., Ltd</td><td>NK-WF0001</td><td>40 mg/kg, administered intraperitoneally</td></tr><tr><td>Suture line(4-0)</td><td>ETHICON</td><td>VCP422H</td><td>suture the injury</td></tr><tr><td>Three-limb tube</td><td>Shandong Xinghua Co, Ltd</td><td>VAB3T</td><td>for urodynamic measurement</td></tr><tr><td>Trace infusion pump</td><td>Zhejiang Smith Medical Instrument Co., Ltd</td><td>20162540335</td><td>Pump the saline at a speed of 0.2ml/min for urodynamic measurement</td></tr><tr><td>Urodynamic measurement equipment</td><td>Medical Measurement SystemsB.V.</td><td>08-0467</td><td>urodynamic measurement equipment can not only help the diagnosis of dysuria, but also provide objective materials for treatment and therapeutic effect. It is the most commonly used examination method in clinical diagnosis and treatment of lower urinary tract functional diseases</td></tr><tr><td>Wistar Rats</td><td>HFK Biotechnology Co.Ltd,Beijing ,China</td><td>SCXK2012-0023</td><td>200-220g</td></tr></tbody></table>

detrusor underactivity model in rats by conus medullaris transection

L'obiettivo del protocollo presentato era quello di stabilire un modello di sottoattività del detrusore (DU) nel ratto attraverso la transezione del cono midollare. La laminectomia è stata eseguita in un totale di 40 ratti Wistar femmina (gruppo di controllo: 10 ratti; gruppo di test: 30 ratti) del peso di 200-220 g, e il cono midollare è stato transettato al livello L4-L5 nel gruppo di test. Tutti i ratti sono stati alloggiati e nutriti nelle stesse condizioni ambientali per sei settimane. Nel gruppo di test, lo svuotamento delle urine è stato eseguito due volte al giorno per sei settimane ed è stato registrato il volume medio residuo di urina. Un cistometrogramma è stato eseguito in entrambi i gruppi. Sono stati registrati e calcolati la capacità cistometrica massima (MCC), la pressione di apertura del detrusore (DOP) e la conformità della vescica. Il gruppo di test ha mostrato una significativa ritenzione urinaria dopo l'intervento chirurgico, sia durante che dopo lo shock spinale. Tuttavia, nessuna anomalia è stata osservata nel gruppo di controllo. Rispetto al gruppo di controllo, l'MCC e la conformità della vescica nel gruppo di prova erano significativamente superiori a quelli del gruppo di prova (3,24 ± 2,261 ml contro 1,04 ± 0,571 ml; 0,43 ± 0,578 ml / cmH 2 O contro 0,032 ± 0,016 ml / cmH 2 O), mentre il DOP nel gruppo di prova era inferiore al controllo (20,28 ± 14,022 cmH 2 O contro 35 ± 13,258 cmH2 O). Questo metodo per stabilire un modello animale di uranio impoverito mediante la transezione del cono midollare offre un'eccellente opportunità per comprendere meglio la fisiopatologia dell'uranio impoverito.

L'intera procedura della transezione del cono midollare può essere completata entro 45 minuti da chirurghi esperti. Il nostro laboratorio ha eseguito oltre 100 casi di interventi chirurgici di transezione del cono midollare. Il tasso di successo è superiore al 95%, come definito dalla sopravvivenza dei ratti e dall'induzione riuscita dell'uranio impoverito. Il test urodinamico ha confermato l'induzione dell'uranio impoverito.
Sulla base della nostra esperienza, l'induzione dell'uranio impove...

Watch this Scientific Journal Video about Detrusor Underactivity Model in Rats by Conus Medullaris Transection at JoVE.com

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Presentiamo un metodo per stabilire un modello di sottoattività del detrusore mediante transezione del conus medullaris nei ratti. La sottoattività del detrusore è stata stimolata con successo in questi animali. Il modello può essere utilizzato per studiare la funzione del tratto urinario.

Modello di sottoattività del detrusore nei ratti mediante transezione di Conus medullaris

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

detrusor-underactivity-model-in-rats-by-conus-medullaris-transection

Research

JoVE Journal

Neuroscience

2.0K Views.  Capital Medical University. Presentiamo un metodo per stabilire un modello di sottoattività del detrusore mediante transezione del conus medullaris nei ratti. La sottoattività del detrusore è stata stimolata con successo in questi animali. Il modello può essere utilizzato per studiare la funzione del tratto urinario.

Video: Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

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.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Valutazione di Biomateriali per l&#39;ampliamento vescicale con analisi cistometrici in modelli di roditori vari

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Ricerca

Bioingegneria

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.

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.

L&#39;uso di cistometria in piccoli roditori: A Study of Chemosensation vescica

	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 ...

Medicina

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.

Vescica urinaria Distensione Evocati visceromotoria Risposte come un modello per il dolore della vescica nei topi

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

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

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.

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,&#160;and pathology.

Vescica muscolo liscio Striscia contrattilità come un metodo per valutare Lower Urinary Tract Farmacologia

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

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.

Nerve-sparing Mid-ostruzione uretrale (Nemo) nel femminile piccoli roditori

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

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Valutazione della procedura per eseguire la cystometria sveglia in un modello di mouse

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in hydrostatic pressure or mechanical stretch and from cultured urothelial cells upon hydrostatic pressure changes, stretch, cell swelling, or drag forces, and in bladder lumen at end of filling. Such findings led to the assumption that these mediators are also released in suburothelium (SubU)/lamina propria (LP) during bladder filling, where they affect cells deep in the bladder wall to ultimately regulate bladder excitability. There are at least two obvious limitations in such studies: 1) none of these approaches provide direct information about the presence of mediators in SubU/LP, and 2) the stimuli used are not physiological and do not recapitulate authentic filling of the bladder. Here, we discuss a procedure that enables direct access to the suburothelial surface of the bladder mucosa in the course of bladder filling. The murine detrusor-free preparation we created closely resembles filling of the intact bladder and allows pressure-volume studies to be performed on the bladder in the absence of confounding signaling from spinal reflexes and detrusor smooth muscle. Using the novel detrusor-free bladder model, we recently demonstrated that intravesical measurements of mediators cannot be used as a proxy to what has been released or present in the SubU/LP during bladder filling. The model enables examination of urothelium-derived signaling molecules that are released, generated by metabolism and/or transported into the SubU/LP during the course of bladder filling to transmit information to neurons and smooth muscle of the bladder and regulate its excitability during continence and micturition.

A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

The detrusor-free bladder model enables direct access to the suburothelium to study local mechanisms for regulation of biologically active mediator availability in suburothelium/lamina propria during storage and voiding of urine. The preparation closely resembles filling of an intact bladder and allows pressure-volume studies to be performed without systemic influences.

Un modello di vescica murine decentralizzato (Ex Vivo) con il muscolo del detrusore rimosso per l'accesso diretto al Suburothelium durante il riempimento della vescica

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

Cistometrici ed esterni misure dello sfintere uretrale in ratti svegli con catetere impiantato ed elettrodi consentendo misurazioni ripetute

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Biologia

Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is necessary, although in some cases, it is inappropriate or needs to be optimized to repair a given stricture. Therefore, developing innovative procedures and evaluating their putative success in experimental models is crucial to fit the clinical need. With this goal, this study describes a protocol in which urethral stricture was induced by electrocautery in Wistar rats. Urethral reconstruction was performed 1 week later with a buccal mucosa graft, harvested from the lower lip, and placed in a ventral onlay fashion. A retrograde urethrogram showed a significant improvement in urethral diameter after urethroplasty compared to the respective value after stricture induction. Additionally, the graft placement was assessed by blood perfusion analysis using laser Doppler. As expected, a dark blue area corresponded to the non-vascularized buccal mucosa graft. This procedure can successfully simulate the normal pathophysiological process of urethral injury and tissue modulation, as well as urethral reconstruction using a buccal mucosa graft in a reproducible manner, and serve as the basis for future research based on tissue engineering or urethral grafts.

In the present protocol, a urethral stricture induction was developed in Wistar rats, followed by urethral reconstruction with a buccal mucosa graft. A retrograde urethrogram and laser Doppler assessment were performed, validating urethral reconstruction (after stricture formation) and graft placement.

Induzione della stenosi uretrale seguita da uretroplastica dell'innesto della mucosa buccale in un modello di ratto

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is ...

Efficient Rat Model for Urethral Stricture: Electric Knife Method

Iatrogenic Injury Recapitulated: Electroexcision Technique for Urethral Stricture Modeling in Rats

Urethral stricture (US) is a common clinical condition in urology, characterized by high prevalence and morbidity across all ages. Current treatments for US, such as urethral dilatation and internal urethrotomy, fail to fully resolve the condition and are associated with high rates of recurrence and complications. 
Additionally, the pathogenesis of US is not well understood. To explore the pathogenesis of US and develop new therapeutic strategies, it is crucial to establish a standardized rat model that accurately reflects the clinical manifestations. This study outlines a straightforward and repeatable method for inducing US in rats using a high-frequency electric knife. The method involves making a longitudinal incision with the electric knife set to a unipolar mixed cutting mode at 4 W, which inflicts significant urethral damage. Histopathological analysis shows thickening of the urothelium, inflammatory infiltration, and disorganized collagen fibers. This model effectively replicates iatrogenic injury through electroexcision in the rat urethra. In summary, this study successfully establishes a new, efficient, and stable rat model of US that closely mimics the clinical scenario, providing a valuable tool for further research into the mechanisms and novel treatments for US.

In this study, we present a novel, efficient, and stable rat model of urethral stricture created through electroexcision of the rat urethra, which effectively simulates iatrogenic injury observed in clinical settings.

Urethral stricture (US) is a common clinical condition in urology, characterized by high prevalence and morbidity across all ages. Current treatments for ...

Modello di sottoattività del detrusore nei ratti mediante transezione di Conus medullaris

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Valutazione di Biomateriali per l'ampliamento vescicale con analisi cistometrici in modelli di roditori vari

L'uso di cistometria in piccoli roditori: A Study of Chemosensation vescica