Questo lavoro è stato supportato anche da finanziamenti del governo federale belga (IUAP P6/28), la Fondazione-Fiandre (FWO) (G.0565.07 e G.0686.09), la ricerca europea Astellas Foundation Award 2009 e il Consiglio di ricerca del KU Leuven (GOA 2009/07, e EF/95/010 PFV/10/006). PU e NOI siamo dottorandi della Research Foundation-Fiandre (FWO). MB è un borsista Marie Curie. DDR un fondamentale-clinica collega del FWO.

1. Animali da laboratorio <ol><li> Animali (topi, ratti) sono alloggiati in una struttura specializzata animale con un 12-hr ciclo luce-buio e ad libitum accesso all&#39;acqua e pellet cibo standard. Sia l&#39;età e il sesso degli animali sono parametri importanti che dovrebbero essere standardizzate in base alle esigenze. Noi di solito eseguire cistometria in 10 - 12 settimana animali vecchi femmine 5,6. </li><li> Tutti gli esperimenti sugli animali siano effettuati in conformità con le linee guida comunitarie dell&#39;Unione europea del Consiglio e approvato dal comitato etico locale. </li></ol> 2. Anestesia <ol><li> Anestesia isoflurano viene utilizzato per eseguire piccoli interventi chirurgici, poiché è facile da dosare a causa della sua breve emivita. Gas di scarico adeguata evacuazione è necessaria quando si utilizza l&#39;isoflurano. <ol class="lalpha"><li> Animali siano posti in una scatola chiusa, gasati con isoflurano 5% in ossigeno puro (1 l / min). </li><li> Dopo l&#39;induzione, l&#39;anestesia al l appropriatoevel è mantenuto da una piccola maschera (un taglio siringa da 10 ml) di 0,8 - 1 l / min di ossigeno con 1 - 1,5% di isoflurano. </li></ol></li><li> Per i topi, l&#39;anestesia uretano viene utilizzato durante le registrazioni funzionali (cfr. infra). Anestesia uretano viene utilizzato durante le registrazioni funzionali perché, a differenza di molti altri anestetici, il riflesso della minzione non è soppresso da questo agente anestetico 4. Uretano 1,2 g di peso corporeo / kg viene somministrato per via sottocutanea (sc) 60 min prima dell&#39;inizio delle registrazioni. Se necessario, la dose di uretano necessario per ottenere l&#39;anestesia adeguata può essere titolato con la somministrazione di dosi supplementari (0,1 g / kg) per via sottocutanea. Va notato, tuttavia, che l&#39;anestesia supplementare può alterare l&#39;efficienza svuotamento e di conseguenza la frequenza della minzione 7,8. Così, le registrazioni cistometrici devono essere attentamente monitorati ed eliminato ogni volta deviazioni significative dal comportamento di controllo siano rispettate. Anestetici non deve essere somministrato durante la provacomposti vengono applicati evitare errori di interpretazione dei dati. </li></ol> 3. Procedura chirurgica - Impianto catetere vescicale <ol><li> Strumenti chirurgici vengono sterilizzati in autoclave prima dell&#39;uso. Radere l&#39;addome dell&#39;animale, disinfettare con etanolo al 70% e di eseguire una laparotomia mediana bassa per esporre la vescica. </li><li> Sotto un microscopio operatorio, mettere una borsa di sutura nella cupola della vescica utilizzando un non-assorbibile, sutura monofilamento (dimensioni 6-0). </li><li> Eseguire una cistostomia piccola (preferibilmente usare un ago da 18 G) all&#39;interno della stringa borsa e inserire un catetere di polietilene PE 50, con un bracciale piccolo alla fine (ottenuta riscaldando cautamente il tubo) attraverso questo foro. Nelle nostre mani, sottili tubi PE (10) hanno una resistenza maggiore e più variabile. Il tubo può essere disinfettato mettendolo in una soluzione di etanolo al 70% o sterilizzati in autoclave. Prima dell&#39;impianto, il tubo deve essere lavata con soluzione salina. </li><li> Fissare la str borsazione attorno al tubo con un nodo di un chirurgo. Estrarre il catetere è posizionato delicatamente verso l&#39;esterno fino a campana punta destra sotto la sutura. Inserire un pezzo di 18 catetere G verso la sutura per evitare perdite. </li><li> Infondere un piccolo volume di soluzione fisiologica (100 - 200 pl) delicatamente per controllare eventuali perdite. Se le perdite sono presenti, una sutura supplementari vanno riportate. </li><li> Chiudere i muscoli addominali utilizzando monofilamento, lasciando un passaggio per il catetere nella parte superiore dell&#39;incisione. </li><li> Tunnel il tubo alla regione interscapolare utilizzando un&#39;asta cava di metallo, impedendo così agli animali di mordere il tubo durante l&#39;esperimento. </li><li> La pelle è chiuso con non assorbibili, monofilamento. Il catetere impiantato viene lavata con soluzione salina per verificare la permeabilità. </li><li> Prima di risvegliare gli animali, gli analgesici sono somministrate per via sottocutanea (buprenorfina, 0,05 mg / kg). </li></ol> 4. Installazione e cistometria <ol><li> A seconda della sperimentale Questi,su (e le specie utilizzate) cistometria viene eseguita in sveglio (di restrizioni o di sfrenatezza) o uretano-anestetizzato animali. Considerando che cistometria è perfettamente realizzabile nei ratti coscienti, che tendono a mantenere la calma in un ambiente sobrio, di solito eseguire in anestesia cistometria uretano (1,2 mg / kg per via sottocutanea) quando si utilizza topi 3,5,6,9. </li><li> In topi anestetizzati, temperatura corporea è costantemente monitorata e mantenuta a 36,5 ° C ± 0,2 ° C per mezzo di una sonda di temperatura ed una lampada di riscaldamento. </li><li> Il set-up è calibrato prima di ogni esperimento secondo le istruzioni del fabbricante. </li><li> Il catetere impiantato è collegata da un luer stub di un 3-way rubinetto, che è collegato al trasduttore di pressione (Edwards Lifesciences) su un lato e una pompa per infusione, dall&#39;altro (Harvard Apparatus). Il trasduttore di pressione è collegato tramite un amplificatore (78534c monitor, Hewlett Packard) al sistema di acquisizione dati (DI-730-USB, Dataq strumenti) e un computer. Windaq / Lite software può essere utilizzato per la registrazione. </li><li> La pompa di infusione viene avviata, consentendo l&#39;infusione di soluzione salina o PBS a, per esempio, 100 pl / min (nei ratti) e 20 microlitri / min (nei topi). Come tale la vescica viene riempita a velocità costante, mentre la pressione intravescicale viene registrato. </li><li> Figura 5 illustra una traccia tipica pressione: durante l&#39;infusione di fluido, vi è un accumulo di pressione lenta nella vescica, finché un certo volume / pressione viene raggiunto, la soglia di minzione. Una volta che viene raggiunta questa soglia, la vescica si contraggono e successivamente lo sfintere urinario si apre, consentendo il passaggio di urina attraverso l&#39;uretra. Come tale, la vescica si svuota, la contrazione si arresta e la pressione scende nuovamente al livello &quot;basale&quot;. </li><li> Come il ratto viene posto in una gabbia metabolica e sopra una bilancia digitale, il volume annullato possono essere misurati simultaneamente, fornendo informazioni riguardanti il ​​volume annullata. A causa dei piccoli volumi annullate in mice, può essere molto impegnativo tramite questo sistema. Pertanto, il volume svuotato è determinato utilizzando piccole carte da filtro pesati prima e dopo svuotamento. </li><li> Tipicamente, si lascia il sistema equilibrare per 30 minuti, seguito da un periodo di registrazione di 30 min. Quindi, i farmaci possono essere somministrati per via sistemica o per via endovescicale e altri 30 minuti vengono registrati. </li><li> I dati registrati possono essere esportati come file. Csv che possono essere importati in un software specializzato per l&#39;analisi quantitativa. Si utilizzano in genere di origine (OriginLab Corporation, MA, USA). </li><li> Dopo gli esperimenti, gli animali sono sacrificati mediante dislocazione cervicale (topi) o intossicazione da CO 2 (ratti). </li></ol> 5. Risultati rappresentativi <img alt="Figura 1" src="/files/ftp_upload/3869/3869fig1.jpg" /> Figura 1. Panoramica laparotomia. A) Posizionare il ratto in posizione supina. B) Shave e asettico preparazione del sito chirurgico. C) incisione della pelle. D) Incisione of i muscoli addominali, e l&#39;esposizione della vescica. <img alt="Figura 2" src="/files/ftp_upload/3869/3869fig2.jpg" /> Figura 2. Una borsa di sutura. <img alt="Figura 3" src="/files/ftp_upload/3869/3869fig3.jpg" /> Figura 3. L&#39;impianto del catetere. <img alt="Figura 4" src="/files/ftp_upload/3869/3869fig4.jpg" /> Figura 4. Tunnelling del catetere. Esempi di misure di pressione intravescicale ottenuta durante la perfusione di soluzione salina in un ratto cosciente e un mouse anestetizzato sono mostrati in Figura 5. Parametri multipli può essere estratto dal segnale di pressione (ad esempio l&#39;intervallo intercontractile, la pressione di riferimento e la pressione di soglia). Per una descrizione completa di questi parametri, vedere Andersson et al. (Rif. 4) e Yoshiyama et al. (<html> rif 10). Abbiamo recentemente cistometria utilizzato per identificare i bersagli molecolari di olio di senape (MO), un composto altamente reattivo che è stato a lungo utilizzato in modelli sperimentali di infiammazione ed iperalgesia di organi viscerali come la vescica urinaria 11,12. Infusione intravescicale di 10 mM MO indotto un forte aumento della frequenza di minzione (diminuzione nell&#39;intervallo intercontractile) in sorci di tipo selvatico (Figura 6A, B) e la diminuzione del volume annullata 6. È interessante notare che, MO indotto cambiamenti simili in topi deficienti del recettore TRPA1 MO. Al contrario, MO prodotto cambiamenti molto più deboli dei parametri cistometrici TRPV1 in topi KO rispetto ai topi WT ed è stato senza alcun effetto a Trpa1/Trpv1 topi KO. Insieme a misurazioni del rilascio del peptide correlato al gene della calcitonina (CGRP) 6, questi dati indicano che dimostrano TRPV1 può giocare un ruolo chiave nella irritazione viscerale indotta da MO. ve_content &quot;&gt; <img alt="Figura 5" src="/files/ftp_upload/3869/3869fig5.jpg" /> Figura 5. Tracce rappresentativi di pressione intravescicale registrata in un ratto conscio femmina (A) e in un mouse anestetizzato femmina (B). La pressione minima è definita come la &quot;pressione basale&quot; (frecce rosse). La pressione alla fine della fase di riempimento è contrassegnata con frecce blu. Il volume del liquido infuso tra questi punti, diviso per la differenza di pressione, permette di calcolare la compliance della parete della vescica (compliance = infuso volume / (pressione di soglia -. Pressione basale) Il &quot;intervallo intercontractile&quot; (ICI) è il tempo tra due contrazioni svuotamento. <img alt="Figura 6" src="/files/ftp_upload/3869/3869fig6.jpg" /> Figura 6. Effetti dell&#39;applicazione intravescicale di olio di senape sul modello cistometria in wild type e topi knockout TRPA1, TRPV1 e Trpa1/Trpv1.(A) gli esempi rappresentativi di variazioni di pressione intravescicale registrate nel WT, KO TRPA1, TRPV1 KO e Trpa1/Trpv1 topi KO in risposta alla infusione di soluzione fisiologica e 10 mM MO. (B) Naturalmente il tempo della frequenza media di svuotamento istantaneo, prima e durante l&#39;infusione intravescicale di MO. Per tutti i topi, i dati sono stati normalizzati alla frequenza media ottenuta durante l&#39;infusione salina. Questi dati sono adattato da Everaerts et al. (Rif 6), con il permesso di Elsevier.

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urodynamic+properties+and+neurotransmitter+dependence+of+urinary+bladder+contractility+in+the+BK+channel+deletion+model+of+overactive+bladder.">Urodynamic properties and neurotransmitter dependence of urinary bladder contractility in the BK channel deletion model of overactive bladder.</a> Am. J. Physiol. Renal. Physiol. 289, 604-610 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

La tecnica qui presentata cistometria permette di effettuare misure in vivo della funzione della vescica in modelli animali. I ratti sono probabilmente il modello animale più utilizzato. I topi sono più difficili da gestire, ma offrono il vantaggio di utilizzare animali geneticamente manipolati. A causa della difficoltà tecnica di utilizzare topi coscienti, che tendono ad essere molto attivo con conseguente allentamento del catetere impiantato e variazioni delle pressioni intra-addominale che possono influen...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Nome commerciale, società, </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> uretano </td><td> Uretano, Sigma-Aldrich </td><td> 315419 </td><td> cancerogeno del gruppo 2B </td></tr><tr><td> isoflurano </td><td> Isoba, Schering-Plough Animal Health </td><td></td><td></td></tr><tr><td> polietilene catetere </td><td> Intramedic tubi in polietilene PE50, Becton Dickinson </td><td> 427411 </td><td></td></tr><tr><td> microscopio chirurgico </td><td> Op-Mi 6, Carl Zeiss </td><td> Op-Mi sei </td><td></td></tr><tr><td> borsa di sutura </td><td> Prolene 6/0, Ethicon </td><td> 8610H </td><td></td></tr><tr><td> fascia e sutura della pelle </td><td> Ethilon 4/0 o 5/0, Ethicon </td><td> 662G o 661G </td><td></td></tr><tr><td> analgesici postoperatori </td><td> Temgesic, Schering-Plough Animal Health </td><td></td><td> dosaggio per i ratti: 0,05 mg / kg </td></tr><tr><td> amplificatore </td><td> 78534c monitor, Hewlett Packard </td><td></td><td></td></tr><tr><td> bilance analitiche e il bilancio di acquisizione dati software </td><td> FZ 300i, A &amp; D </td><td> FZ-300i </td><td></td></tr><tr><td> pompe per infusione </td><td> pompa 33, Harvard apparecchi </td><td> HA33 </td><td></td></tr><tr><td> cistometria Sistema di registrazione </td><td> Strumenti Dataq, DI-730 serie e Windaq / Lite </td><td> DI-730-USB Windaq / Lite </td><td></td></tr><tr><td> Temperatura di registrazione </td><td> Fluke 52 Termometro KJ </td><td> 52 KJ </td><td></td></tr><tr><td> Trasduttori di pressione </td><td> Edwards Lifesciences, monitoraggio pressione impostata </td><td> T322247A </td><td></td></tr></tbody></table>

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

<p class="jove_content"&gt; Le basse vie urinarie (LUT) funge da serbatoio dinamica che è in grado di immagazzinare l&#39;urina e di espellere efficacemente in un momento opportuno. Durante la memorizzazione urine, tuttavia, la vescica è esposto per periodi prolungati di prodotti di scarto. Agendo come una barriera a tenuta, il rivestimento epiteliale LUT, dell&#39;urotelio, evita il riassorbimento di sostanze nocive. Inoltre, le sostanze chimiche nocive stimolare innervazione nocicettiva della vescica e di avviare annullare contrazioni che espellere il contenuto della vescica. È interessante notare che la sensibilità della vescica a sostanze chimiche nocive è stato utilizzato con successo nella pratica clinica, per via endovescicale infondendo il TRPV1 capsaicina agonista per il trattamento della vescica neurogena iperattività<sup&gt; 1</sup&gt;. Ciò sottolinea il vantaggio di vedere la vescica come un organo chemiosensoriali e richiede ulteriori ricerche cliniche. Tuttavia, le questioni etiche limitano fortemente la possibilità di eseguire, su soggetti umani, le misure invasive che sono necessarie per svelare le basi molecolari della LUT farmacologia clinica. Un modo per superare questa limitazione è l&#39;uso di modelli animali<sup&gt; 2</sup&gt;. Qui si descrive l&#39;implementazione di cistometria in topi e ratti, una tecnica che permette di misurare la pressione intravescicale in condizioni di perfusione vescica controllata.</p&gt;<p class="jove_content"&gt; Dopo laparotomia, un catetere viene impiantato nella cupola vescica e tunnel per via sottocutanea nella regione interscapolare. Quindi la vescica può essere riempito a velocità controllata, mentre l&#39;uretra è lasciato libero per minzione. Durante i cicli ripetitivi di riempimento e svuotamento, la pressione intravescicale può essere misurata tramite il catetere impiantato. Come tale, le variazioni di pressione possono essere quantificati e analizzati. Inoltre, la misurazione simultanea del volume consente di distinguere le contrazioni annullata svuotamento di non svuotamento contrazioni<sup&gt; 3</sup&gt;.</p&gt;<p class="jove_content"&gt; Importante, a causa delle differenze di controllo della minzione tra roditori ed esseri umani, misurazioni cistometrici in questi animali hanno un limitato valore traslazionale<sup&gt; 4</sup&gt;. Tuttavia, sono abbastanza strumentale nello studio della fisiopatologia vescica e farmacologia sperimentali pre-clinici impostazioni. Recenti ricerche utilizzando questa tecnica ha rivelato il ruolo chiave di nuovi attori molecolari nelle proprietà meccano-e chemo-sensoriali della vescica.</p

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

Cistometria è una tecnica efficace per misurare la funzione della vescica di piccoli animali<em&gt; In vivo</em&gt;. La vescica è continuamente infuso a velocità controllate tramite un catetere intravescicale, mentre l&#39;uretra viene lasciata libera per la minzione. Questo permette ripetitiva riempimento e lo svuotamento della vescica, mentre la pressione intravescicale e volume annullato vengono registrati.

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 ...

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22.3K Views.  KU Leuven, Belgium. Cistometria è una tecnica efficace per misurare la funzione della vescica di piccoli animali In vivo. La vescica è continuamente infuso a velocità controllate tramite un catetere intravescicale, mentre l'uretra viene lasciata libera per la minzione. Questo permette ripetitiva riempimento e lo svuotamento della vescica, mentre la pressione intravescicale e volume annullato vengono registrati.

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.

Topo iniezione parete della vescica

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

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Medicina

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.

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di PTSD

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.

Neuroendocrini umani cella linee tumorali come un modello tridimensionale per lo studio dei diritti dell&#39;uomo e terapia dei tumori neuroendocrini

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.

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 ...

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.

Arto Remote ischemica precondizionamento: una tecnica neuroprotettivo nei roditori

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.

Esecuzione sottoretinico Iniezioni in roditori di agire cellule retiniche epitelio pigmentato in sospensione

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.

Valutazione dell&#39;efficacia della<em&gt; H. pylori</em&gt; Proteine ​​HP-NAP come strumento terapeutico per il trattamento del cancro della vescica in un ortotopico Modello murino

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.

L&#39;uso di ultra-alto campo MRI in piccolo roditore modelli di malattia del rene policistico per<em&gt; In Vivo</em&gt; Fenotipizzazione 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 ...

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.

Uso di un modello di maiale per lo studio della neurotossicità dello sviluppo sviluppato da anestetici (AIDN): un approccio di neuroscienza transazionale

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.

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 ...