Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

NOTA: Questo protocollo è stato esaminato e approvato dalla University of Pittsburgh Istituzionale Animal Care and Use Committee. 1. Preparazione della soluzione di Krebs Modified e Drug Test Aliquote <ol><li> Rendere 6 L di soluzione di Krebs modificato contenente (in mM): NaCl 117,9, 4.7 KCl, 25 NaHCO 3, 1.3 NaH 2 PO 4, 1.2 MgSO4, CaCl 2 2,5, 11,1 D-glucosio, 2 butirrato di sodio, acetato di sodio 20 , 0.004 nifedipina (per bloccare le contrazioni muscolari spontanee), e 0,003 indometacina (per bloccare la sintesi di prostaglandine endogene). Utilizzare soluzioni Krebs ghiaccio freddi e caldi per la dissezione dei tessuti e la registrazione singola fibra, rispettivamente. </li><li> Preparare eventuali soluzioni chimiche (ad esempio, IS, la capsaicina, ATP) in aliquote a concentrazioni desiderate. </li></ol> 2. Dissezione del Tissue colon-retto-nervo <ol><li> Anestetizzare e eutanasia topi maschi (6 - 8 settimane di vita, 20 - 30 g) inuna camera di CO 2 ad una velocità di flusso che sposta 10 - 30% del volume della camera per minuto fino topi smettere di respirare completamente come indicato dall&#39;assenza di movimento del torace. </li><li> Immediatamente dopo l&#39;eutanasia, Exsanguinate tagliando aperta camera toracica, perforando l&#39;atrio destro, e immergendo la carcassa del mouse in un ampio volume di (~ 500 ml) di ghiacciata soluzione Krebs (4 ° C) gorgogliare con CarboGen (95% O 2 , 5% di CO 2). </li><li> Rimuovere accuratamente tutti i visceri, ma gli organi del colon e pelvica. Transetto il mouse nella metà attraverso il segmento vertebrale T12 leggermente sopra il diaframma toracico e trasferire il mezzo caudale di una camera di dissezione contenente ghiacciata, bolle soluzione Krebs. </li><li> Sotto uno stereomicroscopio, rimuovere la vescica e gli organi riproduttivi per sezionare i loro incroci per l&#39;uretra, e rimuovere la discesa / aorta addominale fino a quando non si biforca nelle arterie iliache comuni. Liberare il PN o il LSN dal loro surrounding tessuti per via smussa e seguire il nervo da fuori cresta iliaca fino al suo punto di ingresso ventrale nel L6 e S1 la colonna vertebrale (per PN) o T13 e L1 la colonna vertebrale (per LSN). </li><li> Tagliare la sinfisi pubica e destra e giunti acetabolari sinistra, e togliere l&#39;osso iliaco. Gratuito con attenzione sia la PN o LSN dal muscolo attaccato e del tessuto connettivo dal vicino al colon-retto fino dove il nervo entra la colonna vertebrale. </li><li> Resecare attentamente l&#39;osso iliaco per esporre il colon-retto distale. Sezionare il colon-retto distale con il PN collegato o LSN in continuum. </li><li> Trasferire il colon retto con il nervo allegata al vano bagno di camera di tessuti. Rimuovere eccessivo tessuto connettivo da ulteriori dissezione, e aprire il colon-retto longitudinalmente lungo il confine anti-mesenterica. </li><li> Con lato mucosa rivolto verso l&#39;alto, pin bordo mesenterica del colon-retto adiacente al vano di registrazione nella base di silicone della chamber e fissare la lunghezza del colon-retto antimesenterico ad un rastrello di ganci collegati ad un attuatore forza (illustrata nella figura 1 e fotografato in Figura 2A). </li><li> Estrarre il PN o LSN nel vano registrazione, che è collegato al vano vasca da un foro mouse e cancello. Adagiare il tronco nervoso su un piccolo specchio di vetro nel vano registrazione, che fornisce una superficie idrofila per il nervo di aderire. Superfuse vano bagno con calda (30 - 32 ° C), soluzione di Krebs ossigenata e riempire il vano di registrazione con olio minerale. </li></ol> 3. Single-fibra Registrazione e localizzazione del campo recettivo <ol><li> Rimuovere con cautela indietro il epinevrio (guaina del nervo) dal PN o LSN allo stereomicroscopio ad alto ingrandimento (50 - 60X). Utilizzando una pinza sottile, prendere in giro il tronco nervoso in 5-8 fasci nervosi di ~ 100 micron di spessore. </li><li> Collocare il platino-iridioelettrodi a contatto con la soluzione Krebs nella camera tessuti. Posizionare in sequenza i singoli fasci nervosi sull&#39;elettrodo registrazione fatta dello stesso materiale. </li><li> Usare un pennello morbido per evocare gli AP delle afferenze colorettali delicatamente accarezzando su e giù per la superficie del colon-retto. Individuare il fascio nervoso (s) che innervano il colon attraverso rilevabile AP (potenziale d&#39;azione) registrazioni. NOTA: Il PN e LSN anche innervano la vescica urinaria e di altri organi pelvici. </li><li> Utilizzare un paio di 30 punte degli aghi G di dividere ulteriormente il fascio nervoso in sottili filamenti fascicolo di ~ 10 micron di spessore e posizionare un individuo filamento sul elettrodo di registrazione. </li><li> Posizionare l&#39;elettrodo concentrico con punta arrotondata perpendicolare alla superficie della mucosa per eccitare elettricamente terminazioni afferenti a intensità suprathreshold stimolo (10 mA magnitudo 0,5 durata msec @ 0,3 Hz), che produce un mm di raggio di diffusione attuale ~ 2. Spostare l&#39;elettrodo sistematicamente (~ 1,5 mm passi) Lungo la lunghezza e la larghezza del colon-retto appiattita localizzare terminazioni ricettive. </li><li> Quando un desinenza afferente è eccitato, regolare la posizione degli elettrodi di individuare il sito di attivazione (campo recettivo, RF) che richiede intensità dello stimolo minimo (soglia di stimolo). Scartare terminazioni con una soglia di stimolo&gt; 3 mA 19. </li><li> Calcolare la velocità di conduzione (CV) da 1) la distanza tra l&#39;elettrodo di stimolazione al campo recettivo (RF) e il sito registrazione e 2) il ritardo di conduzione (ad esempio, la Figura 2B) tra il manufatto stimolo e l&#39;inizio del potenziale d&#39;azione . CV (m / sec) = distanza (mm) / ritardo di conduzione (msec). </li></ol> 4. Classificazione funzionale di meccanosensibili colorettale afferenze <ol><li> Dopo aver individuato un RF da stimolazione elettrica, si applicano le seguenti tre stimoli meccanici alla RF: <ol><li> Effettuare lo stimolo tastatura premendo la punta di un calibramonofilamento ted von Frey-come il nylon (0,4 e 1 g di forza) perpendicolarmente verso la RF sul colon-retto appiattita. </li><li> Effettuare lo stimolo accarezzando accarezzando delicatamente la mucosa del colon-retto con un bel filo di nylon filamento (10 mg forza) per generare un piccolo sforzo di taglio superficiale alla RF. </li><li> Condurre il tratto circonferenziale con una forza attuatore controllato dal computer, che offre una forza tratto rampa (0-170 milioni di euro a 5 mN / sec) in direzione circonferenziale lungo il bordo anti-mesenterica del colon-retto attraverso la rake di ganci descritti al punto 2.8 . </li></ol></li><li> Classificare afferenze come sierose (solo rispondere a smussare sondare), della mucosa (rispondere alle mucose accarezzare e smussare sondare), muscolare (rispondere tratto circonferenziale e smussare sondare) muscolare / mucose (rispondere tratto circonferenziale, mucosa carezze e smussare sondare), o MIA (non reattivo a qualsiasi dei tre stimoli meccanici). </li><li> Per afferenze mesenterici (solo inl&#39;innervazione LSN), che sono difficili da attivare selettivamente dalla stimolazione elettrica, individuare le loro terminazioni ricettive da accarezzare meccanica / sondare del mesentere. </li><li> Per afferenze tratto sensibili (muscolare e muscolo-mucose), determinare la soglia di risposta, che è definita come la forza che evoca il primo AP durante tratto rampa. </li><li> Per afferenze sierose, registrare le loro risposte alla crescente livelli di puntata sondare il campo recettivo guidato dalla forza attuatore controllato dal computer. </li></ol> 5. Chemical Applicazione / Modulazione di Endings ricettive <ol><li> Registrare una risposta di base ad uno stimolo meccanico (cioè, la risposta al tratto rampa, puntata di sondaggio, o mucose carezze). </li><li> Coat il bordo inferiore di un pezzo di tubo (ottone o acciaio inossidabile, altezza 10 mm e 4 x 4 mm 2 quadrata o 4 - diametro di 5 mm) con vaselina e posizionarlo sopra il campo recettivo sul colon-retto. </li><li> Rimuoverela soluzione Krebs all&#39;interno del tubo, ed esporre il finale ricettivo da 3 - 5 minuti a 170 microlitri della soluzione contenente la sostanza chimica (s) da testare. </li><li> Monitorare la risposta del afferente durante l&#39;applicazione chimica (alcune afferenze sono chemiosensibile). </li><li> Rimuovere la soluzione chimica e il tubo di interrompere l&#39;azione della sostanza chimica. Da 4 - 6 min, testare la risposta afferenti allo stesso stimolo meccanico nella risposta basale. </li><li> Riapplicare di nuovo lo stimolo meccanico dopo periodo sufficiente di wash-out (&gt; 15 min). </li></ol> 6. picchi Registrazione e discriminante AP <ol><li> Digitalizzare i segnali elettrici registrati da assoni a 20 kHz e salvare i dati su un computer. Monitorare il segnale in linea da un monitor audio. </li><li> Analizzare le punte AP off-line e discriminare singole unità basate su analisi delle componenti principali delle singole forme d&#39;onda picco 25. NOTA: Un record deve contenere non più thun due unità attive facilmente discriminabili. </li></ol>

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The authors have nothing to disclose.

La vitro preparazione colon-retto-nervo qui descritta ha dimostrato di essere un approccio efficace per studiare le funzioni di codifica neurale delle singole afferenze del colon-retto, che integra ben altri approcci non funzionali (ad esempio, studi cellulari, molecolari e istologiche) sui neuroni sensoriali viscerali ( vedi recensione 27 per i dettagli). Meccanismi neuronali che contribuiscono alla nocicezione e ipersensibilità colorettale a lungo termine sono stati rivelati e ma...

Il dolore e ipersensibilità sono le lamentele predominanti dei pazienti affetti da disturbi gastrointestinali funzionali, tra cui la sindrome dell&#39;intestino irritabile (IBS), che esistono in assenza di apparenti cause patobiologico o danni ai tessuti. Ad esempio, i pazienti IBS presentano ipersensibilità, comprese reazioni accresciute a rettale distensione palloncino e una maggiore sensibilità durante la normale funzione intestinale, così come ipersensibilità di rinvio somatiche (ossia, la tenerezza alla palpazione della zona addominale) 1. Perché il targeting afferenze colorettali ha dimostrato di essere efficace per alleviare il dolore e ipersensibilità in pazienti con IBS (ad esempio, l&#39;instillazione intra-rettale di anestetici locali 2,3; ingestione orale della guanilato ciclasi C-agonisti linaclotide 4-6), una migliore comprensione della innervazione afferente del colon-retto è importante. Afferenze viscerali, tra cui afferenze del colon-retto, Sono in grado di rispondere alle modalità chimici / di nutrienti e termiche (ad esempio, 7-9). Tuttavia, afferenze viscerali che rispondono a stimoli meccanici (cioè, afferenze meccanosensibili) sono stati i più studiato a fondo, perché gli stimoli meccanici (ad esempio, distensione luminale, tratto) sono quelle che generalmente danno luogo a sensazioni coscienti, tra cui il disagio e il dolore 10-16. Inoltre, i visceri sono innervate da afferenze meccanicamente insensibili (MIAS), comunemente definiti silenziosi o dormire nocicettori 17. In condizioni fisiologiche normali, MIAS non rispondere alla stimolazione meccanica o abbia una grande risposta soglie 18, ma possono diventare attivi e acquisire mechanosensitivity in condizioni fisiopatologiche e contribuire alla ipersensibilità. Utilizzando la preparazione in vitro e il protocollo descritto qui, abbiamo sviluppato ed impiegato una strategia di stimolo elettrico al mare<html> RCH per terminazioni ricettive, atti a identificare imparziale di entrambe le terminazioni meccanosensibili e MIA nel colon-retto 19. L&#39;innervazione del colon-retto è derivato da splanchnic lombare (LSN) e del nervo pelvico (PN) percorsi, e include afferenze del colon che possono essere classificati in cinque classi meccanosensibili (sierose, mucose, muscoloso, muscoloso-mucosa, mesenterici) e una classe MIA 20. Utilizzando questa preparazione in vitro, abbiamo scoperto che MIAS colorettali mechanosensitivity acquisita (sensibilizzazione) a seguito di una breve esposizione dei loro campi recettivi ad una zuppa infiammatoria (IS), che ha sensibilizzato il 71% del MIAS nella via PN e il 23% del MIAS nella via LSN 19. Abbiamo anche documentato sensibilizzazione a lungo termine (fino a 28 giorni) del MIAS nel contesto di lunga durata ipersensibilità viscerale comportamentale (cioè, nei topi che ricevono trattamenti intracolonic con zymosan 21 o acido 2,4,6-trinitrobenzensulfonico (TNBS) 22) . jove_content &quot;&gt; Tra afferenze meccanosensibili, afferenze muscolari e muscolo-mucose sono le uniche classi che tonicamente codificano tratto circonferenziale del colon-retto (cioè, sono stiro-sensitive) e Subserve la codifica dei nocivi distensione colorettale 23,24. Utilizzando un controllato dal computer Attuatore di forza, abbiamo applicato uno standard, omogenea, e riproducibile tratto rampa in direzione circonferenziale del tessuto colorettale appiattita e ulteriormente classificati afferenti tratto-sensibili, bassa soglia e ad alta soglia 23. Inoltre, l&#39;andamento temporale della sensibilizzazione di stiramento afferenze -sensitive dopo zymosan intracolonic 21 o 22 TNBS trattamento corrisponde alla insorgenza, la persistenza, e / o il recupero dei comportamentale ipersensibilità viscerale, suggerendo un ruolo di afferenze colorettali tratto sensibile nel dolore viscerale e ipersensibilità.

<table border="0" cellpadding="0" cellspacing="0" width="1173">
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:372px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica MZ16 stereo microscope&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica IC D camera</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amplifier</td>
			<td>World Precision instruments, Inc.</td>
			<td>SYS-DAM80&#160;</td>
			<td>Low-noise differential amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Two-compartment tissue chamber</td>
			<td style="width:291px;">Custom made</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Power1401</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;">Power1401</td>
			<td>Data acquisition, analog signal input/out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spike2 v5.02</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;"></td>
			<td>Software package that works with the Power1401</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Audio monitor</td>
			<td>Natus</td>
			<td style="width:137px;">Am 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Square pulse stimulator</td>
			<td>Natus</td>
			<td style="width:137px;">S48</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Photoelectric isolation unit</td>
			<td>Natus</td>
			<td style="width:137px;">PSIU6</td>
			<td>Stimulus isolation to reduce noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concentric bipolar microelectrode</td>
			<td style="width:291px;">FHC Inc.</td>
			<td style="width:137px;">CBFFG75</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-mode lever system</td>
			<td>Aurora Scientific Inc.</td>
			<td style="width:137px;">Series 300C&#160;</td>
			<td>To deliver mechanical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>forceps with fine tips</td>
		</tr>
	</tbody>
</table>

in vitro functional characterization of mouse colorectal afferent endings

Questo video dimostra in dettaglio una singola fibra protocollo registrazione elettrofisiologica in vitro utilizzando un preparato del mouse colon-retto-nervo. L&#39;approccio permette l&#39;identificazione imparziale e caratterizzazione funzionale delle singole afferenze del colon-retto. Registrazioni extracellulari di potenziali d&#39;azione propagate (AP) che provengono da uno o pochi afferente (cioè, singolo-fibra) campi recettivi (RFS) del colon-retto sono costituiti da fasci di fibre nervose presi in giro. Il colon-retto viene rimosso sia con il bacino (PN) o splanchnic lombare (LSN) nervo collegato e aperta longitudinalmente. Il tessuto viene posto in una camera di registrazione, bloccato piatta e perfuso con soluzione di Krebs ossigenata. Stimolazione elettrica focale viene utilizzato per individuare le terminazioni afferenti colorettali, che vengono ulteriormente testati da tre stimoli meccanici distinti (smussare sondaggio, mucosa carezze e tratto circonferenziale) di categorizzare funzionalmente le afferenze in cinque mechanosensiclassi tive. Endings rispondono a nessuno di questi stimoli meccanici sono classificati come afferenze meccanico-insensitive (MIAS). Sia meccanosensibili e MIAS possono essere valutati per la sensibilizzazione (ad esempio, la risposta maggiore, soglia ridotta, e / o acquisizione di mechanosensitivity) per esposizioni localizzate del RFs ai prodotti chimici (per esempio, la zuppa infiammatoria (IS), la capsaicina, l&#39;adenosina trifosfato (ATP)). Descriviamo l&#39;apparecchio e la preparazione registrazione colorectum-nervo, raccolta di colon-retto con annesso PN o LSN, identificazione del RFs nel colon-retto, registrazione singola fibra da fasci nervosi, e l&#39;applicazione localizzata di prodotti chimici per l&#39;RF. Inoltre, le sfide della preparazione e l&#39;applicazione di stimolazione meccanica standardizzati vengono anche discussi.

La configurazione è illustrata nella figura 1. Esso comprende una camera di tessuto su misura che ospita il colon retto in un vano vasca silicone rivestito e il nervo attaccato in un vano d&#39;olio minerale contigui. La camera a due compartimenti stato lavorato da un blocco solido di plastica acrilica da una macchina CNC; il fondo di entrambi gli scomparti è stato successivamente rivestito con silicone ferma per consentire una facile pin giù del tessuto colorettale. AP extracellulari da fasci nervos...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

In vitro Caratterizzazione funzionale del topo colorettale Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

in-vitro-functional-characterization-mouse-colorectal-afferent

Research

JoVE Journal

Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

9.5K Views.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

Video: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

L&#39;uso di fibroblasti umani primari per il monitoraggio fenotipi mitocondriali nel campo della malattia di Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Ricerca

Medicina

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

In vitro organoide Cultura di primari tumori del colon mouse

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Isolamento e caratterizzazione funzionale di Human ventricolare Cardiomyocytes da campioni freschi chirurgici

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Istituzione e caratterizzazione di UTI e CAUTI in un modello murino

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Valutazione del Cardiac morfologiche e funzionali Variazioni modello murino di trasversale aortica costrizione da parte ecocardiografica Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Longitudinale<em&gt; In Vivo</em&gt; Imaging del Cerebrovasculature: Rilevanza per le malattie del sistema nervoso centrale

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

preparazione e<em&gt; In Vitro</em&gt; Caratterizzazione di magnetizzato miR-modificato cellule endoteliali

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Sviluppo e caratterizzazione funzionale delle cellule dendritiche tollerogeniche murino

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

Caratterizzazione funzionale di varianti RYR1 umane espresse endogenamente

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Occlusion of the Great and Small Saphenous Vein Using Copolymeric Glue Based on N-Butyl Cyanoacrylate and Methacryloxy Sulfolane

We present the preliminary results of a longitudinal observational study aimed at evaluating the effectiveness and safety at different short- and long-term follow-ups of vascular occlusion of the great saphenous vein (GSV) and small saphenous vein (SSV) affected by severe pathological reflux, using an innovative modified cyanoacrylate surgical glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS). Ninety patients, prospectively recruited for 1 year, underwent the study with EcoColor-Doppler (ECD) to evaluate the maximum diameters of the GSV and SSV in the orthostatic position and the reflux time (RT). An RT greater than 0.5 s was considered pathologic. Clinical, etiology, anatomy, and pathophysiology (CEAP) assessment was used for the complete evaluation of each patient in the study. All the patients were treated by NBCA+MS glue to obtain vein occlusion and observed before treatment (baseline; T0), within 6 h after treatment (T1), 1 month after treatment (T2), 3 months after treatment (T3), 6 months after treatment (T4), and 1 year after treatment (T5). Chi-square (&#967;) analysis was performed to evaluate the effectiveness and safety of the treatment. All the patients participated in the entire duration of the study. Complete occlusion was maintained in 100% of patients at T1, 98.9% at T2 and T3, and 97.8% at T4 and T5 (p &lt; 0.001). None of the patients suffered from post-surgical thrombosis. No blue hyperpigmentation, or paresthesia was observed during the entire observation period. Immediately after treatment, 7.7% of patients needed painkillers; 1 week after treatment, 100% of patients returned to normal life. Vascular occlusion of the great or small saphenous vein using NBCA+MS glue is a safe procedure with persistent benefits after a 1 year follow-up. This procedure can be performed with local anesthesia, allowing a quick return to normal life. Thanks to its low invasiveness, the treatment is not painful.

Here, we present a protocol to treat the great saphenous vein (GSV) and the small saphenous vein (SSV) affected by severe pathological reflux, using an original sclerosing and embolizing cyanoacrylate-based glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS).