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><tbody><tr><td>Leica MZ16 stereo microscope</td><td>Leica Microsystems Inc.</td><td></td><td></td></tr><tr><td>Leica IC D camera</td><td>Leica Microsystems Inc.</td><td></td><td></td></tr><tr><td>Amplifier</td><td>World Precision instruments, Inc.</td><td>SYS-DAM80</td><td>Low-noise differential amplifier</td></tr><tr><td>Two-compartment tissue chamber</td><td>Custom made</td><td></td><td></td></tr><tr><td>Power1401</td><td>Cambridge Electronic Design Limited</td><td>Power1401</td><td>Data acquisition, analog signal input/out</td></tr><tr><td>Spike2 v5.02</td><td>Cambridge Electronic Design Limited</td><td></td><td>Software package that works with the Power1401</td></tr><tr><td>Audio monitor</td><td>Natus</td><td>Am 8</td><td></td></tr><tr><td>Square pulse stimulator</td><td>Natus</td><td>S48</td><td>To deliver electrical stimuli</td></tr><tr><td>Photoelectric isolation unit</td><td>Natus</td><td>PSIU6</td><td>Stimulus isolation to reduce noise</td></tr><tr><td>Concentric bipolar microelectrode</td><td>FHC Inc.</td><td>CBFFG75</td><td>To deliver electrical stimuli</td></tr><tr><td>Dual-mode lever system</td><td>Aurora Scientific Inc.</td><td>Series 300C</td><td>To deliver mechanical stimuli</td></tr><tr><td>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 ...

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Universidad de Cartagena UDEC

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

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

La colonscopia flessibile in topi per valutare la gravità della colite e colon-retto Tumori Utilizzo di un sistema Validated endoscopica Scoring

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Ricerca

Medicina

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Un<em&gt; In-vitro</em&gt; Preparazione dei neuroni enterici isolati e glia dal plesso mienterico del topo adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Neuroscienze

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

Registrazione combinata di uscita meccanicamente stimolata afferenti ed nervo terminale Etichettatura in mouse follicolo pilifero lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

In Vitro registrazione di mesenterica afferenti attività del nervo nel topo digiunale e segmenti del colon

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

In Vitro Caratterizzazione delle proprietà elettrofisiologiche di fibre afferenti coliche in ratti

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog skin. Since then, this technique has been applied to many different tissues to study the physiological parameters of transport across membranes. The Ussing chamber method is preferable to other methods because native tissue can be used, making it more applicable to what is happening in vivo. However, because native tissue is used, throughput is low, time is limited, and tissue preparation requires skill and training. These chambers have been used to study specific transporter proteins in various tissues, understand disease pathophysiology such as in Cystic Fibrosis, study drug transport and uptake, and especially contributed to the understanding of nutrient transport in the intestine. Given the whole epithelial transport process of a tissue, not only transepithelial pathways, but also paracellular pathways are important. Tight junctions are a key determinant of tissue specific paracellular permeability across the intestine. In this article, the Ussing chamber technique will be used to assess paracellular permselectivity of ions by measuring transepithelial conductance and dilution potentials.

Intestinal epithelium confers not only nutrient absorption but protection against noxious substances. The apical-most epithelial intercellular junction, i.e., the tight junction, regulates paracellular solute and ion permeability. Here, a protocol for the preparation of mucosal sheets and assessment of the ion selectivity of tight junctions using Ussing chamber technique is described.

Valutazione funzionale della barriera a giunzione stretta intestinale e della permeabilità agli ioni nel tessuto nativo mediante la tecnica della camera di Ussing

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog ...

Biologia

Generation of Mouse Proximal Colon Loop Model: A Surgical Procedure to Generate Proximal Colon Loop and Measure Intestinal Permeability by Detection of Fluorescent Markers in Serum Following Intraluminal Injection

Source: Boerner, K. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/62093">Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.</a> J. Vis. Exp. (2021).
In this video, we describe a surgical procedure to generate mouse proximal colon loop or pcLoop segment followed by intraluminal injection of fluorescent markers into the segment to study the intestinal epithelial permeability.

Generazione di un modello di ansa del colon prossimale di topo: una procedura chirurgica per generare un'ansa del colon prossimale e misurare la permeabilità intestinale rilevando marcatori fluorescenti nel siero dopo l'iniezione intraluminale

Source: Boerner, K. et al. Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized ...

JoVE Encyclopedia of Experiments

Rodent Models

Adult

Electrophysiological Recording from Colonic Afferent Nerves in an Ex Vivo Rat Colon

Source: Meng, Y. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56090">In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats.</a> J. Vis. Exp. (2017)
This video demonstrates a method to record the activity of colonic afferent nerves in an ex-vivo rat colon, cannulated at both ends and infused with saline to increase intraluminal pressure. As pressure increases, the nerve signal rises, indicating its stimulation.

Registrazione elettrofisiologica dai nervi afferenti del colon in un colon di ratto ex vivo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Neuroscience

Neurophysiology

Electrophysiology Techniques

Recording of Mesenteric Afferent Nerve Activity in an Ex Vivo Mouse Jejunum Segment

In this procedure, using a micromanipulator, lower the tip of the suction electrode into the organ bath. Then, gently aspirate some Krebs solution from the organ bath so that the tip of the electrode is submerged in the Krebs solution. Ensure that the Krebs solution covers the wire electrode inside the suction electrode.
Next, position the tip of the suction electrode immediately next to the transected afferent nerve strand, and draw the transected nerve strand into the capillary over its entire length. Maneuver the tip of the electrode towards the adipose tissue, and aspirate it into the glass capillary with the plunger, thereby mechanically sealing the nerve in the capillary from the contents of the organ bath.
To verify the recording of afferent nerve activity, perform a ramp distension-induced increase in afferent firing by closing the output port to lead to a gradual rise in pressure in the intestinal segment. Only perform the desired experimental protocol when three consecutive ramp distensions yield a reproducible multi-unit discharge.

In vitro Caratterizzazione funzionale del topo colorettale Afferent Endings

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