Name: in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats
Uploaded: 2017-09-27T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

Denne protokol blev støttet af forskningstilskud fra National Natural Science Foundation of China (#31171066, #81270464) og Sino-tysk Science Center (GZ919).

forsøgsplan rapporteret her er blevet godkendt af de dyre etiske udvalg af Shanghai Jiaotong University School of Medicine (# SYXK2013-0050). dissektion af colorectum med intakt ganglion og nerve stammen tager mindst 15 minutter for en person, der er ret erfarne i denne teknik. Det er derfor nødvendigt at holde de dyr i live men under dyb anæstesi, mens udføre dissektioner, for at sikre levedygtigheden af væv til efterfølgende elektrofysiologiske optagelse.
 1. forberedelse af Perfusion l...

<ol>
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Protokollen præsenteres her er en forholdsvis ligetil Eksperimentel metode til at vurdere de elektrofysiologiske egenskaber af den Colon afferenter af rotter. Protokol (fra væv dissektion at opsætte nerve optagelse) tager normalt omkring 2 timer at fuldføre. Væv samling (trin 3) og forberedelse af de suge elektrode (trin 5) er de kritiske trin. Det er afgørende at kunne finde PG, LSN og PN og passe på ikke for at beskadige ganglion og nerver i løbet af væv dissektion. Spidsen af glas pipette skal brudt og skrå ...

Mave-tarmkanalen (GIT) er rigt innerveres med extrinsic afferente nerver at formidle sensoriske signaler fra tarmen til centralnervesystemet og at bidrage til den gut-hjerne interaktion. Ændrede ophidselse af disse ydre afferenter, samt ændrede centrale behandling af afferente indgange, ligger til grund for viscerale smerter og andre symptomer på GI betingelser, herunder funktionelle og inflammatoriske tarm sygdomme1. Sensorisk information fra colorectum formidles hovedsagelig gennem den thoracolumbale/hypogastric og lumbosacral/bækken nerver (PN)2. Der har været en øget interesse i at studere de elektrofysiologiske egenskaber af disse primære afferente fibre i gnavere sygdomsmodeller. Men, i vivo elektrofysiologiske optagelser af den Colon afferenter i gnavere er en teknisk udfordring og kræver betydelige kirurgiske færdigheder. Derudover kan hæmodynamiske ændringer, væv bevægelse og narkose også påvirke nerve aktivitet og følsomhed til at teste stimuli i vivo. Derfor, i de senere år et stigende antal studier har ansat i vitro (ex vivo) præparater af forskellige arter, herunder mus, rotter, marsvin og mennesker, til at undersøge mekanismerne af sensoriske transduktion i Colon afferenter og ændrede ophidselse i sygdomstilstande. 3 , 4 , 5 , 6 , 7 , 8
To typer af ex vivo Colon forberedelse er primært blevet rapporteret: "flat-ark" forberedelse5,9,10 og "tube" forberedelse3,4. En video protokol for "flat-ark" murine colorectum forberedelse har været tidligere udgivet11. I denne protokol, mus-colorectum med PN) eller lumbal splanknisk nerver (LSN) fastgjort, er fældet og superfused i et væv kammer. Colorectum er skåret åben på langs, og nerve bundt er udvidet til en optagelse rum fyldt med paraffinolie. Nerve aktivitet registreres ved hjælp af en monopolære platinum-iridium elektrode. Protokollen giver mulighed for identifikation af de receptive felter af individuelle afferente fibre ved hjælp af uvildige elektrisk stimulation. Det lokaliserer anvendelsen af kemiske stimuli, samt anvendelse af forskellige mekaniske stimulation paradigmer (fx fokale slimhinde sondering og omkredsen stretch), at afferent nerveender. Fordi nerve skal udvides til at omfatte en separat kammer fra væv kammer, er det afgørende at holde den vedlagte nerve relativt lang; den succesfulde dissektion af nerverne udgør en udfordring for de nye til denne metode. For nylig, Nullens et al. offentliggjort en video protokol til in vitro- optagelse af den mesenteriallymfeknuderne afferenter i murine jejunal og Colon segmenter12. I denne "tube" forberedelse holdes gut segment med mesenterium knyttet intakt, således at for sorterede udspiling og intra - og ekstra-luminale administration af forskellige kemikalier. Da mesenterium nerve er registreret ved hjælp af en suge elektrode, som kan være placeret tæt på væv, kan afferente aktivitet registreres, selv om mesenterium nerve er forholdsvis kort. Dog mesenterium nerve består af blandede bestande af vagus og spinal afferente fibre, der innerverer jejunum eller thoracolumbale hypogastric. Lumbosacral bækken afferenter innerverer de colorectum, som ikke kan blive udsat for i denne protokol. Vi præsenterer her, en detaljeret protokol for den elektrofysiologiske optagelse af rotte Colon afferenter ved hjælp af "tube" colorectum forberedelse med en intakt PG. Denne metode kan give mulighed for karakterisering af de funktionelle egenskaber af lumbal splanknisk (hypogastric) og lumbosacral bækken afferenter.

<table width="889">
	<tbody>
		<tr height="40">
			<td height="40" style="width:215px;height:40px;">Sodium Pentobarbital</td>
			<td style="width:123px;">Shanghai Westang Bio-Tech</td>
			<td style="width:344px;">B558</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Capsaicin</td>
			<td>Sigma</td>
			<td>M2028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode puller</td>
			<td>MicroData Instrument Inc</td>
			<td>PMP107</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurolog System (Bioamplifier)</td>
			<td>Digitimer, Ltd</td>
			<td>Neurolog System</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">A/D converter</td>
			<td>Cambridge Electronic Design</td>
			<td>Micro1401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data processing software</td>
			<td>Cambridge Electronic Design</td>
			<td>Spike2 version 6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silver wire</td>
			<td>World Precision Instruments</td>
			<td>EP12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass tubes</td>
			<td>World Precision Instruments</td>
			<td>1B150-4</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode holder</td>
			<td>World Precision Instruments</td>
			<td>MEH3SBW</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heating bath</td>
			<td>Grant</td>
			<td>GR150</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>Zoom2000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>World Precision Instruments</td>
			<td>PZMIII-BS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cigarette lighter</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical tools</td>
			<td>World Precision Instruments</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Insect pins</td>
			<td>home-made from 0.1 mm stainless steel wire</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Three way manipulator</td>
			<td>World Precision Instruments</td>
			<td>KITF-R</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rats</td>
			<td>Any</td>
			<td>NA</td>
			<td>Any strain/sex can be used.</td>
		</tr>
	</tbody>
</table>

in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats

Dysfunktion af Colon sensoriske nerver har været impliceret i Patofysiologi af flere fælles betingelser, herunder funktionelle og inflammatoriske tarmsygdomme og diabetes. Her, beskriver vi en protokol til in vitro- karakterisering af de elektrofysiologiske egenskaber af Colon afferenter i rotter. Colorectum, med den intakte bækken ganglion (PG) knyttet til, er fjernet fra rotte; superfused med carbogenated Krebs løsning i optagelse kammer; og kanylerede i de mundtlige og anal ender til giver mulighed for udspiling. Et fint nerve bundt stammer fra PG er identificeret, og den multiunit afferente nerve aktivitet registreres ved hjælp af en suge elektrode. Udspiling af Colon segmentet fremkalder gradvise stigninger i multiunit decharge. En principal komponent analyse er udført for at differentiere lav-tærskel, høj-tærskel og wide-dynamic range afferente fibre. Kemisk følsomhed af Colon afferenter kan studeres gennem bad eller intraluminal administrationen af testen forbindelser. Denne protokol kan ændres for anvendelse til andre arter, som mus og marsvin, og at studere forskellene i de elektrofysiologiske egenskaber af thoracolumbale/hypogastric og lumbosacral/bækken afferenter af den faldende kolon i normal og patologiske tilstande.

Figur 1 er en skematisk illustration af eksperimentel opsætning for ex vivo "tube" colorectum forberedelse, med en repræsentant optagelse fra en nerve distale til PG. Nerven indeholdt formentlig en blanding af bækken og lumbal splanknisk afferenter. I præparater fra normale rotter har Colon afferente nerverne typisk et lavt niveau af uregelmæssig spontan aktivitet. Rampe udspiling af tyktarmen inducerer en gradvis stigning i fyring sats (<strong...

Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

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

Unormal sensoriske funktion bag viscerale smerter og andre symptomer på funktionelle og inflammatoriske tarmsygdomme. En protokol til den elektrofysiologiske optagelse af Colon afferente nerver i en ex vivo rotte colorectum forberedelse præsenteres her.

In Vitro Karakterisering af de elektrofysiologiske egenskaber af Colon afferente fibre i rotter

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

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

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