Name: assessing changes in volatile general anesthetic sensitivity of mice after local or systemic pharmacological intervention
Uploaded: 2013-10-16T21:00:00
Description: Watch this Scientific Journal Video about Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention at JoVE.com

Dette arbejde blev støttet af R01 GM088156 og T32 HL007713-18. Vi vil gerne takke Bill Pennie og Michael Carman fra University of Pennsylvania Research Instrumentering Shop for deres hjælp i at samle vores stabilitetsrefleks apparat.

Alle procedurer, der involverer dyr, der er skitseret heri er blevet godkendt af University of Pennsylvania Institutional Animal Care og brug Udvalg. 1.. Oversigt over testapparaturet <ol><li> Testapparatet består af 24 klare akryl cylindriske kamre 10 cm i længden og 5 cm i diameter (samlet volumen på 200 ml). Denne størrelse er passende for en typisk 25 g voksen mus. Chambers har porte i hver ende for gas indløb og udløb. Udløbsenden er aftagelige, så at dyrene let kan fyldes i k...

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Selvom vurderingen af ​​LORR i en enkelt mus er en tilsyneladende enkel opgave, er det ikke desto mindre vigtigt at opretholde samme fysiologiske forhold mellem fag for at indsamle pålidelige data fra en gruppe af dyr. Den stramt reguleret, høj kapacitet LORR apparat præsenteres her giver en måde at standardisere eksperimenter og maksimere effektiviteten. Ved at følge de grundlæggende principper i varmeregulering og lige flow distribution, kan dette system let genskabt og tilpasses til at passe individuelle ek...

Generel anæstesi er defineret ved deres evne til at forårsage en reversibel tilstand af hypnose i en bred vifte af arter, men en forklaring på, hvordan sådan en forskelligartet klasse af lægemidler kan alle fremkalde en særegen endpoint fortsat undvigende. En række teorier er blevet postuleret i årenes løb, startende med Meyer-Overton sammenhæng mellem bedøvelsesmiddel potens og lipid opløselighed, som foreslog generelle forstyrrelser membran som grundlag for hypnose 1,2. Flere nye oplysninger tyder på, at protein-targets påvirker neuronal signalering bidrager til bedøvende virkninger. Mus har vist sig at være en uundværlig model for at udforske disse teorier på grund af homologi mellem murine og humane bedøvelsesmiddel lydhørhed. Selvom en mus ikke kan blive spurgt om sin subjektive bevidsthed under generel anæstesi, visse primitive reflekser tjene som nyttige surrogat mål for gnaver hypnose. I de første par dage efter fødslen, mus udvikler en refleksiv oprettende hhvonse der forhindrer dem i at blive passivt placeret i rygleje 3. Dosen af anæstesi, hvor en mus mister sin opretningsrefleksen korrelerer godt med humane hypnotiske doser 4. Vurdering af tab af opretningsrefleks (LORR) er blevet en udbredt standard laboratorium til at teste bedøvelsesmiddel følsomhed i mus såvel som en række andre arter, herunder rotte, marsvin, kanin, fritte, får, hunde og 5-8. Den dosis af en given anæstetisk hvor LORR vil forekomme for medlemmer af en art er yderst ensartet, men det kan forskydes væsentligt af miljøfaktorer. For eksempel søvnmangel rotter er mere følsomme over for både flygtige og intravenøse anæstetika 9 og rotter med høj aerob kapacitet er mindre følsomme over for isofluran 10. Hypotermi er også blevet vist at nedsætte dosis af talrige anæstetika kræves for hypnose i et stort spektrum af arter 11-14. Forpålideligt identificere den bedøvende dosis, hvor LORR optræder i en gruppe af forsøgsdyr, er det afgørende, at vurderingen miljø følges nøje for at minimere stress, vedligeholde euthermia og levere lige mængder af stof til alle fag. Ikke overraskende, er genetiske faktorer også kendt for at ændre bedøvelsesmiddel følsomhed 15-18. Derfor bør omhyggelig overvejelse også vægt på at kontrollere for genetiske baggrund 19. Vi har udviklet et apparat, der sikrer ens gasformigt anæstetisk levering til hver af 24 mus under opretholdelse af en konstant 37 ° C miljø. Den gennemsigtige cylindriske design af vores eksponering kamre giver mulighed for hurtig LORR vurdering og nem integration af telemetriløsninger fysiologiske målinger. Har vist, at dette system til at måle isofluran, halothan og sevofluran induktion EC 50 og tid til fremkomst i vildtype-mus 20. Vi har også anvendtdette system til at observere ændringer i bedøvelsesmiddel følsomhed i mus med genetiske mutationer og målrettede hypothalamus læsioner 21-23. Her beskriver vi to måder, hvorpå bedøvelsesmiddel sensitivitet kan vurderes efter en farmakologisk intervention ved hjælp af vores apparat kontrolleret miljø. Steady-state fænotypebestemmelse af flygtigt anæstetikum, induktion og fremkomsten følsomhed kræver 8-10 timer og er dermed bedst skræddersyet til undersøgelser, hvor eksperimentelle betingelser ikke ændrer sig, såsom kronisk eller langtidsvirkende farmakologiske interventioner. Men for korttidsvirkende behandlinger, hvis virkninger spreder sig betydeligt over tid, vi også præsentere en simpel procedure til at vurdere ændringer i stabilitetsrefleks efter stereotaktisk målrettede microinjections eller intravenøse lægemiddelbehandlinger, der væsentligt påvirker bedøvelsesmiddel fremkomst. Disse tests udgør en lille delmængde af de potentielle anvendelser for kontrolleret miljø system, som kan tilpasses til en række subjects i en række arter til at modtage alle typer af inhaleret terapeutisk.

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			<td height="42" style="height:42px;width:115px;">Name of the Reagent</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalogue Number</td>
			<td style="width:171px;">Comments</td>
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			<td height="21" style="height:21px;width:115px;">Oxygen</td>
			<td style="width:88px;">Airgas</td>
			<td style="width:88px;">OX300</td>
			<td style="width:171px;"></td>
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			<td height="42" style="height:42px;width:115px;">Isoflurane</td>
			<td style="width:88px;">Butler Schein</td>
			<td style="width:88px;"></td>
			<td style="width:171px;">Any volatile anesthetic of interest may be substituted</td>
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			<td height="42" style="height:42px;width:115px;">Name of Material</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalogue Number</td>
			<td style="width:171px;">Comments</td>
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			<td height="42" style="height:42px;width:115px;">Mass flow meter- 10 SLPM</td>
			<td style="width:88px;">Omega Engineering</td>
			<td style="width:88px;">FMA-A2309</td>
			<td style="width:171px;"></td>
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			<td height="42" style="height:42px;width:115px;">Mass flow meter- 500 SCCM</td>
			<td style="width:88px;">Omega Engineering</td>
			<td style="width:88px;">FMA-A2305</td>
			<td style="width:171px;"></td>
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			<td height="63" style="height:63px;width:115px;">Anesthetic agent analyzer/gas indicator</td>
			<td style="width:88px;">AM Bickford</td>
			<td style="width:88px;">FI-21 Riken</td>
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			<td height="42" style="height:42px;width:115px;">Heating water pump</td>
			<td style="width:88px;">Fisher Scientific</td>
			<td style="width:88px;">13-874-175</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Temperature transponders</td>
			<td style="width:88px;">BMDS</td>
			<td style="width:88px;">IPTT-300</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">RF temperature reader</td>
			<td style="width:88px;">BMDS</td>
			<td style="width:88px;">DAS-6007</td>
			<td style="width:171px;"></td>
		</tr>
	</tbody>
</table>

assessing changes in volatile general anesthetic sensitivity of mice after local or systemic pharmacological intervention

En ønskelig endpoint af generel anæstesi er den tilstand af bevidstløshed, også kendt som hypnose. Definition af hypnotisk tilstand hos dyr er mindre ligetil end det er i menneskelige patienter. En meget brugt adfærdsmæssige surrogat for hypnose hos gnavere er tabet af stabilitetsrefleks (LORR), eller det sted, hvor dyret ikke længere reagerer på deres medfødte instinkt for at undgå sårbarhed dorsale tilbagelænethed. Vi har udviklet et system til vurdering LORR i 24 mus på samme tid, mens omhyggeligt kontrollere for potentielle confounds, herunder temperatursvingninger og varierende gasstrømme. Disse kamre tillader pålidelig vurdering af bedøvelsesmiddel følsomhed som målt ved latenstid for at vende tilbage til den oprettende refleks (RORR) efter en fast bedøvelsesmiddel eksponering. Alternativt bruge trinvise stigninger (eller fald) i anæstesikoncentrationen, kamrene også muliggøre fastlæggelse af en befolkning følsomhed over for induktion (eller fremkomst) som målt vedEC 50 og Hill skråning. Endelig kan de kontrollerede miljøkamre her beskrevne tilpasses til en bred vifte af alternative anvendelser, herunder inhaleret levering af andre lægemidler, toksikologiske undersøgelser, og samtidig real-time overvågning af vitale tegn.

Figur 1 viser anvendeligheden af den trinvise LORR assay til bestemmelse af langsigtede effekter af en farmakologisk intervention. Ibotensyre (IBA) er en agonist af glutamaterge N-methyl-D-aspartat (NMDA)-receptor, der ofte bruges som et excitotoksin at forårsage permanente neuronale læsioner. Her injicerede vi 10 nl 1% IBA bilateralt i ventrolaterale præoptiske område (VLPO) af C57BL/6J-mus en uge før test. Størstedelen af neuroner i denne kerne udviser lave satser for fyring under vågenhed og s...

Watch this Scientific Journal Video about Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention at JoVE.com

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

Tab af stabilitetsrefleksen har længe fungeret som en standard adfærdsmæssige surrogat for bevidstløshed, også kaldet hypnose, i forsøgsdyr. Ændringer i flygtigt anæstetikum følsomhed forårsaget af farmakologiske midler kan påvises med en nøje kontrolleret high-throughput vurdering system, som kan tilpasses til levering af enhver inhaleret terapeutisk.

Vurdere ændringer i flygtige narkose Følsomhed af mus efter lokale eller systemiske farmakologisk intervention

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

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