Name: a high-performance liquid chromatography measurement of kynurenine and kynurenic acid: relating biochemistry to cognition and sleep in rats
Uploaded: 2018-08-19T13:00:00
Description: Watch this Scientific Journal Video about A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats at JoVE.com

Den foreliggende undersøgelse blev delvis finansieret af National Institutes of Health (R01 NS102209) og en donation fra Clare E. Forbes Trust.

Vores eksperimentelle protokoller blev godkendt ved University of Maryland institutionelle dyrs pleje og brug udvalget og fulgte den National Institutes of Health's Guide til pleje og anvendelse af forsøgsdyr.
Bemærk: Voksne mandlige Wistar rotter (250-350 g) blev brugt i alle eksperimenter. Separat kohorter af dyr blev brugt til biokemiske analyser, adfærdsmæssige eksperimenter og søvn-vågner optagelser. Dyrene har været opstaldet i en temperatur-kontrolleret facilitet på Maryland psy...

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Til en pålidelig vurdering af KYNA i hjernen efter en perifer kynurenine administration er det afgørende at kombinere og fortolke biokemiske og funktionelle eksperimenter. Vi præsenterer her, en detaljeret protokol, der tillader nye brugere at etablere effektive metoder til måling af plasma kynurenine og hjernens KYNA af rotter. Måling af kynurenine i plasma bekræftet den nøjagtige injektion og måling af metabolitten KYNA bekræfter de novo -syntesen i hjernen. Der er flere fordele ved den beskrevne fluo...

KP er ansvarlig for nedværdigende næsten 95% af den essentielle aminosyre tryptophan1. I pattedyr hjernen metaboliseres kynurenine tages astrocytter i neuroactive lille molekyle KYNA primært af enzymet kynurenine aminotransferase (KAT) II2. KYNA fungerer som en antagonist på NMDA og α7nACh receptorer i hjernen2,3,4, og også mål signalering receptorer herunder aryl kulbrinte receptor (AHR) og G-protein koblet receptor 35 (GPR35)5 ,6. I forsøgsdyr, har stigninger i hjernen KYNA vist sig at forringe deres kognitive præstationer i en række adfærdsmæssige assays2,7,8,9,10 . En spirende hypotese foreslår, at KYNA spiller en integrerende rolle i modulerende kognitive funktioner af påvirker søvn-vågner adfærd11, dermed yderligere understøtte rollen, som astrocyte-afledte molekyler i modulerende neurobiologi af søvn og kognition12.
Klinisk, er stigninger i KYNA blevet fundet i cerebrospinalvæsken og post mortem hjernevæv fra patienter med skizofreni13,14,15,16, en invaliderende psykiatrisk lidelse karakteriseret ved kognitive funktionsnedsættelser. Patienter med skizofreni er også ofte plaget af søvnforstyrrelser, der kan forværre sygdom17. Forståelse af KP metabolisme og KYNA rolle i modulerende en sammenhæng mellem søvn og kognition, især mellem indlæring og hukommelse, kan føre til udvikling af nye terapier til behandling af disse dårlige resultater i skizofreni og andre psykiatriske sygdomme.
En pålidelig og ensartet metode til måling af KP metabolitter er vigtigt at sikre, at forskningen kommer fra forskellige institutioner kan integreres i den videnskabelige forståelse af KP biologi. I øjeblikket, beskrive vi metoden til at måle kynurenine i rotte plasma og KYNA i hjernen, rotte af high-performance væskekromatografi (HPLC). Denne protokol, som gør brug af en fluorimetri påvisning i overværelse af Zn2 +, blev først udviklet af Shibata18 og mere for nylig tilpasset og optimeret til at derivatize med 500 mM zink acetat som efter kolonne reagens, giver mulighed for påvisning af endogene, nanomolar mængder KYNA i hjernen11.
For at stimulere de novo endogene KYNA produktion, som beskrevet i denne protokol, sprøjtes direkte bioprecursor kynurenine intraperitoneal (i.p.) i rotter. I kombination med biokemiske vurderinger for at bestemme graden af KYNA produktion, virkningerne af en kynurenine udfordring på Hippocampus-afhængige hukommelse (passiv undgåelse paradigme) og søvn-vågner arkitektur (EEG- og EMG signaler) er også undersøgte11. En kombination af disse metoder giver mulighed for undersøgelse af de biokemiske og funktionelle konsekvenser af en kynurenine udfordring i vivo i rotter.

<table>
	<tbody>
		<tr>
			<td>Wistar rats</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td>adult male, 250-350 g</td>
		</tr>
		<tr>
			<td>L-kynurenine sulfate</td>
			<td>Sai Advantium</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ReproSil-Pur C18 column (4 x 150 mm)</td>
			<td>Dr. Maisch GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EZ Clips</td>
			<td>Stoelting Co.</td>
			<td>59022</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting materials screws</td>
			<td>PlasticsOne</td>
			<td>00-96 X 1/16</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonabsorbable Sutures</td>
			<td>MedRep Express</td>
			<td>699B</td>
			<td>CP Medical Monomid Black Nylon Sutures, 4-0, P-3, 18&#34;, BOX of 12</td>
		</tr>
		<tr>
			<td>Absorbable Sutures</td>
			<td>Ethicon</td>
			<td>J310H</td>
			<td>4-0 Coated Vicryl Violet 1X27&#39;&#39; SH-1</td>
		</tr>
		<tr>
			<td>Dental Cement</td>
			<td>Stoelting Co.</td>
			<td>51458</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill Bit</td>
			<td>Stoelting Co.</td>
			<td>514551</td>
			<td>0.45 mm</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Alliance HPLC system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E2695 separation module</td>
			<td>Waters</td>
			<td>176269503</td>
			<td></td>
		</tr>
		<tr>
			<td>2475 fluorescence detector</td>
			<td>Waters</td>
			<td>186247500</td>
			<td></td>
		</tr>
		<tr>
			<td>post-column reagent manager</td>
			<td>Waters</td>
			<td>725000556</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenovo computer</td>
			<td>Waters</td>
			<td>668000249</td>
			<td></td>
		</tr>
		<tr>
			<td>Empower software</td>
			<td>Waters</td>
			<td>176706100</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Passive avoidance box for rat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Extra tall MDF sound attenuating cubicle</td>
			<td>MedAssociates</td>
			<td>ENV-018MD</td>
			<td>Interior: 22&#34;W x 22&#34;H x 16&#34;D</td>
		</tr>
		<tr>
			<td>Center channel modulator shuttle box chamber</td>
			<td>MedAssociates</td>
			<td>ENV-010MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel grid floor for rat</td>
			<td>MedAssociates</td>
			<td>ENV-010MB-GF</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto guillotine door</td>
			<td>MedAssociates</td>
			<td>ENV-010B-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick disconnect shuttle grid floor harness for rat</td>
			<td>MedAssociates</td>
			<td>ENV-010MB-QD</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulus light, 1&#34; white lens, mounted on modular panel</td>
			<td>MedAssociates</td>
			<td>ENV-221M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonalert module with volume control for rat chamber</td>
			<td>MedAssociates</td>
			<td>ENV-223AM</td>
			<td></td>
		</tr>
		<tr>
			<td>SmartCtrl 8 input/16 output package</td>
			<td>MedAssociates</td>
			<td>DIG-716P2</td>
			<td></td>
		</tr>
		<tr>
			<td>8 Channel IR control for shuttle boxes</td>
			<td>MedAssociates</td>
			<td>ENV-253C</td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared source and dectector array strips</td>
			<td>MedAssociates</td>
			<td>ENV-256</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop interface cabinet, 120 V 60 Hz</td>
			<td>MedAssociates</td>
			<td>SG-6080C</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual range constant current aversive stimulation module</td>
			<td>MedAssociates</td>
			<td>ENV-410B</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid state grid floor scrambler module</td>
			<td>MedAssociates</td>
			<td>ENV-412</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual A/B shock control module</td>
			<td>MedAssociates</td>
			<td>ENV-415</td>
			<td></td>
		</tr>
		<tr>
			<td>2&#39; 3-Pin mini-molex extension</td>
			<td>MedAssociates</td>
			<td>SG-216A-2</td>
			<td></td>
		</tr>
		<tr>
			<td>10&#39; Shock output cable, DB-9 M/F</td>
			<td>MedAssociates</td>
			<td>SG-219G-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Shuttle shock control cable 15&#39;, 6</td>
			<td>MedAssociates</td>
			<td>SG-219SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Small tabletop cabinet and power supply, 120 V 60 Hz</td>
			<td>MedAssociates</td>
			<td>SG-6080D</td>
			<td></td>
		</tr>
		<tr>
			<td>PCI interface package</td>
			<td>MedAssociates</td>
			<td>DIG-700P2-R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Shuttle box avoidance utility package</td>
			<td>MedAssociates</td>
			<td>SOF-700RA-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sleep-Wake Monitoring Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ponehmah software</td>
			<td>Data Sciences International (DSI)</td>
			<td>PNP-P3P-610</td>
			<td></td>
		</tr>
		<tr>
			<td>MX2 8 Source Acquisition interface</td>
			<td>Data Sciences International (DSI)</td>
			<td>PNM-P3P-MX204</td>
			<td></td>
		</tr>
		<tr>
			<td>Dell computer, Optiplex 7020, Windows 7, 64 bit</td>
			<td>Data Sciences International (DSI)</td>
			<td>271-0112-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Dell 19&#34; computer monitor</td>
			<td>Data Sciences International (DSI)</td>
			<td>271-0113-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Receivers for plastic cages, 8x</td>
			<td>Data Sciences International (DSI)</td>
			<td>272-6001-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cisco RV130 VPN router</td>
			<td>Data Sciences International (DSI)</td>
			<td>RV130</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix 2.0</td>
			<td>Data Sciences International (DSI)</td>
			<td>271-0119-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Network switch</td>
			<td>Data Sciences International (DSI)</td>
			<td>SG200-08P</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuroscore software</td>
			<td>Data Sciences International (DSI)</td>
			<td>271-0171-CFG</td>
			<td></td>
		</tr>
		<tr>
			<td>Two biopotential channels transmitter, model TL11M2-F40-EET</td>
			<td>Data Sciences International (DSI)</td>
			<td>270-0134-001</td>
			<td></td>
		</tr>
	</tbody>
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a high-performance liquid chromatography measurement of kynurenine and kynurenic acid: relating biochemistry to cognition and sleep in rats

Kynurenine vej (KP) af tryptophan nedbrydning har været impliceret i psykiatriske lidelser. Specifikt, astrocyte-afledte metabolit kynurenic syre (KYNA), en antagonist på begge N-methyl-d-aspartat (NMDA) og α7 nicotinsyre acetylcholin (α7nACh) receptorer, har været impliceret i kognitive processer i sundhed og sygdom. Som KYNA niveauer er forhøjede i hjernen hos patienter med skizofreni, er en fejlfunktion på glutamatergic og kolinerge receptorer menes at være kausalt relateret til kognitiv dysfunktion, en core domæne af psykopatologi af sygdommen. KYNA kan spille en pathophysiologically betydelig rolle hos personer med skizofreni. Det er muligt at ophøje endogene KYNA i gnavere hjernen ved at behandle dyr med direkte bioprecursor kynurenine, og prækliniske undersøgelser på rotter har vist, at akutte stigninger i KYNA kan påvirke deres indlæring og hukommelse processer. Den nuværende protokol beskriver denne eksperimentelle tilgang i detaljer og kombinerer en) en biokemisk analyse af kynurenine i blodet og hjernen KYNA dannelse (ved hjælp af high-performance væskekromatografi), b) adfærdsmæssige test for at sonde den hippocampus-afhængige kontekstuelle hukommelse (passiv undgåelse paradigme), og c) en vurdering af søvn-vågne opførsel [telemetric recordings kombinerer elektroencefalografi (EEG) og electromyogram (EMG) signaler] i rotter. Tilsammen en sammenhæng mellem forhøjet KYNA, søvn og kognition er studeret, og denne protokol beskriver i detaljer en eksperimenterende tilgang til forståelsen af funktionen resultater af kynurenine højde og KYNA dannelse i vivo i rotter. Resultater, der opnås gennem variationer af denne protokol vil teste hypotesen at KP og KYNA tjene centrale roller i modulerende søvn og kognition i sundhed og sygdom stater.

For at godkende brugen af en intraperitoneal kynurenine injektion som en metode til at ophøje hjernen KYNA, blev en HPLC-analysen af væv udført. Standard kurver (figur 1) blev bygget ved hjælp af det tilknyttede programmel og tilladt for kvantificering af vævsprøver. Repræsentative kromatogrammer for kynurenine og KYNA er præsenteret i figur 2. Kynurenine blev observeret på en retentionstid på 6 min., og KYNA havde en r...

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A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats

En High-performance væskekromatografi måling af Kynurenine og Kynurenic syre: vedrørende biokemi kognition og søvn hos rotter

Ændringer i kynurenine vej (KP) neuroactive metabolitter er impliceret i psykiatriske sygdomme. Undersøge de funktionelle resultater i en ændret kynurenine pathway stofskifte- vivo i gnavere kan hjælpe med at belyse roman terapeutiske tilgange. Den nuværende protokol kombinerer biokemiske og adfærdsmæssige tilgange for at undersøge virkningen af en akut kynurenine udfordring i rotter.

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite ...

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