Name: an improved method for collection of cerebrospinal fluid from anesthetized mice
Uploaded: 2018-03-19T13:00:00
Description: Watch this Scientific Journal Video about An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice at JoVE.com

Dette arbejde blev støttet af National Natural Science Foundation of China (81650110527, 81371400) og nationale nøglen grundlæggende forskning Program af Kina (2013CB530900).

Alle dyreforsøg blev udfoeres af samfundet politikker for neurovidenskab (USA) og Fudan University (Shanghai, Kina) etiske udvalg. Denne procedure er for en ikke-overlevelse kirurgi.
1. opsætning af CSF samling apparater
<ol><li>Trække glasset kapillær (indvendig diameter 0,75 mm, ydre diameter 1.0 mm) med en mikropipette puller (som vist i Liu et al. 10; skærpet kapillær er vist i figur 1).</li>
	<li>Sted en skærpet glas ...

<ol>
	<li>Anoop, A., Singh, P. K., Jacob, R. S., Maji, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF+Biomarkers+for+Alzheimer's+Disease+Diagnosis.">CSF Biomarkers for Alzheimer's Disease Diagnosis.</a> Int. J. Alzheimers. Dis. 2010, 1-12 (2010).</li><li>Blennow, K., Hampel, H., Weiner, M., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+and+plasma+biomarkers+in+Alzheimer+disease.">Cerebrospinal fluid and plasma biomarkers in Alzheimer disease.</a> Nat. Rev. Neurol. 6 (3), 131-144 (2010).</li><li>Schoonenboom, N. S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+markers+for+differential+dementia+diagnosis+in+a+large+memory+clinic+cohort.">Cerebrospinal fluid markers for differential dementia diagnosis in a large memory clinic cohort.</a> Neurology. 78 (1), 47-54 (2012).</li><li>Molinuevo, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+use+of+cerebrospinal+fluid+biomarker+testing+for+Alzheimer's+disease+diagnosis:+A+consensus+paper+from+the+Alzheimer's+Biomarkers+Standardization+Initiative.">The clinical use of cerebrospinal fluid biomarker testing for Alzheimer's disease diagnosis: A consensus paper from the Alzheimer's Biomarkers Standardization Initiative.</a> Alzheimer's Dement. 10 (6), 808-817 (2014).</li><li>Fagan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF+biomarkers+of+Alzheimer's+disease:+impact+on+disease+concept,+diagnosis,+and+clinical+trial+design.">CSF biomarkers of Alzheimer's disease: impact on disease concept, diagnosis, and clinical trial design.</a> Adv. Geriatr. 2014, 1-14 (2014).</li><li>Ramautar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiling+of+mouse+cerebrospinal+fluid+by+sheathless+CE-MS.">Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS.</a> Anal. Bioanal. Chem. 404 (10), 2895-2900 (2012).</li><li>Liu, L., Herukka, S., Minkeviciene, R., Vangreon, T., Tanila, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+observation+on+CSF+A&#946;42+levels+in+young+to+middle-aged+amyloid+precursor+protein/presenilin-1+doubly+transgenic+mice.">Longitudinal observation on CSF A&#946;42 levels in young to middle-aged amyloid precursor protein/presenilin-1 doubly transgenic mice.</a> Neurobiol. Dis. 17 (3), 516-523 (2004).</li><li>Schelle, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+tau+increase+in+cerebrospinal+fluid+of+APP+transgenic+mice+suggests+downstream+effect+of+BACE1+inhibition.">Prevention of tau increase in cerebrospinal fluid of APP transgenic mice suggests downstream effect of BACE1 inhibition.</a> Alzheimer's Dement. , (2016).</li><li>You, J. -. S., Gelfanova, V., Knierman, M. D., Witzmann, F. A., Wang, M., Hale, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+blood+contamination+on+the+proteome+of+cerebrospinal+fluid.">The impact of blood contamination on the proteome of cerebrospinal fluid.</a> Proteomics. 5 (1), 290-296 (2005).</li><li>Liu, L., Duff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Technique+for+Serial+Collection+of+Cerebrospinal+Fluid+from+the+Cisterna+Magna+in+Mouse.">A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse.</a> J. Vis. Exp. (21), (2008).</li><li>Maia, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+amyloid-&#946;+and+Tau+in+the+cerebrospinal+fluid+of+transgenic+mice+overexpressing+amyloid+precursor+protein.">Changes in amyloid-&#946; and Tau in the cerebrospinal fluid of transgenic mice overexpressing amyloid precursor protein.</a> Sci. Transl. Med. 5 (194), 194re2 (2013).</li><li>Oshio, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+cerebrospinal+fluid+production+and+intracranial+pressure+in+mice+lacking+choroid+plexus+water+channel+Aquaporin-1.">Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin-1.</a> FASEB J. , (2004).</li></ol>

Denne protokol beskriver i detaljer en teknik, der forbedrer den nuværende metoder10 af CSF samling at minimere forurening fra blod og den rigelige samling af CSF (gennemsnitligt ~ 10-15 µL kan opnås) fra mus. Når bryde den kapillære tip, spidsen af kapillar bør ikke være for lille (som dengang CSF vil være uddraget meget langsomt) eller for stor (vil ikke blive fint nok til at indsamle EFSR'EN og væv kan blive indgivet i kapillar). Bør udvises forsigtighed ved dissekere område for CSF ...

CSF er en værdifuld kropsvæske for analyse i neuroscience research. CSF er primært fremstillet af blodplasma, som indeholder få celler (ingen røde blodlegemer og kun et par hvide blodlegemer) og er næsten protein-fri. Det er en af væskerne i tæt kontakt med det centrale nervesystem (CNS) og det kan passere mange elektrolytter fra hjerne og rygmarv til det perifere system. Hos mennesker, CSF prøver kan blive indsamlet til støtte i diagnosticering af sygdom eller til forskningsformål i kliniske forsøg, som en rygmarvsprøve (eller lumbalpunktur) er en mindre invasiv procedure: CSF væske kan afspejle ændringer i CNS uden at have direkte adgang til disse væv. I de seneste år, til forskningsformål i klinikken, fremstillet CSF prøver således af patienter i neurodegenerative sygdomme som Alzheimers sygdom og andre demenssygdomme1,2,3. Der er mange biomarkør assays, som er blevet udviklet ved hjælp af CSF prøver at potentielt støtte i diagnosticering af sygdomme i klinikken2,3. Men der er meget debat om pålideligheden af disse assays til at producere konsekvent, følsomme resultater for at specifikt diagnosticere sygdommen4,5. Så er der et stort behov for udvikling af bedre assays og mål, som kan findes i EFSR'EN, at støtte i at fremstille en standard teknik til at diagnosticere neurodegenerative sygdomme med større følsomhed og specificitet. På grund af den potentielle betydning af menneskelige CSF prøver i sygdom er samlingen af CSF fra gnavere i neurovidenskab forskning også af interesse.
Mus er vigtigt dyr i biologiske og medicinske forskning og mulighed for afprøvning af potentielle terapeutiske forbindelser og proof-of-concept undersøgelser før humane kliniske forsøg. Men i mus, det er svært at få CSF prøver på grund af dets nærhed til hjernen i et lille dyr, som den sædvanlige metode af CSF samling i mus er at få det via cisterna magna, en åbning mellem cerebellum og dorsale flade af medulla oblongata. Dette medfører vanskeligheder i indsamling af CSF prøver som dette område er svært at dissekere til og i umiddelbar nærhed af blodkar, øger risikoen for kontaminering fra blodlegemer. På grund af disse vanskeligheder, de fleste forskere kan kun få en lille mængde af CSF for analyse (normalt angivet som 5-7 µL) og kontaminering af CSF prøver af blod celler er en primær bekymring for analyser6,7,8 , 9. blod kontaminering kan overskygge resultaterne og ikke virkelig afspejler tilstanden af CNS. Desuden begrænset prøve indsamles kan påvirke forskning som det sædvanlige beløb indsamlet fra mus er nok kun én måling (i to eller tre eksemplarer) ved hjælp af enzym-forbundet immunosorbent assay (ELISA). Således er CSF prøver normalt samles fra flere mus for at have nok prøve at køre flere assays. Udvikle en protokol til den rigelige, uforurenet samling af CSF fra mus ønskes høj grad og vil være en fordel i at forbedre neurovidenskab forskning ved hjælp af gnavere.
I denne protokol, en teknik til den rigelige (et gennemsnit på 10-15 µL) samling af CSF fra bedøvede mus er beskrevet i detaljer og forbedrer på et aktuelt kendte metode til CSF samling at minimere forurening fra blod10. Et robust protokol for CSF samling vil støtte i udviklingen af CSF-baserede biomarkør assays, som kunne bruges til at støtte i diagnosticering af sygdomme, samt forbedre forskningen i de mekanismer, der ligger til grund for sygdomme, der rammer CNS.

<table>
	<tbody>
		<tr>
			<td>Chloral hydrate (used as anesthetic)</td>
			<td>Sinopharm Chemicals Reagen Co. Ltd.</td>
			<td>30037517</td>
			<td>CAS number 302-17-0.</td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>66 vision technology</td>
			<td>54002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting curved forceps</td>
			<td>66 vision technology</td>
			<td>53072</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting straight forceps</td>
			<td>66 vision technology</td>
			<td>53070</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse adapter (with ear bars)</td>
			<td>Made in-house.</td>
			<td>N/A</td>
			<td>Similar equipment available from World Precision Instruments.</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Meiji Labax</td>
			<td>Model 15381</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>M3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic base for micromanipulator</td>
			<td>Kanetec</td>
			<td>MB-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>Model P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (1ml)</td>
			<td>Tansoole</td>
			<td>02024692</td>
			<td>For 1ml.</td>
		</tr>
		<tr>
			<td>Microtubes (1.5ml)</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor Cocktail Set III EDTA-free</td>
			<td>Calbiochem</td>
			<td>539134</td>
			<td></td>
		</tr>
		<tr>
			<td>Human A&#946;42 ELISA kit</td>
			<td>Invitrogen</td>
			<td>KHB3441</td>
			<td></td>
		</tr>
		<tr>
			<td>Piping (teflon tubing)</td>
			<td>World Precision Instruments</td>
			<td>MMP-KIT</td>
			<td>Obtained from a microinjection kit and attached to the capillary holder and syringe.</td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Tiangen Biotech</td>
			<td>OSE-MC8</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton buds</td>
			<td>Obtained from any household store/pharmacy.</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

an improved method for collection of cerebrospinal fluid from anesthetized mice

Cerebrospinalvæske (CSF) er en værdifuld kropsvæske for analyse i neuroscience research. Det er en af væskerne i tætteste kontakt med det centrale nervesystem og dermed kan bruges til at analysere hjernen eller rygmarven syge tilstand uden direkte adgang til disse væv. Dog i mus er det vanskeligt at opnå fra cisterna magna på grund af dets nærhed til blodkar, som ofte forurener prøverne. Området for CSF samling i mus er også vanskeligt at dissekere til og ofte kun små prøver fremstilles (højst 5-7 µL eller mindre). Denne protokol beskriver i detaljer en teknik, der forbedrer på nuværende metoder til indsamling for at minimere forurening fra blodet og give mulighed for rigelig indsamling af CSF (i gennemsnit 10-15 µL kan afhentes). Denne teknik kan anvendes med andre dissektion metoder til indsamling af væv fra mus, da det ikke påvirker nogen væv under CSF udvinding. Således, hjernen og rygmarven påvirkes ikke med denne teknik og forblive intakt. Med større CSF prøvetagning og renhed, flere analyser kan bruges med denne væske til yderligere støtte neurovidenskab forskning og bedre forståelse af sygdomme, som rammer hjernen og rygmarven.

Anvendelse af proceduren beskrevet her (figur 1 og figur 2), bør CSF straks indsamles i kapillar klart (figur 2E), ikke pink eller røde. Hvis der er en lyserød til rød skær til væske indsamlet i kapillar, så var der forurening med blod.
Som et eksempel på anvendelsen af CSF prøven indsamles med denne metode, niveauer af protein amy...

Watch this Scientific Journal Video about An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice at JoVE.com

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

Denne protokol beskriver en forbedret teknik for rigelige indsamling af cerebrospinalvæske (CSF) med ingen forurening fra blod. Med større prøvetagning og renhed, kan flere analyser udføres ved hjælp af CSF for at fremme vores forståelse af sygdomme, der påvirker hjernen og rygmarven.

En forbedret metode til indsamling af cerebrospinalvæsken fra bedøvede mus

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

The overall goal of this procedure is to improve collection of cerebrospinal fluid from mice without contamination from blood. This method provides an easy and reliable way to collect CSF for monitoring the biomarkers of neural diseases. The main advantage of this technique is that the addition with the micromanipulator to aid in CSF collection from mice decreases the contamination of blood during collection.Begin by placing a pulled glass capillary into a capillary holder which is firmly mounted on a micromanipulator. Next, while viewing through a dissecting microscope, use straight forceps to break the tip of the sharpened capillary so that the inner diameter of the broken tip is about 10 to 20 microns. Then attach a thin tube and a syringe to the other end of the capillary holder, connect the thin tube and the syringe with a three-way valve and shift the three-way valve to open.Then plunge the syringe to expel any contaminants and create positive pressure in the capillary tube. Repeat this a few times by disconnecting and reconnecting the syringe to the three-way valve. Then draw up air to 100 to 200 microliters before the final reconnection of the syringe with the three-way valve.Move the micromanipulator with the glass capillary to the side to prevent damage during the surgical procedure. Before beginning the surgery, ensure that the mouse has been fully anesthetized by pinching all four limbs and observing the absence of a reaction. Then use dissecting scissors to cut and trim the hair at the back of the head of the mouse from above the eyes between the ears to the bottom of the neck.Secure the head of the mouse using a stereotaxic frame mouse adapter. Make sure the head cannot move and is secured tightly in the adapter by gently pushing down onto the head of the mouse. Use scissors and curved forceps to cut through the mouse skin across the back of the neck and then down the middle of the skull between the ears and eyes of the mouse.Pull the skin and tissue apart and out of the area over the cisterna magna. While viewing through the dissecting microscope, carefully dissect away the last thin layers of muscle over the base of the skull using forceps and move these layers of muscle to the side and out of the area of interest. Once the dura over the cisterna magna is exposed, use a wet cotton bud to wipe the membrane clean and then use a dry cotton bud to dry the area.The cisterna magna appears triangular with one to two large blood vessels running through the area. Either side of or between the blood vessels is optimal for capillary insertion and CSF collection. Once the cisterna magna is exposed, use the micromanipulator to align the glass capillary to the back of the mouse head so that the sharpened point is just behind the membrane at a 30 to 45 degree angle.Next, plunge the syringe once or a few times and then move the plunger back 100 to 200 microliters to make sure there are no contaminants and there is negative pressure in the glass capillary. Then move the capillary tip closer to the membrane until resistance can be seen, but without puncturing the membrane. Knock on the micromanipulator controls to gently tap the capillary tube through the membrane.CSF should automatically be drawn into the capillary tube once the opening has been punctured. Leave the capillary in place to slowly collect CSF. If the CSF has stopped flowing, slowly draw the syringe up approximately 50 microliters to create more negative pressure in the capillary.Ten to 15 microliters of CSF is collected in 10 to 30 minutes. Once the desired volume of CSF has been collected, close the tubing by shifting the three-way valve to close and remove the syringe connected to the tubing. Open and then close the tubing to create equalized pressure in the capillary, the tubing, and the area outside the capillary.Then reconnect the syringe before gently removing the glass capillary from the mouse using micromanipulator. Next, place the collection tube containing one microliter of 20X protease inhibitor under the sharpened point of the glass capillary. Open the tubing and gently plunge the syringe.The CSF should flow out of the capillary and into the collection tube. After collection, pulse centrifuge the CSF to mix the sample with the protease inhibitor at the bottom of the collection tube. CSF samples can be aliquoted and then stored for further analysis at minus 80 degrees Celsius.CSF collected from APP/PS1 mice demonstrated a significant increase in levels of human A beta 42 at 12 months of age. In wild type mice, no human A beta 42 was detected. Once mastered, this procedure can be done in one hour if performed properly.While attempting this procedure, it is important to remember to avoid blood vessels when inserting the capillary and to only make minor adjustments with the micromanipulator during CSF collection to avoid contamination with blood.

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