Name: a method for the measurement of salivary gland function in mice
Uploaded: 2018-01-25T14:30:00
Description: Watch this Scientific Journal Video about A Method for the Measurement of Salivary Gland Function in Mice at JoVE.com

Undersøgelsen blev støttet af en bevilling fra National Institutes of Health, nationale Institut for Dental og Craniofacial forskning (DE025030).

Protokollen beskrevet nedenfor blev godkendt af det institutionelle Animal Care og brug udvalg og det følger de etiske retningslinier af National Institutes of Health. Alle data præsenteres i denne rapport blev genereret ved hjælp af kvindelige mus, der var 10-12 ugens i alder. De følgende stammer af mus blev anvendt: C57BL/6, BALB/c, DBA1, 129S og (B6XA/J) F1. Alle mus blev opstaldet i barriere bure (5 dyr pr. bur) i specifikt patogenfrie betingelser og leveret foder og vand ad libitum.
1. ...

<ol>
	<li>Proctor, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+salivary+secretion.">The physiology of salivary secretion.</a> Periodontol 2000. 70 (1), 11-25 (2016).</li><li>Fox, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sj&#246;gren's+syndrome.">Sj&#246;gren's syndrome.</a> Lancet. 366 (9482), 321-331 (2005).</li><li>Eisbruch, A., Kim, H. M., Terrell, J. E., Marsh, L. H., Dawson, L. A., Ship, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xerostomia+and+its+predictors+following+parotid-sparing+irradiation+of+head-and-neck+cancer.">Xerostomia and its predictors following parotid-sparing irradiation of head-and-neck cancer.</a> Int J Radiat Oncol Biol Phys. 50 (3), 695-704 (2001).</li><li>Marmary, Y., Fox, P. C., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+secretion+rates+from+mouse+and+rat+parotid+glands+are+markedly+different+following+pilocarpine+stimulation.">Fluid secretion rates from mouse and rat parotid glands are markedly different following pilocarpine stimulation.</a> Comp Biochem Physiol A Comp Physiol. 88 (2), 307-310 (1987).</li><li>Lin, A. L., Johnson, D. A., Wu, Y., Wong, G., Ebersole, J. L., Yeh, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+short-term+gamma-irradiation+effects+on+mouse+salivary+gland+function+using+a+new+saliva+collection+device.">Measuring short-term gamma-irradiation effects on mouse salivary gland function using a new saliva collection device.</a> Arch Oral Biol. 46 (11), 1085-1089 (2001).</li><li>Scofield, R. H., Asfa, S., Obeso, D., Jonsson, R., Kurien, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+short+peptides+from+the+60-kDa+Ro+antigen+recapitulates+the+serological+and+pathological+findings+as+well+as+the+salivary+gland+dysfunction+of+Sjogren's+syndrome.">Immunization with short peptides from the 60-kDa Ro antigen recapitulates the serological and pathological findings as well as the salivary gland dysfunction of Sjogren's syndrome.</a> J Immunol. 175 (12), 8409-8414 (2005).</li><li>Deshmukh, U. S., Nandula, S. R., Thimmalapura, P. R., Scindia, Y. M., Bagavant, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+innate+immune+responses+through+Toll-like+receptor+3+causes+a+rapid+loss+of+salivary+gland+function.">Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function.</a> J Oral Pathol Med. 38 (1), 42-47 (2009).</li><li>Deshmukh, U. S., Ohyama, Y., Bagavant, H., Guo, X., Gaskin, F., Fu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+stimuli+accelerate+Sj&#246;gren's+syndrome-like+disease+in+(NZB+x+NZW)F1+mice.">Inflammatory stimuli accelerate Sj&#246;gren's syndrome-like disease in (NZB x NZW)F1 mice.</a> Arthritis Rheum. 58 (5), 1318-1323 (2008).</li><li>Szczerba, B. 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=Interaction+between+innate+immunity+and+Ro52-induced+antibody+causes+Sj&#246;gren's+syndrome-like+disorder+in+mice.">Interaction between innate immunity and Ro52-induced antibody causes Sj&#246;gren's syndrome-like disorder in mice.</a> Ann Rheum Dis. 75 (3), 617-622 (2016).</li><li>Takakura, A., Moreira, T., Laitano, S., De Luca J&#250;nior, L., Renzi, A., Menani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+muscarinic+receptors+signal+pilocarpine-induced+salivation.">Central muscarinic receptors signal pilocarpine-induced salivation.</a> J Dent Res. 82 (12), 993-997 (2003).</li><li>Yao, C., 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=Lipopolysaccharide-induced+elevation+and+secretion+of+interleukin-1beta+in+the+submandibular+gland+of+male+mice.">Lipopolysaccharide-induced elevation and secretion of interleukin-1beta in the submandibular gland of male mice.</a> Immunology. 116 (2), 213-222 (2005).</li><li>Knudsen, J., Nauntofte, B., Josipovic, M., Engelholm, S. A., Hyldegaard, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+isoflurane+anesthesia+and+pilocarpine+on+rat+parotid+saliva+flow.">Effects of isoflurane anesthesia and pilocarpine on rat parotid saliva flow.</a> Radiat Res. 176 (1), 84-88 (2011).</li><li>Kohrs, R., Durieux, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketamine:+teaching+an+old+drug+new+tricks.">Ketamine: teaching an old drug new tricks.</a> Anesth Analg. 87 (5), 1186-1193 (1998).</li><li>Kozaki, T., Hashiguchi, N., Kaji, Y., Yasukouchi, A., Tochihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+saliva+collection+using+cotton+swab+on+cortisol+enzyme+immunoassay.">Effects of saliva collection using cotton swab on cortisol enzyme immunoassay.</a> Eur J Appl Physiol. 107 (6), 743-746 (2009).</li></ol>

Spyt produktion er kompleks og påvirkes af mange faktorer. Måle spytkirtel-funktion i forsøgsdyr kan derfor være en udfordring. En ekstra udfordring er, at gentagne målinger af spytkirtel funktion i de samme dyr er forpligtet til at etablere udbrud af sygdom eller til at demonstrere opsving efter behandling.
Vatpind metode beskrevet i denne betænkning er teknisk simpelt at udføre med flere muligheder for at repræsentere dataene. De opnåede resultater er reproducerbare, med lille Inter...

Spytkirtlerne producerer spyt som svar på en række forskellige neurologiske og mekaniske stimuli1. Stimuli sker gennem det sympatiske og parasympatiske nervesystem til de adrenerge og kolinerge receptorer i kirtlen. Pilocarpin er en kolinerge, para-sympatomimetisk middel, der virker overvejende på muskarine receptorer. I spytkirtel inducerer det produktionen af spyt ved at handle på muskarine acetylcholin receptor M31. Spyt produktion efter pilocarpin administration er en indikator for evne til spytkirtlerne til at reagere på stimulering og er almindeligt anvendt som en foranstaltning af spytkirtel-funktion.
Nøjagtig måling af spyt produktion er kritisk i studiet af spytkirtel sygdomme herunder Sjögrens syndrom2 og stråling skade efter hoved og hals kræft behandling3. Flere forskellige metoder har været udviklet til at måle spyt produktion i gnavere. Disse omfatter direkte cannulation af ekskretionsorganerne spyt kanal4, samling af spyt fra mundhulen under vakuum5og samling ved hjælp af glas kapillærer6 eller en mikropipette7,8, 9. direkte cannulation af spyt kanalen giver den mest præcise og rene spyt. Men dette er en teknisk udfordrende procedure, og potentialet for duktalt skade udelukker gentagne spyt samlinger fra de samme dyr. Indsamling spyt under vakuum kan føre til varierende resultater som følge af tørring af spyt fra røret. Dette tab er yderligere overdrevet i mus med nedsat spytsekretion. Glas kapillærer tillader indsamling af spyt for efterfølgende analyser, men ændringer i viskositeten af spyttet udskilles i den syge tilstand forhindrer effektiv fyldning af kapillar. Yderligere, forsøg på at feje mundhulen for at indsamle resterende spyt kan føre til skade. Med pipette overfoeres metode giver mulighed for komplet samling, og til den samme importør, det er bemærkelsesværdigt konsekvente mellem eksperimenter. Af ukendte årsager viser denne metode dog betydelig variation mellem forskellige operatører. Derfor for at foretage passende sammenligninger, bliver det bydende nødvendigt, at den samme operatør udfører alle eksperimenter relateret til et bestemt projekt. Klart, dette udgør en stor ulempe for et laboratorium.
For at overvinde disse problemer, blev en procedure udviklet at kombinerer spyt samling metoder anvendes til mennesker og gnavere. Vatpind metode, beskrevet nedenfor til at måle pilocarpin-induceret spyt volumen er enkel, reproducerbar, ikke påvirket af operatøren, og kan udføres flere gange i den samme dyr. Derudover gør det muligt at indsamle spyt for efterfølgende analyser af proteiner, immunglobuliner eller andre biomolekyler.

<table>
	<tbody>
		<tr>
			<td>Chemicals for saliva collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pilocarpine hydrochloride</td>
			<td>Alfa Aesar, Tewksbury, MA, USA</td>
			<td>B21410</td>
			<td>Dilute in sterile isotonic saline. Store single use aliquots of 100X stock at -80oC</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Anesthesia Mix solution</td>
			<td></td>
			<td></td>
			<td>Mix 1 mL ketamine hydrochloride + 0.5 mL xylazine + 8.5 mL sterile isotonic saline in a sterile vial. Can be used for 3 months.</td>
		</tr>
		<tr>
			<td>Zetamine (Ketamine hydrochloride)</td>
			<td>Vet One, Boise, Idaho, USA</td>
			<td>C3N VT1</td>
			<td>Stock is 100 mg/mL</td>
		</tr>
		<tr>
			<td>Anased (Xylazine)</td>
			<td>Med-Vet International, Mettawa, IL, USA</td>
			<td>RXANASED-20</td>
			<td>Stock is 20 mg/mL</td>
		</tr>
		<tr>
			<td>Isotonic sterile saline</td>
			<td>Vet One, Boise, Idaho, USA</td>
			<td>501032</td>
			<td>Used as diluent</td>
		</tr>
		<tr>
			<td>Artificial tears (lubricant ophthalmic ointment)</td>
			<td>Henry Schien, Dublin, OH, USA</td>
			<td>48272</td>
			<td>Used to prevent eyes from drying during the procedure</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials for saliva collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SalivaBio Children&#39;s swab</td>
			<td>Salimetrics, LLC Carlsbad, CA, USA</td>
			<td>N/A</td>
			<td>Individually wrapped swabs</td>
		</tr>
		<tr>
			<td>50 mL polypropylene tubes</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>89004-364</td>
			<td>Cut off the bottom 1 cm of the tube. Make sure that the cut edge is smooth.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 0.6 mL</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>87003-290</td>
			<td>Make holes in the bottom of tube with heated 18 gauge needles</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2 mL</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>87003-298</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes with permanently attached needles</td>
			<td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td>
			<td>324702</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge regular bevel needles</td>
			<td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel blade #11</td>
			<td>Integra York Inc PA, USA</td>
			<td>4-311</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissecting forceps</td>
			<td>Roboz, Gaithersburg, MD, USA</td>
			<td>RS-5139</td>
			<td>Serrated angular 0.8 mm tip, 4&#34; length</td>
		</tr>
		<tr>
			<td>Label tape</td>
			<td>Santa Cruz Biotechnology, Dallas, TX, USA</td>
			<td>sc-224487</td>
			<td></td>
		</tr>
		<tr>
			<td>3 channel timer</td>
			<td>Amazon.com, Seattle, WA, USA</td>
			<td>B06W2KCYVN</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo, Columbus OH, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals/ reagents for inducing salivary dysfunction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LPS</td>
			<td>Invivogen, San Deigo, CA, USA</td>
			<td>tlrl-b5lps</td>
			<td>Dissolve in endotoxin free water, and store stock solution at 5 mg/mL . dilute in sterile HBSS 100 ug/mL - inject 100 uL/ moue ip</td>
		</tr>
		<tr>
			<td>Imject Alum adjuvant</td>
			<td>Thermo Scientific</td>
			<td>77161</td>
			<td>Dilute 1:1 in sterile saline. Inject intraperitoneally 0.1 mL/mouse</td>
		</tr>
		<tr>
			<td>Rabbit anti-Ro52 antiserum</td>
			<td>Generated in lab</td>
			<td></td>
			<td>Immunization of rabbits with recombinant mouse Ro52</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals/ Kits for saliva analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Salivary lysozyme estimation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EnzChek Lysozyme Assay Kit</td>
			<td>Molecular Probes, Eugene, OR, USA</td>
			<td>E-22013</td>
			<td>Used as per manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Salivary IgA estimation by sandwich ELISA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgA</td>
			<td>Southern Biotech, Birmingham, AL, USA</td>
			<td>0106-01</td>
			<td>Standards for sandwich ELISA - range 30 ng/mL to 0.5 ng/mL</td>
		</tr>
		<tr>
			<td>Goat anti- mouse IgA unlabeled</td>
			<td>Southern Biotech, Birmingham, AL, USA</td>
			<td>1040-01</td>
			<td>Coat at 1 ug/mL in bicarbonate buffer</td>
		</tr>
		<tr>
			<td>Goat anti- mouse IgA HRP</td>
			<td>Southern Biotech, Birmingham, AL, USA</td>
			<td>1040-05</td>
			<td>Detection antibody used at 1:4000 dilution</td>
		</tr>
		<tr>
			<td>TMB substrate</td>
			<td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td>
			<td>555214</td>
			<td>Used as per manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Immulon 4HBX Microtiter 96 well plates</td>
			<td>Thermo Scientific, Rochester, NY, USA</td>
			<td>3855</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Salivary protein estimation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay Dye Reagent Concentrate</td>
			<td>BioRad, Hercules, CA, USA</td>
			<td>5000006</td>
			<td>For protein estimation as per manufacturer&#39;s instructions</td>
		</tr>
	</tbody>
</table>

a method for the measurement of salivary gland function in mice

Patienter med Sjögrens syndrom, en autoimmun sygdom, der påvirker eksokrine kirtler, udvikle spytkirtel betændelse og har reduceret spyt produktion. På samme måde, spyt produktion er alvorligt kompromitteret i patienter, der får strålebehandling for hoved og hals kræft. Gnavere modeller, udviklet til at efterligne disse kliniske tilstande, lette forståelsen af sygdom patogenese og mulighed for udvikling af nye terapeutiske strategier. Derfor er evne til præcist, reproducerbar, og gentagne gange måle spytkirtel-funktion i dyremodeller kritisk. Bygning på procedurer tidligere beskrevet i litteraturen, blev en metode udviklet som opfylder disse kriterier og blev brugt til at evaluere spytkirtel funktion i mus. En yderligere fordel ved denne nye metode er at det er let behersker, og har lidt Inter operator variation. Spytkirtel funktion er vurderet som den mængde (vægt eller volumen) eller sats (mL/min) af spyt fremstillet i svar til pilocarpin stimulation. De indsamlede spyt er en god kilde til analyser af proteinindholdet, immunoglobulin koncentrationer og andre biomolekyler.

I modelsystemer eksperimentelle musen bruges evnen til at producere spyt som svar på pilocarpin stimulation som en indikator for spytkirtel-funktion. Spyt produktion blev målt i 11 uger gamle C57BL/6 hunmus af metoden vatpind. I figur 1A, er resultaterne udtrykt som mængden af spyt (mg) indsamlet efter stigende doser af pilocarpin. På dosis af 1 mg/kg legemsvægt begyndte nogle af mus, udstiller nød (ufrivillig rysten og skælven). Dermed blev højere do...

Watch this Scientific Journal Video about A Method for the Measurement of Salivary Gland Function in Mice at JoVE.com

A Method for the Measurement of Salivary Gland Function in Mice

Spytkirtel hypofunction er en hyppig følge af autoimmune sygdomme og stråling terapi. Reproducerbare evaluering af spytkirtel funktion i musemodeller af disse sygdomme er en teknisk udfordring. Her beskrives en simpel metode til nøjagtig og reproducerbar måling af spyt produktion i mus.

En metode til måling af spytkirtel funktion i mus

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

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