Name: subcutaneous neurotrophin 4 infusion using osmotic pumps or direct muscular injection enhances aging rat laryngeal muscles
Uploaded: 2017-06-13T15:00:00
Description: Watch this Scientific Journal Video about Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles at JoVE.com

Dette arbeidet ble støttet av tilskudd fra Nasjonalt institutt for døvhet og andre kommunikasjonsforstyrrelser (R21DC010806 til CAM og JCS og R01DC011285 til CAM).

Bruk mannlige Fischer 344-Brown Norway-rotter ved 6 og 30 måneder for denne protokollen. Rotter ble oppnådd fra National Institute of Aging gnagere koloni. Vi brukte rotter for denne studien fordi strukturen til råttens strupehode ligner den menneskelige, funksjonelt betjening for luftveisbeskyttelse og artsspesifikke vokaliseringer. Denne studien ble utført i samsvar med PHS-politikken for human omsorg og bruk av laboratoriedyr, NIH-veiledningen for pleie og bruk av laboratoriedyr, og dyrevelferdsloven (7 USC et se...

<ol>
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Laryngeal muskler er sårbare for de ugunstige effektene av aldring. Tidligere studier har vist endringer i aldring av strupehalsmuskler som inkluderer endringer i fiberstørrelse, totalt antall fibre, regenerativ evne, NMJ-størrelse og kvantitetsendringer, i tillegg til variasjoner i kontraktil funksjon og myosin isoformskift 4 , 11 , 27 , 30 , 31 . Aging l...

Laryngeal muskler kontrakt raskt og konsekvent, og er sårbare for skadelige effekter av aldring. Denne konstante aktiviteten antas å bidra til taleproblemer eller dysfagi observert hos personer over 65 år 1 , 2 , 3 , 4 , 5 , 6 , 7 . Flere molekylære og patofysiologiske mekanismer bidrar til denne aldersrelaterte dysfunksjonen. Disse mekanismene kan omfatte remodeling av laryngeal mucosa, muskelfiberatrofi eller tap, mangel på regenerering av muskler eller atrofi som forårsaker bøyning av vokalfoldene og manglende evne til glottis-lukning 8 , 9 , 10 , 11 . Det er ingen bevist medisinsk terapi på dette tidspunktet som kan comForhindre eller rehabilitere disse aldersrelaterte endringene i disse musklene. Modulering av effektiviteten av nevromuskulær overføring kan i stor grad påvirke nevromotorisk ytelse. Familien til nevrotrofiner inkluderer nervevækstfaktor (NGF), hjerneavledet nervevækstfaktor (BDNF), neurotropin 3 (NTF3) og NTF4 12 , 13 . Neurotropiner har vist seg å modulere synaptisk effekt 1 , 4 . Hepatocyt vekstfaktor, transformerende vekstfaktor beta og fibroblastvekstfaktor, har nylig vært brukt hos mennesker til behandling av vokalfold arrdannelse 15-17. NTF4 regulerer også NMJ effektivitet; Mus mangler NTF4 viser demonterte NMJs 11 , 18 , 19 . Disse studiene fører til lovende effekter av behandlereT av aldring av laryngeale muskelforstyrrelser og denervering med vekstfaktorer. Direkte injeksjonsterapi til vevene i vokalfoldene er ikke enestående i mennesker. For eksempel brukes lokale injeksjoner av botulinumtoksin for tiden som en effektiv behandling for nevrologiske bevegelsesforstyrrelser som påvirker musklene i strupehodet, for eksempel spasmodisk dysfoni og bilateral tilbakevendende laryngeal nerve lammelse 20 , 21 . Hyaluronsyre hydrogel er en annen injiserbar, som brukes til å behandle vokalfallsskrem og glottesuffisiens 22 , 23 . Injeksjons laryngoplastikk kan brukes til å behandle en rekke kommunikasjonsforstyrrelser 24 . Disse direkte injeksjonsmetodene gir løfte om å forbedre vokalfunksjonen og svelge i aldrende populasjoner.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurotrophin 4</td>
			<td>Pepro Tech</td>
			<td>450-04</td>
			<td>200ng in 50&#956;l</td>
		</tr>
		<tr>
			<td>Alzet Osmotic Pump</td>
			<td>DURECT Corporation</td>
			<td>2001D</td>
			<td></td>
		</tr>
		<tr>
			<td>30&#176; endoscope</td>
			<td>Stoltz</td>
			<td>61029D</td>
			<td></td>
		</tr>
		<tr>
			<td>50mm 30 gauge 100-&#956;l syringe</td>
			<td>Hamilton</td>
			<td>84850 and 201812</td>
			<td></td>
		</tr>
		<tr>
			<td>saline (sodium chloride solution)</td>
			<td>Sigma-Aldrich</td>
			<td>S8776</td>
			<td></td>
		</tr>
		<tr>
			<td>ketamine hydrochloride</td>
			<td>Henry Schein</td>
			<td>56344</td>
			<td></td>
		</tr>
		<tr>
			<td>xylazine hydrochloride</td>
			<td>Henry Schein</td>
			<td>33198</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G 5/8 needle</td>
			<td>Becton-Dickinson</td>
			<td>305901</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Becton-Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic ointment</td>
			<td>Henry Schein</td>
			<td>8897</td>
			<td></td>
		</tr>
		<tr>
			<td>clippers</td>
			<td>Oster</td>
			<td>44-018</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Decon</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>iodine (Betadine)</td>
			<td>Purdue Pharma L.P.</td>
			<td>606404</td>
			<td></td>
		</tr>
		<tr>
			<td>heating pad</td>
			<td>Sunbeam</td>
			<td>731-5</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 nylon suture thread</td>
			<td>AD Surgical</td>
			<td>PMN-518R6</td>
			<td></td>
		</tr>
		<tr>
			<td>crile hemostat</td>
			<td>Fine Science Tools</td>
			<td>13005-14</td>
			<td></td>
		</tr>
		<tr>
			<td>delicate suture tying forceps</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>meloxicam</td>
			<td>Henry Schein</td>
			<td>49756</td>
			<td></td>
		</tr>
		<tr>
			<td>carprofen</td>
			<td>Merritt Veterinary Supplies</td>
			<td>148700</td>
			<td></td>
		</tr>
		<tr>
			<td>antibiotic ointment</td>
			<td>Henry Schein</td>
			<td>57110</td>
			<td></td>
		</tr>
		<tr>
			<td>acepromizine Aceproject</td>
			<td>Henry Schein</td>
			<td>3845</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane Isothesia</td>
			<td>Henry Schein</td>
			<td>50033</td>
			<td></td>
		</tr>
		<tr>
			<td>induction box (anesthetizing box)</td>
			<td>Harvard Apparatus</td>
			<td>50-0116</td>
			<td></td>
		</tr>
		<tr>
			<td>oxygen compressed tank</td>
			<td>Scott Gross</td>
			<td>UN1072</td>
			<td></td>
		</tr>
		<tr>
			<td>plexiglas platform</td>
			<td>Small Parts Inc (Amazon)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rubber tipped forceps</td>
			<td>Fine science tools rubber</td>
			<td>11075-00</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid rubber for forceps above</td>
			<td>Lowe&#39;s</td>
			<td>42518</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic spectula (BD syringe cut to length)</td>
			<td>Becton-Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>halogen light source rhino-laryngeal stroboscope</td>
			<td>Kay-Pentax</td>
			<td>RLS 9100 B</td>
			<td></td>
		</tr>
		<tr>
			<td>video recorder</td>
			<td>Kay-Pentax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>cryostat Mictotom HM525</td>
			<td>Thermo Scientific</td>
			<td>HM 525</td>
			<td></td>
		</tr>
		<tr>
			<td>Gill 1 hematoxylin</td>
			<td>VWR</td>
			<td>10143-142</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon eosin-Y alcoholic</td>
			<td>Thermo Fisher Scientific</td>
			<td>6766007</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-sodium channel Nav1.5 antibody produced in rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>S0819</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas red-X phalloidin</td>
			<td>Sigma-Aldrich</td>
			<td>T7471</td>
			<td></td>
		</tr>
		<tr>
			<td>alpha- bungarotoxin alexa fluor 488 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>B-13422</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal anaesthesia machine</td>
			<td>Smiths Medical</td>
			<td>CDS 9000</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcutaneous neurotrophin 4 infusion using osmotic pumps or direct muscular injection enhances aging rat laryngeal muscles

Laryngeal dysfunksjon hos eldre er en viktig årsak til funksjonshemning, fra stemmeforstyrrelser til dysfagi og tap av luftveisbeskyttende reflekser. Få, om noen, finnes terapier som målretter aldersrelatert larynxmuskulær dysfunksjon. Neurotropiner er involvert i muskelinnervering og differensiering av nevromuskulære veikryss (NMJ). Det antas at nevrotrofiner forsterker nevromuskulær overføring ved å øke nevrotransmitterutløsningen. De nevromuskulære veikryssene (NMJs) blir mindre og mindre rik på aldring av rotte laryngeale muskler, med bevis på funksjonell denervering. Vi undersøkte effekten av NTF4 for fremtidig klinisk bruk som terapeutisk for å forbedre funksjonen i aldring av larynxmuskulaturen. Her gir vi den detaljerte protokollen for systemisk bruk og direkte injeksjon av NTF4 for å undersøke evnen til å aldre rotte laryngeal muskel til remodel som svar på NTF4 applikasjon. I denne metoden fikk rotter enten NTF4 enten systematisk via oSmotisk pumpe eller ved direkte injeksjon gjennom vokalfoldene. Laryngeale muskler ble deretter dissekert og brukt for histologisk undersøkelse av morfologi og aldersrelatert denervation.

Rottene ble euthanisert etter 2 uker med osmotisk pumpeinfusjon eller 1 uke etter direkte injeksjon av NTF4. Larynger ble høstet, plassert i krybeskyttende middel (30% sukrose og 70% fosfatbuffert saltvann) og deretter serielt snittet i 10mm bredder med en kryostat. Aldring av larynxmusklene påvirkes av administrasjon av NTF4 25 . I tillegg til ung og gammel rotte, sammenlignet vi injisert og ikke-injisert side av thyroarytenoidmusklene. Vanligvis ser vi en endr...

Watch this Scientific Journal Video about Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles at JoVE.com

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Her presenterer vi en protokoll for å beskrive bruken av nevrotrofin 4 (NTF4) systematisk og direkte for å remodel rotte aldrende laryngeale muskler.

Subkutan Neurotropin 4 infusjon ved bruk av osmotiske pumper eller direkte muskelsprøyting forbedrer aldring rotte larynx muskler

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if ...

Research

JoVE Journal

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