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 arbejde blev støttet af tilskud fra National Institute for Døvhed og Andre kommunikationsforstyrrelser (R21DC010806 til CAM og JCS og R01DC011285 til CAM).

Brug mandlige Fischer 344-Brown Norway rotter ved 6 og 30 måneder for denne protokol. Rotter blev opnået fra National Institute of Aging gnaverkoloni. Vi anvendte rotter til denne undersøgelse, fordi strukturen af ​​rotte strubehovedet ligner den hos det menneskelige, funktionelt tjenende til luftvejsbeskyttelse og artsspecifikke vokaliseringer. Denne undersøgelse blev udført i overensstemmelse med PHS-politikken for human pleje og brug af laboratoriedyr, NIH-vejledningen til pleje og brug af laboratoriedyr og ...

<ol>
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Neurosci. 24 (1), 677-736 (2012).</li><li>Gonzalez, 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=Disruption+of+Trkb+mediated+signaling+induces+disassembly+of+postsynaptic+receptor+clusters+at+neuromuscular+junctions.">Disruption of Trkb mediated signaling induces disassembly of postsynaptic receptor clusters at neuromuscular junctions.</a> Neuron. 24 (3), 567-583 (1999).</li><li>Hirano, S., Kishimoto, Y., Suehiro, A., Kanemaru, S., Ito, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+aged+vocal+folds:+first+human+case+treated+with+fibroblast+growth+factor.">Regeneration of aged vocal folds: first human case treated with fibroblast growth factor.</a> Laryngoscope. 118 (12), 2254-2259 (2008).</li><li>Branski, R. 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F., Homberger, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy and dissection of the rat. , (1997).</li><li>Ohno, T., Hirano, S., Rousseau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+changes+in+the+expression+and+deposition+of+vocal+fold+collagen+and+hyaluronan.">Age-associated changes in the expression and deposition of vocal fold collagen and hyaluronan.</a> Ann. Oto. Rhinol. Laryngol. 25 (1), 192-197 (2009).</li><li>Stemple, J. 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=Enhancement+of+aging+rat+laryngeal+muscles+with+endogenous+growth+factor+treatment.">Enhancement of aging rat laryngeal muscles with endogenous growth factor treatment.</a> Physiol. 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Laryngeale muskler er sårbare overfor de ugunstige virkninger af aldring. Tidligere undersøgelser har vist ændringer i aldring i laryngeale muskler, der omfatter ændringer i fiberstørrelse, totalt antal fibre, regenerativ evne, NMJ størrelse og mængdeændringer, ud over variationer i kontraktil funktion og myosin isoformskift 4 , 11 , 27 , 30 , 31 . Ag...

Laryngeale muskler samler sig hurtigt og konsekvent og er sårbare overfor de skadelige virkninger af aldring. Denne konstante aktivitet antages at bidrage til talproblemer eller dysfagi observeret hos personer over 65 år 1 , 2 , 3 , 4 , 5 , 6 , 7 . Flere molekylære og patofysiologiske mekanismer bidrager til denne aldersrelaterede dysfunktion. Disse mekanismer kan omfatte remodeling af larynxslimhinde, muskelfiberatrofi eller tab, mangel på regenerering af muskelfibre eller atrofi, der forårsager bøjning af vokalfoldene og manglende evne til glottislukning 8 , 9 , 10 , 11 . Der er ingen bevist medicinsk terapi på dette tidspunkt, som kan comForhindre eller rehabilitere disse aldersrelaterede ændringer i disse muskler. Modulation af effektiviteten af ​​neuromuskulær transmission kan i høj grad påvirke neuromotorisk ydeevne. Familien af ​​neurotrophins indbefatter nervevækstfaktor (NGF), hjerneafledt nervevækstfaktor (BDNF), neurotrophin 3 (NTF3) og NTF4 12 , 13 . Neurotropiner har vist sig at modulere synaptisk virkning 1 , 4 . Hepatocytvækstfaktor, transformerende vækstfaktor beta og fibroblastvækstfaktor er for nylig blevet anvendt til mennesker til behandling af vokal foldearrring 15-17 . NTF4 regulerer også NMJ effektivitet; Mus mangler NTF4 viser disassembled NMJs 11 , 18 , 19 . Disse undersøgelser fører til lovende virkninger af behandlereT af aldrende larynx muskelforstyrrelser og denervation med vækstfaktorer. Direkte injektionsbehandling til vævene i vokalfoldene er ikke hidtil uset hos mennesker. Lokale injektioner af botulinumtoksin anvendes for tiden som en effektiv behandling af neurologiske bevægelsesforstyrrelser, som påvirker musklerne i strubehovedet, såsom spasmodisk dysfoni og bilateral tilbagevendende larynx-nerveslamning 20 , 21 . Hyaluronsyre hydrogel er en anden injicerbar, som bruges til at behandle vokal fold skræmme og glottisk insufficiens 22 , 23 . Injektionslaryngoplastik kan bruges til at behandle en række kommunikationsforstyrrelser 24 . Disse direkte injektionsmetoder holder løfte om at forbedre vokalfunktionen og sluge i aldrende befolkninger.

<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 dysfunktion hos ældre er en væsentlig årsag til handicap, fra stemmeforstyrrelser til dysfagi og tab af luftvejsbeskyttelsesreflekser. Få, hvis nogen, findes terapier, der målretter aldersrelateret larynx muskel dysfunktion. Neurotropiner er involveret i muskelinnervation og differentiering af neuromuskulære krydsninger (NMJ&#39;er). Det antages, at neurotrophiner forbedrer neuromuskulær transmission ved at øge neurotransmitterfrigivelsen. De neuromuskulære krydsninger (NMJ&#39;er) bliver mindre og mindre rigelige i ældning af rotte laryngeale muskler med evidens for funktionel denervation. Vi undersøgte virkningerne af NTF4 til fremtidig klinisk anvendelse som terapeutisk for at forbedre funktionen i aldrende humane larynxmuskler. Her giver vi den detaljerede protokol til systemisk anvendelse og direkte indsprøjtning af NTF4 for at undersøge evnen til ældning af rotte laryngeal muskel til remodel som svar på NTF4 applikation. I denne metode modtog rotter enten NTF4 enten systematisk via oSmotisk pumpe eller ved direkte indsprøjtning gennem vokalfoldene. Laryngeale muskler blev derefter dissekeret og anvendt til histologisk undersøgelse af morfologi og aldersrelateret denervation.

Rotterne blev euthaniseret efter 2 uger med osmotisk pumpeinfusion eller 1 uge efter direkte injektion af NTF4. Larynger blev høstet, anbragt i kryobeskyttelsesmiddel (30% saccharose og 70% phosphatpufret saltopløsning) og derefter serielt snittet i 10 μm bredder med en kryostat. Aldring i larynx muskler påvirkes af administration af NTF4 25 . Ud over den unge og gamle rotte sammenlignede vi den injicerede og ikke-injicerede side af thyroarytenoidmusklerne. Ty...

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 præsenterer vi en protokol for at beskrive brugen af ​​neurotrophin 4 (NTF4) systemisk og direkte til ombygning af rotte aldrende laryngeale muskler.

Subkutan neurotrofin 4 infusion ved hjælp af osmotiske pumper eller direkte muskelsprøjtning 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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