This work was supported by grants from the National Institute on Deafness and Other communication Disorders (R21DC010806 to C.A.M. and J.C.S and R01DC011285 to C.A.M.).

Use male Fischer 344-Brown Norway rats at 6 and 30 months of age for this protocol. Rats were obtained from the National Institute of Aging rodent colony. We used rats for this study because the structure of the rat larynx is similar to that of the human, functionally serving for airway protection and species-specific vocalizations This study was performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act (7...

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The authors have nothing to disclose.

Laryngeal muscles are vulnerable to the unfavorable effects of aging. Previous studies have demonstrated changes in aging laryngeal muscles that include changes in fiber size, total number of fibers, regenerative ability, NMJ size and quantity changes, in addition to variations in contractile function and myosin isoform shifts 4,11,27,30,31. Aging laryngeal mu...

Laryngeal muscles contract rapidly and consistently, and are vulnerable to the adverse effects of aging. This constant activity is thought to contribute to voice problems or dysphagia observed in persons over 65 years of age 1,2,3,4,5,6,7. Several molecular and pathophysiologic mechanisms contribute to this age-related dysfunction. These mechanisms can include remodeling of laryngeal mucosa, muscle fiber atrophy or loss, lack of muscle fiber regeneration or atrophy which causes bowing of the vocal folds and inability of glottis closure 8,9,10,11. There is no proven medical therapy at this time that can completely prevent or rehabilitate these age-related changes in these muscles.
Modulation of the effectiveness of neuromuscular transmission can greatly influence neuromotor performance. The family of neurotrophins include nerve growth factor (NGF), brain derived nerve growth factor (BDNF), neurotrophin 3 (NTF3) and NTF4 12,13. Neurotrophins have been shown to modulate synaptic efficacy1,4. Hepatocyte growth factor, transforming growth factor beta and fibroblast growth factor have recently been used in humans for the treatment of vocal fold scarring 15-17. NTF4 also regulates NMJ effectiveness; mice lacking NTF4 show disassembled NMJs 11,18,19. These studies lead to promising effects of treatment of aging laryngeal muscle disorders and denervation with growth factors.
Direct injection therapeutics to the tissues of the vocal folds are not unprecedented in humans. For example, local injections of botulinum toxin are currently used as an effective treatment for neurological movement disorders that affect the muscles in the larynx, such as spasmodic dysphonia and bilateral recurrent laryngeal nerve paralysis 20,21. Hyaluronic acid hydrogel is another injectable, which is used to treat vocal fold scaring and glottal insufficiency 22,23. Injection laryngoplasty can be used to treat a variety of communication disorders 24. These direct injection methods hold promise to improve vocal function and swallowing in aging populations.

<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 dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if any, therapies exist that target age-related laryngeal muscle dysfunction. Neurotrophins are involved in muscle innervation and differentiation of neuromuscular junctions (NMJs). It is thought that neurotrophins enhance neuromuscular transmission by increasing neurotransmitter release. The neuromuscular junctions (NMJs) become smaller and less abundant in aging rat laryngeal muscles, with evidence of functional denervation. We explored the effects of NTF4 for future clinical use as a therapeutic to improve function in aging human laryngeal muscles. Here, we provide the detailed protocol for systemic application and direct injection of NTF4 to investigate the ability of aging rat laryngeal muscle to remodel in response to NTF4 application. In this method, rats either received NTF4 either systemically via osmotic pump or by direct injection through the vocal folds. Laryngeal muscles were then dissected and used for histological examination of morphology and age-related denervation.

The rats were euthanized after 2 weeks of osmotic pump infusion or 1 week after direct injection of NTF4. Larynges were harvested, placed in cryoprotectant (30% sucrose and 70% phosphate buffered saline) and then serially sectioned in 10- &#181;m widths with a cryostat. Aging laryngeal muscles are affected by administration of NTF4 25. In addition to young and old rat, we compared the injected and non-injected side of the thyroarytenoid muscles. Typically we see a ...

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

Here, we present a protocol to describe the use of neurotrophin 4 (NTF4) systemically and directly to remodel rat aging laryngeal muscles.

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

The overall goal of this protocol is to deliver Neurotrophin 4 for the induction of aging rat laryngeal muscle remodeling. This method can help answer key questions related to laryngeal muscle function. For example, can age related muscle atrophy be reversed to improve vocal function.The main advantage of this technique is that it can be easily translated to human models of vocal aging. We can use injection methods that are typically used by otolaryngologists in humans to inject the solutions that are necessary to improve vocal function. Generally individuals new to this method will struggle because of the delicacy of the implantation and injection procedures.Demonstrating the procedure will be Tanya Seward, a technician from the department of physiology and Richard Andreatta, an associate professor in communication sciences and disorders, both of the University of Kentucky. To implant the pump, first place the rat in the ventral position in an aseptic surgical area. Wet the back and neck with iodine.Then scrub the dorsal aspect of the neck with 70%ethanol. Next move the animal onto a 34 degree Celsius heating pad and administer pre-anesthetic medication. Fill the aseptically prepared osmotic pumps with 50 microliters of Neurotrophin 4.And use a scalpel to make a two centimeter wide, horizontal incision through the skin just posterior to the space between the scapulae. Life the posterior edge of the incision with forceps and gently insert the tips of the hemostat posterior to the incision. When the tip is approximately two centimeters posterior to the incision, open the handles on the hemostat to make a hollow pocket subcutaneous to the incision site.Insert the pump into the pocket, orienting the end of the delivery portal such that any other interaction between the treatment and the healing of the pocket incision site is minimized. Then, use a 5-0 nylon suture thread, hemostat and forceps to close the pump placement incision and monitor the animal until it is fully alert and ambulatory. 30 minutes before the inject, warm aseptically prepared 50 microliter doses of the Neurotrophin or saline in a 25 degree Celsius water bath.When the treatments are ready, recline an anesthetized rat in the supine position on a plexiglass platform and use the guide wire to suspend the animal via the top frontal incisors. Next, attach a 30 gauge, 50 millimeter, 100 microliter syringe to a 1.9 millimeter 30 degrees sinus endoscope. And attach the syringe assembly to a jig on the outer wall of the endoscope.When the syringe is secure, load the treatment and attach the endoscope assembly to the camera. Then, use rubber tipped forceps to extend the tongue to the side and insert a plastic speculum to maintain oral patency. And turn on the halogen light source to the endoscope.With the video recorder on, immerse the distal end of the endoscope in warm water for a few seconds to minimize the development of condensation on the glass tip. And carefully insert the needle into the area of the left vocal fold, using visual feedback from the monitor as a guide. When the vocal fold is fully visible, inject the needle into the left thyroid arytenoid lateral to the white, medial edge of the vocal fold.And depress the plunger to deliver the treatment. If the epiglottis is stuck closed, preventing a clear view of the larynx, use an upward sweeping motion with the syringe tip to gently unstick the tissue. Then withdraw the needle and place the animal in its home cage on a heating pad until full recumbency.Typically, a change in fiber size is observed with age in Neurotrophin 4 treated animals. A lower lever of fibrosis and fiber generation is also observed in aged Neurotrophin treated animals. While the quantity of neural muscular junctions is increased.Once mastered, the osmotic pump and the injection procedure, can each be completed in one hour if they're performed properly. While attempting this procedure, it's important to remember to use a slow and gentle with the needle to prevent scratching and injury around the laryngeal area. Though this method can provide insight into the mechanisms of vocal fold function during the aging process, it can also be applied to other systems such as the pharynx and swallowing related disorders.

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Research

JoVE Journal

Developmental Biology

8.3K visualizações.  University of Kentucky. The overall goal of this protocol is to deliver Neurotrophin 4 for the induction of aging rat laryngeal muscle remodeling. This method can help answer key questions related to laryngeal muscle function. For example, can age related muscle atrophy be reversed to improve vocal function.The main advantage of this technique is that it can be easily translated to human models of vocal aging. We can use injection methods that are typically used by otolaryngologists in humans to inject the so...