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 U.S.C. et seq.); the animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Kentucky.
1. Anesthesia of Rats
<ol>
	<li>Prepare the anesthetics by mixing ketamine hydrochloride (dissociative anesthetic) and xylazine hydrochloride (sedative and analgesic) in buffered saline. The concentrations of ketamine and xylazine in the final solution are 100 mg/8 mg per kg body weight respectively.</li>
	<li>Inject the anesthetics into the rat by intraperitoneal administration using a syringe with a 25 G needle.</li>
	<li>Determine that the rat is sufficiently anesthetized by pinching the toe or foot with forceps. If the rat does not react to the pinch, then surgery can begin. If the rat reacts to the toe pinch with reflex or muscle contractions, then wait 1-2 min and repeat the pinch test. If the rat reacts again, replace the rat with a new animal and repeat procedure beginning from step 1.2.</li>
	<li>Apply ophthalmic ointment to the rat&#39;s eyes, after the rat is immobile, to prevent the corneas from drying out.</li>
</ol>
2. Osmotic Pump Implantation
<ol>
	<li>Place the rat ventral on the aseptic surgical area. &#160;Administer meloxicam as a preanesthetic medication. Administer intraperitoneally at a dosage of 1-4 mg/kg of body weight using a syringe with a 25 G needle.</li>
	<li>&#160;Use clippers to remove an approximately 1&#34; x 1&#34; square of fur from back of neck, and approximately 1&#34; caudal of space between shoulders. Shave as close to the skin as possible.</li>
	<li>Wet the back and neck with disinfecting ethanol (70%).</li>
	<li>After shaving, scrub the dorsal aspect of the neck with&#160;3 scrubs in succession of iodine-alcohol finishing with alcohol.</li>
	<li>Maintain the body temperature of the rat by placing it on a heating pad set to 34 &#176;C.</li>
	<li>Fill aseptically prepared osmotic pumps with either 50 &#181;L of NTF4 or saline for systemic NTF4 treatment (Figure 1).
	<ol>
		<li>Use a scalpel to make a horizontal incision approximately 2 cm wide through the skin, just cranial&#160;to the space between the scapulae. Lift the posterior edge of the incision with forceps with one hand while inserting the tip of hemostats and gently pushing posterior to the incision.</li>
		<li>After the tip of the hemostats is approximately 2 cm cranial&#160;to the incision, open the handles on the hemostats, expanding the tips to form a hollow &#34;pocket&#34; subcutaneous to the incision site. This will be the placement site for the pump.</li>
	</ol>
	</li>
	<li>Orient the pump delivery portal end first upon insertion to minimize any interaction of the NTF4 and the healing of the pocket incision site.</li>
	<li>Deliver 50 &#181;L of NTF4 saline for either 7 14 days. The 7 day group received 6.72 mg/day of NTF4 for a total dose of 47.04 mg. The 14-day group received 6.72 mg/day for a total dose of 94.08 mg of NTF425.</li>
	<li>Use 5-0 nylon suture thread, hemostats and forceps to close the incision made for pump placement.</li>
	<li>Observe the rats for a minimum of 30 min as they recover from anesthesia. Criteria for completion of monitoring include the animal becoming active, moving about the cage, drinking water, and beginning other normal activities such as grooming.</li>
	<li>Monitor animals daily for the first week by observing the healing of surgical site, normal feed and water consumption and passing of urine/feces, and any abnormal behavioral signs of stress, pain, or other post-operative complications.</li>
	<li>If the rat appears to be in pain or distress, provide the rat with a 5 mg/kg subcutaneous injection of carprofen once every 24 h for up to 5 days to relieve pain.</li>
	<li>If there appears to be an infection, consult a veterinarian to ensue that the wound heals properly.</li>
	<li>Depending on which experimental group the rat in in, remove the 5-0 nylon suture 7-10 days following surgery to prevent irritation from the thread.</li>
</ol>
3. Anesthesia of Rats for Direct Injection
<ol>
	<li>Withhold food from the rats the night before the procedure. This ensures that there is no food to block the endoscope or injection needle.</li>
	<li>Weigh rats and prepare acepromazine 1-2 mg/kg body weight. Inject intramuscularly (the IM location is the left thyroarytenoid muscle).</li>
	<li>Place the rat in the induction box. Induce anesthesia in the induction box with 5% isoflurane and 1 L O2.</li>
	<li>Move the rat to a nose cone with 2% isoflurane and 600 mL O2.</li>
	<li>Determine that the rat is sufficiently anesthetized by pinching the toe or foot with forceps. If the rat does not react to the pinch, then the injection protocol can begin. If the rat reacts to the toe pinch with reflex or muscle contractions, then wait 1-2 min and repeat the pinch test. If the rat reacts again, replace the rat with a new animal and repeat procedure beginning from step 3.4.</li>
</ol>
4. Direct Injection and Visualization
<ol>
	<li>Place aseptically prepared 50-&#181;L dosages containing NTF4 or saline in a H2O bath set to 25&#176; C for 30 min before injection.</li>
	<li>Place the rat in a supine and reclined position on a plexiglass platform (Figure 2). Suspend the rat in the reclined posture from their frontal top incisors via a guide wire strung across the top of the platform.</li>
	<li>Attach a 50 mm, 30 gauge, 100 &#181;L syringe to a 1.9 mm, 30&#176; sinus endoscope (Figure 3). 
	NOTE: The syringe assembly is attached via a jig that holds the cannula firmly to the outer wall of the endoscope. The endoscope allows for visualization of the vocal folds and guidance of the syringe intraorally. The position of the cannula tip is adjusted prior to each animal to ensure that the tip is fully and clearly visible via the endoscopic view (Figure 4).</li>
	<li>Use a rubber-tipped pair of forceps to extend the tongue and move it laterally. Afterward, insert a plastic speculum to maintain oral patency. Make the speculum from a 5 mL plastic syringe barrel that is cut to a length of 1.5 to 2 cm, with the cut edges deburred and polished smooth.</li>
	<li>Turn off the lights in the room and attach a halogen light source to the endoscope. Turn on the video recorder to capture the procedure.</li>
	<li>Immerse the distal end of the endoscope in warm water for a few seconds to minimize the development of condensation on the glass tip when inserted into the mouth of the rat.</li>
	<li>Using visual feedback from the monitor, carefully guide the needle to the area of the left vocal fold.</li>
	<li>Time the injection of the solution with the inspiratory phase of the animals&#39; respiration cycle to fully access the vocal fold. During the inspiratory phase of respiration, the vocal fold is fully exposed.
	<ol>
		<li>Once the vocal fold is fully visible, insert the needle into the left thyroarytenoid, found lateral to the white medial edge of the vocal fold. With the needle in place, deliver the injectate through depression of the syringe.</li>
	</ol>
	</li>
	<li>Turn off the halogen light source on the endoscope and the video player, and turn back on the room lights.</li>
	<li>Return the rat to its home cage and place on a heating pad.</li>
	<li>Allow the rat to recover before removal from the heating pad. Replace food and water in the cage.</li>
	<li>Monitor rats for 7 days after the injection and then euthanize. Remove the larynges for cryosectioning24.</li>
</ol>
5. Euthanization of Rats
<ol>
	<li>Anesthetize rats with ketamine hydrochloride and xylazine hydrochloride (100 mg/8 mg per kg body weight injected intraperitoneal injection).</li>
	<li>Euthanize by exsanguination following a medial thoracotomy.</li>
</ol>

<ol>
	<li>Trupe, E. H., 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=Correlates+and+consequences+of+eating+dependency+in+institutionalized+elderly.">Correlates and consequences of eating dependency in institutionalized elderly.</a> J. Am. Geriatrics Society. 34 (3), 192-198 (1986).</li><li>Ward, P. H., Colton, R., McConnell, F., Malmgren, L., Kashima, H., Woodson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+of+the+voice+and+swallowing.">Aging of the voice and swallowing.</a> Otolaryngol. Head Neck Surg. 100 (4), 283-286 (1989).</li><li>Gay, E. G., Kronenfeld, J. J., Dettmann, F. 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+comparison+on+the+effect+of+regulation+on+health+care+for+the+older+American:+a+tale+of+two+states.">A comparison on the effect of regulation on health care for the older American: a tale of two states.</a> Gerontologist. 34 (6), 787-796 (1994).</li><li>Hagen, P., Lyons, G. D., Nuss, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysphonia+in+the+elderly:diagnosis+and+management+of+age-related+voice+changes.">Dysphonia in the elderly:diagnosis and management of age-related voice changes.</a> South. Med. J. 89 (2), 204-207 (1996).</li><li>Broniatowski, 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=Current+evaluation+and+treatment+of+patients+with+swallowing+disorders.">Current evaluation and treatment of patients with swallowing disorders.</a> Otolaryngol. Head Neck Surgery. 120 (4), 464-473 (1999).</li><li>Lundy, D. S., 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=Aspiration:+cause+and+implications.">Aspiration: cause and implications.</a> Otolaryngol. Head Neck Surg. 120 (4), 474-478 (1999).</li><li>Schindler, J. S., Kelly, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swallowing+disorders+in+the+elderly.">Swallowing disorders in the elderly.</a> Laryngoscope. 112 (4), 589-602 (2002).</li><li>Gambino, D. R., Malmgren, L. T., Gacek, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+the+neuromuscular+junctions+in+the+human+posterior+cricoarytenoid+muscles:+a+quantitative+study.">Age-related changes in the neuromuscular junctions in the human posterior cricoarytenoid muscles: a quantitative study.</a> Laryngoscope. 100 (3), 262-268 (1990).</li><li>Sinard, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aging+voice:+how+to+differentiate+disease+from+normal+changes.">The aging voice: how to differentiate disease from normal changes.</a> Geriatrics. 53 (7), 76-79 (1998).</li><li>Baker, K. K., Olson-Raming, L., Sapir, S., Luschei, E. S., Smith, 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=Control+of+vocal+loudness+in+young+and+old+adults.">Control of vocal loudness in young and old adults.</a> J. Speech Lang. Hear Res. 44 (2), 297-305 (2001).</li><li>McMullen, C. A., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+morphological+evidence+of+age-related+denervation+in+rat+laryngeal+muscles.">Functional and morphological evidence of age-related denervation in rat laryngeal muscles.</a> J. Gerontol. A. Biol. Sci. Med. Sci. 64 (4), 435-442 (2009).</li><li>Lai, K., Ip, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+signaling+of+new+players+at+the+neuromuscular+junction.">Postsynaptic signaling of new players at the neuromuscular junction.</a> J. Neurocytol. 32 (5-8), 727-741 (2003).</li><li>Huang, E. J., Reichardt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins:+roles+in+neuronal+development+and+function.">Neurotrophins: roles in neuronal development and function.</a> Annu. Rev. 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. 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=Effects+of+transforming+growth+factor-beta+1+on+human+vocal+fold+fibroblasts.">Effects of transforming growth factor-beta 1 on human vocal fold fibroblasts.</a> Ann. Oto. Rhinol. Laryngol. 118 (3), 218-226 (2009).</li><li>Kishimoto, Y., 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=Effects+of+exogenous+hepatocyte+growth+factor+on+vocal+fold+fibroblasts.">Effects of exogenous hepatocyte growth factor on vocal fold fibroblasts.</a> Ann. Otol. Rhinol. Laryngol. 118 (8), 606-611 (2009).</li><li>Belluardo, N., 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=Neuromuscular+junction+disassembly+and+muscle+fatigue+in+mice+lacking+neurotrophin-4.">Neuromuscular junction disassembly and muscle fatigue in mice lacking neurotrophin-4.</a> Mol. Cell. Neurosci. 18 (1), 56-57 (2001).</li><li>Johnson, A. M., Ciucci, M. R., Connor, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vocal+training+mitigates+age-related+changes+within+the+vocal+mechanisms+in+the+old+rat.">Vocal training mitigates age-related changes within the vocal mechanisms in the old rat.</a> J. Gerontol. A Biol. Sci. Med. Sci. 68 (12), 1458-1468 (2013).</li><li>Roscow, D. E., Parikh, P., Vivero, R. J., Casiano, R. R., Lundy, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+initial+dosing+of+botulinum+toxin+in+treatment+of+adductor+spasmodic+dysphonia.">Considerations for initial dosing of botulinum toxin in treatment of adductor spasmodic dysphonia.</a> Oto. HeadNeck Surg. 148 (6), 1003-1006 (2013).</li><li>Benninger, M. S., Hanick, A., Hicks, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cricothyroid+muscle+botulinum+toxin+injection+to+improve+airway+for+bilateral+recurrent+laryngeal+nerve+paralysis,+A+case+series.">Cricothyroid muscle botulinum toxin injection to improve airway for bilateral recurrent laryngeal nerve paralysis, A case series.</a> J. Voice. 30 (1), 96-99 (2016).</li><li>Coppoolse, J. M. S., 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=An+in+vivo+study+of+composite+microgels+based+on+hyaluronic+acid+and+gelatin+for+the+reconstruction+of+surgically+injured+rat+vocal+folds.">An in vivo study of composite microgels based on hyaluronic acid and gelatin for the reconstruction of surgically injured rat vocal folds.</a> J. Speech Lang Hear Res. 57 (2), S658-S673 (2014).</li><li>Herteg&#229;rd, S., 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=Cross-linked+hyaluronan+used+as+augmentation+substance+for+treatment+of+glottal+insufficiency:+Safety+aspects+and+vocal+fold+function.">Cross-linked hyaluronan used as augmentation substance for treatment of glottal insufficiency: Safety aspects and vocal fold function.</a> Laryngoscope. 112 (12), 2211-2219 (2002).</li><li>Walker, W. 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. Rep. 4 (10), e12798 (2016).</li><li>McMullen, C. A., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+dysfunction+and+altered+metabolic+profile+of+the+aging+rat+thyroarytenoid+muscle.">Contractile dysfunction and altered metabolic profile of the aging rat thyroarytenoid muscle.</a> J. Appl. Phys. 100 (2), 602-608 (2006).</li><li>Engle, W. K., Cunningham, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+examination+of+muscle+tissue.+An+improved+trichrome+method+for+fresh-frozen+biopsy+section.">Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy section.</a> Neurology. (13), 919-923 (1963).</li><li>Sheehan, D. C., Hrapchack, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+and+practice+of+histotechnology.">Theory and practice of histotechnology.</a> Battelle. , (1980).</li><li>Kulakowski, S. A., Parker, S. D., Personius, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+TrkB+expression+results+in+precocious+age-like+changes+in+neuromuscular+structure,+neurotransmission,+and+muscle+function.">Reduced TrkB expression results in precocious age-like changes in neuromuscular structure, neurotransmission, and muscle function.</a> J. Appl. Phys. 111 (3), 844-852 (2011).</li><li>Nishida, N., 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=Age-related+change+in+rat+intrinsic+laryngeal+muscles:+analysis+of+muscle+fibers,+muscle+fiber+proteins+and+subneural+apparatuses.">Age-related change in rat intrinsic laryngeal muscles: analysis of muscle fibers, muscle fiber proteins and subneural apparatuses.</a> Eur Arch Otorhinolaryngol. 270 (3), 975-984 (2013).</li></ol>

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 ...

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

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 ...

subcutaneous-neurotrophin-4-infusion-using-osmotic-pumps-or-direct

Research

JoVE Journal

Developmental Biology

8.3K Views.  University of Kentucky. Here, we present a protocol to describe the use of neurotrophin 4 (NTF4) systemically and directly to remodel rat aging laryngeal muscles.

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

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...