Name: Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles (Video) | JoVE Journal
Uploaded: 2017-06-13T15:00:00.000+00:00
Duration: 5 min 50 s
Description: Discover how to deliver Neurotrophin 4 using osmotic pumps and direct injection to remodel aging rat laryngeal muscles. This translational method improves vocal function and addresses age-related muscle atrophy.

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5 minutes

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>

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</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><tbody><tr><td>Neurotrophin 4</td><td>Pepro Tech</td><td>450-04</td><td>200 ng in 50 &amp;mu;L</td></tr><tr><td>Alzet Osmotic Pump</td><td>DURECT Corporation</td><td>2001D</td><td></td></tr><tr><td>30 &amp;deg; endoscope</td><td>Stoltz</td><td>61029D</td><td></td></tr><tr><td>50 mm 30 gauge 100-&amp;mu;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&#039;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

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- µm 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 chang...

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

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Source: Andreatta, R. D. et. al., Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles. J. Vis. Exp. (2017)This video demonstrates neurotrophin 4 (NTF4) infusion in an aged rat model to restore laryngeal muscle function affected by aging-related denervation and reduced neuromuscular junctions. NTF4 triggers nerve regeneration and promotes neuromuscular junction formation, highlighting its therapeutic potential for laryngeal...

Neurotrophin 4 Infusion for Laryngeal Nerve Regeneration in an Aged Rat

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury.

An Implantable System For Chronic In Vivo Electromyography

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

We present a protocol for direct vagus nerve injection in rats, enabling drug delivery directly into the nerve without post-injection complications. This method applies to preclinical neurological studies involving autonomic nervous system manipulation. It can be used for direct nerve injection for other nerves in rats and other species, with necessary modifications.

Direct Vagus Nerve Injection Protocol For Rats

Discover how to deliver Neurotrophin 4 using osmotic pumps and direct injection to remodel aging rat laryngeal muscles. This translational method improves vocal function and addresses age-related muscle atrophy.

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

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