The authors want to thank Mr. Cagney Gentry for his audiovisual support in filming the procedure. We also want to acknowledge the Hefner VA medical center for academic support in pursuit of this project.

All procedures were performed at Wake Forest School of Medicine Clarkson Campus for animal studies. The Institutional Animal Care and Use Committee (IACUC) was consulted for ethical considerations and details of the procedures was submitted for review. Wake Forest IACUC approved our study protocol and all procedures were done under IACUC approved protocol #A17-147.
1. Preparing the infusion device
<ol>
	<li>Cut size 10 Polyethylene Tube (PET10) into 25 cm lengths using a pair of scissors.</li>
	<li>Mark PET10 at 1 cm and 2 cm from one end using a black marker.</li>
	<li>Prefill 0.5 mL of Ad5-EGFP solution (109 viral particles/mL) into a 1 mL (tuberculin) syringe.</li>
	<li>Slide non-marked end of PET10 tube over 29-31 G needle attached to a syringe. It is generally easier to perform this task under magnification.</li>
	<li>Infuse the solution into the PET10 until tube is completely full (visible drop at free end).</li>
	<li>Use full standard PPE, including&#160;surgical scrubs, long sleeved gown, impermeable gloves, surgical mask, face shield, hair bonnet, and shoe covers.</li>
</ol>
2. Preparing the animal
NOTE: Cynomolgus macaques were used for the video demonstration. The anatomy of other non-human and human primates is very similar, and the protocol should be translatable to other species.
<ol>
	<li>Inject subcutaneously 0.05 mg/kg of atropine 15 min prior to the procedure to minimize salivary secretions and optimize distribution and retention of the infusate.</li>
	<li>Provide anesthesia using 5 mL syringes with intramuscular ketamine/midazolam (10-15 mg/kg of ketamine and 0.01-0.05 mg/kg of midazolam). Confirm proper anesthesia when the sedated animal becomes unconscious and is unable to react to stimuli.</li>
</ol>
3. Performing the procedure
<ol>
	<li>Use oral retractors to brace open mouth.
	<ol>
		<li>Place the rubber pad of one end of the retractor on the hard palate behind the upper teeth on the side of the mouth opposite to the gland that will be infused. Place the rubber pad of the other end on the lower canine on the same side as upper retractor. Gently allow the spring action of the retractor to expand and open the mouth.</li>
	</ol>
	</li>
	<li>Identify the parotid papilla, the opening of Stensen&#39;s Duct, on the posterior cheek, adjacent to the upper 2nd molar. This is best visualized using dental loops for magnification.</li>
	<li>Gently dilate the parotid papilla with the point of the conical dilator. It is best to place the point of the dilator into the center or opening of the papilla and then gently rotate it back and forth. The point should slowly enter the papilla and dilate it over approximately 20 - 30 s of gentle rotating.</li>
	<li>Insert the PET10 tubing into the dilated parotid papilla. This is best achieved by holding the marked end of the PET10 tube with tweezers approximately 0.5 cm from the distal end and gently inserting the tip of the tube into the dilated papilla.
	<ol>
		<li>Gently advance the tube, which is often facilitated by small rotating movements to help the tube slide, followed by readjustment of the tweezers 0.5 cm proximal to the previous grip. Repeat this until the 2 cm mark reaches the parotid papilla.</li>
	</ol>
	</li>
	<li>Apply cyanoacrylate on the cheek around the papilla and the inserted tube and wait for it to dry (no specific amount recorded, just enough to seal the entrance of Stensen&#39;s duct papilla). This typically takes less than a minute and helps seal the parotid papilla and reduce spillage of infusate back in the oral cavity.</li>
	<li>Slowly push the syringe content over 5 min at a rate of 100 &#181;L/min. This slow infusion rate minimizes risk of duct injury due to sudden increase in intra-ductal pressure.</li>
	<li>Leave PET10 in place for at least 5 min after infusion is complete. Keep the duct sealed and allow the infusate to remain in the parotid gland.</li>
	<li>Remove PET10 with gentle traction. The cyanoacrylate will pull free with the tube.</li>
	<li>Repeat steps 3.2 through 3.8 on the opposite side.</li>
	<li>Slowly release oral retractors after both parotids have been infused and both PET10 tubes removed. 
	NOTE: The whole procedure for both sides should take less than 30 min.</li>
</ol>
4. Post-procedural care
<ol>
	<li>After infusion is completed and Stensen&#39;s duct decannulated, observe animals until anesthesia effect wears off (usually between 20-30 min post-procedure).</li>
	<li>Offer the animal drinks and then food after they are fully awake and resume routine care.</li>
</ol>

<ol>
	<li>Isenman, L., Liebow, C., Rothman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretion+of+mammalian+digestive+enzymes+by+exocrine+glands.">The secretion of mammalian digestive enzymes by exocrine glands.</a> The American Journal of Physiology. 276, 223-232 (1999).</li><li>Perez, P., 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=Salivary+epithelial+cells:+An+unassuming+target+site+for+gene+therapeutics.">Salivary epithelial cells: An unassuming target site for gene therapeutics.</a> The International Journal of Biochemistry &amp; Cell Biology. 42, 773-777 (2010).</li><li>Kochel, T. J., 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=A+dengue+virus+serotype-1+DNA+vaccine+induces+virus+neutralizing+antibodies+and+provides+protection+from+viral+challenge+in+Aotus+monkeys.">A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys.</a> Vaccine. 18, 3166-3173 (2000).</li><li>Ponzio, T. A., Sanders, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+salivary+gland+as+a+target+for+enhancing+immunization+response.">The salivary gland as a target for enhancing immunization response.</a> Tropical Diseases, Travel Medicine and Vaccines. 3, 4 (2017).</li><li>Baum, B. J., 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=Early+responses+to+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+for+radiation-induced+salivary+hypofunction.">Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 19403-19407 (2012).</li><li>Voutetakis, A., 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=Sorting+of+Transgenic+Secretory+Proteins+in+Rhesus+Macaque+Parotid+Glands+After+Adenovirus-Mediated+Gene+Transfer.">Sorting of Transgenic Secretory Proteins in Rhesus Macaque Parotid Glands After Adenovirus-Mediated Gene Transfer.</a> Human Gene Therapy. 19, 1401-1405 (2008).</li><li>Niedzinski, E. J., 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=Enhanced+systemic+transgene+expression+after+nonviral+salivary+gland+transfection+using+a+novel+endonuclease+inhibitor/DNA+formulation.">Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation.</a> Gene Therapy. 10, 2133-2138 (2003).</li><li>Niedzinski, E. J., 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=Zinc+Enhancement+of+Nonviral+Salivary+Gland+Transfection.">Zinc Enhancement of Nonviral Salivary Gland Transfection.</a> Molecular Therapy. 7, 396-400 (2003).</li><li>Samuni, Y., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+in+salivary+glands:+From+the+bench+to+the+clinic.">Gene delivery in salivary glands: From the bench to the clinic.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. , (2011).</li><li>Voutetakis, A., 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=Adeno-Associated+Virus+Serotype+2-Mediated+Gene+Transfer+to+The+Parotid+Glands+of+Nonhuman+Primates.">Adeno-Associated Virus Serotype 2-Mediated Gene Transfer to The Parotid Glands of Nonhuman Primates.</a> Human Gene Therapy. 18, 142-150 (2007).</li></ol>

The authors have nothing to disclose.

Here we describe a protocol of retrograde infusion into the parotid gland through Stensen&#39;s Duct. The methodology described offers guidance that can potentially be used by researchers exploring the utility of salivary tissue as a site for gene therapy and other applications.
There are multiple critical steps to ensure the success of the procedure. First and foremost, all the procedural steps should be completed gently. Forceful bracing of the mouth could result in mandibular subluxation. F...

While salivary glands are well known for their exocrine production of saliva, researchers have long recognized their ability to secrete proteins directly into the bloodstream1, making them a potential target for gene therapy for systemic administration, such as replacement hormones or antibody production. In fact, salivary glands offer several advantages over other tissue targets, such as the inherent ability to produce proteins for secretion (a property muscles lack), heavy encapsulation that can limit vector diffusion, and well-differentiated tissue providing stability for non-integrating vectors. Furthermore, in the event of a serious adverse event, salivary glands are not critical for life and can be surgically removed. While not immediately intuitive, parotid glands are also easily accessible from the mouth through their main excretory duct, Stensen&#39;s Duct2.
Given the advantages of salivary tissue for gene therapy, there is increasing interest in exploring this tissue target. Numerous studies have already been performed in rodent, canine, and non-human primate models and at least one human clinical trial is underway3,4,5. To further explore and develop the utility of this tissue target for gene therapy purposes, more non-human primate studies will need to be performed. This paper describes a method for accessing the parotid glands through Stensen&#39;s Duct to deliver a vectored gene for transduction in the non-human primate model. To visibly demonstrate the delivery of the infusate and the anatomy of the duct as it enters the gland, fluoroscopy using radiocontrast was performed. To demonstrate successful transduction of a vector, an Adenovirus serotype 5 (Ad5) vectored egfp gene was used. Ad5 is a well-described vector capable of transducing salivary tissue. Although it is too immunogenic for ultimate clinical use, an Ad5 vector was chosen for this demonstration study to assure efficient transduction. Evaluating Enhanced Green Fluorescent Protein (EGFP) production is a well-described method to demonstrate successful transcription and translation of a vectored gene following transduction and was done here.

<table><tbody><tr><td>500 &amp;micro;L U100 syringes with 30-gauge needles</td><td>Becton Dickinson</td><td>328466</td><td>fixed needle for less waste</td></tr><tr><td>Adhesive (e.g., Ethicon Dermabond)</td><td>Various</td><td></td><td>Cyanoacrylate adhesive to seal and keep the tubing in the duct during infusion.</td></tr><tr><td>Atropine injectable solution</td><td>Patterson Veterinary</td><td>07 869-6061</td><td>Atropine inj. 0.54 mg/mL</td></tr><tr><td>BD Ultra-Fine Insulin Syringes 30G</td><td>Walmart</td><td>N/A</td><td>Avilable in 0.5 mL and 1.0 mL sizes.</td></tr><tr><td>Cyanoacrylate (medical glue)</td><td>Ethicon</td><td>DNX12</td><td>Dermabond topical skin adhesive</td></tr><tr><td>Dental loops with light</td><td>Amazon (DDP)</td><td>B012M3IV80</td><td>Used to enhance visualization of Stensen&#039;s duct papilla</td></tr><tr><td>Infant Lacrimal Dilator</td><td>Surgipro</td><td>SPOI-137</td><td></td></tr><tr><td>Ketamine injectable solution</td><td>Patterson Veterinary</td><td>07-803-6637</td><td>Ketaset inj. 100 mg/mL</td></tr><tr><td>Lacrimal Dilator</td><td>Surgipro</td><td>SPOI-132</td><td>Used to dialate the Stensen&#039;s duct.</td></tr><tr><td>Midazolam injectable solution</td><td>Patterson Veterinary</td><td>07 890-6698</td><td>Midazolam inj. 5mg/mL</td></tr><tr><td>Pair of scissors</td><td>Amazon (DDP)</td><td>N/A</td><td>Used to cut PET10 tube</td></tr><tr><td>Polyethylene Tubing (PE-10)</td><td>Scientific Comodities, Inc</td><td>BB31695-PE/1</td><td>Tubing connecting the 30G syringe and inserted into the duct.</td></tr><tr><td>Q-tips</td><td>Walmart</td><td>N/A</td><td>Used to spread cyanoacrylate on the cheek</td></tr><tr><td>Size 10 Polyethylene Tube (PET 10)</td><td>Scientific Commodities</td><td>BB31695-PE/1</td><td>low density polyethylene tubing</td></tr><tr><td>Small Animal Mouth Opener</td><td>Amazon (DDP)</td><td>B01F3LVJXC</td><td>Used to keep the animal&#039;s mouth open.</td></tr><tr><td>Tweezers</td><td>Amazon (DDP)</td><td>N/A</td><td>Used to insert PET10 tube into Stenson&#039;s duct</td></tr><tr><td>Zinc Chloride</td><td>Sigma-Aldrich</td><td>7646-86-7</td><td>Included in plasmid DNA infusates</td></tr></tbody></table>

retrograde parotid gland infusion through stensen's duct in a non-human primate for vectored gene delivery

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of secreting proteins into the bloodstream and are easily accessible, making them potentially superior tissue sites for replacement hormone production or vaccination by gene transfer. Suggested methods for gene delivery include transcutaneous injection and retrograde infusion through salivary ducts. We demonstrate how to perform Retrograde Salivary Gland Infusion (RSGI) in non-human primates. We describe the important anatomic landmarks including identification of the parotid papilla, an atraumatic method of cannulating and sealing Stensen&#39;s Duct utilizing basic dental tools, polyethylene tubing, and cyanoacrylate, and the appropriate rate of infusion. While this is the least traumatic method of delivery, the method is still limited by the volume able to be delivered (&#60;0.5 mL) and the potential for trauma to the duct and gland. We demonstrate using fluoroscopy that an infusate can be fully delivered into the gland, and further demonstrate by immunohistochemistry the transduction of a typical vector and expression of the delivered gene.

Successful procedure, transduction and transcription 
Figure 1 shows the parotid papilla adjacent to the 2nd molar on the posterior superior cheek. The image also shows the correct placement of the mouth brace, one rubber end on the hard palate and the other rubber end on the ipsilateral canine. Figure 2 shows an image taken after successful cannulation of the parotid papilla at the 2 cm mark on the PET10. Figu...

Watch this Scientific Journal Video about Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery at JoVE.com

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery

Salivary glands have been proposed as a tissue target site for gene therapy, especially in the area of vaccination by gene transfer. We demonstrate gene delivery in a non-human primate model utilizing retrograde parotid infusion.

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of ...

retrograde-parotid-gland-infusion-through-stensen-s-duct-non-human

Research

JoVE Journal

Immunology and Infection

3.1K Views.  University of Florida. Dit protocol is belangrijk omdat het de procedure van retrograde speekselklierinfusie in detail demonstreert. De procedure kan om verschillende medische redenen worden gebruikt, zoals vectored immunoprofylaxe of gentherapie. Deze techniek is gemakkelijk te leren en te reproduceren.De auteurs zijn erin geslaagd om deze procedure meerdere keren met succes uit te voeren, ondanks het feit dat ze geen kaakchirurgen zijn, noch een dergelijke achtergrond hebben. Gebruik na het verdoven tandlo...

Video: Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

Medicine

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Neuroscience

Effective Systemic Delivery in Mice Using Retrobulbar Injection Technique

Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for systemic delivery with fewer limitations. First, tail vein injections (TVIs) are limited to adult mice where the size of the tail vein is suitable for access. Being restricted to treating adult mice can be problematic when dealing with mouse models that do not survive to adulthood. Second, TVIs are not feasible for mouse models with growth retardation phenotypes where the mice never achieve the size of adult wildtype mice. Therefore, retrobulbar injections can be successfully used to treat both young and small adult mice. Lastly, retrobulbar injections are performed under anesthesia, which is less stressful on the mice than TVIs that are commonly performed without anesthesia. This article presents a protocol and detailed instructions for retrobulbar injections that can be used for systemic delivery to small and young mice.

This article provides a protocol and an accompanying video for the retrobulbar sinus injection of up to a total volume of 150 &#181;L for postnatal, juvenile, and runted adult mice. This procedure is particularly well suited for the injection of small mice (15 g) when tail vein injection is not feasible.

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for ...

Genetics

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Bioengineering

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented.

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery

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