Research reported in this publication was supported by the National Institute of Dental and Craniofacial Research (NIDCR) and the National Cancer Institute (NCI) of the National Institutes of Health under Award Number R56 DE025098, UG3 DE027695, and F30 CA206296. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the NSF DMR 1206219 and the IADR Innovation in Oral Care Award (2016). 
We would like to thank Dr. Eri Maruyama and Andrew Hollomon for their assistance with saliva collection. We would like to thank Pei-Lun Weng for his assistance with gland dissection. We would like to thank Matthew Ingalls for his assistance in figure preparation. We would like to thank Dr. Elaine Smolock and Emily Wu for critical reading of this manuscript.

All in vivo procedures outlined below were approved by the University Committee on Animal Resources at the University of Rochester, Rochester. NY.
1. Preparation
<ol>
	<li>Using an analytical balance, weigh 20 mg of pilocarpine. Dissolve it in 2 mL of sterile saline in a microcentrifuge tube. 
	NOTE: Because pilocarpine is light sensitive and loses activity over time, this solution should be prepared the day of injection, and protected from light until administered.</li>
	<li>Using an analytical balance, weigh and identify all collection tubes and glass capillaries. The number of collection tubes and glass capillaries should match&#160;the number of mice.</li>
</ol>
2. Tracheostomy
<ol>
	<li>Weigh C57/BL6 mice using an analytical balance.</li>
	<li>Using a 0.5 mL syringe with 29G x &#189;&#34; needle, anesthetize mice with an intraperitoneally injected sterile saline solution of 100 mg/kg ketamine and 10 mg/kg xylazine. Proceed to the following step when the mouse no longer responds to stimuli (i.e.&#160;absence of&#160;the pedal withdrawal reflex), which generally occurs within 5 to 10 min following injection.</li>
	<li>After dispensing lubricant on a cotton-tipped applicator, gently apply to eyes and place the mouse in a supine position on the stage.&#160;The anesthetized animal must be kept on warm surface. 
	NOTE: All steps are performed at room temperature.</li>
	<li>Secure the nose, limbs, and tail to the stage using surgical tape.</li>
	<li>Clean and wet the neck with an alcohol wipe. 
	NOTE: This will help prevent fur from entering subsequent incisions.</li>
	<li>Raising the ventral midline neck skin with forceps, make a small, superficial cut using dissecting scissors. Guide the scissors into the opening and slowly open the blades subcutaneously to separate tissue planes.</li>
	<li>Keeping the skin raised, carefully cut to 1 cm below the mouth.</li>
	<li>Make two lateral incisions at the inferior and superior aspects of the first cut. Using forceps, gently reflect away the skin to enable access to head and neck structures.</li>
	<li>Using the dissecting microscope at 8X magnification, visualize the submandibular glands (midline: Figure 1).</li>
	<li>Using fine forceps, gently lift the glands to expose the four infrahyoid (strap) muscles overlying the trachea. Avoid tearing or disrupting the surrounding vasculature or excretory ducts.</li>
	<li>If collecting saliva from more than one mouse, repeat steps 2.1 - 2.10 with each mouse before continuing. 
	NOTE: This procedure can be safely performed on up to 5 mice concurrently.</li>
	<li>With small dissecting scissors, remove the medial portion of strap muscles while staying as close to midline as possible. Cut only as much as necessary to visualize trachea and keep strap muscles out of the way during the procedure. Avoid nicking any nearby vessels because if there is volume depletion secondary to significant hemorrhage, pilocarpine will not be effective at inducing secretion.</li>
	<li>Reflect the strap muscles away from the trachea.</li>
	<li>Visualize the larynx, trachea, and thyroid gland. Ensure that the trachea is clear of overlying tissue.</li>
	<li>Make a horizontal incision in the trachea inferior/posterior to the thyroid using small dissecting scissors. Ensure that the airway is patent and clear of fluid.</li>
</ol>
3. Saliva Collection
<ol>
	<li>Remove the tape near mouth and angle dissection stage downward (45&#176;, cranially) to assist with saliva flow.</li>
	<li>Using a 0.5 mL syringe with 29G x &#189;&#34; needle, perform intraperitoneal injection of 10 &#181;L/g body weight pilocarpine for a total dose of 100 mg/kg.</li>
	<li>Start a timer. If dosing multiple mice, reduce lead time by moving quickly and by having an assistant simultaneously inject the remaining mice.</li>
	<li>Using standard forceps, open the mouth for capillary tube access.</li>
	<li>Once a bead of saliva is observed in the mouth, place the proximal end of the capillary tube into the fluid, with the distal end placed into a collecting tube positioned below the dissecting stage (both tubes pre-weighed). 
	NOTE: In C57/BL6 female mice 6 - 10 weeks of age, gross salivation occurs within 2 min of pilocarpine injection.</li>
	<li>If saliva is building in the mouth, but not flowing through the tube, reattempt previous step. Ensure that the tube is not placed directly against a mucosal surface, which can obstruct the inlet.</li>
	<li>Collect saliva for a total of 12 min following pilocarpine stimulation. Ensure that the stoma remains clear. 
	NOTE: Increased tidal volume and secretions (salivary, urinary, fecal) are normal during this time.</li>
	<li>If mice have depressed saliva function (e.g., due to injury or intervention), transfer the remaining fluid from the mouth to the collection tube using a P200 micropipette. If collecting from multiple mice, ensure that tips are changed.</li>
	<li>Record the weight of collected saliva plus tubes 12 min after pilocarpine injection.</li>
</ol>
4. Gland Dissection
<ol>
	<li>Euthanize mice by CO2 euthanasia&#160;(as described in the AVMA Guidelines for Euthanasia of Animals: 2013 Edition) followed by thoracotomy, taking care to preserve the structures of the head and neck. For this reason, avoid cervical dislocation as a method of euthanasia.</li>
	<li>Place the mouse in a supine position on the stage. Reposition glands in the original site over the trachea (Figure 1).</li>
	<li>Using a dissecting microscope under 8X magnification, visualize the major glands: the PG, SMG, and SLG. To distinguish the glands during the dissection, identify that the PG is diffuse, lying lateral to the SMG and SLG. 
	NOTE: If dissecting the PG, this should be done first because it is the least defined and most likely to be torn.</li>
	<li>Grasp the tail of the PG with forceps, pulling it away from underlying structures, and gently apply tension before cutting the head of the PG with dissecting scissors.</li>
	<li>Breach the capsule surrounding the SMGs using forceps. Gently separate the left and right SMG.</li>
	<li>Free the dorsal aspect of the SMG from adhered nonglandular tissue using forceps.</li>
	<li>Remove the SMG from the neck by drawing Wharton&#39;s duct taut with forceps and cutting the duct with dissecting scissors.</li>
	<li>Gently separate the SLG from the SMG using forceps. 
	NOTE: The SLG is in the superolateral aspect of the SMG.</li>
	<li>Using an analytic balance, record SMG weights.</li>
</ol>

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The authors have nothing to disclose.

We present a multistep method to assess salivary gland function, which can be applied to study gland injury and therapeutics. Our procedure involves tracheostomy, saliva collection, and gland dissection, each of which has experimental applications that can support an integrated study of salivary gland biology. For example, murine tracheostomy has been used for general airway management during procedures obstructing the oral cavity.
Proper dissection and tracheal incision are required for pilocarpine stimulated saliva secretion at 100 mg/kg. Alternatively, a reduced pilocarpine dose could avoid the need for tracheostomy and therefore be used for longitudinal assessment of saliva secretion16.
In addition to recording saliva secretion, this protocol also provides an accurate, consistent means to collect saliva for additional analyses. Such studies include rheology, proteomics, enzyme activity, saliva osmolarity25,26,27, and biomarker discovery28,29.
Finally, standardized dissection methods for the major salivary glands have utility beyond saliva collection. There is an interest in understanding gland development and response to injury. For effective investigations, it is important to perform consistent, clean dissections of the major salivary glands. Clean and efficient gland isolation is critical for cultures derived from primary cells that are mechanically and enzymatically dissociated from the major salivary glands30,31, and for histologic and immunohistochemical preparations. Moreover, when staining for secreted proteins in gland tissue sections, it is critical to consider and control for the likely depletion of these products following stimulated saliva collection.
Quantifying salivary gland function in investigations of injury or intervention is a vital experimental technique. Gland histology is informative and may elicit key observations, yet conclusions are best supported by clear functional data.

Salivary gland disorders include syndromes of dysregulated or impaired secretion leading to either overproduction (sialorrhea) or underproduction (xerostomia and hyposalivation) of saliva1. In both cases, there is an interest in improving our understanding of salivary gland biology towards the end goal of therapeutic development2.
The salivary glands are highly radiosensitive organs, and are often damaged during head and neck cancer radiotherapy, leading to permanent dry mouth (xerostomia)3,4. Unlike other radiosensitive tissues, however, salivary gland turnover rate is relatively low, and the mechanism of secretory loss is poorly understood5,6. In this unique injury setting, tissue regeneration and radioprotection strategies require salivary functional assessment. Experimentally, murine saliva collection is a particularly valuable tool in evaluating the gland response to both radiation and therapeutic agents.
Here, we present a method for performing and quantifying stimulated saliva secretion using pilocarpine, a potent muscarinic agonist7. Pilocarpine stimulates the autonomic nervous system to induce gland secretion8,9. To complete this test appropriately, a tracheostomy is required to ensure that the mouse maintains a patent airway throughout the procedure, and to reduce the risks of choking and aspiration from pooled secretions in the oral cavity10.
This is a terminal procedure, culminating in the removal of the three major salivary glands: the parotid (PG), the submandibular (SMG), and the sublingual (SLG). For functional studies, gland weights are recorded and are often used to normalize saliva measurement11,12,13. This data is particularly important in radiation studies, wherein gland atrophy is an expected outcome14,15
There is variability in the literature with regards to how stimulated saliva secretion is performed and reported16. For example, pilocarpine doses within the literature span at least three orders of magnitude17,18,19,20,21,22,23. Here, we present an optimized high dose pilocarpine protocol with the intent of improved reproducibility in method execution, as well as providing a modular platform of techniques (tracheostomy, saliva collection, and gland dissection) that can be adapted as needed.
In addition to protocol demonstration, we include representative functional data of saliva flow at 2 weeks following fractionated radiation (4 doses of 6.85 Gy) to the SMG region.

<table border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Pilocarpine hydrochloride</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P6503</td>
			<td style="width:225px;">Pilocarpine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Student Vannas Spring Scissors</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">91500-9</td>
			<td>Spring Scissors for Tracheostomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Sterile Saline Solution</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">RDI30296H</td>
			<td>Saline</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:219px;">Dumont #7 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11274-20</td>
			<td>Curved Forceps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:219px;">Dumont #5 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11251-10</td>
			<td>Straight Forceps</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:219px;">Standard Pattern Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11000-12</td>
			<td>Blunt Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Fine Scissors- Tungsten Carbide</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">14568-09</td>
			<td>Dissection Scissors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Microhematocrit Heparinized Capillary Tubes</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:119px;">22362566</td>
			<td>Capillary tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Lubricant Eye Ointment</td>
			<td style="width:128px;">Refresh</td>
			<td style="width:119px;">N/A</td>
			<td>Refresh Lacri-Lube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Goat polyclonal anti-Nkcc1</td>
			<td style="width:128px;">Santa Cruz Biotech</td>
			<td style="width:119px;">SC-21545</td>
			<td>Nkcc1 Antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">GraphPad Prism</td>
			<td style="width:128px;">GraphPad</td>
			<td style="width:119px;">ver6.0</td>
			<td>Statistical Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Cotton tipped applicator</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td>Applicator for eye ointment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">0.5cc Insulin Syringe, 29G x 1/2&#34;</td>
			<td style="width:128px;">BD</td>
			<td style="width:119px;">7629</td>
			<td>Syringe for intraperitoneal injection</td>
		</tr>
	</tbody>
</table>

murine salivary functional assessment via pilocarpine stimulation following fractionated radiation

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).

When performing high dose pilocarpine stimulated saliva collection, it is important to maintain the airway to prevent aspiration or choking from secretions in the oral cavity. A schematic of the tracheostomy is provided (Figure 1). Following tracheal incision, the stoma must remain clear of tissue and fluids.
To enhance capillary action during saliva collection, mice should be positioned with their heads downwards at a 45&#176; angle. These steps can be performed on &#62;1 mouse, though it is not recommended to exceed 5 mice at one time (Figure 2). Once collection is completed, it is recommended that tubes be weighed quickly to avoid evaporative losses.
Following saliva collection, mice are euthanized. Cervical dislocation should not be performed, as it may damage head and neck structures. Mice are returned to the dissecting stage, and the glands should be repositioned over the trachea, as originally located. Gland dissection (Figure 3) should begin with the PG, due to both its position and diffuse consistency. The SMG and SLG are firm, and can be removed easily.
Following 4 fractions of 6.85 Gy radiation to the murine SMG, saliva function significantly decreases by 2 weeks (Figure 4). Both total saliva secretion, and secretion normalized to gland wet weight were plotted. Using either metric, salivary functional capacity is reduced, as expected, at 2 weeks following irradiation24.
Gland tissue can be fixed and sectioned for histological stains and/or immunohistochemistry. Hematoxylin and Eosin staining (H &#38; E) shows similar gross SMG architecture both with or without pilocarpine stimulation (Figure 5). Staining for Nkcc1, a membrane sodium-potassium-chloride transporter, shows similar cell morphology irrespective of pilocarpine stimulation (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57522/57522fig1.jpg" /> 
Figure 1. Tracheostomy schematic. (A)&#160;After opening the neck region, visualize the major structures. (B)&#160;To complete a tracheostomy, gently lift, but do not detach, the SMGs without disrupting neighboring structures, including blood supply, innervation, and excretory ducts. Cut the strap muscles to expose tracheal cartilage before making an incision into the trachea. Leave SMG in place and trachea exposed during entire saliva collection procedure. <a href="/files/ftp_upload/57522/57522fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57522/57522fig2.jpg" /> 
Figure 2. Saliva collection. Following pilocarpine stimulation, angle mice downwards and collect saliva through capillary tubes into pre-weighed collection tubes, which are placed below the dissection stage. <a href="/files/ftp_upload/57522/57522fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57522/57522fig3.jpg" /> 
Figure 3. Major salivary gland dissection. Once mice have been euthanized, reposition and separate the left and right SMG. Carefully separate each gland from surrounding nonglandular tissues and blood vessels. The PG is a diffuse, lateral structure. The SMG and SLG are joined, but can be cleanly separated with forceps. <a href="/files/ftp_upload/57522/57522fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57522/57522fig4.jpg" /> 
Figure 4. Representative secretion data. Two weeks following fractionated radiation (4 doses of 6.85 Gy) to the SMG, pilocarpine-stimulated saliva secretion was measured. Salivary function is reported as total saliva volume (A) and total saliva volume (B) normalized to total SMG wet weight. As expected, there is a significant decrease in the functional capacity of the SMG (mean &#177; SD **p &#60;0.01 using two-tailed unpaired t-test). <a href="/files/ftp_upload/57522/57522fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57522/57522fig5.jpg" /> 
Figure 5. Hematoxylin and Eosin SMG staining. SMG tissue was harvested from a mouse after pilocarpine stimulation (100 mg/kg, Right) compared to control, untreated mouse SMG (Left). H &#38; E staining shows gross gland structure at 10X&#160;(Scale bars: 200 &#181;m) (A, B) and 40X (Scale bars: 50 &#181;m) (C, D). <a href="/files/ftp_upload/57522/57522fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57522/57522fig6.jpg" /> 
Figure 6. Nkcc1 SMG Immunohistochemistry. SMG tissue was harvested from a mouse after pilocarpine stimulation (100 mg/kg, Right) compared to control, untreated mouse SMG (Left). Nkcc1 is a membrane protein and can be used to evaluate cellular morphology. DAPI is used as a nuclear counterstain (Scale bars: 75 &#181;m). <a href="/files/ftp_upload/57522/57522fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation at JoVE.com

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...

murine-salivary-functional-assessment-via-pilocarpine-stimulation

Nanyang Technological University

Research

JoVE Journal

Medicine

11.0K Views.  University of Rochester. We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

Video: Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

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 ...

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

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. ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...