Name: retroductal submandibular gland instillation and localized fractionated irradiation in a rat model of salivary hypofunction
Uploaded: 2016-04-24T15:00:00
Description: Watch this Scientific Journal Video about Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction at JoVE.com

We thank Dr. John Chang (Radiation Oncology, LSU Health Science Center) for assistance with radiation dose measurements. The study was supported by the American Cancer Society (Grant number: 116945-RSG-09-038-01-CCE), National Institute of Health (Grant number: R21CA173162) and the Feist-Weiller Cancer Center.

All procedures were approved by the LSU Health, Shreveport, Animal Care and Use Committee and were in accordance with the NIH guidelines for the care and use of laboratory animals.
1. Cannulation of Rat Submandibular Salivary Glands
<ol>
	<li>Preparation of Syringe-Tubing Assembly
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		<li>Cut a 10 cm length of PE10 polyethylene tubing with a scalpel. Hold both ends of the tubing between the index finger and thumb. Heat the middle section of the tubing above a gentle flam...

<ol>
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J., Goldenberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+complications+of+radiotherapy.">Oral complications of radiotherapy.</a> Lancet Oncol. 7 (2), 175-183 (2006).</li><li>Rodrigues, N. 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=A+prospective+study+of+salivary+gland+function+in+lymphoma+patients+receiving+head+and+neck+irradiation.">A prospective study of salivary gland function in lymphoma patients receiving head and neck irradiation.</a> Int J Radiat Oncol Biol Phys. 75 (4), 1079-1083 (2009).</li><li>Coracin, F. L., 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=Major+salivary+gland+damage+in+allogeneic+hematopoietic+progenitor+cell+transplantation+assessed+by+scintigraphic+methods.">Major salivary gland damage in allogeneic hematopoietic progenitor cell transplantation assessed by scintigraphic methods.</a> Bone Marrow Transplant. 37 (10), 955-959 (2006).</li><li>Jensen, S. B., 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+systematic+review+of+salivary+gland+hypofunction+and+xerostomia+induced+by+cancer+therapies:+management+strategies+and+economic+impact.">A systematic review of salivary gland hypofunction and xerostomia induced by cancer therapies: management strategies and economic impact.</a> Support Care Cancer. 18 (8), 1061-1079 (2010).</li><li>de Castro, G., Federico, M. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+irradiation-induced+salivary+hypofunction+by+microvessel+protection+in+mouse+salivary+glands.">Prevention of irradiation-induced salivary hypofunction by microvessel protection in mouse salivary glands.</a> Mol Ther. 15 (12), 2101-2106 (2007).</li><li>Zheng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+radiation-induced+salivary+hypofunction+following+hKGF+gene+delivery+to+murine+submandibular+glands.">Prevention of radiation-induced salivary hypofunction following hKGF gene delivery to murine submandibular glands.</a> Clin Cancer Res. 17 (9), 2842-2851 (2011).</li><li>Palaniyandi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+delivery+of+Tousled+kinase+for+the+protection+salivary+glands+against+ionizing+radiation+damage.">Adenoviral delivery of Tousled kinase for the protection salivary glands against ionizing radiation damage.</a> Gene Ther. 18 (3), 275-282 (2011).</li><li>Baum, B. J., Voutetakis, A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+glands:+novel+target+sites+for+gene+therapeutics.">Salivary glands: novel target sites for gene therapeutics.</a> Trends Mol Med. 10 (12), 585-590 (2004).</li><li>Limesand, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor-1+preserves+salivary+gland+function+after+fractionated+radiation.">Insulin-like growth factor-1 preserves salivary gland function after fractionated radiation.</a> Int J Radiat Oncol Biol Phys. 78 (2), 579-586 (2010).</li><li>Timiri Shanmugam, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+AAV9-TLK1B+administration+ameliorates+fractionated+radiation-induced+xerostomia.">Recombinant AAV9-TLK1B administration ameliorates fractionated radiation-induced xerostomia.</a> Hum Gene Ther. 24 (6), 604-612 (2013).</li><li>Coppes, R. P., Vissink, A., Konings, A. W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+radiosensitivity+of+rat+parotid+and+submandibular+glands+after+different+radiation+schedules.">Comparison of radiosensitivity of rat parotid and submandibular glands after different radiation schedules.</a> Radiother Oncol. 63 (3), 321-328 (2002).</li><li>Sunavala-Dossabhoy, G., Palaniyandi, S., Richardson, C., De Benedetti, A., Schrott, L., Caldito, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAT-mediated+delivery+of+Tousled+protein+to+salivary+glands+protects+against+radiation-induced+hypofunction.">TAT-mediated delivery of Tousled protein to salivary glands protects against radiation-induced hypofunction.</a> Int J Radiat Oncol Biol Phys. 84 (1), 257-265 (2012).</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=Transfer+of+the+AQP1+cDNA+for+the+correction+of+radiation-induced+salivary+hypofunction.">Transfer of the AQP1 cDNA for the correction of radiation-induced salivary hypofunction.</a> Biochim Biophys Acta. 1758 (8), 1071-1077 (2006).</li><li>Tran, S. D., 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=Paracrine+effects+of+bone+marrow+soup+restore+organ+function,+regeneration,+and+repair+in+salivary+glands+damaged+by+irradiation.">Paracrine effects of bone marrow soup restore organ function, regeneration, and repair in salivary glands damaged by irradiation.</a> PLoS One. 8 (4), e61632 (2013).</li><li>Nanduri, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salisphere+derived+c-Kit++cell+transplantation+restores+tissue+homeostasis+in+irradiated+salivary+gland.">Salisphere derived c-Kit+ cell transplantation restores tissue homeostasis in irradiated salivary gland.</a> Radiother Oncol. 108 (3), 458-463 (2013).</li><li>Arany, S., Benoit, D. S., Dewhurst, S., Ovitt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-mediated+gene+silencing+confers+radioprotection+to+salivary+glands+in+vivo.">Nanoparticle-mediated gene silencing confers radioprotection to salivary glands in vivo.</a> Mol Ther. 21 (6), 1182-1194 (2013).</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=Reengineered+salivary+glands+are+stable+endogenous+bioreactors+for+systemic+gene+therapeutics.">Reengineered salivary glands are stable endogenous bioreactors for systemic gene therapeutics.</a> Proc Natl Acad Sci U S A. 101 (9), 3053-3058 (2004).</li></ol>

Salivary glands often receive radiation doses beyond the threshold of tissue recovery in patients undergoing radiotherapy for head neck cancer, elective ablation of neck nodes, or regional hematologic malignancies. Although the fluid-secreting acinar cells of the gland are terminally differentiated, they are paradoxically sensitive to radiation. The secretory function drops within the first weeks of radiation, and irreversible gland damage results in a chronic low saliva output. To combat poor gland function and oral dry...

Collateral destruction of healthy tissues accounts for a number of deleterious side-effects of cancer treatments. A part or whole of the major salivary glands that lie with the radiation fields are inevitably destroyed. Therefore, most patients undergoing radiotherapy for head and neck cancer, cervical lymphoma, or full-body radiation before bone marrow transplantation suffer one of the most common and persistent adverse effects of radiation, salivary gland hypofunction2-6.
The fluid-producing acinar cells of the salivary glands are acutely sensitive to radiation. Damage to the salivary glands causes a drastic diminishing of salivary flow, a condition referred to as salivary hypofunction. The chronic reduction in salivary flow impairs key oral activities such as mastication, swallowing, speech, and taste, but the morbid sequelae of intense pain, mucosal tears, dysphagia, opportunistic infections, and dental caries worsens a patient&#39;s well-being and function2,3.
Since radiotherapy-associated salivary cell loss is irreversible, there is no corrective treatment of xerostomia. Current treatment that focuses on assuaging symptoms with artificial salivary substitutes and prosecretory drugs is ineffective for long-term relief6. Although improved radiation delivery techniques have helped diminish the severity of the condition, normal tissue toxicity and its complications remain a limiting factor in cancer treatment6,7. Pre-emptive measures to prevent radiotherapy-associated complications are, therefore, becoming the norm. Radio-protective agents that scavenge free radical oxygen species, foster cell repopulation, or enhance DNA repair are being explored to avert salivary hypofunction 8-11.
Secretions of exocrine salivary glands drain into the mouth through the main excretory ducts. Intra-oral cannulation of the excretory ducts for injection of contrast agents is done routinely as an outpatient procedure. Utilizing a similar approach, salivary glands can be directly targeted for localized treatment12. Apart from reducing the risk of systemic side-effects, retroductal gland instillation has added benefits. The monolayer arrangement of salivary cells around the ductal tree allows targeting of all salivary epithelial cells, and the fibrous encapsulation of the gland acts as a barrier to reduce unwanted therapeutic spread. In essence, salivary glands are optimally suited for targeted treatment of gland afflictions such as radiation-induced salivary hypofunction.
Conventional radiation for cancer treatment is delivered in small doses (1.8 - 2.5 Gy/fraction/day, five days a week) for a period of weeks. Therefore, a radio-protective therapeutic that shows efficacy against a protracted radiation scheme in experimental models has greater clinical bearing. Compromised salivary function after fractionated radiation has been recorded in small animals, but radiation source, dose fraction, and protocols used are varied9,10,13.
This report establishes methods for retroductal delivery to and localized radiation of rat submandibular glands using patient-relevant radiation source and dose fraction.

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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.

Adapting a minimally invasive sialography technique, local treatment of major salivary glands is feasible. Retroductal administration in rat submandibular salivary glands was attempted by intra-oral cannulation of Wharton&#39;s ducts (Figure 2). The salivary ducts of Wharton open on the sub-lingual papillae located on the floor of the mouth, but the orifices are not readily visible. Insertion of the cannula was, therefore, done by gentle probing. To avoid untoward bleedin...

Watch this Scientific Journal Video about Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction at JoVE.com

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

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. ...

The overall goal of this procedure is to retroductally administer therapeutics to the submandibular glands to assess their efficacy in combating salivary disfunction. This method can help answer key questions in the field of salivary gland disorders such as what therapies can protect against or restore salivary gland function against radiation. The main advantage of this technique is that salivary glands can be treated locally thereby reducing the risk of nontarget tissue side effects.Generally people new to this method will struggle because the duct openings are not visible and the cannula has to be manipulated blindly into the duct. Before beginning the procedure cut a ten centimeter length of PE 10 polyethylene tubing with a scalpal, then grasp both ends of the tubing between the index finger and thumb of each hand and heat the middle section of the tubing above a gentle flame carefully pulling both sides to stretch the softened tubing to double it's length. Next use a scalpal to cut the tubing in the middle at a 45 degree angle to obtain two cannuli each with a tapered end.Stretch the non-tapered end of one of the cannuli over the 29 gauge needle of 0.5cc insulin syringe. Then remove the cannula and draw 220 microliters of saline solution into the syringe. Tap the syringe shaft to dislodge any air bubbles and reattach the cannula over the needle.Depress the plunger to expell the air from the cannula and to ensure a free flow of solution through the tubing and adjust the volume of saline in the syringe to 200 microliters. Now confirm the appropriate level of sedation of the rat by toe pinch and palpable reflex and apply eye lubricant over the exposed corneas. Place the amimal on a specially designed restraining platform and engage the upper incisor teeth on the transverse bar.Loop a rubber band around the lower incisors to pull down the jaw. Anchoring it to the platform. Next pass a sterile suture through the tongue and lift it up to raise the floor of the mouth.Then use a hemostat to clamp the sutures and pass the hemostat over the transverse bar. Next use a custom built cheek spreader to expand the cheeks and a disecting microscope to locate the sublingual papilli on the floor of the mouth. Using delicate forcebs grasp the tapered end of the preformed PE 10 tubing and gently manipulate the tip of the cannula into the ductal orifice on the sublingual papilla.Then thread the cannula three to five millimeters into the duct making sure that the cannula passes throught the duct without obstruction. Before instilling the saline inject atrophine subcutaniously into the scruff of the animal's neck. Ten minutes after the injection secure the cannula to the duct orifice with a drop of cyanoacrylate.After the glue has dried slowly depress the syringe plunger at 50 microliters per minute until 200 microliters of the saline solution has been instilled into the gland. If you encounter back preesure during instillation stop and then withdraw the tubing then recanulate with a new cannula. Then clamp the tubing with a hemostat and carefully remove the syringe.Leave the tubing in the duct for 30 to 60 minutes until the animal regains conciousness. Then remove the suture that holds the tongue and transfer the animal into a separate cage under a heat lamp until it is fully recovered. To locally apply fractionated irradiation to the submandibular gland.Columnate a three centimeter slit of radiation field that emcompasses the area from the lower border of the manible to the top of the sternum of the anesthetized rat in the supine position. After placing a one centimeter tissue equivilant bolus over the region. Adjust the distance between the radiation source and the top of the bolus to 100 centimetes using a six megavolt photon beam on the linear accelerator.Irradiate the animal with 2.5 grays for the appropriate length of exposure. At the end of the radiation exposure period warm the animal during it's recovery as just demonstated. During the initial experiments the proper placement of the cannula was confirmed by infusing the gland with hematoxylin or tryplan blue solution and evaluating the gland staining in euthanized animals.The submandibular glands were studied because they can be selectively irradiated with columnated radiation to the neck avoiding severe oral off target effects that can effect the animals health and experimental outcome. Analysis of the effects of this fractionated radiation scheme on the salivary flow at eight weeks post radiation demonstrates that daily exposure to 2.5 grey increments precipitously reduces the salivary flow. Within your 10 fold reduction in the saliva output in irradiated animals compared to non-irradiated animals.Once mastered this technique can be completed in a few minutes if it is performed properly. While attempting this procedure it is important to remember to ensure the unrestricted passage of cannula into the excratory duct of salivary gland. After it's development this technique paved the way for researchers in the field of salivary gland disorders to explore treatments for radiation and autoimmune salivary gland afflictions.After watching this video you should have a good understanding of how to perform retroductal installation in the rat's submandibular gland and to locally irradiate the glands to assess the the therapeutic efficacy of radiation toxicity amelioration. Don't forget that working with gene and protein therapeutics can be extremely dangerous and the precautions such as avoiding needle pricks and minimizing skin exposure to biological agents should alwasys be taken while performing this procedure.

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Medicine

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 Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

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 ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

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).

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

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 ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.