This study was supported by the Department of Veterans Affairs (VA-ORD BLR&#38;D Merit I01BX004536), and the National Institute of Health grants # 1R01DK105183, DK120394, and DK118529 to HW. We thank Dr. Hongju Wu for sharing technical experience.

All procedures were conducted with the approval of the Institutional Animal Care and Use Committees at the Medical University of South Carolina and the Ralph H. Johnson Medical Center. C57BL/6J male mice between 8-10 weeks of age were used in this study. Mice were housed under a standard 12 light/ 12 dark cycle with ad libitumaccess to food and water.
1. Preparation of TNBS solution for injection
<ol>
	<li>Prepare 10% ethanol in 0.9% saline. Dissolve stock TNBS (see Table of Material...

<ol>
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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=Histone+deacetylase+2+is+involved+in+&#181;-opioid+receptor+suppression+in+the+spinal+dorsal+horn+in+a+rat+model+of+chronic+pancreatitis+pain.">Histone deacetylase 2 is involved in &#181;-opioid receptor suppression in the spinal dorsal horn in a rat model of chronic pancreatitis pain.</a> Molecular Medicine Reports. 17 (2), 2803-2810 (2018).</li><li>Gui, F., 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=Trypsin+activity+governs+increased+susceptibility+to+pancreatitis+in+mice+expressing+human+PRSS1R122H.">Trypsin activity governs increased susceptibility to pancreatitis in mice expressing human PRSS1R122H.</a> The Journal of Clinical Investigation. 130 (1), 189-202 (2020).</li><li>Sun, Z., 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=Adipose+Stem+Cell+Therapy+Mitigates+Chronic+Pancreatitis+via+Differentiation+into+Acinar-like+Cells+in+Mice.">Adipose Stem Cell Therapy Mitigates Chronic Pancreatitis via Differentiation into Acinar-like Cells in Mice.</a> Molecular Therapy. 25 (11), 2490-2501 (2017).</li><li>Aghdassi, A. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+duct+pressure+in+chronic+pancreatitis.">Pancreatic duct pressure in chronic pancreatitis.</a> The American Journal of Surgery. 144 (3), 313-316 (1982).</li><li>Zhao, J. B., Liao, D. H., Nissen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pancreatitis:+can+it+be+translated+to+human+pain+study.">Animal models of pancreatitis: can it be translated to human pain study.</a> World Journal of Gastroenterology. 19 (42), 7222-7230 (2013).</li><li>Winston, J. H., He, Z. J., Shenoy, M., Xiao, S. Y., Pasricha, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+behavioral+changes+in+nociception+in+a+novel+rat+model+of+chronic+pancreatitis+for+the+study+of+pain.">Molecular and behavioral changes in nociception in a novel rat model of chronic pancreatitis for the study of pain.</a> Pain. 117 (1-2), 214-222 (2005).</li><li>Cattaruzza, F., 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=Transient+receptor+potential+ankyrin+1+mediates+chronic+pancreatitis+pain+in+mice.">Transient receptor potential ankyrin 1 mediates chronic pancreatitis pain in mice.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 304 (11), 1002-1012 (2013).</li><li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+insular+cortex+mediates+hyperalgesia+induced+by+chronic+pancreatitis+in+rats.">Anterior insular cortex mediates hyperalgesia induced by chronic pancreatitis in rats.</a> Molecular Brain. 12 (1), 76 (2019).</li><li>Puig-Div&#237;, V., 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=Induction+of+chronic+pancreatic+disease+by+trinitrobenzene+sulfonic+acid+infusion+into+rat+pancreatic+ducts.">Induction of chronic pancreatic disease by trinitrobenzene sulfonic acid infusion into rat pancreatic ducts.</a> Pancreas. 13 (4), 417-424 (1996).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAX4+Gene+Transfer+Induces+alpha-to-beta+Cell+Phenotypic+Conversion+and+Confers+Therapeutic+Benefits+for+Diabetes+Treatment.">PAX4 Gene Transfer Induces alpha-to-beta Cell Phenotypic Conversion and Confers Therapeutic Benefits for Diabetes Treatment.</a> Molecular Therapy. 24 (2), 251-260 (2016).</li><li>Ceppa, E. 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=Serine+proteases+mediate+inflammatory+pain+in+acute+pancreatitis.">Serine proteases mediate inflammatory pain in acute pancreatitis.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 300 (6), 1033-1042 (2011).</li><li>Puig-Divi, V., 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=Induction+of+chronic+pancreatic+disease+by+trinitrobenzene+sulfonic+acid+infusion+into+rat+pancreatic+ducts.">Induction of chronic pancreatic disease by trinitrobenzene sulfonic acid infusion into rat pancreatic ducts.</a> Pancreas. 13 (4), 417-424 (1996).</li><li>Xu, G. Y., Winston, J. H., Shenoy, M., Yin, H., Pasricha, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+excitability+and+suppression+of+A-type+K++current+of+pancreas-specific+afferent+neurons+in+a+rat+model+of+chronic+pancreatitis.">Enhanced excitability and suppression of A-type K+ current of pancreas-specific afferent neurons in a rat model of chronic pancreatitis.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 291 (3), 424-431 (2006).</li><li>Drewes, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pain+in+chronic+pancreatitis:+the+role+of+neuropathic+pain+mechanisms.">Pain in chronic pancreatitis: the role of neuropathic pain mechanisms.</a> Gut. 57 (11), 1616-1627 (2008).</li></ol>

Bile duct infusion of TNBS to induce chronic pancreatitis is technically challenging in mice, as up to 22.5% of mice can die within 3-4 days of drug infusion10. Here, this report refined the procedure based on previous studies and reduced early mouse mortality to &#60;10%. For example, the increased drug volume (from 35 &#956;L to 50&#956;L) can ensure the drugs reach&#160;the whole pancreas. Using an insulin syringe and a smaller needle size (31G) reduces potential damage to the pancreatic duct a...

Chronic pancreatitis (CP) is a chronic inflammatory disease characterized by the atrophy of the pancreas, fibrosis, abdominal pain, and eventual loss of both exocrine and endocrine functions1. Current medical and surgical treatments are not curative but are undertaken to relieve symptoms that are the consequence of the disease: refractory abdominal pain, endocrine and exocrine dysfunction. Therefore, more effective treatments are urgently needed2. Animal models provide an essential tool for developing a better understanding of the disease and investigating potential therapeutics3. Multiple mouse models for CP have been developed, of which cerulein and/or alcohol models are commonly used. Cerulein, an oligopeptide stimulating pancreatic secretion, has been shown to reproducibly induce a CP model featuring pancreatic atrophy, fibrosis, among others4. Another common model uses serial injections of L-arginine, which produces exocrine insufficiency similar to that observed in human patients5. CP can also be induced by complete or partial pancreatic duct ligation, as well as pancreatic duct hypertension6,7. Despite the variety of animal models available for CP, none of these models effectively reproduces the abdominal pain experienced by CP patients8.
Previous studies showed that local pancreatic injection of 2,4,6 -trinitrobenzene sulfonic acid (TNBS) replicates the persistent pain experienced by CP patients9,10,11. TNBS-treated mice demonstrated abdominal hypersensitivity and increased pain-related behaviors as well as a &#34;generalized hypersensitivity&#34; to painful stimuli, a phenomenon that has been observed in CP patients10. In addition to accurately mimicking CP pain, the TNBS model also replicates other pathological features of the human condition such as fibrosis, mononuclear cell infiltration, and replacement of acinar cells with fatty tissue10,12. However, TNBS infusion via bile duct is a technically challenging procedure in mice that may cause death. To our knowledge, there is no visual protocol to show how bile duct infusion is performed. In this article, we demonstrate the procedure of the bile duction infusion of TNBS to generate a CP mouse model. This procedure will help generate valuable animal models for the study of CP and other pancreatic diseases and can be used to infuse other materials (e.g., virus, cells) into the pancreas13.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Neutral buffered formalin v/v</td>
			<td>Fisher Scientific</td>
			<td>23426796</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads, sterile</td>
			<td>Fisher Scientific</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Anesthesia system</td>
			<td>VetEquip, Inc.</td>
			<td>901806</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride, injection</td>
			<td>Par Sterile Products, LLC</td>
			<td>NDC 42023-179-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>0553859A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, absolute (200 proof), molecular biology grade</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra fine Micro Dissecting scissors 4&#8221; straight sharp</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe forceps 4&#8221; extra delicate tip</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated pad</td>
			<td>Amazon</td>
			<td>B07HMKMBKM</td>
			<td></td>
		</tr>
		<tr>
			<td>Hegar-Baumgartner Needle Holder 5.25&#8221;</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-7850</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe with 31-gauge needle</td>
			<td>BD</td>
			<td>324909</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine prep pads</td>
			<td>Fisher Scientific</td>
			<td>19-027048</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Critical Care</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro clip applying forceps 5.5&#8221;</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5410</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro clip, straight strong curved 1x6mm</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5433</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro clip, straight, 0.75mm clip width</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5420</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrylsulfonic acid solution, TNBS, 1M in H2O</td>
			<td>Millipore Sigma</td>
			<td>92822-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Suture 4-0</td>
			<td>Med-Vet International</td>
			<td>MV-8683</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Suture 5-0</td>
			<td>Med-Vet International</td>
			<td>MV-8661</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride, 0.9% intravenous solution</td>
			<td>VWR</td>
			<td>2B1322Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical drape, sterile</td>
			<td>Med-Vet International</td>
			<td>DR1826</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Cassette</td>
			<td>Fisher Scientific</td>
			<td>22-272416</td>
			<td></td>
		</tr>
		<tr>
			<td>Von Frey filaments</td>
			<td>Bioseb</td>
			<td>EB2-VFF</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse model for chronic pancreatitis via bile duct tnbs infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

The bile duct infusion procedures were optimized to reduce mouse mortality associated with this procedure10. TNBS was first given in a total volume of 35 &#956;L or 50 &#956;L. Injection of TNBS in a volume of 50 &#956;L could reach the whole pancreas and induce a more homogeneous disease phenotype (Figure 1B). In addition, injection of TNBS using insulin syringe with 31G needle could better control infusion speed relative to regular syringes and needle sizes. Freshly...

Watch this Scientific Journal Video about A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion at JoVE.com

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Chronic pancreatitis is a complex disease involving pancreatic inflammation, fibrosis, abdominal pain, and other symptoms. The bile duct TNBS infusion model replicates the neuropathic pain observed in chronic pancreatitis patients. This bile duct TNBS infusion protocol has been optimized to induce the generation of a chronical pancreatitis model in mice.As bile duct drug fusion in mice is technically challenging, its visualization will be helpful to researchers new to the method. Before and two and three weeks after the surgery, use Von Frey monofilaments to apply different levels of force in ascending order to the upper abdominal area of an eight to 10 week old, C57 black six mouse, 10 times for one to two seconds per application. Consider raising retraction or licking of the abdomen as a positive response.Next, load an insulin syringe equipped with a 31 gauge needle with 50 microliters of a freshly prepared 0.75 percent TNBS solution. Load a second syringe with 50 microliters of 10 percent ethanol in saline as a vehicle control and place both syringes on ice protected from light. After confirming a lack of response to pedal reflex, remove the hair from the abdominal region of the anesthetized mouse by shaving or applying hair removal cream and place the mouse on a heated surgical pad under a dissecting microscope.Apply veterinary ointment to each eye, and disinfect the surgical site with three sequential two percent iodine and three 70 percent ethanol wipes use micro scissors to make a 0.5 to one centimeter incision in one continuous motion and use cotton swabs to gently expose the duodenum and to locate the common bile duct. To prevent the flow of the treatment into the liver and gallbladder, use the microscope to place a straight micro hemoclip over the proximal common duct, and insert the needle of the TNBS loaded syringe through the papilla of vater into the pancreatic duct. Use a curved micro hemoclip to secure the needle, and infuse the solution into the pancreatic duct over the course of one minute.When all of the solution has been delivered, carefully remove the micro clamps and confirm that the duodenum has returned to its original position. Before closure of the abdominal cavity, add 500 microliters of 36 to 37 degrees Celsius sterile saline to help the duodenum return to its original position and to assist with the recovery of peristalsis. Use a continuous suture with a five-O stitch to close the incision in the muscle layer, and close the skin using an interrupted suture with a four-O stitch.Then place the mouse in a recovery cage on a heating pad with monitoring until full recumbency. Monitor mouse health and behavior daily during the first week post-surgery for signs of distress, such as vocalizing, a hunched back posture or reduce locomotion and measure the body weight every other day. As observed, the injection of 50 microliters of ink as demonstrated results in the infiltration of the entire pancreas, allowing the induction of a more homogenous disease phenotype.In this representative analysis, in the first three days after surgery, the mice in the control group lost around six percent of their original body weight before gradually recovering and gaining over 100%of their original body weight by day 21. In contrast, mice that received TNBS lost about 15 percent of their original body weight during the first five days after surgery and regained just under 100 percent of their original body weight by day 21. Compared to control mice, the TNBS-injected animals showed an increased abdominal mechanical hypersensitivity during the second and third weeks after surgery.The size and weight per body weight of the pancreases, were significantly reduced in TNBS mice compared to pancreases harvested from control animals, suggesting a marked pancreatic atrophy in TNBS-treated animals consistent with the findings in humans with severe and longterm chronic pancreatitis. Histological analysis of pancreatic tissue sections from control ethanol-treated mice showed a normal morphology while tissue sections from TNBS-treated mice exhibited vacuous realization with a massive loss of acinar cells that were replaced by fat cell infiltration and fibrosis. Preventing the injected TNBS from entering the gal bladder and the intestine is important as a TNBS has tissue damaging effects.During the infusion, keep the hand as stable as possible to avoid puncture-acting that bile duct. In addition to inducing chronical pancreatitis development, this bile duct infusion mice art can be used for the development of the other pancreatic disease models. This TNBS chronic pancreatitis mouse model can be used to study both the pathogenesis and treatment of chronic pancreatitis as well as other pancreatic diseases.

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3.7K ビュー.  Medical University of South Carolina. 慢性膵炎は、膵炎、線維症、腹痛、その他の症状を伴う複雑な疾患です。胆管TNBS注入モデルは慢性膵炎患者で観察される神経因性疼痛を複製する。この胆管TNBS注入プロトコルは、マウスの慢性膵炎モデルの生成を誘導するために最適化されています。マウスの胆管薬物融合は技術的に困難であるため、その視覚化は、この方法に新しい研究者に役立ちます。手術?...

ビデオ: A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

acute biliary pancreatitis model: a method to generate acute biliary pancreatitis mouse model via pancreatic duct infusion

This video describes the procedure for the generation of a mouse model with acute biliary pancreatitis via pancreatic infusion of sodium taurocholate. This model helps study pathophysiological changes induced by sodium taurocholate in the...

Acute Biliary Pancreatitis Model: A Method to Generate Acute Biliary Pancreatitis Mouse Model via Pancreatic Duct Infusion

To perform the laparotomy with minimal exposure of the cavity, using a tractor pull the liver towards the head approximately 1 centimeter from the intestine and locate the region of the pancreas and the duodenum. Using forceps, lift the liver towards the animal head. By gently pulling the portion of the small intestine, fix the two lateral ends with a 6-0 polypropylene suture for a better view of the distal portion of the common bile duct...

determining bile duct density in the mouse liver

Determining Bile Duct Density in the Mouse Liver

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary...

partial bile duct ligation in the mouse: a controlled model of localized obstructive cholestasis

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in...

establishment of a mouse severe acute pancreatitis model using retrograde injection of sodium taurocholate into the biliopancreatic duct

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient...

a model of chronic nutrient infusion in the rat

A Model of Chronic Nutrient Infusion in the Rat

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...

extrahepatic bile duct and gall bladder dissection in nine-day-old mouse neonates

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile...

robotic lateral pancreaticojejunostomy for chronic pancreatitis

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

Robotic lateral pancreaticojejunostomy (RLPJ) may be used in patients with painful, morphine dependent, chronic pancreatitis and a dilated main pancreatic duct. We describe a standardized and reproducible technique for RLPJ, which includes transection of the gastroduodenal...

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3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating...

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

Retrograde Parotid Gland Infusion through Stensen&#039;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...

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

preparing a mice model of severe acute pancreatitis via a combination of caerulein and lipopolysaccharide intraperitoneal injection

Author Spotlight: Identifying the Therapeutic Targets for Developing New Treatments for Severe Acute Pancreatitis in Mice

Intraperitoneal drug administration is a safe and effective non-invasive approach for inducing pancreatic injury. This study compared five distinct intraperitoneal injection protocols on mice to induce varying degrees of pancreatic injury and established a model of severe pancreatic injury to investigate the pathological changes and treatment strategies for severe acute pancreatitis...

Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

cannulation of the mouse submandibular salivary gland via the wharton's duct

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton&#039;s Duct

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...

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

pancreatic duct infusion: an effective and selective method of drug and viral delivery

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented...

laparoscopic common bile duct exploration in patients with a previous history of biliary tract surgery

Laparoscopic Common Bile Duct Exploration in Patients with a Previous History of Biliary Tract Surgery

Programmed surgery based on surgical methods and anatomical markers helps shorten the operation time, reduce complications, and improve the safety of the surgery. This study investigated and summarized the surgical methods and anatomic markers of laparoscopic common bile duct exploration in patients with a history of biliary tract...

a mouse model of chronic liver fibrosis for the study of biliary atresia

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA...

continuous drug infusion system in mouse model: a surgical procedure to implant micro-osmotic pump infusion system in the mouse brain for continuous drug delivery

In this video, we demonstrate the assembly of a micro-osmotic pump brain infusion system followed by its surgical implantation in the mouse skull. This system enables continuous delivery of potential drugs and facilitates the study of their effects on the brain and brain...

Continuous Drug Infusion System in Mouse Model: A Surgical Procedure to Implant Micro-osmotic Pump Infusion System in the Mouse Brain for Continuous Drug Delivery

Then, use surgical scissors to cut the catheter tubes to desired length. Using a blunt-end brain infusion needle, attach the tubes to the brain infusion kit. Then, fill the tubes in the infusion kit with the drug solution. After assembling the kit, attach it to the micro-osmotic pump. Finally, to prevent the pump from drying out, immerse the osmotic pump brain infusion setting in aCSF within a sterilized 50-milliliter...

a mouse model for pathogen-induced chronic inflammation at local and systemic sites

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic...

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 ...

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

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  ...

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

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 ...