This work was supported by grants from the National Institutes of Health (NIH) to S.S.H. (R01-DK078640), from the Howard Hughes Medical Institute (HHMI) through the HHMI/Vanderbilt University Certificate Program in Molecular Medicine to T.J.W. (GRDOT56006779), the Vanderbilt Diabetes Research and Training Center (P30-DK020593) and the Vanderbilt Digestive Disease Research Center (P30-DK058404) providing Core Services.

1. Prepare Cannula
<ol>
 <li>Warm a 1-inch long section of PE10 tubing with your fingertips and stretch it so that the tubing becomes thin. 
 Note: the size of the vessel or duct to be cannulated will determine the degree of stretching required. The cannula must be well-stretched for bile duct casts but may only be required to be moderately stretched to fit within the larger portal vein.</li>
 <li>Cut the stretched tubing at a diagonal to generate a beveled tip.</li>
 <li>Cut the other edge of the tubing to create a 5-6 inch segment.</li>
 <li>Insert a 32 gauge, &frac12; inch hypodermic needle into the non-beveled edge of the tubing.</li>
</ol>
2. Prepare Syringe with PBS
<ol>
 <li>Fill a 3 ml syringe with PBS.</li>
 <li>Screw the syringe onto the hypodermic needle with attached pulled PE10 tubing previously prepared.</li>
 <li>Flick syringe and push syringe plunger to ensure that PBS has filled the needle and tubing and there are no air bubbles in the syringe.</li>
 <li>Place the prepared syringe with PBS into a syringe-holder shaped out of modeling clay. This holder will keep the syringe steady while the tubing is inserted into the duct.</li>
</ol>
3. Other Set-up
<ol>
 <li>Weigh 0.1 g of catalyst into a small container, e.g., one well of a 24-well plate.</li>
 <li>Pull 1 ml resin into a 3 ml syringe. 
 Note: relative amounts of catalyst and resin may be adjusted to alter curing time. Decreasing amount of catalyst may, however, increase cast shrinkage and lead to a less desirable cast. 
 Note: when handling resin, take precautions to avoid contact with resin or inhalation of the resin fumes. Always wear gloves and perform steps involving resin in a well-ventilated area, such as a chemical fume hood</li>
 <li>Cut a piece of 5.0 silk suture of approximately 5 inches to be used as a ligature.</li>
</ol>
4. Prepare Mouse
<ol>
 <li>Sacrifice adult mouse. Lay mouse on its back and open up the abdominal cavity.</li>
 <li>Lay mouse on dissecting scope platform. Position a 15 ml conical tube perpendicularly underneath the mouse to increase visibility and accessibility to liver.</li>
 <li>Wet a cotton swab with PBS and use it to flip the liver upwards and away from you to expose the dorsal view of the liver along with the extrahepatic portal vein or common bile duct.</li>
 <li>For portal vein casts, you can prevent blood clotting by flushing the vein with room temperature PBS. Attach a needle to a 3 ml syringe filled with PBS and insert needle into extrahepatic portal vein as far from the liver as you can. Gently push PBS through into the portal vein. Make a nick in the common cardinal vein to drain blood. Use a wet cotton swab to massage liver, enhancing blood drainage. This step is not recommended for the bile duct and may not be necessary for all vascular systems. 
 Note: this step can also be performed with 4% paraformaldehyde instead of PBS. Use of 4% paraformaldehyde may increase vascular integrity and result in a more precise cast, specifically of smaller vascular structures.</li>
</ol>
5. Cannulate Extrahepatic Portal vein or Common Bile Duct
<ol>
 <li>Use curved forceps to pass the surgical suture ligature underneath the portal vein or bile duct, approximately &frac14; inch from the liver.</li>
 <li>Tie the suture into a loose common knot. DO NOT tighten the knot.</li>
 <li>If you are working with an open-ended system, it may be helpful to tie and tighten a ligature at the other end of the system. If you have opened the common cardinal vein to drain the blood from the portal vein, tie a ligature around it to increase pressure within the portal vein and prevent resin leakage into the central hepatic vein.</li>
 <li>Use spring scissors to make a small cut in the portal vein or bile duct, approximately &frac14; inch below the suture knot. This cut should penetrate no more than halfway through the diameter of the portal vein or bile duct.</li>
 <li>Using #5 forceps, hold the portal vein or bile duct at the site of the cut and insert beveled end of the tubing into the bile duct.</li>
 <li>Push tip of tubing past the pre-tied ligature and towards the liver.</li>
 <li>Test for cannulation accuracy by injecting PBS through the cannula and watching to see if it enters the portal vein or bile duct.</li>
 <li>Tighten the ligature to hold the cannula in place.</li>
</ol>
6. Resin Cast the Portal Vein or Intrahepatic Bile Duct
<ol>
 <li>Add pre-measured 1 ml resin to pre-measured 0.1 g catalyst and mix thoroughly. Avoid generating air bubbles.</li>
 <li>Draw resin-catalyst mixture back into the 3 ml syringe. Remove any air bubbles by gently flicking the syringe and expelling bubbles. 
 Note: for casting small structures, it may be helpful to use a vacuum chamber to remove very small air bubbles from the resin and enhance cast quality. To allow for time to perform this step, it will be necessary to decrease the catalyst/resin ratio from that suggested above in order to slow resin curing.</li>
 <li>Carefully replace PBS-containing syringe with resin-containing syringe, leaving the 32 gauge needle attached to the cannula.</li>
 <li>Slowly and gently push resin into the portal vein or bile duct until resistance increases and liver is filled.</li>
 <li>Use wet cotton swab to gently massage liver and encourage an even fill.</li>
 <li>Remove cannula from the structure and quickly re-tighten the ligature to prevent resin leakage.</li>
 <li>Allow mouse to lay flat at room temperature for 20 min while the resin cures, then remove liver.</li>
 <li>Proceed with either clearing or macerating the liver.</li>
</ol>
7. Clear Liver for Visualization in situ
<ol>
 <li>Fix the liver in 4% paraformaldehyde overnight at 4 &deg;C, rocking.</li>
 <li>Wash the liver in PBS for at least four hr, rocking. At this point, you may separate the liver lobes if desired.</li>
 <li>Dehydrate in 50% methanol and then in 100% methanol for at least four hr each, rocking at room temperature.</li>
 <li>Clear the liver in a 1:2 solution of benzyl alcohol and benzyl benzoate (BABB) for at least 24 hr, rocking. Longer wash times may be needed. If the liver does not clear completely after 2-3 days, move back to 100% methanol for 24 hr and repeat BABB clearing.</li>
 <li>Submerge liver in BABB in a glass Petri dish for imaging in situ.</li>
</ol>
8. Macerate Liver Tissue
<ol>
 <li>Remove liver from mouse and put into a 50 ml conical tube with water. Wait 1 hr for complete resin curing. Separate liver lobes if desired.</li>
 <li>Pour off water and move liver to a glass bottle. Submerge in 15% KOH. Leave overnight at room temperature. Do not rock.</li>
 <li>Carefully pour off KOH and wash cast in distilled water. Note: the cast is very fragile and should be handled with extreme care. Avoid disrupting the cast when adding or removing fluid from the tube.</li>
 <li>When resin cast is clean, gently lay on a Kimwipe to dry, then transfer to a container to be stored.</li>
</ol>
9. Representative Results
A successful cast of the portal vein or bile duct will show a continuous network of progressively smaller branches extending from the hilum to the liver periphery.
Figure 1 shows a portal vein cast as visualized in situ after BABB clearing. Figure 1A shows the cast of the entire left liver lobe and the shape and size of the cast. Figure 1B shows a close-up view of the same cast, demonstrating the penetration of the resin into the smallest portal vein branches at the liver periphery.
Figure 2 shows a portal vein cast that has undergone KOH maceration. Figure 2A shows the entire left liver lobe and Figure 2B is a close up of the small peripheral branches.
This technique has also been successfully applied to the intrahepatic bile duct and published using both the BABB clearing and KOH maceration techniques1,2,7.
Common problems include incomplete fills, bubbles in the resin, and overflow into surrounding systems or tissues. Figure 3 demonstrates how to recognize an incomplete fill (Figure 3A), bubbles (Figure 3A), and an overflow of resin from the portal vein to the central vein (Figure 3B).
All images are taken using a Leica MZ 16 FA stereoscope and QImaging RETIGA 4000R camera.
<img alt="Figure 1" src="/files/ftp_upload/4272/4272fig1.jpg" /> 
 Figure 1. BABB-cleared resin cast of hepatic left lobe portal vein. A. Resin cast shows a hierarchical branching structure extending from the hilum to the liver periphery. Resin cast appears in yellow color within blue-tinted liver lobe. B. A close up shows that resin fill extends to small branches in the liver periphery. Resin is whitish through cleared liver. Scale bar = 1 mm.
<img alt="Figure 2" src="/files/ftp_upload/4272/4272fig2.jpg" /> 
 Figure 2. Tissue-macerated resin cast of hepatic left lobe portal vein. A. Resin cast of whole liver lobe shows many branches of varying sizes. B. Close-up shows small branches in periphery of liver. Feathering appearance of small branches represents resin filling of sinusoidal spaces and may or may not be visible on resin casts and will increase the apparent branch density if present. Resin is white in color. Scale bar = 1 mm.
<img alt="Figure 3" src="/files/ftp_upload/4272/4272fig3.jpg" /> 
 Figure 3. Common problems with resin casts. A. An incomplete fill of the left lobe portal vein is apparent by some or all cast branches failing to extend to the liver periphery. This cast also displays bubbles, visible as breaks in the continuous cast (arrow). B. Due to the proximity of the portal vein and central vein branches in some places, the portal vein resin will often enter and fill the central vein. This can be recognized when there are two distinct hilar branches (arrows) and two branching structures that have different patterns and overlap. Discriminating between technical errors and true morphological alterations can be done in two ways. First, the cast structure should be compared to the structure expected based on 2-dimensional analysis or other methods. Second, it is important to perform several replicates, and when possible, quantify the structure and perform statistical analysis to determine reproducibility and if there is a statistically significant morphological alteration between and within different experimental and control arms.

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No conflicts of interest declared.

We have described specific examples for how the portal vein and intrahepatic bile duct systems of the liver can be cast, but this technique can be applied to virtually any other ductal or vascular system with slight adaptations. Previous work has demonstrated the feasibility of this technique in multiple species, including mouse1,2,7-9, duckling10,11, rabbit12,13, dog14, and pig15, and in many organs, including nasal gland14, heart16, bladder<su...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>PE10 polyethylene tubing</td>
 <td>Fisher Scientific</td>
 <td>1417012P</td>
 <td></td>
 </tr>
 <tr>
 <td>5-0 surgical black braided silk</td>
 <td>Roboz Surgical</td>
 <td>SUT-15-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Steriject 32 G x 13 mm needle</td>
 <td>Air-tite</td>
 <td>TSK3213</td>
 <td></td>
 </tr>
 <tr>
 <td>Spring scissors</td>
 <td>Fine Science Tools</td>
 <td>15000-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Number 5 forceps</td>
 <td>Fine Science Tools</td>
 <td>91150-20</td>
 <td></td>
 </tr>
 <tr>
 <td>Mercox II kit</td>
 <td>Ladd Research</td>
 <td>21247</td>
 <td></td>
 </tr>
 <tr>
 <td>Benzyl alcohol</td>
 <td>Fisher Scientific</td>
 <td>1816-04</td>
 <td>Only required for BABB-clearing</td>
 </tr>
 <tr>
 <td>Benzyl benzoate</td>
 <td>MP Biomedicals, LLC</td>
 <td>154839</td>
 <td>Only required for BABB-clearing</td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline (PBS)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Modeling clay</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Scale</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Laboratory scissors</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml cap polypropylene tubes</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>4% paraformaldahyde</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>15% potassium hydroxide (KOH)</td>
 <td></td>
 <td></td>
 <td>Only required for maceration</td>
 </tr>
 <tr>
 <td>Razor blade</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>100% methanol</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>3 ml luer lock syringe</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

3-dimensional resin casting and imaging of mouse portal vein or intrahepatic bile duct system

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.

Watch this Scientific Journal Video about 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System at JoVE.com

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 architecture.

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

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17.1K Views.  Vanderbilt University. 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.

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

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

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 ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

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 ...

Ablation of Ischemic Ventricular Tachycardia Using a Multipolar Catheter and 3-dimensional Mapping System for High-density Electro-anatomical Reconstruction

Ventricular tachycardia (VT) in patients with ischemic cardiomyopathy mainly results from endocardial scars after myocardial infarction; those scars represent zones of slow conduction that allow the occurrence and maintenance of reentrant circuits. Catheter ablation enables substrate modification of those low voltage areas and thus can help to alter the scar tissue in such a way that arrhythmias cannot appear anymore. Hospitalizations of concerned patients decrease, quality of life and outcome rise. Consequently, VT ablation represents a growing field in electrophysiology, especially for patients with endocardial scars in ischemic heart disease after myocardial infarction. However, ablation of ventricular tachycardia remains one of the most challenging procedures in the electrophysiology lab. Precise scar definition and localization of abnormal potentials are critical for ablation success. The following manuscript describes the use of a multipolar mapping catheter and 3-dimensional (3D) mapping system to create a high density electro-anatomical map of the left ventricle including a precise scar representation as well as mapping of fractionated and late potentials in order to allow a highly accurate substrate modification.

With the following protocol, we provide an approach to Ventricular Tachycardia (VT) ablation using high density mapping with a multipolar catheter and 3D mapping system enhancing the success of the procedure.

Ventricular tachycardia (VT) in patients with ischemic cardiomyopathy mainly results from endocardial scars after myocardial infarction; those scars ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

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.

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. ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

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

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation of the extrahepatic bile duct (EBD) in mouse neonates without damaging the bile duct during preparation. Because of its exceptionally close preparation compared to the cholangiocytes cell line and harvesting of the entire extrahepatic bile duct system (EBDS), the described approach is extremely useful in researching animal models of newborn bile duct disorders, such as biliary atresia. After euthanasia, the peritoneal cavity was accessed, and the bile duct system, duodenum, and liver were extracted with the unique En-bloc-Resection (EbR). The extracted sample is placed on a foam mat, and the EBD is dissected from contaminating cells atraumatically without necessary touch. The dissection of the entire EBDS is a significant advantage of this method. Caution must be taken due to the small size and amount of bile duct tissue. Using the described technique, there is no damage to the cholangiocytes. Further, the purity of the technique is reproducible (n = 10). Therefore, optimally comparable samples can be harvested. Furthermore, no bile duct tissue is harmed, because any contact with the bile duct system can be avoided during preparation, leaving the bile fluid inside the gall bladder. Most importantly, while performing the final gall bladder and bile duct dissection, atraumatic microinstruments were used only slightly lateral of the bile duct without squeezing it. This is the key to a clean and intact sample, and essential for further histological investigation or the isolation of cholangiocytes. To summarize, the described innovative dissection technique enables especially inexperienced operators with the necessary equipment to isolate the EBDS as cleanly as possible.

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 duct.

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation ...