This research is supported by Tom Peterson Foundation and NIH grant R01-HL119780 (Jain, MK).

Animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Case Western Reserve University School of Medicine and was conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (8th Edition, 2011). Mice were euthanized by methods compliant with the American Veterinary Medical Association (AVMA) Guidelines for Euthanasia of Animals (2013 Edition). C57BL/6J, male, 8 - 16 week old mice were used in this protocol. Mice were housed in a 12 h dark/light cycle environment.
1. Pre-operative Preparation
<ol>
	<li>Transfer mice to a clean cage 24 h before the procedure. Allow animals free access to water. Replace chow food with soft food (gel dietary supplement) 4 - 6 h before the surgery to reduce the contents of the small intestine for surgery.</li>
	<li>Sterilize all surgical instruments and prepare the single-use sterile surgical materials. Clean the surgery area and the anesthetic nose cone with 70% ethanol.</li>
	<li>Set up a dissection microscope, isoflurane anesthetic vaporizer, instruments, and a temperature-controlled small animal surgical table for body temperature maintenance. Organize instruments, sutures, and syringes in a location for free access during the surgery.</li>
	<li>Set up a light source to provide enough light for the surgical area.</li>
	<li>Prepare sterile 0.9% saline in 5 mL syringe for intestinal and abdominal cleaning.</li>
</ol>
2. Ileectomy and Anastomosis
<ol>
	<li>Anesthetize mice with isoflurane (2 - 3%) in a small animal incubation chamber. Determine adequate anesthetization using the toe pinch technique while the animal is on isoflurane.</li>
	<li>Remove abdominal hair by applying hair removal product and wipe hair away using surgical sponges while maintaining anesthesia. Place the mouse on a temperature-controlled small animal surgical table (Figure 1A) to maintain body temperature at 37 &#176;C. Maintain anesthesia with isoflurane (1 - 2%) through a facemask. Treat the mouse eyes with ocular ointment.</li>
	<li>Clean the skin using povidone-iodine and 70% alcohol and cover the surgical area of the abdomen with sterile surgical gauze (Figure 1B).</li>
	<li>Make a midline abdominal incision using a surgical scalpel once anesthesia is in effect. Use a cotton-tipped applicator to protect the intestine and pull mouse abdominal muscle with retractors to fully open and expose the abdominal cavity (Figure 1C).</li>
	<li>Locate the cecum. Starting from the cecum, carefully move the connected ileum and part of the jejunum out of abdominal cavity (Figure 1D). 
	NOTE: The cecum can be easily identified due to its large size even after fasting.</li>
	<li>Ligate the upper branch of the superior mesenteric artery with a 7-0 silk suture to occlude the blood supplying the ileal segment that is to be excised. The ileal color changes from pink to dark purple after ligation. (Figure 1E - F).</li>
	<li>Depending on the purpose of the experiment, using scissors, excise and remove 50% or 90% of ileum. 
	NOTE: For sham surgery, do not perform the superior mesenteric artery ligation and do not remove the ileum.</li>
	<li>Flush the lumen of both ileal ends with 0.9% saline. 
	NOTE: As the remaining intact ileum is still receiving a normal blood supply from the superior mesenteric artery, a small amount of blood will be flushed out during the process. This also indicates that the blood supply to the ileal ends is normal and ensures no ischemia during the anastomosis procedure (Figure 1G).</li>
	<li>Locate the mesenteries on the side of both ileal ends. Align the mesenteries and suture the ileal ends together using 8-0 suture (Figure 1G - H).</li>
	<li>Suture the contralateral side of the ileum to keep the ileum anastomosed in a natural manner (Figure 1I).</li>
	<li>Suture the upper and lower sides between the two original sutures to thoroughly join the two ileal ends together (Figure 1J).</li>
	<li>Confirm that there is no leakage from the anastomosis site after finishing the three-step suturing procedure (Figure 1G-I). Return the cecum and the small intestine into the abdominal cavity to the original anatomical location. Wash the surgery area with warm 0.9% saline using a blunt needle. (Figure 1K).</li>
	<li>Close the incision of the abdominal muscle layer with 6-0 suture. Align the abdominal skin incision using forceps and suture the abdominal skin to facilitate optimal wound healing (Figure 1L).</li>
</ol>
3. Post-operative Care
<ol>
	<li>Transfer the post-surgery mice to an intensive care unit for recovery. House them in a paper-bedding cage on a temperature-controlled heating pad to continue the post-surgery recovery overnight. Supply mice with soft food in addition to regular food and water.</li>
	<li>Administrate buprenorphine (0.05 - 0.1 mg/kg) with subcutaneous injection every 8-12 h for analgesia. 
	NOTE: Euthanize mice by CO2 if severely sick.</li>
	<li>At the end-point, sacrifice mice using overdosed isoflurane and harvest samples as needed (section 4).</li>
</ol>
4. Evaluation of Ileectomy-induced Bile Overaccumulation
<ol>
	<li>Weigh and dissect the mice one day after a resection of 0% (sham), 50%, or 90% of ileum.</li>
	<li>Remove the GI tract and weigh it. Calculate the GI weight to body weight ratio to evaluate the severity of bile salt malabsorption and overaccumulation.</li>
	<li>Transfer the GI tracts into 15 mL conical tubes and cut them into short segments using scissors. After cutting, centrifuge at 3,000 x g for 10 min. Transfer the supernatant (GI fluid containing bile salts) to a clean tube.</li>
	<li>Measure the total volume and weight of GI fluid and calculate the fluid weight to GI tract weight ratio to further assess bile overaccumulation in the GI tract.</li>
	<li>Determine the total bile amount in the supernatant by bile acid assay as described in reference 8.</li>
</ol>

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The authors have nothing to disclose.

In order to perform a successful ileectomy, the superior mesenteric artery must be ligated in advance to block the blood supply to the resecting segment. The ischemic ileal segment will turn dark purple after ligation. The ileal segment must then be completely resected. A normal blood supply must be ensured for the retained ends. This is essential to avoid bleeding and improper removal, which can easily cause surgical failure due to ischemic necrosis after suturing. During the anastomosis, it is important to join the ile...

In modern biomedical research, genetically manipulated animal models are widely utilized to glean insights into human diseases. In particular, tissue or cell-specific gain-and-loss functions of genes have been used to study molecular regulation as well as induced biological effects. Despite the advancements in manipulating target genes in vivo, there are lingering limitations. First, many cell or tissue specific deletions will affect multiple organs. For example, epithelial gene deletion will eliminate expression in epithelia of multiple tissues. Further, even if deletion is restricted to a specific tissue, spatial control is rarely feasible. For example, in a tissue like the intestine, distinct segments carry out very specific functions that cannot be manipulated with precision in vivo. In these situations, resection of the gene-containing tissues is considered to be a more efficient studying approach to determine the mechanistic and functional significance of tissue communication.
Ileectomy is mostly used in patients with Crohn&#39;s and inflammatory diseases involving the distal ileum 1,2,3. The ileum typically produces several energy storage hormones like fibroblast growth factor 15/19 (FGF15/19), peptide YY (PYY), and glucagon-like peptide 1/2 (GLP1/2); these hormones play important local and endocrine roles in many biological functions4,5,6. Among these hormones, FGF15 has been identified as a robust endocrine inhibitor of bile acid synthesis in the liver. Once reabsorbed into ileal enterocytes, bile acids activate the nuclear receptor farnesoid X receptor (FXR) to stimulate Fgf15 expression, which subsequently leads to feedback inhibition of hepatic bile acid synthesis 7. In a recent study, we introduced the mouse ileectomy model in order to study the ileal kruppel-like factor 15 (KLF15)-Fgf15 signaling axis that regulates circadian bile acid production in the liver 8. Most importantly, we introduced a novel family, the kruppel-like factors, particularly KLF15, into bile acid biology. Based on functional studies including ileectomy surgery, we determined that KLF15 upregulates bile acid synthesis via an indirect non-hepatic mechanism. Finally, ileal KLF15 is also identified as the first endogenous negative regulator of Fgf15.
The intestinal segments descending from proximal to distal regions are responsible for absorption of different nutrients. The ileum is the major segment responsible for bile acid and vitamin B12 (VB12) absorption 9. An earlier study employed a mouse model of proximal gut resection to study short bowel syndrome; various resection lengths, diets, and suture types were proposed to maintain an optimal post-surgery survival rate 10. Furthermore, a more recent review indicates that ileal resection typically results in more severe disease than other gastrointestinal (GI) segment resections because of the decreased adaptive capacity of the remaining tract 11. This topic has gained intensive interests of basic and clinical research groups, whereas the understanding of recovery and the effective therapeutic approaches are still limited.
Bile acid diarrhea results from imbalances in bile acid homeostasis in the enterohepatic circulation 12,13. It can be a consequence of ileal resection, gastrointestinal disease, or a result of idiopathic bile acid malabsorption. More than 80% of patients have been found to present with diarrhea after undergoing ileal resection 14. Ileectomy has the potential to be an important surgery model for the investigation of bile acid diarrhea. In this study, a series of ileal resections provide a gradient assessment of FGF15 deficiency as well as intestinal bile salt malabsorption, overaccumulation, and toxic damages.

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		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Dissection microscope</td>
			<td style="width:195px;">Olympus</td>
			<td style="width:97px;">SZ61</td>
			<td style="width:183px;">For surgery&#160;</td>
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		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Animal temperature controller</td>
			<td style="width:195px;">Physitemp Instruments, Inc.</td>
			<td style="width:97px;">TCAT-2LV</td>
			<td style="width:183px;">For body temperature control</td>
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		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Isoflurane anesthetic vaporizer</td>
			<td style="width:195px;">VetEquip</td>
			<td style="width:97px;">&#160;911104</td>
			<td style="width:183px;">For anesthesia</td>
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			<td height="21" style="width:248px;height:21px;">Dissection forceps</td>
			<td style="width:195px;">&#160;Fine Science Tools, Inc.</td>
			<td style="width:97px;">11274-20</td>
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			<td height="21" style="width:248px;height:21px;">Scissors</td>
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			<td style="width:97px;">14084-08</td>
			<td style="width:183px;">For surgery&#160;</td>
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			<td height="21" style="width:248px;height:21px;">Needle holder</td>
			<td style="width:195px;">&#160;Roboz Surgical Instrument Co.</td>
			<td style="width:97px;">RS-7882</td>
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			<td height="21" style="width:248px;height:21px;">Micro knives-needle blade</td>
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			<td style="width:97px;">10318-14</td>
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			<td height="21" style="width:248px;height:21px;">6-0 monofilament suture</td>
			<td style="width:195px;">Ethicon</td>
			<td style="width:97px;">1698G</td>
			<td style="width:183px;">For abdominal skin closure</td>
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		<tr height="21">
			<td height="21" style="width:248px;height:21px;">7-0 silk suture</td>
			<td style="width:195px;">Ethicon</td>
			<td style="width:97px;">766G</td>
			<td style="width:183px;">For ligation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">8-0 monofilament suture</td>
			<td style="width:195px;">Ethicon</td>
			<td style="width:97px;">1714G</td>
			<td style="width:183px;">For anastomosis</td>
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			<td height="21" style="width:248px;height:21px;">Surgical sponges</td>
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			<td style="width:97px;">3333</td>
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			<td style="width:97px;">23-400-118</td>
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			<td height="21" style="width:248px;height:21px;">Isoflurane</td>
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			<td style="width:97px;">66794-013-25</td>
			<td style="width:183px;">For anasthesia</td>
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			<td height="21" style="width:248px;height:21px;">Buprenorphine hydrochloride</td>
			<td style="width:195px;">Reckitt-Benckiser Pharmaceuticals</td>
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			<td height="21" style="width:248px;height:21px;">Povidone iodine prep solution</td>
			<td style="width:195px;">Dynarex Corp.</td>
			<td style="width:97px;">1413</td>
			<td style="width:183px;">For skin preparation</td>
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			<td height="21" style="width:248px;height:21px;">Puralube vet ointment</td>
			<td style="width:195px;">Dechra Veterinary Products</td>
			<td style="width:97px;">17033-211-38</td>
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			<td height="21" style="width:248px;height:21px;">Hair remover lotion</td>
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			<td style="width:183px;">For skin preparation</td>
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			<td height="21" style="width:248px;height:21px;">Intensive care unit</td>
			<td style="width:195px;">ThermoCare</td>
			<td style="width:97px;">FW-1</td>
			<td style="width:183px;">For post-surgery recovery</td>
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			<td height="21" style="width:248px;height:21px;">DietGel recovery</td>
			<td style="width:195px;">ClearH2O</td>
			<td style="width:97px;">72-06-5022</td>
			<td style="width:183px;">For post-surgery recovery</td>
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			<td height="21" style="width:248px;height:21px;">Aurum total RNA fatty and fibrous tissue kit</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:97px;">7326830</td>
			<td style="width:183px;">For RNA isolation</td>
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		<tr height="31">
			<td height="31" style="width:248px;height:31px;">iScript reverse transcription supermix for RT-qPCR</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:97px;">1708841</td>
			<td style="width:183px;">For reverse transcription assay</td>
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			<td height="21" style="width:248px;height:21px;">TaqMan fast advanced master mix</td>
			<td style="width:195px;">Applied Biosystems/Life Technologies</td>
			<td style="width:97px;">4444965</td>
			<td style="width:183px;">For QPCR analysis</td>
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			<td height="21" style="width:248px;height:21px;">Total bile acid assay kit</td>
			<td style="width:195px;">Genzyme Diagnostic</td>
			<td style="width:97px;">DZ042A-K01</td>
			<td style="width:183px;">For bile acid assay</td>
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			<td height="21" style="width:248px;height:21px;">C57BL/6J&#160;</td>
			<td style="width:195px;">The Jackson Laboratory</td>
			<td style="width:97px;"></td>
			<td style="width:183px;"></td>
		</tr>
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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.

The procedures for ileectomy are shown in Figure 1. The first step includes preparing mouse abdominal skin, making an abdominal incision, and using retractors to fully expose the intestine (Figure 1A-C). Next, the mouse cecum was located (Figure 1D); given that its size and shape make it an easily identifiable landmark. The cecum, ileum, and part of the distal jejunu...

Watch this Scientific Journal Video about Ileectomy-induced Bile Overaccumulation in Mouse Intestine at JoVE.com

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

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 ...

ileectomy-induced-bile-overaccumulation-in-mouse-intestine

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Medicine

9.4K Views.  Case Western Reserve University. 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.

Video: Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

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.

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  ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

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 ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...