This work was supported by the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (grant numbers R21 DK093406 and T32 DK067872 to J-P.R.) and a VA Merit award (grant number 1BX002129 to J-P.R.).

The following protocol adheres to guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Maryland School of Medicine (IACUC Protocol #0415011, approved June 18, 2015).
1. Gavaging Mice with 19F-Labeled Bile Acids
<ol>
	<li>Gavage mice with 150 mg/kg body weight 19F-labeled bile acids. Fill a 1-ml syringe to the necessary volume with 19F-labeled bile acid stock solution [cholic acid-trifluoro-acetyl lysine (CA-lys-TFA; in 1:1 polyethylene glycol 400:Dulbecco&#39;s phosphate buffered saline) or cholylsarcosine-trifluoro-N-methyl-acetamide (CA-sar-TFMA; in 60% polyethylene glycol 400 and 40% Dulbecco&#39;s phosphate buffered saline] and attach a 20-gauge 1.5-inch curved bulb-tipped gastric gavage needle. Make sure the gavage needle is long enough to reach the level of the mouse&#8217;s xyphoid cartilage when inserted in the esophagus up to the hub of the needle</li>
	<li>Firmly grasp the animal by the loose skin at the back of the neck between the thumb and index finger and use the remaining fingers to grasp the skin on the lower back and tail.</li>
	<li>Hold the mouse upright and pass the gavage needle along the side and the roof of the mouth into the esophagus and down into the stomach. If resistance is encountered at the pharynx, reposition the needle until the animal &#39;swallows&#39; it &#8212; do not push against resistance.</li>
	<li>If anesthesia is required for gavage, place the mouse in a bell jar containing 5 ml isoflurane and close. When the mouse falls on its side, wait 7 sec, remove the mouse and perform gavage. To protect personnel from anesthetic vapors, use the bell jar only in a fume hood.</li>
	<li>Observe the animal recover from isoflurane in a few min. 
	NOTE: Since isoflurane is metabolized by the liver, a fluorine signal emanating from intact drug or its metabolites excreted into the biliary system and gallbladder can confound fluorine signals from 19F-labeled bile acids15. An alternative is to use ketamine plus xylazine (see section 3.1 for doses).</li>
</ol>
2. Harvesting the Gallbladder, the Liver and Blood for Bile Acid Measurements Using Liquid Chromatography/Mass Spectrometry
<ol>
	<li>To achieve maximal gallbladder filling, fast mice for at least 6 hr before harvesting the organs. Prepare ketamine and xylazine in phosphate-buffered saline (100 &#181;l ketamine, 62.5 &#181;l xylazine, 840 &#181;l PBS).</li>
	<li>Using a 1-ml sterile syringe, inject a mouse subcutaneously 1 hr prior to organ harvest with 15 &#181;l/g body weight of ketamine/xylazine solution (150 mg ketamine and 18 mg xylazine per kg body weight).</li>
	<li>One hr after administering ketamine/xylazine confirm adequate anesthesia by toe pinch and place the anesthetized mouse supine.</li>
	<li>Use 5 or 6 inch scissors to make a midline abdominal skin incision from the pubis to the xyphoid and fine scissors (4 inch) to cut the peritoneal lining and expose abdominal organs &#8212; do not to pierce the diaphragm.</li>
	<li>Grasp the xyphoid process with a 5 inch clamp and lift back across the chest to expose the upper abdominal cavity. Use forceps and a blunt instrument to dissect and move the liver aside, exposing the gallbladder. 
	NOTE: Do not lacerate the liver or touch the gallbladder as the former will cause severe bleeding and the latter may stimulate gallbladder contraction and emptying.</li>
	<li>Place a 4-inch clamp across the common bile duct (Figure 3, dashed arrows). Cut the ligament attaching the superior pole of the gallbladder to the diaphragm and gently move the gallbladder to the right side of the abdomen.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/54597/54597fig3.jpg" /> 
Figure 3: Anatomical and Proton MRI Views of the Mouse Gallbladder. 
The left panel shows the exposed mouse gallbladder to the left of midline after abdominal incision. The clamp grasps the xyphoid process. The bile-filled fasting gallbladder is indicated by the large arrow and the clamped common bile duct by the dashed arrows. [Inset: Excised intact gallbladder with the common bile duct clamped. The ruler is marked in millimeters (mm).] The right panel shows a high-resolution proton density-weighted MRI image of the fasting murine gallbladder (arrow). <a href="http://cloudfront.jove.com/files/ftp_upload/54597/54597fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="7">
	<li>Before excising the gallbladder perform cardiac puncture, harvest blood, and exsanguinate the animal to verify euthanasia. 
	&#8203;NOTE: Harvesting the gallbladder first may lacerate the liver causing cardiovascular collapse and failure to obtain an adequate blood sample (&#8805; 200 &#181;l).</li>
	<li>Expose the underside of the left diaphragm and identify the beating surface of the heart. At the point of maximal cardiac pulsation puncture the diaphragm and heart with a 23-gauge needle attached to a 1-ml syringe.
	<ol>
		<li>Slowly withdraw the syringe while aspirating. When blood starts to fill the syringe, stop withdrawing and maintain suction to collect 0.2 - 0.6 ml blood. Gently rotating the needle or withdrawing it slightly may re-establish flow if it ceases.</li>
		<li>Transfer the blood to a 1.5-ml heparinized tube and centrifuge at 2,000 x g for 15 min. Precipitate the plasma with four parts acetonitrile and centrifuge at 12,000 x g for 10 min. Analyze the supernatant by liquid chromatography/mass spectroscopy (LC/MS/MS)11-1312-1412-1412-1412-14. If necessary, store the plasma at -80 oC before analysis.</li>
	</ol>
	</li>
	<li>Using blunt dissection, free the gallbladder from the liver. Transect the common bile duct below the clamp, remove and weigh the gallbladder, and place it in a 1.5-ml microcentrifuge tube. Harvest the liver.</li>
	<li>Homogenize approximately 100 mg of liver and the entire gallbladder on ice in a size-21 glass tissue homogenizer. Extract with 75% acetonitrile and 25% water (800 &#181;l for liver, 300 &#181;l for gallbladder) and centrifuge at 12,000 x g for 10 min. Dilute extracts as necessary and quantify bile acid contents using LC/MS/MS11-13.</li>
</ol>
3. Live Animal Proton (1H) and Fluorine (19F) Magnetic Resonance Imaging
<ol>
	<li>To achieve maximal gallbladder filling, fast mice for at least 6 hr before imaging. Using ketamine and xylazine, anesthetize mice to prevent motion in the MRI scanner. Prepare a stock solution of ketamine plus xylazine in phosphate buffered saline (130 &#181;l ketamine, 42.5 &#181;l xylazine, 827 &#181;l PBS). One hr before MRI, use a 1-ml sterile syringe to inject a mouse subcutaneously with 5 &#181;l/g body weight of this solution (65 mg ketamine and 4.25 mg xylazine per kg body weight). To prevent dryness under anesthesia apply veterinary ointment to the animal&#39;s eyes.</li>
	<li>After induction with ketamine/xylazine as above, clip a 1.5 cm2 area on left lower half of the mouse abdomen using #40 or finer electric clipper blades. After fur removal, prep the area with 8 - 12% diluted iodine surgical scrub solution and rinse with 70% alcohol &#8212; repeat both steps. Insert a 24-gauge by 0.75-inch needle/catheter subcutaneously and tunnel into the abdominal cavity. Make sure the catheter is not in the cecum or other abdominal organ by pulling back on the plunger &#8212; there should be no blood or fecal material in the catheter.</li>
	<li>Remove the needle and leave the intraperitoneal catheter. Place the mouse on a temperature-controlled thermal pad in the MRI scanner animal chamber.</li>
	<li>Prepare a 1-ml sterile syringe containing ketamine and xylazine in phosphate buffered saline (1,000 &#181;l ketamine, 300 &#181;l xylazine, 6,700 &#181;l PBS) and fill the desired length of 72-inch sterile tubing. Connect the intraperitoneal catheter to the prefilled sterile tubing and extend it away from the MRI scanner. To maintain anesthesia inject 50 &#181;l of this solution every 20 min if the mouse&#8217;s vital signs are stable. 
	&#8203;NOTE: Before imaging, make certain no metals are near the MRI scanner.</li>
	<li>Use a 19F/1H dual-tuned linear volume MRI coil to transmit and receive radio frequency signals at 300.283 MHz for 1H and 282.524 MHz for 19F nuclei.
	<ol>
		<li>Perform system calibration11, 13 and animal localization with three-slice (axial, mid-sagittal, and coronal) scout images using a fast low-angle shot sequence (FLASH). To start the experiment, click the &#39;traffic light&#39; button in the Scan Control window on the software console.</li>
		<li>Acquire multislice 1H MR images using rapid acquisition with relaxation enhancement (RARE) sequence in the cross view of the sample or the body of the animal with repetition time 2,200 msec, echo time 8.9 msec, RARE factor 8, field of view 4 x 4 cm2, slice thickness 1.0 mm, matrix size 266 x 266, in-plane resolution 150 x 150 &#181;m2, and number of averages 6. To start the experiment, click the &#39;traffic light&#39; button in the Scan Control window on the software console.</li>
		<li>Acquire 19F images using a FLASH sequence in the same region of the 1H MRI with repetition time 220 ms, flip angle = 30&#176;, echo time 3.078 ms, matrix size 32 x 32, in-plane resolution 1.25 x 1.25 mm2, slice thickness 4.0 mm, and number of averages 768. To start the experiment, click the &#39;GOP&#39; (Go- On- Pipeline) button in the Spectrometer Control Tool window on the software console.</li>
	</ol>
	</li>
	<li>After MRI, euthanize the mouse with intraperitoneal injection of 15 &#181;l/g body weight ketamine/xylazine solution (150 mg ketamine/18 mg xylazine per kg body weight) followed by cardiac puncture for exsanguination.</li>
	<li>To recover a mouse from anesthesia, remove the intraperitoneal catheter but do not leave the animal unattended until it regains sufficient consciousness to maintain sternal recumbency.</li>
	<li>To measure 19F-labeled bile acid concentrations11-13 from organ harvest, maintain anesthesia with ketamine plus xylazine as described above.</li>
</ol>

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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=In+Vivo+Magnetic+Resonance+Imaging+to+Detect+Biliary+Excretion+of+19F-Labeled+Drug+in+Mice.">In Vivo Magnetic Resonance Imaging to Detect Biliary Excretion of 19F-Labeled Drug in Mice.</a> Drug Metab Dispos. 39, 736-739 (2011).</li><li>Ridlon, J. M., Kang, D. J., Hylemon, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+salt+biotransformations+by+human+intestinal+bacteria.">Bile salt biotransformations by human intestinal bacteria.</a> J Lipid Res. 47, 241-259 (2006).</li><li>Frisch, K., 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=N-methyl-11C]cholylsarcosine,+a+novel+bile+acid+tracer+for+PET/CT+of+hepatic+excretory+function:+radiosynthesis+and+proof-of-concept+studies+in+pigs.">N-methyl-11C]cholylsarcosine, a novel bile acid tracer for PET/CT of hepatic excretory function: radiosynthesis and proof-of-concept studies in pigs.</a> J Nucl Med. 53, 772-778 (2012).</li></ol>

The authors have nothing to disclose.

The synthesis of CA-lys-TFA and CA-sar-TFMA and the in vitro analysis of their transport using stably transfected Madin-Darby canine kidney cells expressing ASBT and human embryonic kidney cells expressing the sodium/taurocholate co-transporting polypeptide (NTCP) are detailed elsewhere13,14. Here, the focus is on oral administration of MFBA by gavage to live animals, followed by harvest of the gallbladder, liver, and blood for analysis of MFBA content, and, notably, imaging MFBA in the gallbladder by...

Along with their classical role as detergents that facilitate fat absorption from the gut, bile acids have emerged as potent signaling molecules affecting multiple organs in addition to those associated with their enterohepatic circulation1,2. In addition to controlling their own metabolism, bile acids modulate several aspects of gastrointestinal physiology (e.g., gut motility and incretin hormone production, colon physiology, and cancer susceptibility) and have systemic effects on vascular tone, glucose and lipid metabolism, and energy utilization. While some of these effects are mediated in the gut, others are due to postprandial changes in systemic bile acid levels, as noted in obese patients or after gastric by-pass surgery. To elucidate the complex metabolic actions of bile acids new technology is required that permits simultaneous monitoring of bile acid levels in different anatomical compartments, in the gastrointestinal tract and metabolic tissues (liver, pancreas, skeletal muscle and adipose). Obtaining such temporal and spatial information requires innovative technology - in vivo imaging using novel bile acid tracers as described here is such a novel approach.
Bile acid composition and distribution in anatomical compartments are regulated by factors that modulate their hepatic synthesis and ileal uptake, including diet, surgery, antibiotic use and changes in gut flora. A key regulator of intestinal bile acid uptake for their enterohepatic circulation3 (Figure 1) is the ileal Apical Sodium-dependent Bile Acid Transporter (ASBT; SLC10A2). Although passive absorption occurs throughout the intestines, ASBT mediates uptake of 95% of intestinal bile acids so that normally there is limited spillage of bile acids into the feces. Asbt-deficient (Slc10a2-/-) mice have increased fecal bile acids and a diminished bile acid pool4.
<img alt="Figure 1" src="/files/ftp_upload/54597/54597fig1.jpg" /> 
Figure 1: Enterohepatic Circulation of Bile Acids. 
Illustration of Enterohepatic Circulation whereby Bile Acids are Synthesized in the Liver, Excreted into the Biliary Tree, Stored in the Gallbladder, Released into the Proximal Small Intestine with Meals, and Actively taken up via ASBT in the Distal Ileum. Whereas small amounts of bile acids are absorbed passively throughout the gut, approximately 95% of intestinal bile acids are transported actively by ASBT resulting in minimal (approximately 5%) loss in the stool which is compensated by a similar amount of new bile acid synthesis in the liver, thereby maintaining a steady-state bile acid pool. The arrows on the right identify factors that may impact native and fluorine-labeled bile acid stability, including gastric acid, pancreatic and intestinal mucosal enzymes, and, most importantly, hydrolytic enzymes released by Clostridial species that colonize the distal small bowel and colon. (Modified with permission16) <a href="http://cloudflare.jove.com/files/ftp_upload/54597/54597fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Bile acid malabsorption can be categorized into three types, each of which increases fecal dihydroxy bile acids, thereby causing intermittent or chronic diarrhea. Type 1 results from gross ileal pathology (e.g., resection, Crohn disease)5. Type 3 results from cholecystectomy, vagotomy, celiac disease, bacterial overgrowth, and pancreatic insufficiency. In contrast, persons with &#39;primary&#39; (Type 2) bile acid malabsorption pose a formidable diagnostic challenge because they lack such antecedent conditions and do not have evidence of pathology in the ileum. Hence, primary bile acid malabsorption is commonly misdiagnosed as diarrhea-predominant irritable bowel syndrome (IBS-D), perhaps the most common reason for gastroenterology-related out-patient visits. It has been estimated that one-third of patients with IBS-D have primary bile acid malabsorption; in the U.S., this may represent several million persons5. Recent insights indicate that primary BAM derives from impaired feedback inhibition of hepatic bile acid synthesis by intestinal fibroblast growth factor-19 (FGF19), not from reduced expression or function of ASBT.
In primary bile acid malabsorption, low plasma levels of FGF19 fail to shut off hepatic bile acid synthesis - the resulting increase in intestinal bile acids saturates bile acid transporters, including ASBT, and the augmented spillage of bile acids into the feces causes diarrhea6 (Figure 2). Mice deficient in Fgf15 (murine FGF19) have an expanded bile acid pool and increased fecal bile acids7.
<img alt="Figure 2" src="/files/ftp_upload/54597/54597fig2.jpg" /> 
Figure 2: Mechanisms of Intestinal Bile Acid Malabsorption. 
Normally, as shown in panel A, approximately 95% of intestinal bile acids are absorbed by active transport in the distal ileum via ASBT. When ASBT expression or activity is diminished (panel B), impaired intestinal bile acid uptake results in spillage of bile acids into the colon. With impaired FGF19 signaling (panel C), the lack of feedback inhibition of hepatic bile acid synthesis results in increased concentrations of intestinal bile acids that overwhelm ASBT transport capacity with spillage of bile acids into the colon. <a href="http://cloudflare.jove.com/files/ftp_upload/54597/54597fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Long-term, chronic elevation in fecal bile acids may promote colon neoplasia. Colon neoplasia arises from progressive mucosal dysplasia associated with somatic gene mutations, but environmental factors that increase fecal bile acids may accelerate and augment this process. In rodents, increased fecal bile acids either as a consequence of exogenous administration or Asbt deficiency promote colon dysplasia and tumor formation8-10.
Notably, provocative findings indicate that commonly-used drugs approved by the Food and Drug Administration (FDA) potently inhibit bile acid transport by ASBT in vitro11. If these drugs reduce small intestinal bile acid transport in vivo and increase fecal bile acid levels, the potential impact on colon pathology would be concerning. Even a small increase in colon pathology attributed to use of such a drug could have a major health impact. A toolkit which can assess the plausibility of these in vitro findings and epidemiologic observations would spur additional research, including post-marketing safety studies.
Despite the need, practical assays to identify people with bile acid malabsorption are lacking. Direct measurement of fecal bile acids was rejected years ago as cumbersome, impractical, and unreliable5. Alternative approaches include measuring retention of a radioactive selenium-labeled cholic acid derivative (75SeHCAT) and plasma levels of 7&#945;-hydroxy-4-cholesten-3-one (C4), or a therapeutic trial of bile acid binders. 75SeHCAT testing has limited availability in Europe and is not FDA-approved or available for use in the U.S. Moreover, even modest radiation exposure (0.26 mSv/75SeHCAT test) from diagnostic testing raises concerns, and bacterial overgrowth and advanced liver disease may confound 75SeHCAT results. C4 testing is potentially attractive since only plasma is required, but it has low positive-predictive value and testing is not widely available. Measuring serum levels of FGF19 has similar limitations. Frequently clinicians resort to a therapeutic trial of bile acid sequestrants, but this approach cannot provide a definitive diagnosis of bile acid malabsorption5.
For these reasons, a novel MRI approach was conceived to measure bile acid transport and distribution in vivo using innovative multi-fluorinated bile acids (MFBA-MRI). MFBA containing three atoms of fluorine (19F), a stable isotope of 100% natural abundance, are transported similarly to native bile acids12, and can be used to visualize bile acid transport with a combination of proton (1H) and fluorine (19F) MRI, a sensitive, safe method without ionizing radiation exposure13,14.

<table><tbody><tr><td>Duall size-21 all glass tissue grinder</td><td>Kimble Chase Life Science, Vineland, NJ</td><td>885351-0022</td><td></td></tr><tr><td>Bruker BioSpec 70/30USR Avance III 7T horizontal bore MR Scanner</td><td>Bruker Biospin MRI GmbH, Germany</td><td></td><td>Use companion Paravision Version 5.1 software (see step 3.5)</td></tr><tr><td>Bruker 40 mm &lt;sup&gt;19&lt;/sup&gt;F/&lt;sup&gt;1&lt;/sup&gt;H dual-tuned linerar volume coil</td><td>Bruker Biospin MRI GmbH, Germany</td><td></td><td>Use companion Paravision Version 5.1 software (see step 3.5)</td></tr><tr><td>Waters Acquity UPLC System with Quadrupole Detector</td><td>Waters Corporation, Milford, MA</td><td></td><td></td></tr><tr><td>Waters Acquity UPLC ethylene bridged hybrid C8 1.7 &amp;mu;m 2.1 x 50 mm column</td><td>Waters Corporation, Milford, MA</td><td></td><td></td></tr><tr><td>Gavage Needle</td><td>Braintree Scientific, INC.</td><td>N-010</td><td>20 G-1.5&quot; curved 2.25 mm ball</td></tr><tr><td>2 Stainless Steel Hemostats&amp;nbsp;</td><td>VWR</td><td>10755-018</td><td>4 and 5 inch, straight</td></tr><tr><td>Ketamine</td><td>MWI Veterinary Supply</td><td>501090</td><td>Ketamin zetamine 100 mg/ml</td></tr><tr><td>Xylazine</td><td>Akorn, Inc.</td><td></td><td>20 mg/ml</td></tr><tr><td>Intraperitoneal Catheter</td><td>Abbott</td><td>AbbocathTM-T.I.V. G720-A01 4535-42</td><td>24-G x 0.75&quot;</td></tr></tbody></table>

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.

The use of MFBA for in vivo MRI to &#39;see&#39; bile acid transport in real time has great potential for both research and clinical use. Moreover, the methods described here for resection of the gallbladder and biochemical analysis of its contents using liquid chromatography and mass spectrometry provide a means of confirming imaging results. However, the validity of these methods requires precise dosing, timing of assays, and localization of the gallbladder for imaging or surgi...

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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.

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 ...

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Research

JoVE Journal

Medicine

11.1K Views.  University of Maryland School of Medicine.  Tools om te diagnosticeren galzuurintermediairen malabsorptie en meet galzuur transport in vivo beperkt zijn. Een innovatieve aanpak in levende dieren wordt beschreven dat de gecombineerde proton (1H) plus fluor (19 F) magnetic resonance imaging gebruikt; Deze nieuwe methodologie heeft translationeel potentieel om te screenen op galzuurintermediairen malabsorptie in de klinische praktijk. 

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

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Immunology and Infection

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Bioengineering

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

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 diseases.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Developmental Biology

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Neuroscience

Quantitative MRI of Endothelial Permeability and (Dys)function in Atherosclerosis

Cardiovascular diseases are the leading causes of death worldwide. A permeable/leaky and dysfunctional endothelium is considered the earliest marker of vascular damage and thought to drive atherosclerosis. A method to identify these changes in vivo would be desirable in the clinic. Magnetic resonance imaging (MRI)-based tools and other technologies have enabled a profound understanding of the role of the endothelium in cardiovascular diseases and risk in vivo. There is, however, a need for reproducible and simple approaches for extracting quantifiable data reflective of endothelial damage from a single imaging study. A non-invasive, easy-to-implement, and quantitative MRI workflow was developed to acquire and analyze images that allow the quantification of two imaging biomarkers of arterial endothelial damage (leakiness/permeability and dysfunction). Here, the protocol describes the application of this method in the brachiocephalic artery of atherosclerotic ApoE-/- mice using a clinical MRI scanner. First, late gadolinium enhancement (LGE) and Modified Look-Locker Inversion Recovery (MOLLI) T1 mapping protocols to quantify endothelial leakage using an albumin-binding probe are described. Second, anatomic, and quantitative blood flow sequences to measure endothelial dysfunction, in response to acetylcholine are described. Importantly, the method outlined here allows the acquisition of high-spatial-resolution 3D images with large volumetric coverage enabling accurate segmentation of vessel wall structures to improve inter- and intra-observer variability and to increase reliability and reproducibility. Additionally, it provides quantitative data without the need for high-temporal resolution for complex kinetic modeling, making it model-independent and even allowing for imaging of highly mobile vessels (coronary arteries). Therefore, the approach simplifies and expedites data analysis. Finally, this method can be implemented on different scanners, can be extended to image different arterial beds, and is clinically applicable for use in humans. This method could be used to diagnose and treat patients with atherosclerosis by adopting a precision-medicine approach.

Quantitative MRI of Endothelial Permeability and Dysfunction in Atherosclerosis

We have developed an accurate, non-invasive, and easy-to-use method to quantify endothelial permeability and dysfunction in the arteries using Magnetic Resonance Imaging (MRI), named qMETRIC. This technique enables assessing vascular damage and cardiovascular risk associated with atherosclerosis in preclinical models and humans.

Cardiovascular diseases are the leading causes of death worldwide. A permeable/leaky and dysfunctional endothelium is considered the earliest marker of ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...

A Comprehensive 3D-Liver Fat Function Distribution Model Using Dixon MRI

A 3D Quantification Technique for Liver Fat Fraction Distribution Analysis Using Dixon Magnetic Resonance Imaging

This study presents a 3D quantification methodology for the distribution of liver fat fraction (LFF) through the utilization of Dixon MRI image analysis. The central aim is to offer a highly accurate and non-invasive means of evaluating liver fat content. The process involves the acquisition of In-phase and Water-phase images from a Dixon sequence. LFF maps are then meticulously computed voxel by voxel by dividing the Lipid Phase images by the In-phase images. Simultaneously, 3D liver contours are extracted from the In-phase images. These crucial components are seamlessly integrated to construct a comprehensive 3D-LFF distribution model. This technique is not limited to healthy livers but extends to those afflicted by hepatic steatosis. The results obtained demonstrate the remarkable effectiveness of this approach in both visualizing and quantifying liver fat content. It distinctly discerns patterns that differentiate between normal and steatotic livers. By harnessing Dixon MRI to extract the 3D structure of the liver, this method offers precise LFF assessments spanning the entirety of the organ, thereby holding great promise for the diagnosis of hepatic steatosis with remarkable effectiveness.

Author Spotlight: A Non-Invasive Tool to Assess and Differentiate Fat Patterns in Liver Using 3D Dixon MRI 

This study introduces a unique 3D quantification method for liver fat fraction (LFF) distribution using Dixon Magnetic Resonance Imaging (Dixon MRI). LFF maps, derived from in-phase and water-phase images, are integrated with 3D liver contours to differentiate LFF patterns between normal and steatotic livers, enabling precise assessment of liver fat content.

This study presents a 3D quantification methodology for the distribution of liver fat fraction (LFF) through the utilization of Dixon MRI image analysis. ...

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

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