We are extremely grateful to the Cystic Fibrosis Foundation (Pilot and Feasibility Award 1810, to ABK), the Rainin Foundation (Synergy Award, to PI GJ Randolph and Co-I ABK), and the National Institutes of Health (R01DK118239, R03DK116011 to ABK) for their support of these studies.

All surgical procedures were approved by the University of Pittsburgh Internal Animal Care and Use Committee [Protocol # 20047008] and comply with the NIH Guide for the Care and Use of Laboratory Animals. C57BL6/J male mice, 8-14 weeks of age, were used for the present study. The mice were obtained from a commercial source (see Table of Materials). All mice were housed on a 12 h light/dark cycle with ad libitum access to standard chow and water.
1. Animal preparation
<ol>
	<li>Depending on the experimental design, feed the mice or allow them to fast. 
	NOTE: An overnight fast is unnecessary unless there are concerns about differences in the rate of stomach emptying.</li>
	<li>Induce anesthesia by 5% isoflurane gas and ensure proper anesthetization by tail and toe pinch. Once anesthetized, keep the mice at a proper anesthetic plane with 2%-3% isoflurane gas and position them with tape on the surgical platform.</li>
	<li>Keep the mice warm on a heated surgical platform that utilizes circulating warm water (see Table of Materials).</li>
</ol>
2. Surgery and experimental design
<ol>
	<li>Start the surgery by applying vet ointment to the eyes, shaving the abdomen, and applying antiseptic surgical scrub (see Table of Materials) to the surgical site. This sterilizes the incision and reduces the generation of airborne fur during the midline incision.</li>
	<li>Maintain a sterile working area by utilizing sterile instruments, drapes, and other equipment and supplies needed.</li>
	<li>Inject the first dose of carprofen (see Table of Materials) for pain relief (i.p.) at a dose of 5 mg/kg.</li>
	<li>Grasp the skin with tweezers and make a midline abdominal incision with small scissors. Cut toward the sternum (never above) and cut down to the inguinal fat. Then, cut through the muscle layer separately using the scissors. 
	NOTE:&#160;Sterilize all equipment before use.&#160;</li>
	<li>Using the retractor, move the peritoneal viscera out of the way until the lymph duct is visible.</li>
	<li>Using a saline-soaked Q-tip (see Table of Materials), move the liver toward the top-right side of the body and the intestines and stomach to the left of the animal.</li>
	<li>Stretch the duodenum toward the left transversely to expose the superior mesenteric artery and the intestinal lymph duct.</li>
	<li>Prepare a 30-40 cm length of cannula tubing by inserting a blunt needle in the cannula tubing (see Table of Materials) and flush a small amount of heparin (1,000 U/L) through the tube using a 1 mL syringe. 
	NOTE: Lymph flow is assisted by gravity to flow down and into the microcentrifuge collection tubes on ice. Depending on where the collection ice bucket is located adjacent to the surgical setup, the length of the cannula tubing may need to be adjusted.</li>
	<li>Use scissors to cut a bevel at the tip of the cannula tubing.</li>
	<li>Make a shallow incision with iris scissors (see Table of Materials) in the lymph duct approximately 5 mm from its appearance in the small intestine.</li>
	<li>Hold the cannula tubing with a pair of fine forceps and gently insert the tip bevel up into the duct. 
	NOTE: It is important not to push the cannula too far into the duct because this may prevent lymph flow when the retracted organs are returned to their original position.</li>
	<li>The lymph duct is much too fragile for the manipulations associated with tying the catheter in with a suture. Instead, use a drop of cyanoacrylate glue (see Table of Materials) to glue the lymph cannula into the mesenteric lymph duct.</li>
	<li>Check for the milky, white lymph (if pre-surgery olive oil gavage was used) or clear lymph (if mice have fasted pre-surgery) that begins to flow immediately through the lymph fistula cannula. 
	NOTE: Check that the lymph cannula is successfully placed and lymph is flowing; continue with the intraduodenal cannula placement.</li>
	<li>Using an 18 G needle, puncture a hole through the pyloric region of the stomach posterior to the pyloric sphincter.</li>
	<li>Insert the duodenal infusion tube (see Table of Materials) through that hole in the stomach to approximately 1-2 mm beyond the pyloric sphincter into the duodenum.</li>
	<li>Secure by a purse-string ligature using silk 5-0 suture (see Table of Materials) with a needle to the stomach and seal with a drop of cyanoacrylate glue to prevent leakage.</li>
	<li>Start the intraduodenal infusion of 5% glucose-0.9% saline at 0.3 mL/h. 
	NOTE: When using mice with small variances in body weight who are roughly 25 g (~8 weeks in age), the 0.3 mL/h infusion of glucose/saline is appropriate. If mice are dramatically larger, the infusion rate must be adjusted upward to account for the change in body weight and blood volume.</li>
	<li>Replace the organs in the body cavity and suture (5-0) the muscle and skin tissues separately. 
	NOTE: The lymph cannula and the intraduodenal feeding tube are both exteriorized through the same incision. There is no precedence for separating the tubes with a suture and no preference for the angle of the exteriorization of the tubes.</li>
	<li>After surgery but prior to the withdrawal of anesthetic, place the mice gently in Snuggle restraints (see Table of Materials) to limit motility. 
	NOTE: The snuggle restraints prevent the mice from grasping&#160;or rotating the head to chew on the stitches and tubings.&#160;</li>
	<li>Then, place the mice in a clear plexiglass box on a rotator with gentle rocking, and warm using a temperature-controlled commercially available amphibian/reptile heating pad adhered to the side of the containment box. Provide humidification also in the form of sterile deionized water containers at the corners of the containment box (see Table of Materials).</li>
	<li>At 30 min before the isoflurane is withdrawn, administer the mice their second pain relief dose (Buprenex, 0.1 mg/kg, i.p.).</li>
	<li>Depending on the experiment, provide the mice with a continuous intraduodenal infusion of 5% glucose-0.9% saline at a rate of 0.3 mL/h for 1 h. 
	NOTE: The 5% glucose-0.9% saline solution is prepared using a sterile saline bag (for human use, pH 7.4) from the pharmacy and a sterile bottle for human use of 50% dextrose, following strict guidelines for sterility. The solution must be made fresh and discarded if past the expiration dates provided on the saline bag or the dextrose bottle.</li>
	<li>Administer the mice with one of the lipids listed in Table 1 as an experimental lipid via an&#160;intraduodenal cannula (see Table of Materials). 
	NOTE: For all data shown in Figure 1, SMOFlipid (20% lipid injectable emulsion, USP, see Table of Materials) was intraduodenally infused. This infusion both pre- and post-intraduodenal lipid bolus replaces lost fluid draining through the lymph duct and is an absolute requirement. The mice are now ready to be infused with an experimental lipid dose.</li>
	<li>Perform the classic lipid infusion of a 0.3 mL lipid emulsion bolus (SMOFlipid 20% lipid injectable emulsion, USP) via the intra-duodenal infusion tube (see Table of Materials).</li>
	<li>Following the bolus infusion of intraduodenal lipids, switch the infusion back to 5% glucose-0.9% saline at 0.3 mL/h continuously through to the end of the experiment.</li>
	<li>Collect the lymph samples in pre-weighed microcentrifuge tubes for 60 min and keep the lymph on ice. Weigh the tubes a second time to establish the weight of lymph that is secreted each 60 min period. 
	NOTE: Expect to collect approximately 50-300 &#181;L of lymph per hour, per mouse in the ~6 h after a 0.3 mL lipid bolus.</li>
	<li>Lymph can flow for up to 6 h after the lipid infusion. At the 6 h time point, euthanize the mice via isoflurane and cervical dislocation. 
	NOTE: A surgeon and/or lab technician should be present to monitor the animals throughout the experiment. During the observation, look out for clots in the lymphatic drainage and changes in&#160;animal behavior that indicate&#160;pain/distress. If the lymph duct is clogged at any point during the experiment, the experiment is terminated, and the animal is euthanized. The animal can technically be kept alive for up to 24 h after surgery (as per IACUC survival surgery rules). However, survival rates decrease over time, and 6 h is a reproducible survival period where lipid absorption still mimics the in vivo physiology, and the animal is not struggling with survival.&#160;</li>
	<li>Perform terminal tissue collection after euthanasia (step 3.1). 
	NOTE:&#160;The difference in dietary lipid absorption profiles in mice kept for 6-h or 24-h post-surgery was recently tested44. We found that the 24-h procedure reduces the animal survival rates and&#160;the excursion of dietary lipids into the lymph. For these reasons, we strongly recommend the 6-h approach. This updated surgical design reduces unnecessary animal death and the potential for animal distress in the postsurgical period. This supports a major goal of the American Association for Accreditation of Laboratory Animal Care, which is animal &#8220;Replacement, Reduction, Refinement&#8221; [ref PMID: 21595115].</li>
</ol>
3. Collection of luminal contents and mucosal tissue
<ol>
	<li>At the end of the 6 h lipid infusion period, sacrifice the mice via isoflurane and cervical dislocation. Tie both ends of the stomach, small intestine, and cecum with 5-0 sutures to avoid leakage of the luminal contents.</li>
	<li>Collect the stomach, cecum, and colon, and place each into a 15 mL glass tube. Divide the small intestine into three or four segments, and collect the luminal contents by rinsing the tissue in 2-3 mL of cold PBS after cutting open longitudinally.</li>
	<li>Remove the intestinal mucosa comprising epithelial cells and associated lamina propria from the muscular layer by scraping each section in 2-3 mL of cold PBS with a curved tweezer.</li>
	<li>Place all tissues, the luminal and mucosal isolates, and the muscular layer in glass tubes and add 8 mL of 2:1 vol/vol CHCl3:MeOH to each tube.</li>
	<li>Determine the radioactivity and/or lipid concentrations by liquid scintillation counting and/or a triglyceride assay kit (see Table of Materials) after Folch extraction45.</li>
</ol>
4. Thin-layer chromatography (TLC)
<ol>
	<li>Extract the total lipids of the luminal and mucosal isolates via a modified Folch extraction45.</li>
	<li>Dry the extracted lipids in the solvent under a nitrogen evaporator and then resuspend them in 2:1 vol/vol of CHCl3:MeOH before loading them onto a TLC silica gel plate (see Table of Materials).</li>
	<li>Separate the lipids using a solvent system of petroleum ether, ethyl ether, and glacial acetic acid (25:5:1 vol/vol/vol). Use iodine vapor for the visualization of different lipids, including standards.</li>
	<li>Scrape the spots on the TLC plate corresponding to monoglyceride/phospholipid, diglycerides, fatty acids, triglyceride, and cholesterol ester into individual scintillation vials before the addition of 4 mL of the scintillation fluid (see Table of Materials) for scintillation counting.</li>
	<li>Express the data as either a percent of the total bolus lipid dose or as a fraction of the total lipid detected for each sample.</li>
</ol>
5. Chylomicron isolation and characterization
<ol>
	<li>Transfer the combined lymph samples from the 6 h post-lipid bolus (step 2.29) to ultracentrifuge tubes, mixed with 0.9% NaCl (300-500 &#181;L), and then carefully overlay with an appropriate volume of 0.87% NaCl (300-500 &#181;L).
	<ol>
		<li>Ultracentrifuge (see Table of Materials) the samples at 110,000 x g at 4&#160;&#176;C for 16 h. Collect the top fraction containing isolated chylomicrons and transfer them to a new microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Determine chylomicron triglyceride, cholesterol, and apoB concentrations following the steps below.
	<ol>
		<li>Determine triglyceride and cholesterol concentrations using a triglyceride and cholesterol assay kit (see Table of Materials).</li>
		<li>Briefly, incubate 2 &#181;L of the samples with 200 &#181;L of enzyme reagent at 37 &#176;C for 5 min in a 96-well plate. Read the plate using a plate reader at 500 nm for triglyceride and 600 nm for cholesterol. 
		NOTE: Standards and blanks were used for the calculation of concentrations. ApoB concentrations were determined using Mouse ApoB ELISA Kit (see Table of Materials).</li>
	</ol>
	</li>
	<li>Determine the chylomicron particle size.
	<ol>
		<li>Use fresh chylomicron samples at triglyceride concentrations of around 40 mg/dL for TEM imaging. Briefly, place 5-10 &#181;L of each sample on a TEM grid and dry.</li>
		<li>Examine the grid with a transmission electron microscope and capture images. Measure the lipoprotein particle sizes and analyze them using ImageJ software (see Table of Materials).</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The original 2 day lymph fistula procedure was described by Bollman et al.25 and practiced by the laboratory of Patrick Tso for the last 45 years26,27. The protocol presented here is a powerful addition to this classic, gold-standard method for identifying, quantifying, and understanding the unique chylous secretions of the small intestine, as well as the in vivo dynamics of dietary nutrient absorption and metabolism, gut hormones, and intestinal immunity.
The advantages of this model include (1) the ability to continuously sample mesenteric lymph throughout the feeding and post-prandial period rather than static sampling at one time point during either absorption, digestion, or secretion; (2) the measurement of gut hormones and cytokines directly in their physiological compartment rather than in blood, where they are diluted and enzymatically degraded17,44; (3) the ability to isolate, quantitate, and characterize the lipoproteins secreted by the small intestine following the ingestion of a lipid meal and the absence of endothelial lipases in the mesenteric lymph, which preserves chylomicron triglyceride concentrations and native chylomicron structure46,47; (4) the ability to directly measure lymph flow rate, the output of triglyceride and cholesterol (or other duodenal-infused compounds), chylomicron composition, and intestinal hormone concentrations. Finally, this protocol allows for collecting relatively large quantities of &#62;50 &#181;L of lymph every hour over a 6 h period. As the fluid is replenished with intra-duodenal saline and glucose infusion, the lymph fistula model is significantly improved over other lymph sampling techniques and results in flowing rather than static pools of mesenteric lymph. As volume is a major hurdle for lipidomic, proteomic, and metabolomic approaches, this is a major strength.
The 1 day mouse lymph fistula protocol described here has several advantages over the original lymph fistula protocol, including a reduction in total experimental animal number from previously described 2 day lymph fistula protocols26,27 because of a higher survival rate after surgery; a reduction in the overall experimental time from 2 days to a single day; and, finally, a reduction in the recovery period for mice from overnight (&#62;18 h), where breakthrough pain or poor post-surgical outcomes may occur, to a more manageable ~6 h post-surgery period.
A feature of this 1 day protocol is the focus on humane considerations and endpoints. These must take the highest priority: (1) animals must receive intraduodenal or IV replacement fluids; (2) they must be kept warm and as pain-free as possible (with post-operative Buprenex and/or carprofen, depending upon the experimental design and the need for avoiding anti-inflammatory effects); (3) bleeding, shaking, diarrhea, or signs of distress are all compelling reasons for a humane endpoint. Per strict IACUC guidelines, isoflurane followed by cervical dislocation is a good endpoint. Surgical survival rates are ~70% for a single day (compared to ~40% for the original 2 day surgery), but investigators should not hesitate to end the experiment at a sign of distress. This should be taken into consideration when planning animal numbers.
In terms of troubleshooting this technique, successful placement of the lymph cannula is the major bottleneck in this surgical procedure. While practicing the surgery, it helps to gavage the mouse with 0.3 mL olive oil approximately 2 h prior to surgery. This will cause the secretion of chylomicrons into the mesenteric lymph duct, making it appear &#34;milky&#34; and more visible. Methylene blue can also be used but is often less obvious than the milky lymph duct. If after the placement of the lymph cannula and its placement with glue the mesenteric lymph is successfully flowing through the cannula into a collection vessel, then one can proceed with the placement of a duodenal infusion tube. Occasionally, the lymph may not flow continuously but may start flowing again when the animal is placed on the rotating table. Critically, watch out for clots within the lymph tubing. These should be massaged out of the tubes to prevent backflow pressure on the lymph duct.
In addition to triglyceride secretion and lymph flow rate, this technique can be used to determine the following lipid absorption kinetics and chylomicron characteristics:
Chylomicron secretion45,48,49 
Immediately following a meal containing fat, there is a transient rise in circulating plasma triglyceride. As triglycerides are inherently hydrophobic, they must first be emulsified to be soluble in blood50. Small intestinal enterocytes carry out this role and package dietary triglyceride into chylomicron emulsion particles51. Chylomicrons contain cholesterol and dietary triglyceride in their core, surrounded by phospholipids and apolipoproteins, including apoB-48, apoA-I, apoA-IV, and apoC-III52,53,54,55. ApoB-48 is the essential structural protein, and the other apolipoproteins have various functions required for chylomicron metabolism and clearance from the blood. To determine the key characteristics of chylomicrons, including their triglyceride and apolipoprotein content, the 1 day lymph fistula technique shown here should be used. The chylomicron secretion rate is the percent of infused 3H-triglyceride that is secreted into the lymph and measured by scintillation by counting hourly lymph samples. This can be combined with detailed chylomicron characterization48,49,56,57,58. Hourly lymph samples can be combined or kept separately. Lymph is transferred to ultracentrifuge tubes, mixed with 0.9% NaCl, and then carefully overlaid with 300-500 &#181;L of 0.87% NaCl. Samples are then ultra-centrifuged at 110,000 x g at 4 &#176;C for 16 h. The top fractions containing isolated chylomicrons are collected and tested for triglyceride concentrations using the triglyceride assay kit. Briefly, 2 &#181;L of the chylomicron (1:10 dilution) is incubated with 200 &#181;L of enzyme reagent at room temperature for 10 min in a 96-well plate. The plate is read by a plate reader at 500 nm, and the standards and blanks are used for the calculation of triglyceride concentrations. Chylomicron size can then be determined by negative staining and transmission electron microscopy (TEM)14,29. Triglyceride and cholesterol can be quantified by chemical assay and apolipoprotein content (apoB-48, A-I, C-II, C-III) by ELISA Kits or Western blot.
Determining the primary site of lipid absorption48,59 
By isolating the luminal and epithelial cell compartments of the duodenum, jejunum, and ileum at 6 h after the infusion of 3H-triglyceride or a radio-labeled mixed meal, the contents are Folch extracted to determine how much of the 3H-triglyceride is absorbed across the epithelial cell membrane (normal) or is retained in the lumen (abnormal) along the length of the small intestine48. These studies are particularly impactful if there are potential differences in GI motility60,61,62, if there is a hypothesis regarding bile acids (highly active throughout lipid absorption and themselves reabsorbed in the ileum)63,64,65,66, or if there is a concern that nutrients are being absorbed in the wrong anatomical location (ileum or even colon)67,68,69,70.
Identifying mechanisms of 3H-triglyceride trafficking into absorptive epithelial cells48 
This is performed by calculating the percent of 3H-triglyceride hydrolyzed to 3H-free fatty acid in the intestinal lumen, absorbed into the mucosa, and re-esterified into intracellular 3H-triglyceride prior to secretion as chylomicrons. This is a powerful marker of absorption/secretion defects since it can be traced to show the movement of dietary triglyceride into its breakdown products and subsequent packaging into chylomicrons. mRNA expression of the fatty acid absorption machinery (CD36, FABPs, ACSLs), re-esterification pathway (MGAT, DGAT, MTTP, apoB), and apolipoproteins (apoC-III, B-48, C-II, A-I, A-IV) can be further quantitated by RT-PCR.
Future applications of this technique are only limited by interest in gut-organ crosstalk, metabolism, immunity, nutrient absorption, environmental dietary contaminants, or any other disease with a role in the GI system. It is likely that many compelling experiments and hypotheses have been stalled because of the difficulty in accessing the critical mesenteric lymph system, and the goal of this visualized protocol is to make this technique more readily available. Isolating chylomicrons and the mesenteric lymph in which they initially reside is a critical part of understanding whole-body metabolism; the 1 day mouse lymph fistula model is a powerful physiological model for studying these events.

The physiological importance of the mesenteric lymphatic system 
The mesenteric lymphatics have been described in some form since ~300 BC when Herophilos described the hepatic portal system and all the &#34;absorptive veins in the intestines&#34;1,2,3,4,5. For more than 1,700 years after this initial description, a defining characteristic of intestinal lymphatics was the presence of milky fluid in the mesenteric lymph shortly after a meal3. It is now known that milky, chylomicron-containing lymph (&#34;chylous&#34; lymph) does not drain into the portal vein and liver but instead travels through the cisterna chyli into the thoracic duct and, ultimately, joins the blood at the left subclavian vein6. Due to this anatomical arrangement, chylous lymph first travels through the heart and lungs before circulating through the rest of the body. This means that the heart and lungs get a &#34;first-pass&#34; at the secretions into the mesenteric lymph7.
A major role of mesenteric lymphatics is their transport of dietary lipids from the small intestine8,9,10. Anatomically, chylomicrons, intestinal immune cells11,12,13,14, gut hormones15,16,17,18, antigens19,20,21, non-chylomicron lipophilic compounds22,23, and excess fluid enter the lacteals underlying the enterocyte basement membrane and are then concentrated through the lymphatic capillaries to the mesenteric lymph nodes. There are likely many unknown mesenteric lymphatic components, including metabolites, antigens, environmental contaminants, nutrients, and signaling molecules.
The components of mesenteric lymph have not been systematically identified, largely due to the difficulty of isolating mesenteric lymph. Accessing the mesenteric lymph has always been a serious challenge because lymph ducts are colorless, and except for after a fatty meal when they become chylous and milky, the mesenteric lymphatics contain colorless lymphatic fluid6,8,9,10.
Current and commons methods for isolating intestinal lymph 
Mesenteric lymph cannot be accessed in humans (except for rare and extreme circumstances where serious GI trauma has occurred)24. In vivo lymph collection is surgically complex and demanding. The original 2 day lymph fistula procedure was described by Bollman et al.25 and has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years26,27. The lymph fistula procedure allows investigators to collect flowing lymph from conscious animals during a 6 h duodenal lipid infusion period.
The lymph fistula model has mainly been used in rats to measure lymph flow rate, the output of triglyceride and cholesterol (or other duodenal-infused compounds), chylomicron composition, and intestinal hormone concentrations. To a lesser extent, this technique can also be used in mice, though surgical survival and lymph volume suffer. Due to the difficulties in seeing mesenteric lymph ducts, historical methods have included cannulating mesenteric lymph in larger animals like mini-pigs28, sheep29, pigs30, dogs31, and rats17. Working with these larger models is resource intensive and does not allow for studies in knock-in or knock-out models.
Alternative&#160;approaches have also been used. Chylomicrons can be isolated from blood in the post-prandial state (though these will be partially hydrolyzed by plasma lipoprotein lipase)32,33,34,35. The thoracic duct can also be cannulated, though the lymph collected there contains an admixture of mesenteric lymph and extra-intestinal lymph drainage26,36. In vitro, Caco-2 cells secrete a chylomicron-like particle in response to fatty acid treatment, and these cells can be co-cultured with relevant lymphatic endothelial or vascular cells37,38. Human and mouse intestinal organoid cultures have been shown to process apical lipids and secrete chylomicrons39,40,41,42. These models are highly advantageous and enable mechanistic insights into small intestinal physiology, but they cannot replicate the complexity, physio-chemical gradients, or dynamic lymph flow of in situ mesenteric lymphatics.
Advantages of the 1 day mouse lymph fistula model presented here 
With respect to these other methods of isolating intestinal lipoproteins, the Tso Lab lymph fistula technique has traditionally been considered the gold-standard technique for measuring the absorption of dietary nutrients into mesenteric lymph. This in vivo technique has the advantage of capturing key physiological aspects of dietary lipid absorption-the dynamic appearance of compounds over the absorption period, which requires the repeated sampling of flowing mesenteric lymph in live animals with duodenal nutrients. This surgical technique also measures gut hormones and cytokines directly in their physiological compartment rather than in blood, where they are diluted and enzymatically degraded17,43.
If the experimental question requires an understanding of lipid secretion dynamics or the dynamic absorption and metabolism of any hydrophobic GI compound or drug, then this technique is not only appropriate but is also the only approach that interpolates the movement of luminal contents from the proximal to the distal gut (stomach to colon) and from the apical to the basolateral surface (luminal contents through enterocyte to lacteals and portal circulation). As this technique employs the luminal delivery of nutrients through the intraduodenal catheter, and because the flowing mesenteric lymph is diverted and collected, the entire absorption apparatus is under experimental control and can be used to qualitatively assess small intestinal absorption profiles.
Presented visually here for the first time is a major update to the Tso Lab lymph fistula model, which (1) reduces the experimental time to a 1 day surgical implantation and experimental collection period; (2) improves mouse survival and animal welfare considerations; and (3) increases the reproducibility of the approach in mice to harness the power of mouse genetic models. This technique must be considered a gold standard for all experimental questions of intestinal secretions, lipoproteins, or dietary lipid absorption and is the best technique for the high-fidelity determination of lipid absorption kinetics and chylomicrons.

<table><tbody><tr><td>0.9% Sodium Chloride Injection, USP&amp;nbsp; sterile</td><td>Hospira, Lake Forest, IL, US</td><td>NDC 0409-4888-06</td><td></td></tr><tr><td>1.7 mL Eppendorf tubes</td><td>Fisher Scientific, Waltham, MA, US</td><td>7200184</td><td></td></tr><tr><td>&lt;sup&gt;14&lt;/sup&gt;C-cholesterol: Cholesterol-[4-14C] (0.1mCi/ mL)</td><td>American Radiolabeled Chemicals Inc., St. Louis, MO, US</td><td>ARC 0857</td><td></td></tr><tr><td>18 G needle</td><td>Becton Dickinson, Franklin Lakes, NJ, US</td><td>305199</td><td></td></tr><tr><td>2 Dumont micro-dissecting forceps</td><td>Fine Science Tools, Foster City, CA, US</td><td>11251-35</td><td></td></tr><tr><td>2 Forceps</td><td>ROBOZ Surgical Instrument Co., Gaithersburg, MD, US</td><td>RS-5130</td><td></td></tr><tr><td>&lt;sup&gt;3&lt;/sup&gt;H-TAG:&amp;nbsp; Triolein [9,10-3H(N)] (3H-TG) (1mCi/ mL)</td><td>American Radiolabeled Chemicals Inc., St. Louis, MO, US</td><td>ART0199</td><td></td></tr><tr><td>&lt;sup&gt;3&lt;/sup&gt;H-TG Triolein [9,10-3H(N)] (&lt;sup&gt;3&lt;/sup&gt;H-TG)</td><td>American Radiolabeled Chemicals, INC. St. Louis</td><td>MO 63146</td><td></td></tr><tr><td>50% Dextrode Injection, USP 25grams/50 mL&amp;nbsp; sterile</td><td>Hospira, Lake Forest, IL, US</td><td>NDC-0409-6648-16</td><td></td></tr><tr><td>Analtech TLC Uniplates: silica gel matrix Z265500-1PAK</td><td>Fisher Scientific, Waltham, MA, US</td><td>11-101-0007</td><td></td></tr><tr><td>BD CareFusion ChloraPrep Swabstick</td><td>Fisher Scientific, Waltham, MA, US</td><td>14-910-501</td><td></td></tr><tr><td>Betadine surgical scrub</td><td>Dynarex Corp., Orangeburg, NY, US</td><td>1201</td><td></td></tr><tr><td>Bevel-cut cannula</td><td>Braintree Scientific.,&amp;nbsp; Braintree , MA, US</td><td>MRE025</td><td></td></tr><tr><td>Buprenorphine HCl Injection Carpuject PFS 0.3mg/mL 10/Bx (Buprenex)</td><td>HenrySchein, Warrendale, PA, US</td><td>1278184</td><td></td></tr><tr><td>C57BL6/J male mice</td><td>Jackson Laboratory, Bar Harbor, Maine</td><td></td><td></td></tr><tr><td>ChloraPrep</td><td>Becton Dickinson, Franklin Lakes, NJ, US</td><td>260100</td><td></td></tr><tr><td>Cholesterol Assay Kit</td><td>FujiFilm Healthcare, Lexington, MA, US</td><td>99902601</td><td></td></tr><tr><td>Cholesterol-[4-14C]</td><td>American Radiolabeled Chemicals, INC. St. Louis</td><td>MO 63146</td><td></td></tr><tr><td>Clear plexiglass box L43cm X W26 X H 21 with temperature-controlled heating pad and humidification</td><td></td><td></td><td>our own design and modifications</td></tr><tr><td>commercially available&amp;nbsp; amphibian/reptile heating pad</td><td>ShenZhen XingHongChang Electric CO., LTD. ShenZhen, China</td><td>XHC-F035D</td><td></td></tr><tr><td>Cotton tip applicators</td><td>Fisher Scientific, Waltham, MA, US</td><td>22363156</td><td></td></tr><tr><td>Duodenal infusion tube - canuala</td><td>Braintree Scientific, Braintree , MA, US</td><td>MRE037</td><td></td></tr><tr><td>Ensure</td><td>Abbott Nutrition, Columbus, OH</td><td></td><td></td></tr><tr><td>Heating pad surgical platform with circulating warm water pump combination</td><td>Patterson Scientific, Waukesha, WI, US</td><td>Gaymar T/Pump Classic</td><td></td></tr><tr><td>Hetarin Sodium Injection, USP 1,000 units/mL sterile</td><td>Mylan, Morgantown, WV, US</td><td>NDC-67457-384-31</td><td></td></tr><tr><td>Image J Software</td><td>National Institute of Health, Bethesda, Maryland,</td><td></td><td></td></tr><tr><td>Iris scissors</td><td>ROBOZ Surgical Instrument Co., Gaithersburg, MD, US</td><td>RS-5602</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Pharma Solutions, Riverview, MI, US</td><td>NDC 66794-017-25</td><td></td></tr><tr><td>Isoflurane induction apparatus and Anesthesia Apparatus</td><td>Patterson Scientific, Waukesha, WI, US</td><td>mouse induction chamber</td><td></td></tr><tr><td>Krazy glue</td><td>Elmer&#039;s products Inc., Columbus, OH, US</td><td>KG484</td><td></td></tr><tr><td>Liposyn III 20% lipid injectable</td><td>Hospira Inc. Lake Forest, Illinois, USA</td><td></td><td></td></tr><tr><td>LS 6500 Multi-Purpose Scintillation Counter</td><td>Beckman Coulter, Brea, CA</td><td></td><td></td></tr><tr><td>Micro-dissecting Spring Scissors</td><td>ROBOZ Surgical Instrument Co., Gaithersburg, MD, US</td><td>RS-5602</td><td></td></tr><tr><td>Mouse apoB ELISA Kit</td><td>ABCAM Inc., Waltham, MA, US</td><td>ab230932-1</td><td></td></tr><tr><td>Needle Holder</td><td>Fine Science Tools, Foster City, CA, US</td><td>12002-12</td><td></td></tr><tr><td>Retractors</td><td>Kent Scientific Co., Torrington, CT, US</td><td>SURGI-5001</td><td></td></tr><tr><td>Rimadyl (Carprofen)</td><td>Zoetis Inc., Kalamazoo MI, US</td><td>4019449</td><td></td></tr><tr><td>Rotating table Barnstead Thermolyne</td><td>Labquake, Z&amp;uuml;rich, Switzerland</td><td>Barnstead Thermolyne</td><td></td></tr><tr><td>SMOFlipid 20% lipid injectable emulsion, USP</td><td>Fresenius Kabi, Warrendale, PA, US</td><td>NDC-63323-820-01</td><td></td></tr><tr><td>Snuggle</td><td>Lomir Biomedical Inc., Notre-Dame-de-l&#039;&amp;Icirc;le-Perrot, QC J7V 7M4, Canada</td><td>MS 02.5PM</td><td></td></tr><tr><td>Surgical Scissors</td><td>ROBOZ Surgical Instrument Co., Gaithersburg, MD, US</td><td>RS-5912SC</td><td></td></tr><tr><td>Suture (5-0 silk with needle)</td><td>DemeTECH, Miami Lakes, FL, US</td><td>DT-719</td><td></td></tr><tr><td>Transmission Electron Microscope (JEOL 1400-FLASH 120KV )</td><td>JEOL, Peabody, MA</td><td></td><td></td></tr><tr><td>Triglyceride Assay Kit</td><td>Randox Laboratories, Crumlin, United Kingdom</td><td>TR210</td><td></td></tr><tr><td>ULTIMA GOLD XR Scintillation Fluid</td><td>Perkin Elmer, Hebron, KY, US</td><td>6013119</td><td></td></tr><tr><td>Ultracentrifuge, rotor S100AT4-497</td><td>SORVALL RC M120 GX</td><td></td><td></td></tr></tbody></table>

the isolation of flowing mesenteric lymph in mice to quantify in vivo kinetics of dietary lipid absorption and chylomicron secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

Figure 2 shows the triglyceride secretion in mesenteric lymph and the average lymph flow rates in the most recent n = 17 wild-type (WT) mice who underwent the single-day lymph fistula procedure described here. As shown in Figure 2A, the triglyceride concentration in lymph increases in response to an intraduodenal bolus of 300 &#181;L of SMOFLipid. Peak triglyceride concentration is reached at ~2-3 h post-bolus and decreases steadily through the 6 h time (immediately before euthanasia). In parallel with triglyceride secretion, lymph flow rate increases from Time 0 bolus infusion through the end of the experiment (Figure 2B).
These results show that the surgical implantation of both the mesenteric lymph duct and intraduodenal cannula have occurred and are representative of a positive control to which other lymphatic contents (hormones, peptides, nutrients) could be benchmarked. If there is no change in triglyceride concentration in lymph in response to bolus intraduodenal lipids, this signals that the surgery has caused significant damage to the small intestine so that lipid absorption capacity is absent, or, in previously unphenotyped genetic models, this would suggest that the mouse has a defect in lipid absorption that is physiologically meaningful.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64338/64338fig01.jpg" /> 
Figure 1: Proposed timeline for the lymph fistula mouse model. T-4, T-3, T-2: Double-cannulation takes approximately 2-4 h, followed by placement of the mice in recovery chambers. T-1: Once the mice are in the recovery period, they receive a continuous duodenal infusion of 5% glucose-0.9% saline. T0: the continuous intraduodenal infusion is switched from 5% glucose-0.9% saline to an infusion of experimental nutrients. T0-T6: Mice receive continuous intraduodenal 5% glucose in saline or, alternatively, continuous nutrients. Lymph is collected hourly during this period. Endpoint: Mice are euthanized, and tissues can be collected. The figure was created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/64338/64338fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64338/64338fig02.jpg" /> 
Figure 2: Mesenteric lymph triglyceride concentration and flow rate. Wild-type mice were fitted with an intraduodenal feeding tube and a mesenteric lymph cannula. Mice received a bolus infusion of 300 &#181;L of lipid. Lymph was collected for 6 h in hourly aliquots and kept on ice. (A) Lymph triglyceride content was determined by a chemical assay in each hourly aliquot of lymph. (B) In the 6 h after bolus infusion, the lymph flow is plotted as grams of lymph secreted per hour. Points are means &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/64338/64338fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Lipid Delivery Methods</td>
			<td colspan="6">Recommendations/Instructions</td>
		</tr>
		<tr>
			<td>Mixed lipid emulsion bolus</td>
			<td colspan="6">A 0.3 mL dose of either (A) Liposyn III 20% lipid injectable or (B) SMOFlipid 20% lipid injectable emulsion, USP, via intra-duodenal infusion tube can be used. These emulsified lipids are useful because they do not need to be prepared in advance, are liquid at room temperature, are sterile, and because they contain an &#8220;easily digestible&#8221; mix of free fatty acids, triglyceride, phospholipids, and sodium taurocholate. This is a good starter infusion for mice that may have pancreatic or biliary insufficiencies.</td>
		</tr>
		<tr>
			<td>Triglyceride bolus</td>
			<td colspan="6">0.3 mL gently warmed olive oil (or purified triolein) as a neutral lipid that reflects a diet rich in unsaturated fat, lard (reflective of a diet with high saturated fat content) or coconut oil (reflective of a diet with high medium chain triglyceride content) can all be given by intra-duodenal infusion tube or oral gavage. If the experimental triglyceride is saturated, it must be warmed to be liquid at room temperature.&#160;</td>
		</tr>
		<tr>
			<td>Mixed meal bolus</td>
			<td colspan="6">0.3 mL Ensure with the formulation containing 0.075 g fat (21.6%), 0.5 g carbohydrate (64.0%), and 0.1125 g protein (14.4%) and reflects a low-fat mixed meal, can be administered via intra-duodenal infusion.</td>
		</tr>
		<tr>
			<td>Radiolabeled lipid bolus</td>
			<td colspan="6">5.0 &#181;Ci 3H-labeled glycerol trioleate and/or 1.0 &#181;Ci 14C-cholesterol in 0.3 mL of olive oil can be given via intra-duodenal infusion. 14C-cholesterol: Cholesterol-[4-14C] with specific activity of 55Ci/mmol and a concentration of 0.1mCi/ mL. 3H-TG Triolein [9,10-3H(N)] (3H-TG) with specific activity of 60Ci/mmol and concentration of 1mCi/mL</td>
		</tr>
		<tr>
			<td>Continuous dose</td>
			<td colspan="6">The infusion of any of the above experimental nutrient formulations at a rate of 0.3 mL/h (rather than 0.3 mL total bolus), will result in a steady output of triglyceride into the lymph. This differs from a bolus infusion, where triglyceride concentration in lymph peaks at ~2&#8211;3 h, and then returns to baseline concentrations at ~6&#8211;8 h.</td>
		</tr>
	</tbody>
</table>
Table 1: Table of lipid infusions.

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The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

the-isolation-flowing-mesenteric-lymph-mice-to-quantify-vivo-kinetics

Nanyang Technological University

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Biology

2.4K Views.  University of Pittsburgh. The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

Video: The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Medicine

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Immunology and Infection

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

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In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

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Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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Isolating Lymphocytes from the Mouse Small Intestinal Immune System

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Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

Assessing Whole-Body Lipid-Handling Capacity in Mice

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors