Funding from the Australian Research Council (ARC) and National Health and Medical Research Council (NHMRC) is gratefully acknowledged.

The studies described in this manuscript were approved by the local animal ethics committee and were conducted in accordance with the Australian and New Zealand Council for the Care of Animals in Research and Teaching guidelines. Prior to beginning any animal procedure, ensure that the appropriate permission is obtained through the local institution/organization. As with all animal surgeries, ensure that the surgery is performed by appropriately trained operators, under aseptic conditions and that anesthetics, analgesics and antibiotics are administered when required to ensure an ethical and successful outcome.
1. Preparations the Day Before the Surgical Procedure
<ol>
	<li>Fast the rat the night before surgery if required. Ensure the rat has free access to water.</li>
	<li>Prepare the solutions to be administered to the rat.
	<ol>
		<li>Prepare a rehydration solution such as sterile saline or Ringers solution.</li>
		<li>Prepare anesthetic solutions as required. The anesthetic used in the experiments described here consisted of&#160; &#8220;Cocktail 1&#8221; prepared by combining 1.9 ml of ketamine 100 mg/ml, 0.5 ml of Xylazine 100 mg/ml, 0.2 ml of acepromazine 10 mg/ml and 2.5 ml of saline and &#8220;Cocktail 2&#8221; consisting of 1 ml of ketamine 100 mg/ml and 0.1 ml acepromazine 10 mg/ml. NOTE: Inhaled isoflurane or sevoflurane may be preferred as it can provide a surgical plane of anaesthesia for a longer period of time.</li>
		<li>Optionally prepare a formulation containing the drug in, for example, a lipid emulsion. Perform appropriate stability studies to assure the stability of the formulation over the period of storage and administration. 
		NOTE: The exact nature of the formulation and drug to be administered will vary with each study as the aim of lymphatic drug transport studies is most often to evaluate the efficiency of lymphatic drug transport as a function of the formulation and drug administered.
		<ol>
			<li>The formulation utilized in the representative experiments in Figure 4 and 5 here is prepared, as described previously43. Briefly, incorporate 14C oleic acid (2-5 &#181;Ci) and halofantrine (200 &#181;g) into 40 mg oleic acid prior to dispersion in 5.6 ml of an aqueous phase consisting of 5 mM sodium taurocholate in phosphate buffered saline (pH 6.9).</li>
			<li>For formulations containing 2-monoolein, add 7.1 mg of 2-monoolein along with other components, to the aqueous phase at the same time as the oleic acid phase containing halofantrine. Prepare the formulations containing 2-monoolein immediately prior to administration to the rat as 2-monoolein is relatively unstable and isomerises to 1-monoolein.</li>
			<li>Subsequently emulsify the formulations by ultrasonication for 2 min at RT. 
			NOTE: As has been described previously43, monitor the stability of the formulations by i) visual inspection of the emulsion under polarized light to ensure that the emulsion is not phase separated, ii) particle size analysis immediately after preparation and following dosing using dynamic light scattering to provide an indication of any phase changes and/or separation, and iii) analysis of halofantrine concentrations using a validated HPLC assay.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare anti-coagulant solution containing 10 mg/ml EDTA in sterile water or 10 IU/ml heparin in saline to flush the lymph cannula. Also prepare anti-coagulant solution containing 2.5 - 10 IU/ml heparin in saline to flush the carotid artery cannula.</li>
	<li>Prepare the polyethylene cannulas for insertion into the mesenteric lymph duct (0.8 mm O.D, 0.5 mm I.D.), carotid artery (0.96 mm O.D, 0.58 mm I.D. or 0.8 mm O.D, 0.5 mm I.D.) and duodenum (0.96 mm O.D, 0.58 mm I.D.)
	<ol>
		<li>Cut the cannulas to the required length (typically 25 - 30 cm for the carotid artery and duodenal cannula and 10 - 15 cm for the lymph cannula) and place a bevel at the tip of the cannulas using a sterile surgical blade (see Figure 1). 
		NOTE: This increases the ease of cannula insertion and reduces the incidence of cannula blockage post-insertion.</li>
		<li>Place a J shape anchor point in the intraduodenal cannula by looping a small portion of the cannula back on itself, heating it with a lighter or hot plate and then cutting to size (see Figure 1).</li>
	</ol>
	</li>
	<li>Attach the lymph cannula to a 25 G needle and syringe containing an anti-coagulant solution (prepared in step 1.3). Fill the lymph cannula with the anti-coagulant solution and leave the solution in the cannula O/N to reduce the incidence of clot formation in the lymph cannula during lymph collection.</li>
	<li>Prepare tubes for lymph and blood sample collection. Pre-weigh lymph collection tubes, label tubes for lymph and blood sample collection and add anti-coagulant solution. Add sufficient anti-coagulant solution to achieve a final concentration of 1 mg/ml EDTA in sterile water or 10 - 20 IU/ml heparin in the collected lymph or blood.</li>
</ol>
2. Preparations Immediately Prior to Commencing the Surgical Procedure
<ol>
	<li>Check all cannulas by flushing with sterile saline or anti-coagulant solution to ensure that they are patent.</li>
	<li>Prepare the rat for the surgical procedure. 
	NOTE: The mesenteric lymph duct is more visible in rats that have eaten or been administered a dose of oil as the mesenteric lymph becomes milky and opaque with higher lipid loading. Some operators, particularly those new to the surgery, therefore find it easier to cannulate the mesenteric lymph duct when rats are pre-dosed with an oil (around 0.1 - 1 ml) such as olive or soybean oil 0.5 - 2 hr prior to commencing surgery. However, pre-dosing oil affects the lymphatic transport of lipids, lipophilic drugs and other factors in lymph such that it may not be ideal to pre-dose oil depending on the aim of the experiment.
	<ol>
		<li>Anesthetize the rat throughout the surgery and sample collection period.
		<ol>
			<li>For example, initiate the anesthesia via subcutaneous injection of 1.5 ml/kg of Cocktail I (from step 1.2.2) into a skin fold at the back of the rats neck using a 1 ml syringe attached to a 25 G needle. Maintain the anaesthesia via intraperitoneal injection of&#160; 0.44 ml/kg of Cocktail 2 (from step 1.2.2) approximately each hour as required, using a 1 ml syringe attached to a 25 G needle.</li>
			<li>Prior to commencing the surgery ensure that the depth of anaesthesia is sufficient by observing respiratory rate, whisker movement, muscle tone and responses to stimuli such as pinching of foot. Administer additional anesthesia as required.</li>
		</ol>
		</li>
		<li>Shave the fur from the surgical regions, which include the right side of the abdomen for lymph and duodenum cannulation, and the neck and left clavicle region for carotid artery cannulation.</li>
		<li>Clean the surgical regions aseptically using povidine-iodine solution or chlorhexidine solution and a 70% ethanol scrub. Repeat this 3 times for each region, finishing with a final cleansing with 70% ethanol.</li>
	</ol>
	</li>
	<li>Place the animal in dorsal recumbence on a clean sheet above a heated surgical pad (37 &#176;C).</li>
</ol>
3. Cannulation of the Mesenteric Lymph Duct
<ol>
	<li>Place the animal with its right side facing toward the operator. Perform the surgery with or without the assistance of a surgical microscope as required.</li>
	<li>Open the top layer of the abdominal muscle wall with a straight 4 cm incision extending from the midline (xyphoid process) to the right flank approximately 2 cm below the ribcage (costal margin) using a sterile scalpel blade (see Figure 2B).</li>
	<li>Open the remaining layers of the abdominal muscle wall from 4 - 5 mm lateral to the midline to the right flank with a small pair of surgical scissors (see Figure 2B).</li>
	<li>Retract the small intestine under the left abdominal muscle wall and keep it in place using 2 - 3 pieces of sterile gauze saturated with normal saline.</li>
	<li>Bridge the rat over a 10 ml plastic syringe placed horizontally under the rat&#8217;s back at the level of right kidney to ease visualisation of the mesenteric lymph duct.</li>
	<li>Locate the superior mesenteric lymph duct: a vessel approximately 0.5 - 1 mm in diameter that is perpendicular to the right kidney and immediately rostral and parallel to the mesenteric artery (a pulsating dark red blood vessel; see Figure 2C). 
	NOTE: In non-fasted or oil pre-dosed rats the vessel is white, opaque and easier to visualise than in fasted rats in which it is quite translucent.</li>
	<li>Inspect the area immediately caudal to the large superior mesenteric lymph duct and the mesenteric artery to determine if a second, smaller accessory lymph duct is present. If present, block the flow of lymph through the duct by severing the duct and occluding with superglue, or tying a suture around the duct, to ensure that the entire volume of lymph flowing from the intestine is collected from the superior mesenteric lymph duct.</li>
	<li>Pass a pair of straight forceps through the peri-renal fat bed at the lower margin of the right kidney, and through the connective tissue layers immediately below the vena cava, in a direction parallel with the superior mesenteric lymph duct.</li>
	<li>Using the tip of the forceps take hold of the lymph cannula and pull it through the peri-renal fat bed with one end immediately adjacent to the mesenteric lymph duct and the other exteriorised from the animal at the level of the right kidney.</li>
	<li>Isolate the mesenteric lymph duct from overlying layers of connective and fat tissue by blunt dissection. Take care not to pierce or damage the lymph duct.</li>
	<li>After isolating the lymph duct, make a small hole in the lymph duct with either micro-scissors, the sharp tip of jeweller&#8217;s forceps or a 25 G needle. Take care not to completely sever the vessel.</li>
	<li>Ensure that the lymph cannula is completely filled with anti-coagulant solution (using a syringe and 25 G needle) with no air gaps. Insert the lymph cannula approximately 2 - 4 mm inside the mesenteric lymph duct via the small hole with the aid of a small pair of forceps.</li>
	<li>Observe the lymph cannula for a couple of minutes. Observe a gradual flow of intestinal lymph from the free collecting end of the cannula if the cannulation is successful. If the cannulation is not successful, repeat steps 3.8 - 3.12.</li>
	<li>If the cannulation is successful, secure the cannula by placing a small drop of veterinary adhesive&#160;over the entrance hole into the lymph duct. Take care not to occlude the vessel via the application of excess veterinary adhesive.</li>
	<li>Whilst waiting for the veterinary adhesive&#160;to set, observe the flow of lymph for several minutes to ensure that the cannulation procedure was successful. Once certain of the success of the cannulation, remove the gauze pieces that were placed in the abdominal cavity carefully and place the small intestine in its original position.</li>
	<li>Place a lymph collection tube containing anti-coagulant (see step 1.6) at the end of the cannula to collect the free flowing lymph.</li>
	<li>Next, cannulate the duodenum for infusion of rehydration solutions and/or formulations.</li>
</ol>
4. Cannulation of the Duodenum
<ol>
	<li>Attach the duodenum cannula to a syringe (e.g., 10 ml) filled with the rehydration solution.</li>
	<li>Identify the duodenum as the bright pink (with more blood vessels) section of the small intestine, which upon gentle downward pulling reveals the stomach.</li>
	<li>Make a small puncture hole in the duodenum approximately 2 cm below the junction of the stomach and duodenum (the pylorus) using a sterile 23 G needle.</li>
	<li>Insert the J shaped hooked end of the duodenal cannula through the puncture hole. Secure in place with a drop of cyanoacrylate glue.</li>
	<li>Commence hydration of the rat by attaching the cannula to the rehydration syringe loaded in an infusion pump. According to the literature rehydration rates range from 0.5 - 3 ml/hr15,25.</li>
	<li>After completion of the lymph and duodenum cannulation, close the incision in the abdominal muscle wall with sutures. Then close the skin incision by applying a few drops of tissue adhesive (cyanoacrylate) along the side of the incision and pinching the skin on both sides of the incision together.</li>
</ol>
5. Cannulation of the Carotid Artery
The carotid artery cannulation may be performed before or after the cannulation of the mesenteric lymph duct. Some operators prefer to cannulate the carotid artery prior to cannulating the lymph duct so as to reduce potentially dislodging the lymph cannula when moving the animal.
<ol>
	<li>Place a mark on the carotid artery cannula 2.5 cm from the bevel tip to identify the portion of the tubing to insert into the artery. Connect the other end of the cannula (i.e., without bevel) to a 23 or 25 G needle attached to a syringe filled with anti-coagulant solution and fill the cannula with the anti-coagulant solution making sure no air gaps are present.</li>
	<li>To facilitate cannulation, place the anaesthetised rat in dorsal recumbency with the neck stretched and head pointing toward the operator. Note that some operators prefer to perform this technique with the rat&#8217;s tail pointing toward the operator.</li>
	<li>Place a 1 - 1.5 cm longitudinal incision through the skin layer above the left of the trachea in the sagittal plane.</li>
	<li>Dissect the subcutaneous connective tissue using blunt tip forceps to expose the underlying paired neck muscles.</li>
	<li>Retract the neck muscles to reveal the left carotid artery (located ~1 cm below the skin surface and pulsing). Clean the overlying connective tissue from the artery by blunt dissection.</li>
	<li>Isolate an ~1 cm section of the artery carefully using blunt tissue forceps, taking care not to damage the adjacent vagal nerve track. Perform this by opening and closing the blunt tip forceps vertically along either side of the artery to free it from the surrounding tissue and vagus nerve.</li>
	<li>Using fine tip forceps, thread two silk sutures underneath the carotid artery and place them at each end of the isolated artery section. Tie off the suture closest to the operator and furthest from the rat&#8217;s heart and clavicle (Figure 3A).</li>
	<li>Occlude blood flow through the artery by placing a pair of straight fine tip forceps underneath the artery (Figure 3B).</li>
	<li>Using the fine tip iris scissors, place a small incision on the top surface of the artery (about 1/3 of the way down from the top of the isolated area). Flush any spilled blood on the surface of the artery with heparinised saline and gently wipe using cotton tips.</li>
	<li>Insert the tip of the cannula into the artery with a pair of curved tip forceps. Once inserted, remove the straight fine tip forceps occluding the blood flow and advance the cannula 2.5 cm into the artery using two pairs of forceps, one to hold the cannula inside the artery to stop blood from leaking back and the other to insert the cannula.</li>
	<li>Use a small artery clamp to hold the cannula inside the artery and remove the straight fine tip forceps that were placed below the artery in step 5.8 to occlude the blood flow.</li>
	<li>Confirm the patency of the cannula by injecting anti-coagulant solution into the cannula and drawing back a small amount of blood (Figure 3C). Then flush the cannula with anticoagulant solution and seal the end using the flame of a lighter or a cannula plug.</li>
	<li>Tie the cannula in place with the two sutures placed under the artery in step 5.8.</li>
	<li>Remove the artery clamp and secure a third suture around the artery and cannula. Close the neck wound with sutures.</li>
</ol>
6. Post-surgical Period and Formulation Infusion
<ol>
	<li>Continue to maintain the rat under anaesthesia and on the heated pad.</li>
	<li>Rehydrate the rat via infusion of rehydration solution into the duodenum for at least 0.5 hr following completion of the cannulations. Observe the lymph flow rate decrease (~0.1 - 0.5 ml/hr) during the initial period following cannulation and soon increase to a steady baseline (0.4 - 2.5 ml/hr depending on rehydration rate).</li>
	<li>Following the recovery period, infuse the formulation of interest (containing lipids, drugs, etc.) into the duodenum via the cannula. Optionally, use an infusion pump to regulate the flow rate of formulation.&#160;NOTE: In the protocol described here the animals remain anaesthetised throughout the surgery and lymph collection period and are euthanised under anaesthesia such that additional analgesia is not required. If the protocol is modified and&#160; animals are enabled to regain consciousness then appropriate analgesia and post-operative care will be required.&#160;</li>
	<li>At the completion of formulation infusion continue to rehydrate the rat at rate of 1.5-3 ml/hr with normal saline into the duodenum to prevent dehydration.</li>
	<li>Continue to keep the rat on the 37&#730;C heated surgical pad to maintain temperature (as per step 2.3), express urine from the bladder via gentle downward push on the lower abdomen as required and reapply ophthalmic ointment every hour (as per step 2.2.3). Regular body position changes are recommended if the animal is to regain consciousness after the procedure.</li>
</ol>
7. Collection of Lymph and Blood Samples to Assess Lipid and Drug Absorption
<ol>
	<li>Collect lymph continuously into tubes containing anti-coagulant. Change tubes at the required time intervals (e.g., hourly).</li>
	<li>Collect blood samples at set time points.
	<ol>
		<li>Occlude blood flow through the cannula by bending the cannula back approximately 2 cm from the sealed end. Unseal the cannula by cutting off the sealed end or removing the cannula plug.</li>
		<li>Using an empty syringe, withdraw heparinised saline from the cannula until blood fills the entire cannula.</li>
		<li>Using a new empty syringe, withdraw the desired volume of blood and place in a tube containing anti-coagulant.</li>
		<li>Using the first syringe, replace the blood taken before sampling to reduce unnecessary blood loss. Before injection, flick the syringe while holding it upright to ensure that no air bubbles enter the cannula.</li>
		<li>Using a syringe, replace the volume of blood removed from the rat with anti-coagulant solution. Ensure that no residual blood is visible in the cannula as any remaining may clot and block the cannula.</li>
		<li>Bend the cannula approximately 2 cm from the unsealed end to block blood flow and remove the syringe. Using the flame of a lighter or a cannula plug, reseal the cannula.</li>
	</ol>
	</li>
	<li>At the completion of blood and lymph collection from the rat,&#160;euthanize&#160;the rat via intraperitoneal administration of &#62;100 mg/kg sodium pentobarbitone.</li>
	<li>Determine lymph flow rate by measuring the mass of lymph collected during each time period.</li>
	<li>Measure the concentration of drug and lipid in lymph and blood using, for example, HPLC, HPLC-MS or using commercial kits, to assess lipid and drug absorption efficiency.</li>
	<li>Calculate mass transport of drug and lipids in lymph from the product of the measured concentrations and volume of lymph collected.</li>
</ol>

The authors have nothing to disclose.

The rat mesenteric lymph cannulation model enables direct quantification of the concentration and rate of transport of various cells and molecules (such as lipids and drugs) from the intestine into the lymph and the changes to these that occur in response to challenge of various substances (diet, antigen, drugs, formulations, etc.)10,27 and disease (cancer, viruses, colitis, insulin resistance, etc.)5-7. The components collected in lymph may also be further used in additional exper...

Lymph flows from the small intestine via a unidirectional process that originates at single lacteals that are contained within each small intestinal villi1. Lacteals are relatively permeable to fluid, macromolecules and cells and lymph formation thus commences with the entry of these factors into lacteals. The initial lymph in the lacteals subsequently flows from the intestine via a network of lymphatic microvessels, collecting (afferent) lymphatic vessels, a series of mesenteric lymph nodes and ultimately the post-nodal (efferent) lymph vessels. Within the nodes, lymph passes through a series of medullary sinuses where exchange occurs with node resident immune cells as well as material entering the node from the blood. All lymph flowing from the small intestine eventually converges into the efferent superior mesenteric lymph duct and subsequently the cisterna chyli. The cisterna chyli also collects lymph draining the caudal peripheral tissues, intestinal, hepatic and lumbar regions and joins the thoracic lymph duct together with lymph from the mediastinum and cranial parts of the body. The thoracic lymph duct empties lymph directly into the venous system at the junction of the left internal jugular and subclavian veins. The protocol described here, which enables the collection of lymph directly from the superior mesenteric lymph duct, thus facilitates the analysis of various factors transiting directly from the intestine to the systemic (general) circulation via the intestinal lymphatic system.
The major physiological functions assigned to the intestinal lymphatic system are to maintain fluid balance, to facilitate lipid and lipophilic molecule absorption, and to enable appropriate immune responses1. Tumor cells and viruses also propagate via the intestinal lymphatics2-4 and key changes occur within the lymphatics in several inflammatory and metabolic pathologies5-7. Cannulation of the mesenteric lymph duct to collect lymph within the mesentery enables an analysis of bulk fluid flow via the intestinal lymphatics as well as quantification of the concentration and transport rate of various cells and molecules. Changes to the concentration or transit of these factors in response to various challenges (e.g., diets, antigens, drugs) and in disease models (e.g., colitis, HIV, diabetes) can also be assessed. Whilst it is impossible to extensively describe each lymph component which may be analysed and compared here, mesenteric lymph simplistically consists of aqueous, lipid and cellular phases. Components of interest in the aqueous phase include peptides and proteins such as antigens or tolerogens8, immune messengers such as cytokines and mast cell mediators9, and metabolic mediators such as incretins10. The cellular fraction of the post-nodal mesenteric lymph consists almost entirely (over 99%) of lymphocytes11. Various immune cells (dendritic cells, mast cells, etc.) enter the pre-nodal mesenteric lymphatics but remain within the node12. If the cells within afferent lymph are of interest, it is possible to collect these cells via removal of the mesenteric lymph nodes several days before cannulation of the mesenteric lymph duct12. In this way the afferent and efferent lymph ducts are directly connected and the lymph cells in afferent lymph pass directly into the mesenteric lymph duct. The transit and phenotype of various immune cells passing through the intestinal lymphatics can thus be examined. Perhaps the most common reason cited for collecting mesenteric lymph to date, however, is to study the intestinal processing, absorption and transport of dietary lipids and lipophilic molecules10.
Following ingestion, dietary lipids are digested (for example, from triglyceride to fatty acids and monoglyceride, phospholipid to fatty acids and lysophospholipid, and cholesterol ester to fatty acid and cholesterol, etc.) and dispersed within the intestinal lumen into small micelle and vesicular structures via the addition of amphiphiles from bile (phospholipids, cholesterol and bile salts) and the action of pancreatic enzymes10,13. From here they are absorbed into enterocytes. A proportion of the absorbed components are re-esterified to form triglycerides, phospholipids and cholesterol esters within the absorptive cells (enterocytes). These re-esterified lipids are assembled from a combination of exogenously ingested lipid components and endogenous lipid components from the secreted bile, mucosal lipid pools or the intestinal blood supply13. From here the esterified lipids are either stored within enterocytes or assembled into intestinal lipoproteins (chylomicrons, very low density lipoproteins (VLDL)) together with various apoproteins and other lipophilic molecules (e.g., vitamins)10,13. After exiting enterocytes, lipoproteins are specifically transported from the intestine to the systemic circulation via the mesenteric lymphatic system as the intestinal lacteals are more permeable to their entry than the intestinal blood capillaries. A proportion of absorbed lipid components are also transported from the intestine to the systemic circulation via the blood capillaries and portal vein as single, non-lipoprotein associated, molecules14 . In general, however, the portal vein transport route is only a significant player in the absorption of short and medium chain length lipids.
The collection of mesenteric lymph thus enables the assessment of the transport of lipoproteins and associated components (lipids, lipophilic molecules, apo-proteins) from the intestine. The lipoproteins can be quantitated and characterized with the advantage that mesenteric lymph lipoproteins, in general, are in a nascent state since they have not been extensively modified by systemic enzymes such as lipoprotein lipase15. Whilst the mesenteric lymph cannulated rat model has perhaps historically been most extensively described for the analysis of lipid/lipoprotein transport from the intestine, an area of expanding interest is the role of lymphatics in the transport of lipophilic drugs, prodrugs and other xenobiotics13,16 which is the focus of the model described here. Lipophilic drugs (generally those with log P &#62; 5 and solubility in long-chain triglyceride &#62; 50 mg/g although exceptions are apparent)17,18, prodrugs19 and other xenobiotics13,16 can gain access to the intestinal lymphatics either passively or by actively integrating into intestinal lipoprotein transport pathways19.
The rat mesenteric lymph cannulation technique thus has many applications. Bollman et al. first described a technique to cannulate the mesenteric lymph duct in rats in 194820. Since then a number of variations on the model have been described. For example, collection can occur when the rat is anaesthetized with various anesthetics21,22, or in the conscious state whilst restrained15 or freely moving23,24. Rats can be administered different rehydration solutions and other substances such as lipids and drug formulations at different rates into the stomach, intestine or parenterally (typically 0 - 5 ml/hr)25. In some studies the thoracic lymph duct rather than mesenteric lymph duct is cannulated to estimate transport from the intestine via the lymphatics although this may overestimate transit from the small intestine, depending on the factor of interest, as the thoracic lymph duct also receives lymph from other regions22,26. Lymph cannulation models have also been described in several other species including mice15,27, mini-pigs12, sheep28,29, pigs30 and dogs31. However, the rat model is the most widely and consistently cited. Detailed protocols for cannulation of the mesenteric lymph duct followed by collection of lymph in conscious25 or anaesthetized22 rats and mice15,27 have been published previously and the interested reader is directed to these protocols. This protocol is the first to demonstrate the technique in a visualized format.
The lymph cannulated rat model has advantages over larger animal models in terms of expense, the ease of the surgery and ethical considerations. When compared to the mouse model, mesenteric lymph cannulation surgery is also easier in the rat although the mouse model enables more detailed studies in transgenic animals27. Nonetheless, there are some limitations of the rat model, particularly those associated with differences in physiology, that limit extrapolation to other pre-clinical and clinical situations. For example, in the rat bile flow is constant and independent of food intake whereas in higher species food or lipids stimulate bile flow32. This creates challenges to obtaining representative pre- and post-prandial environments in the rat that reflect what is seen in larger species and humans. For drug delivery studies, larger species may also be preferred when assessing lymphatic transport after the administration of realistic human dosage forms25. In a recent study, lipid transport rates in the mesenteric lymph were found to be comparable across species (mouse, rat, dog) after administration of an equivalent mass and type of lipid which provides some confidence in extrapolating lipid transport data across species27. However, the transport of a model lipophilic drug, halofantrine, ranked in the order of animal size (i.e., dog &#62; rat &#62; mouse). A scaling factor may thus be required to extrapolate lymphatic drug transport data from rat to other species.
A limitation of lymph cannulation models, in general, is that passive lymph collection directly from a lymphatic duct may modify lymph flow and transport since lymph vessels work against a pressure gradient that is altered once the vessel is cannulated33. The lymph cannulation model can also be difficult to establish in laboratories that are unfamiliar with the technique. Alternate models have thus been described. For instance, the transit of factors via the intestinal lymphatic system, such as lipoproteins and lipophilic molecules, has been indirectly studied via collection of blood. One such model involves comparing blood concentrations of lipids and/or drugs following oral administration in the presence and absence of inhibitors (e.g., colchicine, Pluronic L81, cycloheximide) of intestinal lipoprotein production that block lymphatic transport34. An advantage of models that quantify lymphatic transport indirectly via collection of blood samples is that it enables some evaluation of lymphatic transport in humans as invasive surgery is not required35. However, inhibitors of lymphatic transport are not specific and factors that are transported via the lymphatics are diluted and modified in the systemic circulation which complicates such assessments. In vitro alternatives have also been described. For example, caco-2 cell or isolated enterocyte cultures have been utilized to study in more detail the intestinal secretion of molecules that enter the lymphatics36-38. An advanced in vitro model that is more representative of the human intestinal microenvironment was also recently described39. In this model a lymphatic endothelial cell layer is co-cultured with Caco-2 cells which enables more detailed analysis of the transfer of substances from the intestine into the lymphatics. However, in vitro cell systems lack exchange flow and transfer ie interconnection with an intestinal lumen and underlying blood and lymphatic vascular supply. In an alternate approach, Kassis et al. established a dual-channel (high-speed bright-field video and fluorescence) in situ imaging system which enables quantitative comparisons between vessel contraction, lymph flow and fluorescent lipid concentrations in mesenteric lymphatic vessels33. An advantage of this model over the aforementioned in vitro systems is that it enables accurate tracking of the passage of immune cells through the lymphatics. Absolute measurements of mass lipid (or drug) transport are, however, not yet established using imaging methods. In vitro and in silico approaches to specifically predict the extent of lipophilic drug transport via the intestinal lymphatics have also been published40-42. For example, the ex vivo affinity of several compounds for plasma chylomicrons was found to correlate reasonably well with their lymphatic transport in vivo41. Subsequently, the same group established an in silico model to predict drug affinity for chylomicrons based on multiple physicochemical properties40. Holm et al. also established a relatively complex in silico model to outright predict lymphatic transport of lipophilic compounds on the basis of molecular descriptors42. These models may provide a useful approach to predict the extent of lymphatic transport of unknown drugs. Validation of the models with a wide range of drugs and across different labs will, however, be required to confirm their accuracy and reproducibility.
Cannulation of the mesenteric lymph duct thus remains the only means to directly examine the content of lymph draining the small intestine and the transit rate of the complex array of factors (cells, proteins, peptides, lipids, drugs) in lymph in an in vivo situation. Herein we describe a protocol for cannulation of the mesenteric lymph duct and carotid artery that enables the collection of mesenteric lymph and systemic blood from anaesthetized rats. Representative data demonstrate how the model can be used to examine lipid and drug transport from the intestine via the mesenteric lymphatic system. This is followed by a discussion of difficulties that can be encountered in establishing the model and a troubleshooting guide. Once established the model is a powerful tool to investigate intestinal lymphatic transport.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Sterile saline</td>
			<td>Baxter healthcare</td>
			<td>AHB 1307</td>
			<td>Any brand can be used. Example here is Baxter 100 ml saline bags, box of 50</td>
		</tr>
		<tr >
			<td >70 % ethanol in water</td>
			<td>Any</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Chlorhexidine gluconate solution (Microshield 4)</td>
			<td>Livingstone International</td>
			<td>JJ60243L</td>
			<td>Any brand can be used. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=JJ60243L</td>
		</tr>
		<tr >
			<td >Betadine solution</td>
			<td>Livingstone International</td>
			<td>BU0510</td>
			<td>Any brand can be used. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=BU0510</td>
		</tr>
		<tr >
			<td >Ilium Ketamil (Ketamine 100 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >KETA I 1</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >Ilium Xylazil (Xylazine 100 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >TRO-3828</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >ACP 10 Injection (Acepromazine 10 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >VTG-DACP010020</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >Sodium pentobarbitone</td>
			<td>PROVET VICTORIA&#160;</td>
			<td>24529</td>
			<td>Any brand can be used. Example here is Lethabarb&#174; 325 mg/ml sodium pentobarbitone, Virbac Animal Health. http://www.provet.com.au</td>
		</tr>
		<tr >
			<td >Heparin (35000I.U. in 35 mL)</td>
			<td>Sigma Pharmaceuticals</td>
			<td>337220</td>
			<td>http://sigmaco.com.au/</td>
		</tr>
		<tr >
			<td >Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate</td>
			<td >Sigma-Aldrich</td>
			<td >E1644</td>
			<td>Any brand can be used. Example here is disodium salt of EDTA from Sigma.&#160;</td>
		</tr>
		<tr >
			<td >Polyethylene (PE) cannula o.d. 0.96 mm x i.d. 0.58 mm</td>
			<td>Microtube extensions</td>
			<td>PE8050</td>
			<td>Any brand can be used. Example here is PE tubing 0.8x0.5 mm, 30 m</td>
		</tr>
		<tr >
			<td >Polyethylene (PE) cannula o.d. 0.8 mm x i.d. 0.5 mm</td>
			<td>Microtube extensions</td>
			<td>PE9658</td>
			<td>Any brand can be used. Example here is PE tubing 0.96x0.58 mm, 30 m</td>
		</tr>
		<tr >
			<td >Ruler</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Markers</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Cigarette lighter</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Cyanoacrylate glue</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >23 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN23GX0.75LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 23GX0.75inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&#38;CAT= 
			6&#38;search=DN23GX0.75LV</td>
		</tr>
		<tr >
			<td >25 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN25GX1.0LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 25GX1.0inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search= 
			DN25GX1.0LV</td>
		</tr>
		<tr >
			<td >1 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS01TA</td>
			<td>Any brand can be used. Example here is Terumo syringe 1 ml Slip Tuberculin 100/Box. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=T3SS01TA</td>
		</tr>
		<tr >
			<td >10 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS10SA</td>
			<td>Any brand can be used. Example here is Terumo syringe 10 ml Slip 100/Box. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=T3SS10SA</td>
		</tr>
		<tr >
			<td >Gauze swabs</td>
			<td>Livingstone International</td>
			<td>GSC075</td>
			<td>Any brand can be used and cut to required size. Example here is gauze swabs cotton filled 7.5x7.5 cm, 8 ply. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=GSC075</td>
		</tr>
		<tr >
			<td >Cotton buds</td>
			<td>Livingstone International</td>
			<td>CTAST075DP</td>
			<td>Any brand can be used. Example here is Livingstone cotton applicator plastic double tipped. 75MM. 100/PK. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=CTAST075DP</td>
		</tr>
		<tr >
			<td >Heating pad</td>
			<td>Ratek</td>
			<td>WT1</td>
			<td>Any brand that keeps temperature at 37C can be used. Example here is Ratek warming tray.</td>
		</tr>
		<tr >
			<td >Surgical light</td>
			<td>Harvard Apparatus</td>
			<td>72-0215 with 72-0267</td>
			<td>Any brand can be used. Example here is Harvard apparatus V-Lux 1000 Cold Light Source with Bifurcated Gooseneck Light Guide, Black, 4.7 mm fiber diameter (each arm). http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_50601_ 
			-1_HAI_ProductDetail and&#160; http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_35487_ 
			-1_HAI_ProductDetail___</td>
		</tr>
		<tr >
			<td >Surgical microscope</td>
			<td>Zeiss</td>
			<td>495005-0014-000</td>
			<td>Any brand can be used. Example here is Zeiss Stereomicroscope Stemi 2000-C with Stand S Double Spot and KL 300 LED. https://www.micro-shop.zeiss.com/?l=en&#38;p=us&#38;f=e&#38;i=10143</td>
		</tr>
		<tr >
			<td >Silk suture</td>
			<td>Livingstone International</td>
			<td>DTSK163019F4</td>
			<td>Any brand can be used. Example here is *&#160; 
			Email this item to my friend 
			3/8 Circle Reverse Cut Silk Suture 3/0 Thread 19mm. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=DTSK163019F4</td>
		</tr>
		<tr >
			<td >Scalpel blades</td>
			<td >Fine Science Tools (FST)</td>
			<td >10020-00</td>
			<td>Any brand can be used. Example here is FST Scalpel Blade #20. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=191</td>
		</tr>
		<tr >
			<td >Scalpel handle</td>
			<td >Fine Science Tools (FST)</td>
			<td>10004-13</td>
			<td>Any brand can be used. Example here is FST Scalpel Handle #4. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=298&#38;CategoryId=51</td>
		</tr>
		<tr >
			<td >1 x Small surgical scissors</td>
			<td >Fine Science Tools (FST)</td>
			<td>14060-09</td>
			<td>Any brand can be used. Example here is FST Fine Scissors, 9 cm with 21 mm cutting edge, sharp, straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=40&#38;CategoryId=17</td>
		</tr>
		<tr >
			<td >2 x Forceps with serrated curved tip</td>
			<td >Fine Science Tools (FST)</td>
			<td >11001-13</td>
			<td>Any brand can be used. Example here is FST 13 cm standard pattern forceps with curved 2.8x1.4 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=405&#38;CategoryId=32</td>
		</tr>
		<tr >
			<td >1 x Iridectomy scissors</td>
			<td >Fine Science Tools (FST)</td>
			<td>15000-08</td>
			<td>Any brand can be used. Example here is FST Vannas Spring Scissors - 2.5mm Cutting Edge, Straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=17&#38;CategoryId=16&#160;</td>
		</tr>
		<tr >
			<td >1 x Forceps with straight serated tip</td>
			<td >Fine Science Tools (FST)</td>
			<td>11650-10</td>
			<td>Any brand can be used. Example here is FST Graefe 10 cm straight with serrated 1 x 0.99 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=390&#38;CategoryId=32</td>
		</tr>
		<tr >
			<td >1 x Forceps with smooth sharp straight fine tip</td>
			<td >Fine Science Tools (FST)</td>
			<td>11251-10</td>
			<td>Any brand can be used. Example here is FST Dumont #5 forceps straight 11cm with 0.08 x 0.04mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=335&#38;CategoryId=29</td>
		</tr>
		<tr >
			<td >1 x Forceps with smooth fine curved forceps</td>
			<td >Fine Science Tools (FST)</td>
			<td>11063-07</td>
			<td>Any brand can be used. Example here is FST Delicate Forceps 9 cm with smooth 0.4 x 0.3mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=360</td>
		</tr>
		<tr >
			<td >2 x Hemostats</td>
			<td >Fine Science Tools (FST)</td>
			<td >13010-12</td>
			<td>Any brand can be used. Not all operators use the hemostats. Example here is FST 12 cm Micro-Mosquito Hemostats with 20 mm length x 1.3 mm width serrated, straight tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=377&#38;CategoryId=33</td>
		</tr>
		<tr >
			<td >1 x Suture needle holder</td>
			<td >Fine Science Tools (FST)</td>
			<td>12001-13</td>
			<td>Any brand can be used. Example here is FST 13cm Hasley Needle Holder with 16 mm length x 1.9 mm width tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=254&#38;CategoryId=70</td>
		</tr>
		<tr >
			<td >1 x Artery clamp</td>
			<td >Fine Science Tools (FST)</td>
			<td>18050-28</td>
			<td>Any brand can be used. Example here is FST Bulldog Serrefines straight, 28 mm long, 9x1.6 mm jaw dimension with medium clamp press. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=270&#38;CategoryId=82</td>
		</tr>
		<tr >
			<td >Oleic acid</td>
			<td >Sigma Aldrich</td>
			<td >O1008</td>
			<td>When required, any brand can be used. Example here is 99% pure oleic acid. http://www.sigmaaldrich.com/catalog/product/sial/o1008?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >14C-oleic acid</td>
			<td >Perkin&#160;</td>
			<td>NEC317050UC&#160;</td>
			<td>Any brand can be used. Example here is Oleic Acid, [1-14C]-, 50&#181;Ci (1.85MBq). http://www.perkinelmer.com/Catalog/Product/ID/NEC317050UC</td>
		</tr>
		<tr >
			<td >Sodium taurocholate</td>
			<td >Sigma Aldrich</td>
			<td >T4009</td>
			<td>Any brand can be used. Example here is taurocholic acid sodium salt hydrate &#8805;95% (TLC) . http://www.sigmaaldrich.com/catalog/product/sigma/t4009?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >Halofantrine</td>
			<td >Glaxo Smith Kline</td>
			<td ></td>
			<td>Halofantrine was kindly provided as a gift from Glaxo Smith Kline</td>
		</tr>
		<tr >
			<td >Sodium phosphate monobasic</td>
			<td >Sigma Aldrich</td>
			<td >71507</td>
			<td >Any brand can be used. Example here is sodium phosphate monobasic monohydrate, BioXtra, for molecular biology, &#62;99.5%. http://www.sigmaaldrich.com/catalog/product/sigma/71643?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >Sodium phosphate dibasic</td>
			<td >Sigma Aldrich</td>
			<td >71643</td>
			<td >Any brand can be used. Example here is sodium phosphate dibasic dihydrate, BioUltra, for molecular biology, &#62;99%. http://www.sigmaaldrich.com/catalog/product/sigma/71507?lang=en&#38;region=AU</td>
		</tr>
	</tbody>
</table>

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.

The results of a representative experiment to quantitate the cumulative extent and rate of lipid and drug transport via the lymphatic system following intestinal delivery using the mesenteric lymph cannulation model are shown in Figure 4 and Figure 5. In this experiment, 200 &#181;g of the model lipophilic drug halofantrine was administered into the duodenum of rats over 2 hr in a formulation containing 40 mg oleic acid (including 2 - 5 &#181;Ci 14C-oleic acid) and 7.1 mg 2-mo...

Watch this Scientific Journal Video about The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport at JoVE.com

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

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 ...

the-mesenteric-lymph-duct-cannulated-rat-model-application-to

Research

JoVE Journal

Immunology and Infection

28.2K Views.  Monash University (Parkville Campus). 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.

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

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...

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 ...

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability.
Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.
The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol&#160;describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...