Mesenteric Lymph Duct Cannulation Research Articles

Mesenteric Lymph Duct Cannulation in a Rat Model to Harvest Lymph Fluid

Mesenteric Lymph Duct Cannulation in a Rat Model to Harvest Lymph Fluid

Mesenteric Lymph Duct Cannulation in a Rat Model to Harvest Lymph Fluid

Mesenteric Lymph Duct Cannulation in a Rat Model to Harvest Lymph Fluid

The digestion of diacylglycerol isomers by gastric and pancreatic lipases and its impact on the metabolic pathways for TAG re-synthesis in enterocytes

The specific activities of gastric and pancreatic lipases were measured using triacylglycerols (TAG) from rapeseed oil, purified 1,3-sn-DAG and 1,2(2,3)-sn-DAG produced from this oil, as well as a rapeseed oil enriched with 40% w/w DAG (DAGOIL). Gastric lipase was more active on 1,3-sn-DAG than on 1,2(2,3)-sn-DAG and TAG, whereas pancreatic lipase displayed a reverse selectivity with a higher activity on TAG than on DAG taken as initial substrates. However, in both cases, the highest activities were displayed on DAGOIL. These findings show that DAG mixed with TAG, such as in the course of digestion, is a better substrate for lipases than TAG. The same rapeseed oil acylglycerols were used to investigate intestinal fat absorption in rats with mesenteric lymph duct cannulation. The levels of TAG synthesized in the intestine and total fatty acid concentration in lymph were not different when the rats were fed identical amounts of rapeseed oil TAG, 1,2(2,3)-sn-DAG, 1,3-sn-DAG or DAGOIL. Since the lipolysis of 1,3-sn-DAG by digestive lipases leads to glycerol and not 2-sn-monoacylglycerol (2-sn-MAG) like TAG lipolysis, these results suggest that the re-synthesis of TAG in the enterocytes can entirely occur through the “glycerol-3-phosphate (G3P)” pathway, with the same efficiency as the 2-sn-MAG pathway predominantly involved in the intestinal fat absorption. These findings shed new light on the role played by DAG as intermediate lipolysis products. Depending on their structure, 1,2(2,3)-sn-DAG versus 1,3-sn-DAG, DAG may control the pathway (2-sn-MAG or G3P) by which TAG are re-synthesized in the enterocytes.

Intestinal Lymph Collection via Cannulation of the Mesenteric Lymphatic Duct in Mice

BackgroundWe have optimized a technique for cannulation of mesenteric lymph duct (MLD) in mice. Mice have low rates of intestinal lymph production; the MLDs are smaller and associated with fragile vasculature. Previous protocols for lymph collection based on the open lymph fistula model were associated with low success rates in mice. Bariatric surgery procedures worsen success rates due to postoperative adhesions and GI rearrangement. We have used this procedure to collect mesenteric lymph from mice undergoing bile diversion from gall bladder to ileum (GB-IL). HypothesisWe hypothesize that peptide YY (PYY) levels in mesenteric lymph will increase following nutrient delivery in mice undergoing bile diversion from gall bladder to ileum (GB-IL). Methods and resultsWe observe that cannulation of the MLD using a needled-catheter maintains lymph vessel integrity, prevents excessive lymph leakage, and is less traumatic, leading to high success rates (>95%). PYY levels in mesenteric lymph after GB-IL were significantly higher post nutrient infusion. The procedure takes approximately 20 min; small rodent surgical experience and practice are required for success. ConclusionsIntestinal lymph can be collected from mice, including those undergoing bariatric surgical procedures with high success rates by cannulation of the mesenteric lymph duct.

Rapeseed Lecithin Increases Lymphatic Lipid Output and α-Linolenic Acid Bioavailability in Rats

Soybean lecithin, a plant-based emulsifier widely used in food, is capable of modulating postprandial lipid metabolism. With arising concerns of sustainability, alternative sources of vegetal lecithin are urgently needed, and their metabolic effects must be characterized. We evaluated the impact of increasing doses of rapeseed lecithin (RL), rich in essential α-linolenic acid (ALA), on postprandial lipid metabolism and ALA bioavailability in lymph-cannulated rats. Male Wistar rats (8 weeks old) undergoing a mesenteric lymph duct cannulation were intragastrically administered 1 gof an oil mixture containing 4% ALA and 0, 1, 3, 10, or 30% RL (5 groups). Lymph fractions were collected for 6 h.Lymphlipidsandchylomicrons (CMs) were characterized. The expression of genes implicated in intestinal lipid metabolism was determined in the duodenum at 6 h.Datawasanalyzedusing either sigmoidal or linear mixed-effects models, or one-way ANOVA, where appropriate. RL dose-dependently increased the lymphatic recovery (AUC) of total lipids (1100 μg/mL·hperadditional RL%; P=0.010) and ALA (50 μg/mL·hperadditional RL%; P=0.0076). RL induced a faster appearance of ALA in lymph, as evidenced by the exponential decrease of the rate of appearance of ALA with RL (R2=0.26; P=0.0064). Although the number of CMs was unaffected by RL, CM diameter was increased in the 30%-RL group, compared to the control group (0% RL), by 86% at 3-4 h(P=0.065) and by 81% at 4-6 h(P=0.0002) following administration. This increase was positively correlated with the duodenal mRNA expression of microsomal triglyceride transfer protein (Mttp; ρ= 0.63; P=0.0052). The expression of Mttp and secretion-associated, ras-related GTPase 1 gene homolog B (Sar1b, CM secretion), carnitine palmitoyltransferase IA (Cpt1a) and acyl-coenzyme A oxidase 1 (Acox1, beta-oxidation), and fatty acid desaturase 2 (Fads2, bioconversion of ALA into long-chain n-3 PUFAs) were, respectively, 49%, 29%, 74%, 48%, and 55% higher in the 30%-RL group vs. the control group (P<0.05). In rats, RL enhanced lymphatic lipid output, as well as the rate of appearance of ALA, which may promote its subsequent bioavailability and metabolic fate.

The role of mesenteric lymph exosomal lipid mediators following intestinal ischemia-reperfusion injury on activation of inflammation

Intestinal ischemia caused by hemorrhagic shock is known to induce systemic inflammatory responses. Previous studies have shown that mesenteric lymph (ML) plays a crucial role in gut-mediated inflammation. Lipid mediators, such as lysophosphatidylcholines (LPCs), which contain polyunsaturated fatty acids (PUFAs), are present in the postshock ML. Exosomes are also present in the ML and act as transcellular carriers of lipids; however, their role in postshock systemic inflammation has not been revealed. Here, we aimed to identify changes in lipid mediators in ML exosomes after intestinal ischemia. Male Sprague-Dawley rats underwent laparotomy, followed by ML duct cannulation. Animals were subjected to 60 minutes of intestinal ischemia by superior mesenteric artery clamping, followed by 120 minutes of reperfusion. Mesenteric lymph was obtained before and after intestinal ischemia, and exosomes were isolated from ML by ultracentrifugation. The biological activity of ML exosomes was determined using the monocyte nuclear factor κB (NF-κB) activation assay. Lipids of ML exosomes were extracted and quantified by liquid chromatography/electrospray ionization mass spectrometry. Mesenteric lymph exosome-induced NF-κB activation significantly increased after intestinal ischemia, and lipid analysis revealed a significant increase in the concentration of PUFA-containing LPCs. In addition, PUFA-containing LPCs also induced NF-κB activation. Our results suggest that biologically active lipid mediators in ML exosomes may be involved in the inflammatory response after intestinal ischemia.

Pharmacokinetic and Toxicodynamic Characterization of a Novel Doxorubicin Derivative.

Doxorubicin (Dox) is an effective anti-cancer medication with poor oral bioavailability and systemic toxicities. DoxQ was developed by conjugating Dox to the lymphatically absorbed antioxidant quercetin to improve Dox’s bioavailability and tolerability. The purpose of this study was to characterize the pharmacokinetics and safety of Dox after intravenous (IV) and oral (PO) administration of DoxQ or Dox (10 mg/kg) and investigate the intestinal lymphatic delivery of Dox after PO DoxQ administration in male Sprague–Dawley rats. Drug concentrations in serum, urine, and lymph were quantified by HPLC with fluorescence detection. DoxQ intact IV showed a 5-fold increase in the area under the curve (AUC)—18.6 ± 1.98 compared to 3.97 ± 0.71 μg * h/mL after Dox—and a significant reduction in the volume of distribution (Vss): 0.138 ± 0.015 versus 6.35 ± 1.06 L/kg. The fraction excreted unchanged in urine (fe) of IV DoxQ and Dox was ~5% and ~11%, respectively. Cumulative amounts of Dox in the mesenteric lymph fluid after oral DoxQ were twice as high as Dox in a mesenteric lymph duct cannulation rat model. Oral DoxQ increased AUC of Dox by ~1.5-fold compared to after oral Dox. Concentrations of β-N-Acetylglucosaminidase (NAG) but not cardiac troponin (cTnI) were lower after IV DoxQ than Dox. DoxQ altered the pharmacokinetic disposition of Dox, improved its renal safety and oral bioavailability, and is in part transported through intestinal lymphatics.

Free fatty acid receptor activation alters mesenteric lymphatic flow and fatty acid transport in rats

Enteroendocrine cells express nutrient GPCRs and release gut hormones in response to luminal nutrients. Free fatty acid receptors (FFARs) are activated by luminal long-chain (LCFAs) or short-chain fatty acids (SCFAs). We hypothesized that activation of FFARs may alter lipid absorption. Lymph was collected via mesenteric lymph duct cannulation in anesthetized rats. We examined the effect of bolus perfusion of selective FFAR agonists (10 µM) into the duodenum on lymphatic flow. Fatty acid transport was assessed by in vivo microscopy and by the appearance rate of fluorescent-conjugated LCFA probe (C1-BODIPY-C12 (BDPY), equivalent to C18 oleic acid) into lymph. Luminal BDPY was quickly absorbed by duodenal and jejunal villous cells. Luminal BDPY (25 µM) increased the lymphatic flow rate and was transported into lymph. The CD36 inhibitor sulfosuccinimidyl oleate reduced BDPY transport rate, confirming that BDPY functioned as a LCFA. The selective FFA1 agonist GW9508 enhanced the lymphatic flow rate and BDPY transport, accompanied by glucagon-like peptide-2 (GLP-2) release into lymph. GLP-2 receptor antagonism abolished the GW9508-induced increase of lymphatic flow and BDPY transport. In contrast, the selective FFA2 agonist increased BDPY transport via 5-HT3 activation, consistent with FFA2 expression on EC cells. The selective FFA4 agonist had little effect on lymphatic flow or on BDPY transport. Respective release of GLP-2 and 5-HT via FFA1 and FFA2 activation alters LCFA transport during lipid absorption. Luminal FFARs may be therapeutic targets for obesity and 5-HT-related dyspeptic symptoms. Supported by VA Merit Review, NIH R01 DK54221

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.

Testosterone Depletion or Blockade in Male Rats Protects Against Trauma Hemorrhagic Shock-Induced Distant Organ Injury by Limiting Gut Injury and Subsequent Production of Biologically Active Mesenteric Lymph

We tested the hypothesis that testosterone depletion or blockade in male rats protects against trauma hemorrhagic shock-induced distant organ injury by limiting gut injury and subsequent production of biologically active mesenteric lymph. Male, castrated male, or flutamide-treated rats (25 mg/kg subcutaneously after resuscitation) were subjected to a laparotomy (trauma), mesenteric lymph duct cannulation, and 90 minutes of shock (35 mm Hg) or trauma sham-shock. Mesenteric lymph was collected preshock, during shock, and postshock. Gut injury was determined at 6 hours postshock using ex vivo ileal permeability with fluorescein dextran. Postshock mesenteric lymph was assayed for biological activity in vivo by injection into mice and measuring lung permeability, neutrophil activation, and red blood cell deformability. In vitro neutrophil priming capacity of the lymph was also tested. Castrated and flutamide-treated male rats were significantly protected against trauma hemorrhagic shock (T/HS)-induced gut injury when compared with hormonally intact males. Postshock mesenteric lymph from male rats had a higher capacity to induce lung injury, Neutrophil (PMN) activation, and loss of red blood cell deformability when injected into naïve mice when compared with castrated and flutamide-treated males. The increase in gut injury after T/HS in males directly correlated with the in vitro biological activity of mesenteric lymph to prime neutrophils for an increased respiratory burst. After T/HS, gut protective effects can be observed in males after testosterone blockade or depletion. This reduced gut injury contributes to decreased biological activity of mesenteric lymph leading to attenuated systemic inflammation and distant organ injury.

Lymphatic chylomicron size is inversely related to biliary phospholipid secretion in mice

Biliary phospholipids (PL) stimulate dietary fat absorption by facilitating intraluminal lipid solubilization and by providing surface components for chylomicron (CM) assembly. Impaired hepatic PL availability induces secretion of large very-low-density lipoproteins, but it is unclear whether CM size depends on biliary PL availability. Biliary PL secretion is absent in multidrug resistance protein 2-deficient (Mdr2(-/-)) mice, whereas it is strongly increased in essential fatty acid (EFA)-deficient mice. We investigated lymphatic CM size and composition in mice with absent (Mdr2(-/-)) or enhanced (EFA deficient) biliary PL secretion and in their respective controls under basal conditions and during enteral lipid administration. EFA deficiency was induced by feeding mice a high-fat, EFA-deficient diet for 8 wk. Lymph was collected by mesenteric lymph duct cannulation with or without intraduodenal lipid administration. Lymph was collected in 30-min fractions for up to 4 h, and lymphatic lipoprotein size was determined by dynamic light-scattering techniques. Lymph lipoprotein subfractions were isolated by ultracentrifugation, and lipid composition was measured. Lymphatic CMs were significantly larger in Mdr2(-/-) mice than in Mdr2(+/+) controls either without (+50%) or with (+25%) enteral lipid administration, and molar core-surface ratios were increased [triglyceride (TG)-to-PL ratio: 4.4 +/- 1.4 in Mdr2(-/-) mice vs. 2.7 +/- 0.8 in Mdr2(+/+) mice, P < 0.001]. In contrast, EFA-deficient mice secreted lipoproteins into lymph that were significantly smaller than in EFA-sufficient controls (173 +/- 32 vs. 236 +/- 47 nm), with correspondingly decreased core-surface ratios (TG-to-PL ratio: 3.0 +/- 1.0 in EFA-deficient mice vs. 6.0 +/- 1.9 in EFA-sufficient mice, P < 0.001). CM size increased during fat absorption in both EFA-deficient and EFA-sufficient mice, but the difference between the groups persisted. In conclusion, the present results strongly suggest that the availability of biliary PL is a major determinant of the size of intestinally produced lipoproteins both under basal conditions and during lipid absorption. Altered CM size may have physiological consequences for postprandial CM processing.

Evaluation of a chylomicron flow blocking approach to investigate the intestinal lymphatic transport of lipophilic drugs

The purpose of this study was to examine the feasibility of investigating the lymphatic transport of drugs in vivo utilizing known chylomicron flow blocking substances. Vitamin D 3 (0.5 mg/kg), a model lipophilic molecule, was administered to rats with blocked chylomicron flow, induced by either cycloheximide injection (3 mg/kg), colchicine injection (5 mg/kg) or intraduodenal infusion of pluronic L-81 (1 mg/h). The effect of these experimental models on the absorption of Vitamin D 3 was compared to the outcomes of the mesenteric lymph duct cannulated rat model. The oral d-xylose loading test was used to verify that other intestinal absorptive functions were not affected. Colchicine treatment induced severe adverse effects whereas pluronic L-81 and the cycloheximide models did not affect other absorption pathways and did not cause apparent adverse effects. Vitamin D 3 absorption in these two models was in good correlation to the mesenteric lymph duct cannulation model (25% non-lymphatic relative bioavailability) indicating that the incorporation of the lipophilic molecule into the chylomicron is an essential step in the cascade of lymphatic absorption. Moreover, the data suggest that the drug association with the chylomicron occurs at an early stage of its assembly process. The results also specify that lymphatic absorption and portal blood absorption are separate pathways that are not affected by each other. In conclusion, the chemical blockage of chylomicron flow provides a potential approach for lymphatic transport investigation, and may elucidate processes involving in the absorption of lipophilic compounds.

Intestinal absorption and metabolism of 9-cis-beta-carotene in vivo: biosynthesis of 9-cis-retinoic acid.

This study was done to examine the intestinal absorption and cleavage of 9-cis-beta-carotene in vivo. A micellar solution, containing either no addition or 10 mumol of 9-cis- or all-trans-beta-carotene, was perfused for 2 h through the upper portion of the small intestine of ferrets. The effluent of a mesenteric lymph duct cannulation was collected, as well as intestinal mucosa scrapings, a portal blood sample, and a liver biopsy, both before and after perfusion. Carotenoids and retinoids were measured by reverse-phase, high performance liquid chromatography. 9-Cis- and all-trans-beta-carotene were transported equally well into mesenteric lymph, although the intestinal concentration of the corresponding isomer was tenfold higher after perfusion of the 9-cis- isomer than after perfusion of all-trans-beta-carotene. Regardless of which isomer was used, perfusion of beta-carotene resulted in the biosynthesis of similar amounts of retinoic acid in portal blood, liver, and intestine. However, after the perfusion of all-trans-beta-carotene, all the retinoic acid formed was in the all-trans- form, whereas the perfusion of 9-cis-beta-carotene resulted in the biosynthesis of about 50% of the total retinoic acid as the 9-cis-isomer. We conclude that in the in vivo ferret model, 9-cis-beta-carotene has a good bioavailability and is a precursor of 9-cis-retinoic acid.

Vitamin E enhances the lymphatic transport of β-carotene and its conversion to vitamin A in the ferret

Background/Aims : β-Carotene and α-tocopherol may have either antagonistic or synergistic effects on each other's absorption and metabolism. The effects of both physiological and pharmacological concentrations of α-tocopherol on the absorption and metabolism of β-carotene in ferret intestine were determined. Methods : A high concentration of β-carotene was perfused through the upper portion of the small intestine of ferrets in vivo with varying levels of α-tocopherol. The effluent of a mesenteric lymph duct cannulation, the intestinal mucosa scraping, and portal vein blood were sampled and analyzed by high-performance liquid chromatography. Results: The lymphatic transport of β-carotene was enhanced 4-fold by α-tocopherol at a physiological dose and 12–21-fold at a pharmacological dose. The lymphatic transport of α-tocopherol was linearly ( r = 0.8; P < 0.05) related to the luminal α-tocopherol concentration even in the presence of a high concentration of β-carotene. Furthermore, α-tocopherol increased the conversion of β-carotene into retinol in the intestine in a dose-dependent manner. Conclusions : α-Tocopherol has a positive effect on the intestinal absorption of intact β-carotene and may modulate the metabolic conversion of β-carotene into retinoids.

Intestinal uptake and lymphatic absorption of beta-carotene in ferrets: a model for human beta-carotene metabolism.

To determine the appropriateness of the ferret as a model for human beta-carotene (beta-C) metabolism, we have perfused both 15,15'-beta-[14C]C and unlabeled beta-C through the upper 30-cm portion of the small intestine of ferrets in vivo. The effluents of a mesenteric lymph duct cannulation and a common bile duct cannulation, as well as portal vein blood periodically sampled via an indwelling catheter, were collected. Ten percent (9.5 +/- 0.06%) of the total administered beta-C was taken up by the intestine after a 4-h perfusion. Of the radioactivity taken up, 68.6 +/- 6.5% remained in the intestinal mucosa, 3.2 +/- 0.2% was recovered in the lymph, and 28.2 +/- 6.5% (calculated) was absorbed via the portal system. The total uptake/absorption of beta-C was 12.9 +/- 6.8 nmol.h-1.30 cm intestine-1. Large amounts of unchanged beta-C and relatively small amounts of both beta-apo-12'-carotenal and beta-apo-10'-carotenal were isolated in the intestinal mucosa after a 4-h perfusion with beta-C. Considerable amounts of metabolites more polar than retinol were formed and comprised 35% of the total radioactivity recovered in the intestinal mucosa. Polar metabolites were absorbed mostly into the portal venous system, whereas retinol and retinyl esters were absorbed mainly into the mesenteric lymph. Of the total absorbed radioactivity in lymph, 10 +/- 1.0% appeared as unchanged beta-C, with peak absorption occurring at 3 h after beginning the perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal apolipoprotein B-48 synthesis and lymphatic cholesterol transport are lower in swine fed high fat, high cholesterol diet with soy protein than with casein

Effects of dietary proteins on intestinal lipoproteins were studied in 8 Yorkshire swine fed a high fat, high cholesterol diet with either casein or soy protein. After 5 weeks of feeding, the casein group exhibited moderately elevated levels of serum cholesterol (334 ± 46 mg/dl). The soy protein group showed significantly less hypercholesterolemia as compared to the casein group (122 ± 8 mg/dl). Swine were subjected to cannulation of mesenteric lymph duct under halothane anesthesia. A single dose of 250 μCi [ 14C]cholesterol and 10 MCi [ 3H]leucine was infused into the upper jejunum 2 h after one-fifth of daily food was given. The 3-h lymphatic transport of cholesterol in casein-fed swine was significantly higher than in those fed soy protein. Triglyceride transport values were similar in the 2 groups. The [ 3H]leucine incorporation study revealed that transport of apo B-48 bore a significant positive relationship to transport of cholesterol in both chylomicron and VLDL fractions of mesenteric lymph. A greater apo B-48 secretion with higher specific activity was probably responsible for the greater transport of cholesterol in chylomicrons in casein-fed than in soy protein-fed swine. Similarly, the transport of lymph VLDL cholesterol in swine fed casein or soy protein paralleled the amount of accompanying apo B-48. Dietary proteins probably influence the intestinal synthesis of apo B-48 which in turn affects cholesterol transport into the lymphatics.