A single-day mouse mesenteric lymph surgery in mice: an updated approach to study dietary lipid absorption, chylomicron secretion, and lymphocyte dynamics

Figures

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  Fig. 1. Experimental model for 1-day versus 2-day lymph fistula mouse. A: Double cannulation takes approximately 2–4 h. Mice recover in Snuggle restraints and in a warmed incubator. B: One-day experimental model. One hour prior (−1) to the bolus lipid infusion, mice receive a continuous duodenal infusion of 5% glucose in sterile saline, and a single hour of “fasting” lymph is collected. At time 0, the continuous intraduodenal infusion is switched to a bolus infusion of 0.3 ml SMOFlipid. Immediately after the bolus lipid, the continuous glucose infusion is resumed. Lymph is collected hourly on ice for 6 h after the bolus infusion. At 6 h, mice are euthanized, and tissues were collected. C: Two-day experimental model. Mice receive a continuous duodenal infusion of 5% glucose in sterile saline as in (B), but the infusion continues for an additional ∼18 h. D: On the morning of the second day, a single hour of “fasting” lymph is collected. As in (B), at time 0, the continuous intraduodenal infusion is switched to a bolus infusion of 0.3 ml SMOFlipid. Immediately after the bolus lipid, the continuous glucose infusion is resumed. Lymph is collected hourly on ice for 6 h after the bolus…

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  Fig. 2. Anatomical models and surgical photos for 1-day lymph fistula mouse. A: Cartoon mouse showing key areas of the liver and gastrointestinal tract and in green, the lymphatic vasculature. Red circle orients the site of the main mesenteric lymph duct and the focus on our surgical technique. B: The mesenteric lymph duct is found when the small intestine and liver are gently moved out of the body cavity. Black arrows in cartoon highlight key anatomical and surgical landmarks (clockwise from top of cartoon: liver, mesenteric fat, and clear cannula tubing). Photo shows the same anatomical and surgical landmarks as in cartoon but in white arrows. C: Higher magnification photo of the clear mesenteric cannula (upper white arrow), inserted into the mesenteric lymph duct (lower white arrow). Cartoon shows the mesenteric lymph cannula (upper black arrow, yellow tube) and clear mesenteric lymph duct (lower black arrow, green tube). D: Photo of the blue tip of the inserted duodenal feeding tube at ∼5 cm into proximal duodenum. Cartoon shows the insertion of the duodenal feeding tube through the stomach wall, advanced ∼5 cm into proximal duodenum. E: After implantation of duodenal and mesenteric…

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  Fig. 3. Surgical success and lymph flow comparison between 1-day and 2-day surgical models. A: WT mice were fitted with an intaraduodenal feeding tube and a mesenteric lymph cannula. Mice received a bolus infusion of 300 μl of lipid. Surgical success resulted in the secretion of milky lipid-rich flowing lymph starting ∼15 min after the duodenal lipid infusion and lasting at least 5 h. Data are plotted as the number of successful mice (1 day = 17 and 2 days = 8). N = 48. B: Hourly lymph flow rate in the 6 h after intraduodenal lipid infusion. In open circles, WT mice received lipid on the same day as surgery (1 day). In black squares, WT mice received lipid on second day after surgery (2 days). C: Total volume of lymph secreted by mice in each experimental paradigm. Values are means ± SEM or means + SEM. ∗∗P < 0.005.

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  Fig. 4. Increased TAG secretion in lymph in response to shortened experimental time. A: In open circles, WT mice received intraduodenal lipid on the same day of surgery (1 day), n = 17. In black squares, WT mice received intraduodenal lipid after an overnight recovery period, on the day after surgery (2 days), n = 8. TAG concentrations in lymph in response to lipid bolus were determined by chemical assay. B: Total mass of TAG secreted during the 6 h after intraduodenal bolus. C: In white diamonds, WT mice received lipid on the same day as surgery (day 1). In black diamonds, the same mice received a second intraduodenal lipid dose the day after surgery—experimental model shown in (D). n = 10+, and values are means ± SEM. P < 0.05 or means + SEM. ∗P < 0.05, ∗∗P < 0.005, and ∗∗∗P < 0.0001. Model created with BioRender.com.

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  Fig. 5. Analysis of T lymphocytes in mesenteric lymph before and after a lipid bolus. WT mice were fitted with mesenteric lymph cannula and intraduodenal feeding tube. Mice then received a continuous duodenal infusion of 5% glucose in sterile saline. During this “prelipid bolus” period, clear lymph was collected. At time 0, the continuous intraduodenal infusion is switched to a bolus infusion of 0.3 ml lipid. Immediately after the bolus lipid, the continuous glucose infusion is resumed. “Postlipid bolus” lymph was then collected hourly on ice for 6 h after the bolus infusion. A: Number of cells per volume (1 ml) of lymph fluid collected and quantification of percent of parent population. Data are pooled from three independent experiments (n = 2–3/experiment). B–E: Representative flow cytometric plots showing lymphocytes from equal volumes of prelipid or postlipid bolus lymph. Cells gated: Live lineage: negative for LIVE/DEAD stain, (B) total leukocyte: CD45+ cells gated on live cells, (C) CD4+ cell gated on CD45+ cells, (D) naïve CD4 cells: CD62hi CD44low, and (E) Treg cells: CD25+ Foxp3+ gated on CD4+. Numbers on plots denote cell frequencies (in percentages) of indicated parent…