GSE168755
Age-associated suppression of exploratory activity during sickness is linked to meningeal lymphatic dysfunction and microglia activation [ablation]
The objective of this study is to assess the contribution of meningeal lymphatic vessels to microglia response to peripheral inflammation.  To this end, we sequence CD11b+ cells sorted from whole brain of adult mice with intact meningeal lymphatic vessels or mice with experimental meningeal lymphatic ablation, two hours following 1µg IL-1ß or saline vehicle intraperitoneal injection.  The two-hour timepoint was selected for analysis because that corresponds with the peak of the behavioral response as defined by a decrease in locomotor activity and the connection between behavior and microglia activation is of particular interest in this study. Overall design: Mice were maintained under specific pathogen-free conditions, with controlled temperature and humidity, on 12 h light:dark cycles, and ad libitum access to food and water.  Experiments were all performed during light cycle.  Experimental lymphatic ablation or sham operation was performed on two-month old male C57BL/6J (Jax #000664) mice.  For lymphatic ablation, mice were anesthetized with ketamine (100 mg kg-1) and xylazine (10 mg kg-1), and secured on a stereotaxic frame.  5µL of Visudyne (Valeant Ophtalmics) solution or artificial CSF for sham surgery was delivered via intra-cisterna magna injection and 15 minutes later, the drug was then photoconverted through intact skull using a non-thermal 689-nm wavelength laser (Coherent Opal Photoactivator, Lumenis) at 5 points previously demonstrated to be effective.  Each point was photoconverted for 83 seconds to provide a light dose of 50 J/cm2 at an intensity of 600 mW/cm2.  The mice were then sutured and recovered on heating pad until fully awake.  Following 1-week recovery, Mice were injected i.p. with either 1µg IL-1ß or saline vehicle (5 mice per group, 4 groups +/- Visudyne +/-IL-1ß).  Two hours later mice were euthanized by lethal dose of anesthetic (Euthasol) and perfused with cold PBS.  One whole hemisphere of brain was collected from each mouse and biological replicates were pooled.  Brain tissue was dissociated in 5mL Hank's buffering saline solution with DNaseI (50U/mL) and papain (4U/mL) at 37ºC for 45 minutes.  Cell suspension was then passed through a 70um strainer and CD11b+ cells were magnetically sorted using CD11b MicroBeads, human and mouse (Miltenyi) as per manufacturer's instructions using AutoMACS (Miltenyi) and 2000 cells per condition were targeted for sequencing.