Aging disrupts sympathetic innervation of the thymus

Figures

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  Figure 1.. Immunolabeling and OCT identify sympathetic innervation of the intact mouse thymus (A) Schematic outline of the thymic tissue processing and optical tissue clearing (OTC) protocol. Blood vessel labeling was performed via retro-orbital injection of CD31 and CD144 antibodies, followed by intracardiac perfusion of PBS and 4% paraformaldehyde (PFA). The thymus was removed and post-fixed in 4% PFA and then processed for antibody labeling. After labeling, the thymus lobes were dehydrated in ethanol and cleared in ethyl cinnamate. Imaging was performed on a Nikon CSU-W1 spinning disk confocal microscope. (B) Representative images of thymus lobes before and after clearing with ethyl cinnamate. (C) Representative confocal z stack projection through 200 μm depth of a left whole thymus lobe showing TH+ sympathetic nerves and CD31+/CD144+ blood vessels. (D) Representative confocal z stack projection of the thymus hilum showing multiple TH+ nerve bundles entering the thymus along a large artery. (E) High magnification confocal z stack projection of two large blood vessels, identified as veins and arteries by morphology, innervated by TH+ sympathetic nerves. (F) High magnification confocal z…

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  Figure 2.. Sympathetic innervation of blood vessels ends abruptly in the thymic medulla (A) Representative confocal z stack projection of a 50 μm cryosection demonstrating the loss of β3-tubulin+ innervation of large blood vessels immediately before entering the thymic medulla (white arrowheads). The abrupt ending of nerves occurred at the same location as a reduction in α-SMA+ cells, and the medulla contains non-vascular α-SMA+ cells (green arrowhead). (B) TH+ sympathetic innervation ends (yellow arrowhead) along a blood vessel that projects from the cortex, labeled with the cTEC marker keratin 8, into the medulla, labeled with the mTEC marker keratin 5. (C and D) TH+/β3-tubulin+ sympathetic nerves innervating a large vessel along the cortico-medullary junction, with cortex/medulla denoted by cell density (Hoechst, C) and mTEC marker (keratin 8, D). Representative confocal z stack projection of TH+ sympathetic innervation ending along a blood vessel that enters the medulla (blue arrowheads). Dashed white lines represent cortico-medullary junctions. (E and F) Quantification of TH+ segments per thymic region for all sections (E) and average per mouse (F); n = 3–4 sections per mouse for 4…

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  Figure 3.. Nestin-GFP+ MSCs are innervated by sympathetic nerves in the thymus and express nerve growth factor (A) Representative projection of cleared thymus depicting nestin-GFP+ cells in the deep thymic parenchyma near large blood vessels of the hilum. Inserts demonstrate a TH+ sympathetic nerve projecting along nestin-GFP+ cell bodies and their processes (white arrowheads). (B) Representative confocal z stack projection of the thymic capsule depicting nestin-GFP+ cells and TH+ nerves. The insert depicts another example of TH+ innervation along the cell bodies and processes of capsular nestin-GFP+ cells (white arrowheads). (C) Representative confocal z stack projection of TH+ innervation along nestin-GFP+ cells as they project from the capsule into the subcapsular cortex. Insert showing a nerve bundle and individual fiber (white arrowheads) projecting along the processes of nestin-GFP+ cells. The thymus border, capsule, and sub-capsular cortex are delineated with dashed white lines. The main images in (B) and (C) are presented at the same scale as in (A). (D) Fluorescence-activated cell sorting gating strategy to isolate thymic ECs, mesenchymal stromal cells (MSCs), cTECs, and mTECs. (E)…

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  Figure 4.. Disrupted sympathetic innervation in the aged thymus (A) The whole thymus lobe of an aged mouse (16–24 months old) was cleared and imaged for TH+ sympathetic nerves. (B) Representative confocal z stack projections of TH+ sympathetic innervation of the vasculature of a young mouse (3–5 months old), showing well-defined nerve fibers and large bundles along CD31+/CD144+ blood vessels at 190 μm depth near the hilum. (C) Representative confocal z stack projection of TH+ sympathetic innervation of the vasculature of an aged mouse (regions identical to those in [B]), revealing a collection of tortuous blood vessels and nerves, with a clear thinning of nerve bundles. (D) Representative confocal z stack projection of the aged thymic capsule for TH+ sympathetic nerves. (E and F) Representative confocal z stack projections of TH+ sympathetic nerve fibers projecting from the capsule into the cortex in a young mouse (E) and an absence of these fibers in aging (F). Dotted lines indicate the thymus border. Images presented in (B)–(F) are set at the same scale. (G and H) Quantification of TH+ nerve fiber volume (G) and area (H) as a percent region of interest (ROI), with quantification of the…

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  Figure 5.. Aging impairs axon and synapse morphology in the thymus (A) Representative confocal z stack projection (20 μm cryosection) of a young thymus stained for the general axon marker β3-tubulin and synaptic vesicle marker synaptophysin, depicting innervation of large artery and vein structures. (B) Insert from (A) demonstrating both large bundle and small fiber axonal morphology and synaptic density throughout most axons (white arrowheads); non-axonal synaptophysin expression could also be found throughout the tissue parenchyma (blue arrowheads). (C) Representative confocal z stack projection (20 μm cryosection) of an aged thymus depicting gross innervation of large arteries and veins. (D) Insert from (C) showing fraying of axons (yellow arrowheads) and overall reduction in the amount of synaptophysin in small fiber axonal segments. (E) Quantification of synaptophysin+ synapses within β3-tubulin+ axons for all images (all ROIs) in young and old mice, with data also expressed as an average for each biological replicate (average per animal). (F) Quantification of Ngf mRNA (expressed as relative to Actb/β-actin) from sorted MSCs of young and aged thymi. (G) Quantification of the total…

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  Figure 6.. Large autofluorescent phagocytic macrophages directly contact dystrophic axons in the aged thymus (A) Representative confocal z stack projection from Figure 5C highlighting the direct interaction of autofluorescent phagocytic-like cells in direct contact with along axons in an aged thymus. (B) Insert from (A) showing a group of phagocytic cells in direct contact with a dim β3-tubulin+ axon bundle in the thymic parenchyma (white arrowheads). (C) Insert from (A) showing many phagocytic cells in direct contact with nerve bundles along a large CD31+/CD144+ vein (white arrowheads). (D) Identification of large autofluorescent cells as macrophages by Iba1 labeling in a young thymus. (E) Image from an aged thymus demonstrating large hypertrophic Iba1+ macrophages. (F) Quantification of mRNA (2ddCT) for genes associated with cellular ROS production, inflammatory cytokines, and phagocytosis from sorted young adult and aged macrophages. Data are presented as mean ± standard error of the mean and analyzed with unpaired t test (Nfe2l2, Sod1, Il1b, and Trem2), unpaired Welch’s t test (Cybb, Cyba, Gpx1, Prdx1, Tnfa, Lamp1, and Lamp2), or Mann-Whitney U test (Apoe); n = 10–12/group from 3…

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  Figure 7.. Sympathetic nerve ablation impairs thymic homeostasis, while sympathomimetic treatment partially restores thymopoiesis in aged mice (A) Schematic of the experimental design for neonatal chemical sympathectomy, with neonatal mice treated with 6-OHDA (100 mg/kg) on days 2, 4, 6, 8, and 9 after birth. (B) Example images of cleared thymi from vehicle and 6-OHDA-treated mice at 4 months of age. Scale bar, 100 μm. (C–E) Quantification of total thymocytes, DP thymocytes, early thymic progenitors (ETPs), ECs, and MSCs from vehicle and 6-OHDA-treated mice at 4 (C) and 14 (D) months of age, and in 14-month-old mice treated with vehicle or the β3-adrenergic receptor agonist BRL37344 for 14 days (E). Data are presented as mean ± standard error of the mean and analyzed with unpaired t test (C–E) and unpaired Welch’s t test (E, ECs); n = 3–5/group.