Macrophage-Induced Blood Vessels Guide Schwann Cell-Mediated Regeneration of Peripheral Nerves.
Here we show that blood vessels direct the migrating cords of Schwann cells.Importantly, disrupting the organization of the newly formed blood vessels in vivo, either by inhibiting the angiogenic signal or by re-orienting them, compromises Schwann cell directionality resulting in defective nerve repair.This study provides important insights into how the choreography of multiple cell-types is required for the regeneration of an adult tissue.
Affiliation: MRC Laboratory for Molecular Cell Biology, UCL, Gower Street, London WC1E 6BT, UK.
- Blood Vessels/metabolism*
- Peripheral Nerves/physiology*
- Schwann Cells/metabolism*
- Cell Hypoxia
- Endothelial Cells/metabolism
- Neovascularization, Physiologic
- Rats, Sprague-Dawley
- Vascular Endothelial Growth Factor A/genetics
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figs5: Hypoxia within the Bridge Leads to HIF-1α Stabilization and Expression of the Pro-angiogenic Target Gene, Vegfa, Related to Figure 5(A) Quantification of Figure 5A to show the proportion of hypoxyprobe-1+ cells within the rat bridge (n = 4 animals per group; graph shows mean value ± SEM).(B) Representative longitudinal sections of a rat sciatic nerve bridge and the contralateral uninjured nerve, Day 2 after transection and 30 min after injection of hypoxyprobe-1 (pimonidazole chloride), immunostained to detect hypoxyprobe-1 (green). Nuclei were counterstained with Hoechst (blue). Scale bar = 100 μm. To reconstruct the longitudinal section of the injured nerve (bottom), multiple images from the same sample were acquired using the same microscope settings.(C) Quantification of Figure 5B to show the proportion of hypoxic cells that are macrophages at Day 2 and Day 3 (n = 4 animals per group; graph shows mean value ± SEM).(D) Quantification of the proportion of macrophages (Iba1+) that are hypoxic (hypoxyprobe-1+), Day 2 and Day 3 after injury (n = 4 animals per group, graph shows mean value ± SEM). Note the significant decrease of hypoxic macrophages at Day 3 compared to Day 2.(E) Representative images of a bridge region of a rat sciatic nerve, Day 2 after transection and the contralateral nerve (uncut), immunolabelled to detect macrophages (Iba1+, red) and HIF-1α expression (green). Scale bar = 20 μm. White arrowheads indicate HIF-1α+/Iba-1+ cells. Graph shows quantification of the proportion of HIF-1α+ cells that are macrophages (Iba1+) within the bridge (n = 3 animals, graph shows mean value ± SEM).(F) Representative images of sections of rat sciatic nerve, Day 2 after transection and the contralateral uninjured rat sciatic nerve following in situ hybridization of rat Vegfa mRNA (red) and subsequent immunostaining for macrophages (CD68+, green). Scale bar = 10 μm.(G) Quantitative RT-PCR analysis of Vegfa mRNA isolated from the bridge, the proximal and the distal stump of transected sciatic nerves, Day 2 after injury. Graph shows the Vegfa transcript levels relative to the levels in the proximal stump (n = 8 animals, graph shows mean value ± SEM).(H–K) Representative images of cryosections of rat sciatic nerve, after transection, immunolabelled to detect VEGF-A (green) and macrophages (CD68+, red) to show proximal, bridge and distal regions at Day 2 (H) and the bridge region at Day 2 and Day 3 (J). The proportion of VEGFA+ cells (I) and VEGFA+ macrophages (CD68+) (K) are quantified at Day 2 and Day 3 in the bridge region (n = 3, graphs show mean value ± SEM).(L) Representative images of a bridge region of a rat sciatic nerve, Day 2 or Day 3 after transection, immunostained to detect VEGF-A (green) and macrophages (CD68+, red) or blood vessels (RECA-1+, red) as indicated. Note that VEGF-A is undetectable in the blood vessels. Scale bars = 15 μm.
New blood vessels normally form in response to decreased oxygen levels (hypoxia) within a tissue. Upon hypoxia, the transcription factor HIF-1α is stabilized and initiates a transcriptional response that induces angiogenesis by upregulating pro-angiogenic factors such as VEGF (Krock et al., 2011; Pugh and Ratcliffe, 2003). To test whether the nerve bridge was hypoxic, we injected rats with hypoxyprobe-1 (pimonidazole hydrochloride) that forms immunofluorescent detectable protein adducts in hypoxic conditions (pO2 < 10 mm Hg) (Young and Möller, 2010). Immunostaining of day 2 nerve bridges revealed the presence of large numbers of hypoxic cells prior to its vascularization (Figures 5A and S5A). Hypoxic cells were found only in the bridge and at the tips of both the distal and proximal stumps but not further along the stumps or in the uncut nerve (Figure S5B). The proportion of hypoxic cells decreased substantially by day 3, when the bridge had become vascularized (Figures 5A and S5A), consistent with the new blood vessels resolving the hypoxic environment of this new tissue.