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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figs7: Disorganization of Blood Vessels Leads to Disrupted Schwann Cell Migration and Axonal Regrowth, Related to Figure 7(A) A higher magnification of Figure 7B to show the Schwann cell cords (S100+, green), aligned to the blood vessels (RECA-1+, red). Scale bar = 50 μm.(B) Quantification of Figure 7B to show the alignment between Schwann cells and regrowing axons. Graph shows the mean relative angle ± SD for each animal with the mean between animals shown by the red lines. Rose plots show the distribution of cells for all animals (n = 3 animals for each condition).(C) Images of bridge regions of a control (PBS) and VEGF-treated rat sciatic nerve, Day 6 after injury, immunostained to detect Schwann cells (S100+, green) and endothelial cells (RECA-1+, red). Nuclei were counterstained with Hoechst (blue). Scale bar = 300 μm. The beads are indicated by white asterisks in the VEGF-treated animals. Note the center of the bridge is poorly vascularised in the VEGF-treated mice and the Schwann cell cords fail to enter the bridge.(D) A further example of aberrant regeneration in a VEGF-treated sciatic nerve. Upper panels show images of a bridge region of control (PBS) and VEGF-treated rat sciatic nerves, Day 6 after injury, immunostained to detect Schwann cells (S100+, blue), endothelial cells (RECA-1+, red) and axons (NF+, green). Scale bar = 100 μm. Lower panels show the same images as in the upper panels, filtered to show only the axons (NF+, white). Note the axons in the VEGF-treated nerves are misdirected, toward the beads, into the adjoining muscle.(E) Image of a disconnected nerve following treatment with VEGF-treated beads in which the beads redirect Schwann cell cords from the distal stump. Note the blood vessels (RECA-1+, red) and Schwann cells (S100+, green) are directed away from the bridge into the surrounding muscle. Scale bar = 200 μm. For reconstruction of longitudinal sections shown in (C), (D) and (E), multiple images from the same sample were acquired using the same microscope settings.(F) Images of nerves stained with osmium tetroxide taken from Vegfafl/fl (control) and Vegfafl/flTie2-Cre mice, 6 months following transection. Note the visibly smaller distal stump in the mutant mice. Scale bar = 2mm.(G) Cross sections of a nerve from Vegfafl/fl (control) and Vegfafl/flTie2-Cre mice 6 months following transection and stained with toluidine blue, at low magnification to show the entire nerve (top panels) and at higher magnification to show the indistinguishable structures of the control and mutant nerves (lower panels). Scale bar = 100 μm (top) and 5 μm (bottom).(H) Graph to show the difference in area between the Vegfafl/fl (control) and Vegfafl/flTie2-Cre nerves as in (G), n = 3; graph shows the mean ± SEM.
To address whether VEGF-A-induced blood vessels are sufficient to guide cords of SCs, we redirected the blood vessels to test whether the SCs would follow the blood vessels or continue to cross the bridge. To do this, we implanted heparin beads loaded with recombinant human VEGF165 into muscle adjacent to the proximal side of the injury site, immediately after the transection of the rat sciatic nerve. Six days later, the regenerative process was found to be abnormal in 10 out of 13 of the VEGF-treated animals compared to 1 out of 13 PBS-bead-treated controls. In five of the ten VEGF-treated animals in which abnormal regeneration was observed, a complete failure of the regenerative process was associated with misdirection of the blood vessels, SC cords and the accompanying axons, away from the bridge and into surrounding muscle towards the beads (Figures 7A, 7B, and S7A; quantified in Figures 7C–7F and S7B). Analysis of the bridges in a further five cases showed that the beads had moved into the bridge leading to the formation of disorganized blood vessels close to the beads (Figure S7C). In these cases, SCs migrated into the vascularized areas and either appeared “trapped” or deviated from the normal direction of movement, taking the axons along with them (Figure S7D). Moreover, beads implanted adjacent to the distal stump could also redirect blood vessels and SCs (Figure S7E). Together, these results demonstrate that VEGF-induced blood vessels are sufficient to guide SCs and their accompanying axons during peripheral nerve regeneration.