High-resolution optical mapping of inflammatory macrophages following endovascular arterial injury

Abstract

Purpose: Inflammation following arterial injury mediates vascular restenosis, a leading cause of cardiovascular morbidity. Here we utilize intravital microscopy (IVM) and a dextran-coated nanosensor to spatially map inflammatory macrophages in vivo following endovascular injury of murine carotid arteries.

Procedures: C57Bl/6 mice (n = 23) underwent endovascular guidewire carotid arterial injury. At day 14 or day 28 post-injury, mice underwent fluorescence IVM, 24 h after injection with the near-infrared fluorescent macrophage nanosensor CLIO-VT680. Adventitial collagen was concomitantly imaged using second harmonic generation (SHG) IVM. Correlative fluorescence microscopy and immunohistochemistry were performed.

Results: Two-plane IVM reconstructions detected macrophage inflammation in the arterial wall that was elevated at day 14 compared to day 28 animals (P < 0.05). SHG-based collagen imaging of the outer arterial wall facilitated analysis of the macrophage-rich, inflamed neointima. Histological analyses and fluorescence microscopy data demonstrated increased macrophage infiltration in day 14 compared to day 28 neointima.

Conclusions: We demonstrate that the macrophage response to arterial injury can be imaged in vivo using IVM-based molecular imaging, and shows a higher macrophage influx at day 14 compared to day 28 post-injury.

Fig. 1. Endovascular guidewire injury model in murine carotid arteries

Upper panel shows a schematic representation. (a) Temporary ligations are introduced to the carotid artery. (b) An 0.014″ guidewire is next inserted via the external carotid (EC) artery and traversed along the entire length of common carotid artery up to the carotid bifurcation for 1 minute (distal to head) to induce injury to the vessel. (c) The EC is closed and temporary ligations are removed to allow blood flow via the internal carotid (IC) artery. The lower panel shows the procedure in detail. (d) The IC artery is temporary ligated and 6-0 silk suture is passed underneath the EC artery. (e) Distal to the head, an arterial clamp stops blood flow while passing the guidewire into the artery. (f) Threads are stretched and loops are created to allow the nicked area to be viewed distinctly. (g) The guidewire is introduced into the EC and rotated inside to induce endothelial denudation, while the arterial clamp restricts blood flow during the procedure. (h) Permanent ligation of the EC, followed by release of the clamp and return of blood flow through the IC artery. Arrowhead indicates point of insertion of the guidewire. (i) The surgical incision is sutured closed. EC = external carotid artery, IC = internal carotid artery, CC = common carotid artery.

Fig. 2. Endothelial cell layer and restenosis assessment of injured and control carotid arteries at day 3 and day 14

Endothelial cells were detected by CD31 immunohistochemistry. (a) Day 3 control carotid artery. (b) Day 3 wire-injured carotid artery (A and B - 20 x). (c) Day 14 control carotid artery. (d) Day 14 wire-injured carotid artery (C and D – 10 x). Yellow arrows show re-endothelialized regions; blue arrows show denuded artery lumen. Scale bar, 100 μm.

Fig. 3. Representative coronal reconstruction IVM images of murine carotid arteries

Images were generated by summing 60 coronal slices starting from the luminal edge of adventitial collagen, defined by the SHG signal. A and B show CLIO-VT680 channel images (SHG signals are not displayed). (a) Day 14 and day 28 injured carotid artery. (b) Day 14 and day 28 control carotid artery. Images were pitch- and angle-corrected. Scale bar, 100 μm.

Fig. 4. Axial reconstruction IVM and SHG images of carotid arteries

Axial reconstructions (200 slices * 2 μm thickness each) of carotid arteries were summed starting from the luminal edge of adventitial collagen defined by SHG signal (a) Injured arteries, day 14 and day 28. (b) Contralateral control arteries, day 14 and day 28. Images were pitch- and angle-corrected. Representative summation injured and matched images are shown and were equally windowed. SHG, second harmonic generation. Scale bar, 100 μm.

Fig. 5. Macrophage infiltration in IVM coronal and axial reconstructions of carotid arteries

Quantification of CLIO-VT680 TBR values of the carotid artery at day 14 and day 28 were calculated on summation (a) coronal images and (b) axial images. Mean ± SEM of 5 mice are shown. *P < 0.05.

Fig. 6. Fluorescence microscopy and histological analyses at day 14 and day 28 after endovascular injury

Upper panel, day 14; Lower panel, day 28 representative images. Rows a, c: Mac-3 macrophage immunohistochemistry. The yellow and red dotted lines show measurements of neointimal and medial areas, respectively. Rows b, d – fluorescence microscopy images showing a merged FITC and CLIO-VT680 (macrophage) fluorescence, colored green and red, respectively. (e), Intima-to-media ratio in arteries as a measure of restenosis. (f) Neointimal area in carotid arteries as a measure of restenosis. Mean ± SEM of 4 mice are shown. Each data point represents the measured average of one mouse. *P < 0.05 and **P < 0.001. Scale bars, 100 μm.