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Comparative Study
. 2021 Jan 21;117(2):520-532.
doi: 10.1093/cvr/cvaa037.

Light sheet fluorescence microscopy as a new method for unbiased three-dimensional analysis of vascular injury

Nicholas E Buglak  1   2   3   4 , Jennifer Lucitti  5 , Pablo Ariel  6 , Sophie Maiocchi  1   2   4 , Francis J Miller  5   7 , Edward S M Bahnson  1   2   3   4   8
Affiliations
Comparative Study

Light sheet fluorescence microscopy as a new method for unbiased three-dimensional analysis of vascular injury

Nicholas E Buglak et al. Cardiovasc Res. .

Abstract

Aims: Assessment of preclinical models of vascular disease is paramount in the successful translation of novel treatments. The results of these models have traditionally relied on two-dimensional (2D) histological methodologies. Light sheet fluorescence microscopy (LSFM) is an imaging platform that allows for three-dimensional (3D) visualization of whole organs and tissues. In this study, we describe an improved methodological approach utilizing LSFM for imaging of preclinical vascular injury models while minimizing analysis bias.

Methods and results: The rat carotid artery segmental pressure-controlled balloon injury and mouse carotid artery ligation injury were performed. Arteries were harvested and processed for LSFM imaging and 3D analysis, as well as for 2D area histological analysis. Artery processing for LSFM imaging did not induce vessel shrinkage or expansion and was reversible by rehydrating the artery, allowing for subsequent sectioning and histological staining a posteriori. By generating a volumetric visualization along the length of the arteries, LSFM imaging provided different analysis modalities including volumetric, area, and radial parameters. Thus, LSFM-imaged arteries provided more precise measurements compared to classic histological analysis. Furthermore, LSFM provided additional information as compared to 2D analysis in demonstrating remodelling of the arterial media in regions of hyperplasia and periadventitial neovascularization around the ligated mouse artery.

Conclusion: LSFM provides a novel and robust 3D imaging platform for visualizing and quantifying arterial injury in preclinical models. When compared with classic histology, LSFM outperformed traditional methods in precision and quantitative capabilities. LSFM allows for more comprehensive quantitation as compared to traditional histological methodologies, while minimizing user bias associated with area analysis of alternating, 2D histological artery cross-sections.

Keywords: Light sheet fluorescence microscopy; Neointimal hyperplasia; Restenosis; Vascular disease; Vessel remodelling.

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Figures

Figure 1
Figure 1
Sample processing workflow comparison for H&E or LSFM analysis. Arteries processed for LSFM imaging are able to be rehydrated and cryosectioned for H&E analysis.
Figure 2
Figure 2
Analysis workflow in Bitplane Imaris. Rat carotid artery used as the representative sample. Video 2 overlays the three surfaces with the fluorescent volumetric rendering.
Figure 3
Figure 3
Demonstration of LSFM imaging for rat and mouse injury models. (A) Vessel orientation and figure image colours, unless otherwise stated. (B) Volumetric rendering and slice view of healthy rat artery (L = lumen). (C) Representative balloon-injured rat artery. (D) Representative slice views of an injured rat artery (arrowheads = hyperplasic region). White box indicates region of the surface rendering of the IEL and lumen volumes. (E) Representative cross-section in x-z axis from arrowheads in (D) with H&E-stained image of the same artery after rehydration and cryosectioning. (F) Cross-section of surface rendering of the representative rat [from (D) and (E)) arterial media, hyperplasia, and both surfaces overlaid]. (G) Representative H&E-stained cross-sections of ligated mouse artery processed exclusively for histology. (H) Longitudinal and cross-sectional slice rendering of representative ligated mouse carotid artery (white arrow = ligation site at bifurcation). (I) Representative volume rendering of ligated mouse carotid artery depicting the periadventitial neovascular plexus (white arrow = ligation site at bifurcation and arrowheads = neo-vascular plexus). (J) Slice and volumetric rendering of representative healthy mouse artery.
Figure 4
Figure 4
Macrophage presence in the adventitia of injured mouse carotids. Uninjured and ligated mouse carotid arteries stained for CD31 and CD68 show presence of CD68+ puncta only in the ligated arteries around the ligation site and periadventitially next to the CD31+ neovascular plexus (n = 3, white = CD31, magenta = CD68, green = autofluorescence). (A) 3D rendering healthy mouse carotid arteries. (B) 3D rendering healthy mouse carotid arteries.
Figure 5
Figure 5
Demonstration of LSFM quantification. (A) Rat intima-to-media (I:M) ratio using optical 3D volume compared to a separate cohort of injured rat arteries harvested directly for histological processing (LSFM n = 7, H&E n = 10, n.s. , no significance, Student’s t-test). (B) Mouse I:M ratio using optical volume compared to a separate cohort of ligated mouse arteries harvested directly for histological processing (LSFM n = 6, H&E n = 12). (C) Plot of injured rat artery IEL radius and change (Δ) in radius between the lumen and the IEL along the artery centre (dataset shown in Figure 3D). 99% CI defines the threshold at which the Δ Radius is indicative of neointimal hyperplasia formation. 99% CI determined using the average Δ Radius along the contralateral healthy rat artery (Supplementary material online, Figure S1). (D) Average IEL radii per rat along their hyperplasic region compared to the non-hyperplastic region of the injured artery (N = 5 #P < 0.05, paired t-test). (E) Average IEL radii per rat along their hyperplasic region compared to their contralateral healthy artery (N = 7; #P < 0.05, paired t-test). (F) Plot of medial thickness comparing the hyperplastic region of each injured artery to the respective contralateral healthy control artery (N = 7; #P < 0.05, paired t-test).
Figure 6
Figure 6
Artery processing for LSFM imaging does not cause arterial shrinkage. Comparison between healthy, uninjured arteries processed only for LSFM or only for H&E analysis. (A) Representative cross-section of healthy rat arteries. (B) Average luminal surface area per healthy rat artery (n = 7, n.s., no significance, Student’s t-test). (C) Average luminal and EEL perimeter per healthy rat artery. (D) Representative cross-section of healthy mouse arteries. (E) Average luminal surface area per healthy mouse artery (LSFM n = 6 and H&E n = 8). (F) Average luminal and EEL perimeter per healthy mouse artery.
Figure 7
Figure 7
Histological staining of rehydrated, LSFM-processed rat carotid artery. (A) Immunofluorescent staining for alpha-smooth muscle (αSM) actin. (B) H&E staining of three representative arterial cross-sections.
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