Using digital pathology to analyze the murine cerebrovasculature

Abstract

Research on the cerebrovasculature may provide insights into brain health and disease. Immunohistochemical staining is one way to visualize blood vessels, and digital pathology has the potential to revolutionize the measurement of blood vessel parameters. These tools provide opportunities for translational mouse model research. However, mouse brain tissue presents a formidable set of technical challenges, including potentially high background staining and cross-reactivity of endogenous IgG. Formalin-fixed paraffin-embedded (FFPE) and fixed frozen sections, both of which are widely used, may require different methods. In this study, we optimized blood vessel staining in mouse brain tissue, testing both FFPE and frozen fixed sections. A panel of immunohistochemical blood vessel markers were tested (including CD31, CD34, collagen IV, DP71, and VWF), to evaluate their suitability for digital pathological analysis. Collagen IV provided the best immunostaining results in both FFPE and frozen fixed murine brain sections, with highly-specific staining of large and small blood vessels and low background staining. Subsequent analysis of collagen IV-stained sections showed region and sex-specific differences in vessel density and vessel wall thickness. We conclude that digital pathology provides a useful tool for relatively unbiased analysis of the murine cerebrovasculature, provided proper protein markers are used.

Keywords: Aperio ScanScope; VCID; arteriolosclerosis; immunohistochemistry; microvessel.

Figure 1.

Digital pathology workflow. (Upper Panel) FFPE (on slides) or frozen-fixed (free floating) mouse brain sections were stained for a variety of blood vessel markers. The resulting slides were scanned by the Aperio ScanScope AT2 slide scanner to obtain digitized images. Two regions of interest (cortex and hippocampus), were outlined, then analyzed, using the supplied microvessel algorithm to quantify different vessel parameters, such as vessel density, vessel perimeter, lumen area, and vessel wall thickness. (Middle Panel) I. The blood vessel markers used are specific for a variety of blood vessel structures, including astrocyte endfeet (DP71), elastic lamina (collagen IV), and endothelial cells (CD31, CD34, and von Willebrand Factor). II. A representation of the output measurements in relation to the blood vessel structure. (Lower Panel) I. A representative micrograph with a DAB-stained blood vessel. II. A false color markup of the vessel by the microvessel algorithm, showing the relevant output parameters.

Figure 2.

CD34 staining. Mouse brain sections were stained for CD34 using a variety of antibodies. (a) While QBEnd10 staining showed blood vessel staining, this was indistinguishable to that seen in the negative, anti-mouse secondary antibody control. We next evaluated anti-CD34 antibodies not generated in mouse (b). RAM34 stained only very large blood vessels (red arrow), albeit very lightly (c). ab8158 showed very light, inconsistent blood vessel staining, but also significant staining of neuronal cells (blue arrow: d). ab81289 showed light, inconsistent blood vessel staining, but with moderate background staining (e). MA5-29674 predominantly stained glial cells (blue arrow), but did not stain blood vessels (f). PA5-89536 showed no staining, other than a very high background (g).

Figure 3.

Initial evaluation of non-CD34 blood vessel markers. We tested a variety of potential blood vessel markers for use in mouse brain tissue. (a) CD31 stained blood vessels lightly, but also showed some non-vascular cell staining. (b) Analysis by the microvessel algorithm identified blood vessels, but also cell staining (blue arrows). There were many vessels left unidentified (red arrow). (c) Collagen IV stained blood vessels lightly, with very low background. (d) Given the light staining, the microvessel algorithm clearly missed some visible blood vessels (red arrows). (e) The DP71 stained slide had moderately high background staining, but showed both blood vessel and cell body staining. (f) The microvessel algorithm readily detected blood vessels, but also misidentified cell staining as blood vessels (blue arrows). (g) The von Willebrand Factor (VWF) stained slide had very high background. (h) The background was so high that the algorithm could not identify any blood vessels. The entirety of the displayed region of interest was excluded from analysis (yellow shading). The number of vessels identified by the algorithm (i), as well as the vessel density (j) varies significantly for each antibody. CD31 and DP71 identified the most vessels, possibly due to the non-vessel cell staining. Red boxes indicate the region of interest analyzed by the microvessel algorithm, shown in the second panel for each antibody.

Figure 4.

Optimization of collagen IV staining. We tested different antigen retrieval methods to improve the staining of blood vessels with anti-collagen IV. (a) Low pH heat-mediated antigen retrieval of collagen IV staining led to only light reactivity. (c) High pH heat-mediated antigen retrieval did not improve blood vessel staining. (b, d) Though the microvessel algorithm did detect some vessels, there were many lightly-stained vessels that were not identified (red arrows). (e) Pepsin-mediated retrieval for 10 minutes improved blood vessel staining, while maintaining a very low background. (f) The microvessel algorithm detected more vessels using this method, but still missed some that were lightly stained (red arrows). (g) Pepsin-mediated retrieval for 20 minutes further improved staining of blood vessels, both small and large, while retaining the low background. (h) The microvessel algorithm was able to detect all stained vessels in the region of interest, while minimizing detection of non-vessels. Each of the antigen retrieval methods tested yielded an increase in the number of vessels detected by the algorithm (i), as well as the vessel density (j). Red boxes indicate the region of interest analyzed by the microvessel algorithm, shown in the second panel for each antibody.

Figure 5.

Staining for blood vessels in free-floating sections. We tested a few different blood vessel markers to determine whether they also work for frozen fixed brain sections. (a) CD31 showed light-inconsistent staining of blood vessels, but also stained cells in the dentate gyrus (blue arrow). (b) The microvessel algorithm detected a few blood vessels, but clearly missed some that were lightly stained (red arrows). (c) Collage IV robustly stained large and small blood vessels in both the hippocampus and cortex, which were readily detected by the microvessel algorithm (d). (e) DP71 also stained the blood vessels well, though there was apparent staining of astrocyte cells as well, which were erroneously identified as blood vessels by the microvessel algorithm (blue arrows; f). The algorithm identified more vessels in the DP71 stained slide than the other antibodies, likely due counting non-vessel stained cells (g), yielding a higher apparent blood vessel density (h). Red boxes indicate the region of interest analyzed by the microvessel algorithm, shown in the second panel for each antibody.

Figure 6.

The effect of sex on cerebral microvessels. FFPE sections of mouse brain were stained for microvessels using collagen IV as a marker. Each slide was subsequently scanned using the Aperio ScanScope and analyzed using the microvessel algorithm feature of the associated ImageScope software, drawing regions of interest around the hippocampus and cortex of each section. Representative images for each brain region are shown for both female (a, c) and male (b, d) mice. (e) Vessel density was similar in males and females in the hippocampus, while females had a higher density in the cortex. (f) Vessel perimeter was not significantly different between females and males in either brain region tested. (g) The lumen area trended larger in the males than the females in the hippocampus. (h) Wall thickness was slightly thinner in males compared with females in both brain regions. Vessel walls were thicker in the hippocampus than the cortex of female mice. N = 6/sex. The distribution of data was evaluated by a Shapiro-Wilk test for normality (e, p = 0.017, f, p = 0.23, g, p < 0.0001, h, p = 0.425). Vessel density and lumen are were subsequently analyzed with a Mann-Whitney U test for nonparametric data. Vessel perimeter and wall thickness were subsequently analyzed by ANOVA.