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. 2023 Feb 17;12(4):645.
doi: 10.3390/cells12040645.

High Resolution Multiplex Confocal Imaging of the Neurovascular Unit in Health and Experimental Ischemic Stroke

Jeffrey J Lochhead  1 , Erica I Williams  1 , Elizabeth S Reddell  1 , Emma Dorn  1 , Patrick T Ronaldson  1   2 , Thomas P Davis  1   2
Affiliations

High Resolution Multiplex Confocal Imaging of the Neurovascular Unit in Health and Experimental Ischemic Stroke

Jeffrey J Lochhead et al. Cells. .

Abstract

The neurovascular unit (NVU) is an anatomical group of cells that establishes the blood-brain barrier (BBB) and coordinates cerebral blood flow in association with neuronal function. In cerebral gray matter, cellular constituents of the NVU include endothelial cells and associated pericytes, astrocytes, neurons, and microglia. Dysfunction of the NVU is a common feature of diseases that affect the CNS, such as ischemic stroke. High-level evaluation of these NVU changes requires the use of imaging modalities that can enable the visualization of various cell types under disease conditions. In this study, we applied our confocal microscopy strategy using commercially available labeling reagents to, for the first time, simultaneously investigate associations between endothelial cells, the vascular basal lamina, pericytes, microglia, astrocytes and/or astrocyte end-feet, and neurites in both healthy and ischemic brain tissue. This allowed us to demonstrate ischemia-induced astrocyte activation, neurite loss, and microglial migration toward blood vessels in a single confocal image. Furthermore, our labeling cocktail enabled a precise quantification of changes in neurites and astrocyte reactivity, thereby showing the relationship between different NVU cellular constituents in healthy and diseased brain tissue. The application of our imaging approach for the simultaneous visualization of multiple NVU cell types provides an enhanced understanding of NVU function and pathology, a state-of-the-art advancement that will facilitate the development of more effective treatment strategies for diseases of the CNS that exhibit neurovascular dysfunction, such as ischemic stroke.

Keywords: astrocytes; endothelial cells; ischemic stroke; microglia; neurons; neurovascular unit; pericytes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Schematic showing cells and structures present at the NVU. (B) Confocal micrograph of a pre-capillary arteriole in the caudoputamen stained with the NVU labeling cocktail showing endothelial cells (EC), the basement membrane, (BM), microglia (M), pericytes (P), Astrocyte end-feet (AEF), and neutites (N). Scale bar = 5 μm.
Figure 2
Figure 2
Images of regions in the cortex (A) and the caudoputamen (B) stained with the NVU labeling cocktail showing capillaries (C), microglia (M), pericytes (P), astrocytes (A), neurites (N), and nerve bundles (NB). Scale bar = 10 μm.
Figure 3
Figure 3
Images of capillary cross-section (A) and a longitudinal cross-section of a precapillary arteriole (B) in the cortex showing endothelial cells (EC), the casement membrane (BM), microglia (M), pericytes (P), astrocyte end feet (AEF), the vessel lumen (VL) and neurites (N). Scale bar = 5 μm in (B).
Figure 4
Figure 4
Ischemia-reperfusion injury induces changes in the NVU. The NVU was stained with lectin (A,B), anti-PDGFR-β (C,D), anti-GFAP (E,F) and the Pan Neuronal antibody (G,H), and capillaries from the caudoputamen in the hemisphere ipsilateral to where an MCAO was performed were compared to the contralateral hemisphere. The ipsilateral hemisphere shows an increased association of microglia with capillaries (arrows in B), an increase in GFAP fluorescence (arrow in F), and neurite loss (arrow in H) when compared to the contralateral side. Representative images of TTC-stained coronal sections from control (I) and MCAO (J) treated rats with boxes showing the approximate regions where the ipsilateral (I) and contralateral (C) images were acquired. Fluorescence intensity from the contralateral (C) and ipsilateral (I) regions was quantified for GFAP (K) and Milli-Mark (L) staining (n = 6). Merged images (M,N) and 3D reconstructions (O,P) are shown below. Images are representative of randomly chosen regions of interest in the caudoputament from 6 different rats. Scale bar = 5 μm; * p < 0.05; ** p < 0.01.
Figure 5
Figure 5
Ischemia-reperfusion injury induces changes in the NVU. The NVU was stained with lectin (A,B), anti-PDGFR-β (C,D), anti-GFAP (E,F) and the Pan Neuronal antibody (G,H), and cortical microvessels in the hemisphere ipsilateral to where an MCAO was performed were compared to the contralateral hemisphere. The ipsilateral hemisphere shows an increased association of microglia with capillaries (arrows in B), an increased in GFAP fluorescence (arrows in F), and neurite loss (arrows in H) when compared to the contralateral side. Representative images of TTC-stained coronal sections from control (I) and MCAO treated rats (J) with boxes showing the approximate regions where the ipsilateral (I) and contralateral (C) images were acquired. Fluorescence intensity from the contralateral (C) and ipsilateral (I) regions was quantified for GFAP (K) and Milli-Mark (L) staining (n = 6). Merged images (M,N) and 3D reconstructions (O,P) are shown below. Images are representative of randomly chosen regions of interest in the somatosensory cortex from 6 different rats. Scale bar = 10 μm; * p < 0.05.
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