Aging Impairs Cerebrovascular Reactivity at Preserved Resting Cerebral Arteriolar Tone and Vascular Density in the Laboratory Rat

Abstract

The age-related (mal)adaptive modifications of the cerebral microvascular system have been implicated in cognitive impairment and worse outcomes after ischemic stroke. The magnitude of the hyperemic response to spreading depolarization (SD), a recognized principle of ischemic lesion development has also been found to be reduced by aging. Here, we set out to investigate whether the SD-coupled reactivity of the pial arterioles is subject to aging, and whether concomitant vascular rarefaction may contribute to the age-related insufficiency of the cerebral blood flow (CBF) response. CBF was assessed with laser-speckle contrast analysis (LASCA), and the tone adjustment of pial arterioles was followed with intrinsic optical signal (IOS) imaging at green light illumination through a closed cranial window created over the parietal cortex of isoflurane-anesthetized young (2 months old) and old (18 months old) male Sprague-Dawley rats. Global forebrain ischemia and later reperfusion were induced by the bilateral occlusion and later release of both common carotid arteries. SDs were elicited repeatedly with topical 1M KCl. Pial vascular density was measured in green IOS images of the brain surface, while the density and resting diameter of the cortical penetrating vasculature was estimated with micro-computed tomography of paraformaldehyde-fixed cortical samples. Whilst pial arteriolar dilation in response to SD or ischemia induction were found reduced in the old rat brain, the density and resting diameter of pial cortical vessels, and the degree of SD-related oligemia emerged as variables unaffected by age in our experiments. Spatial flow distribution analysis identified an age-related shift to a greater representation of higher flow ranges in the reperfused cortex. According to our data, impairment of functional arteriolar dilation, at preserved vascular density and resting vascular tone, may be implicated in the age-related deficit of the CBF response to SD, and possibly in the reduced efficacy of neurovascular coupling in the aging brain. SD has been recognized as a potent pathophysiological contributor to ischemic lesion expansion, in part because of the insufficiency of the associated CBF response. Therefore, the age-related impairment of cerebral vasoreactivity as shown here is suggested to contribute to the age-related acceleration of ischemic lesion development.

Keywords: aging; arteriolar diameter; cerebral blood flow; cerebral ischemia; neurovascular coupling; reperfusion; spreading depolarization; vascular density.

Figure 1

Pial vascular density. (A) Representative images (top) of the pial surface of a young and an old preparation at green light illumination (orientation: medial to the top, rostral to the right). The relative area covered by vessels was calculated in computed images (bottom). (B) Vascular density was expressed as the area covered by the pial vascular network relative to the full image size. Individual values are gray symbols; bars show mean ± SEM (n = 11/10, young/old). An independent samples T-test was used to statistically evaluate age-related differences (p < 0.112).

Figure 2

The density of the cerebrocortical penetrating vasculature. (A) A representative 3D micro-CT reconstruction of the penetrating vascular architecture of the frontoparietal cortex of a young preparation. (B) Tissue volume filled by blood vessels, relative to the size of the sample. (C) The mean number of vascular profiles in the sample. (D) The diameter of vascular profiles encountered. Individual values are gray symbols; bars show mean ± SEM (n = 5/4, young/old). A one-way analysis of variance (ANOVA) was used to statistically evaluate age-related differences (*p < 0.05). No statistically significant difference has been found among any of the variables investigated.

Figure 3

Pial arteriolar diameter between subsequent events of the experimental protocol. (A) A green intrinsic optical signal (IOS) image of a representative young preparation demonstrates the ranking of pial arterioles. (B) The Resting diameter of 1st, 2nd and 3rd order pial arterioles in the two age groups. Individual values are gray symbols; bars show mean ± SEM (n = 7/6, 7/7 and 7/3, young/old, 1st, 2nd and 3rd order). A multivariate ANOVA paradigm was used for statistical analysis, considering vessel rank or age as factors (**p < 0.01). (C) Changes of the pial arteriolar diameter of 1st, 2nd and 3rd order arterioles relative to baseline (100%) over the experimental protocol. Sampling times are: (1) baseline—prior to the elicitation of the first spreading depolarization (SD); (2) baseline—prior to ischemia induction; (3) ischemia—minimum vascular diameter after ischemia onset; (4) ischemia—prior to the elicitation of the first SD under ischemia; (5) ischemia—prior to the initiation of reperfusion; (6) reperfusion—maximum vascular diameter after reperfusion initiation; (7) reperfusion—prior to the elicitation of the first SD under reperfusion; and (8) reperfusion—prior to the termination of the experiment. (D) The impact of age on the relative changes of pial arteriolar diameter of 1st, 2nd and 3rd order arterioles. Data are given as mean ± SEM (n = 8/5, young/old). In panels (C,D), a repeated measures paradigm was used for statistical analysis, considering vessel rank or age as factors (*p < 0.05 and **p < 0.01).

Figure 4

Cerebral blood flow (CBF) variation over the experimental protocol. (A) A pseudo-colored, representative baseline CBF map used for single-point analysis (i.e., CBF changes were extracted from a region of interest—ROI, as a function of time). (B) Changes of local CBF relative to baseline (100%) in the two age groups as assessed with single-point analysis. Sampling times over the experimental protocol are: (1) baseline—prior to the elicitation of the first spreading depolarization (SD); (2) baseline—between SDs; (3) ischemia—minimum CBF after ischemia onset; (4) ischemia—prior to the first ischemic SD; (5) ischemia—between SDs; (6) reperfusion—maximum CBF after reperfusion initiation; (7) reperfusion—between SDs; and (8) reperfusion—prior to the termination of the experiment. Data are given as mean ± SEM (n = 9/9, young/old). The statistical analysis relied on a repeated measures paradigm with age as a factor (level of significance: *p < 0.05). (C) Computer-generated, pseudo-colored representative CBF image pairs of a young and an old preparation used for whole field analysis. The pial vasculature is masked (black) to be excluded from the analysis targeting the cortical parenchyma. The sampling time given numerically in the upper left-hand corner corresponds to the numbering used in panel (B). (D) Spatial flow distribution in the CBF maps, demonstrated as the relative area occupied by given CBF ranges (shown at an increment of 10%). The histograms are presented at 10% steps. Data are given as mean ± SEM (n = 9/9, young/old). A one-way ANOVA was used to test age-related differences (*p < 0.05).

Figure 5

Representative traces of the variation in pial arteriolar diameter and local CBF with spreading depolarization (SD) events in a young preparation and maximum dilation of pial arterioles in response to SDs elicited during baseline. (A) A representative field of view over the parietal cortex of a young rat at green light illumination. Pial arteriolar segments used for the measurement of diameter changes in panel (B) are indicated (v1: 1st order, v2: 2nd order, v3: 3rd order arteriole). A ROI designates the origin of the local CBF trace in panel (B). (B) The dilation of pial arterioles in response to SD events (top), with the corresponding changes in local CBF (bottom) in a representative young preparation. (C) Maximum dilation of 1st, 2nd and 3rd order pial arterioles in response to the first and recurrent SDs in normally perfused cortex in the two age groups. Individual values are gray symbols; bars show mean ± SEM (SD1: n = 6–7/3–6, young/old; recurrent SD: n = 11–13/5–13, young/old). A multivariate ANOVA paradigm was used for statistical analysis, considering vessel rank or age as factors (*p < 0.05 and **p < 0.01).

Figure 6

The spatial distribution of CBF at peak hyperemia in response to spreading depolarization (SD) elicited in normally perfused cortex. (A) Representative CBF maps of a young and an old animal demonstrate flow distribution with the passage of peak hyperemia in response to recurrent SD. The SD event was confirmed by the transient negative shift of the DC potential (bottom). Small white spheres in the images show the position of the microelectrodes implanted into the cortex. White dotted lines and arrows indicate the origin and radial direction of propagation of SD. (B) Relative area occupied by given CBF ranges (shown at an increment of 20%) under the passage of peak hyperemia with recurrent SDs triggered in the normally perfused cortex. Data are given as mean ± SD. A MANOVA paradigm was used to test age-related differences. (C) Histograms (mean for each age group) to demonstrate the relationship between CBF and surface area of the cerebral cortex with recurrent SD. (D) CBF at the peak of the histograms shown in panel (C). Individual values are gray symbols; bars show mean ± SEM (n = 10/10, young/old). An independent samples T-test was used to statistically evaluate age-related differences (*p < 0.05).