1.

Introduction

Ischemic cerebrovascular diseases (CVD) and their complications are one of the leading causes of morbidity and mortality worldwide. Ischemic cerebrovascular events, such as ischemic stroke and transient ischemic attack also frequently lead to vascular dementia and persistent cognitive impairment.^1–4 Studies have shown that atherosclerotic disease often plays a causative role in the onset of the ischemic CVDs.^5–7 It is well known that in the presence of elevated plasma low-density lipoprotein (LDL), plaques can build up inside extracranial and intracranial large arteries. As a consequence, the luminal stenosis reduces or even blocks blood supply to the brain, causing oxygen and energy substrate deficiency in cerebral tissue.^8–12 The impact of atherosclerotic disease on cerebral small vessels, however, still remains to be fully elucidated.^13–15

Clinical neuroimaging, such as magnetic resonance imaging and computed tomography, evaluates cerebral small vessel disease-related ischemic parenchymal lesions rather than alterations of the cerebral microvasculature itself due to the limited spatial resolution of the imaging techniques and the small size of vessels.^16–19 High-resolution optical imaging techniques such as intrinsic signal optical imaging (ISOI), optical coherence tomography (OCT), and two-photon microscopy combined with a genetically modified ATX mouse model thus provides an opportunity to investigate the effects of atherosclerotic disease on cerebral microvasculature.^20–23 In this work, we used ${\mathrm{LDLR}}^{-/-}{\mathrm{hApoB}}^{+/+}$ transgenic mice, which have LDL receptor deficiency and express the human apolipoprotein B-100 gene. Previous studies have reported that ATX mice at 3 months of age had no atherosclerotic lesions in the aorta, carotid arteries, or cerebral arteries,^24–26 whereas mice at 6 months of age and older developed extensive atherosclerosis. Learning capacity and cognitive functions were also found to decline significantly in one-year-old ATX mice.^25,27 Systemic chronic inflammation caused by atherosclerosis contributes to cerebral endothelial cell senescence and dysfunction, which further leads to blood-brain barrier (BBB) leakage and capillary microbleeding in ATX mice where atherosclerosis is fully developed.²⁸

The objective of our study was to assess how cerebral hemodynamics, cerebral microvasculature, and cerebral tissue oxygenation were affected by hyperlipidemia-associated atherosclerotic disease. To achieve this goal, multiple optical imaging techniques were used, including ISOI, spectral-domain Doppler OCT, two-photon phosphorescence lifetime microscopy, and line scans with two-photon fluorescence excitation. ISOI was first used to evaluate the hemodynamic response in the somatosensory cortex to whisker stimulation. This parameter reflects the efficiency of blood flow regulation by cerebral vasculature to meet increased metabolic needs of the brain.²⁹ Doppler OCT was then performed to investigate possible structural and functional changes of descending arterioles as well as ascending venules related to atherosclerotic disease. Cerebral tissue oxygenation, a crucial parameter for brain function, can be disturbed by inadequate blood flow in microvasculature. We measured absolute cerebral tissue ${\mathrm{PO}}_2$ using two-photon phosphorescence lifetime imaging coupled with the oxygen quenching phosphorescent dye PtP-C343 to assess the impact of changes in blood flow. Finally, we used two-photon fluorescence line scans to investigate the impact of atherosclerotic disease and its progression on capillary properties.

2.

Materials and Methods

3.

Results

3.1.

Atherosclerosis is Associated with Reduced Hemodynamic Response

The hemodynamic response in the somatosensory cortex following whisker stimulation was measured using ISOI. Figures 1(a)–1(c) show typical images of ${\mathrm{HbO}}_2$, HbR, and HbT concentration changes due to stimulation. Images are shown at 3 s after the beginning of a whisker stimulation. ${\mathrm{HbO}}_2$, HbR, and HbT concentration changes were averaged across the entire somatosensory cortex to get the time courses [Fig. 1(d)]. The peak value of the ${\mathrm{HbO}}_2$ and HbT time series and the inverted peak value of the HbR time series were used to quantify the hemodynamic response amplitude to whisker stimulation. As seen in Fig. 1(e), the old ATX group had significantly smaller amplitude for $\mathrm{\Delta }{\mathrm{HbO}}_2$, $\mathrm{\Delta }\mathrm{HbR}$, and $\mathrm{\Delta }\mathrm{HbT}$ than the young ATX group. Hemodynamic responses were averaged to obtain group-averaged time evolutions of ${\mathrm{HbO}}_2$ and HbR [Fig. 1(f)]. The old ATX group showed a weaker response to whisker deflection than the young ATX group. Another observation was that the response undershoot in ${\mathrm{HbO}}_2$ was present in young mice but disappeared in older mice. However, no obvious difference was found in the time delay between the two groups.

Fig. 1

ISOI used to measure hemodynamic response in somatosensory cortex following whisker stimulation. (a)–(c) Images of relative changes in ${\mathrm{HbO}}_2$, HbR, and HbT acquired 3 s after the start of the stimulus. (d) A typical hemodynamic response obtained by averaging ${\mathrm{HbO}}_2$, HbR, and HbT changes across the somatosensory cortex for a representative mouse. The transparent orange zone represents the 5-s whisker stimulation. The dashed black line corresponds to the moment when images (a)–(c) were acquired. (e) Comparison of the peak value of ${\mathrm{HbO}}_2$, HbR, and HbT concentration changes after each stimulation. The young ATX mice had significantly higher peak values for $\mathrm{\Delta }{\mathrm{HbO}}_2$, $\mathrm{\Delta }\mathrm{HbR}$, and $\mathrm{\Delta }\mathrm{HbT}$ than the old ATX mice. (f) Group-averaged time series of $\mathrm{\Delta }{\mathrm{HbO}}_2$ and $\mathrm{\Delta }\mathrm{HbR}$ for the young and the old ATX mice. Data are shown as $\text{mean}\pm \mathrm{SEM}$. Unpaired $t$-test was performed: ****$p<0.0001$ and ***$p<0.001$. Y, young and O, old.

3.2.

Atherosclerosis is Associated with Reduced Basal Blood Flow and Reduced Arteriolar but not Venular Diameter

Using spectral-domain Doppler OCT, we investigated cerebral blood flow in single descending arterioles and ascending venules, which deliver oxygen-rich blood and collect oxygen-poor blood, respectively. Figure 2(a1) shows a typical en face Doppler OCT image, which was acquired at a depth of $180\text{-}\mu \mathrm{m}$ beneath the cortical surface. Detected arterioles and venules are encircled by red and blue lines in Fig. 2(a2). Vessel diameter, blood velocity, and blood flow for each identified vessel were calculated based on such segmented en face slices using a segmentation algorithm to avoid operator bias. The diameter and mean blood flow of descending arterioles in the old ATX mice decreased by 9.0% and 24.7%, respectively, compared with the young ATX mice [Figs. 2(c) and 2(d)]. Arteriolar diameter and arteriolar mean blood flow showed a decreasing trend with the depth in both groups of ATX mice [Figs. 2(g) and 2(h)]. However, the old ATX group displayed significantly smaller arteriolar lumen size and arteriolar mean blood flow at all measured cortical depths when compared with the young ATX group, which suggests a systemic modulation to cerebral arterioles caused by atherosclerotic disease. Correlation between arteriolar mean blood flow and arteriolar diameter was examined using data obtained from both the young and the old ATX mice. A strong positive correlation was found with a correlation coefficient of 0.96 [Fig. 2(b)], suggesting that narrowed arterioles caused lower mean blood flow. Interestingly, for old ATX mice, arteriolar mean blood flow not only dropped more rapidly with depth than in the young ATX mice but saturated at a lower bound as if it reached a plateau. As depicted in Figs. 2(e) and 2(f)], both diameter and mean blood flow of ascending venules showed no difference between the two groups of ATX mice.

Fig. 2

Arteriolar and venular parameters obtained from Doppler OCT data. (a1) An en face Doppler OCT image obtained at a depth of $180\text{-}\mu \mathrm{m}$ beneath the cortical surface of a representative mouse. The scale bar is $100\mu \mathrm{m}$. (a2) The same Doppler OCT image with identified arterioles and venules encircled by red and blue circles, respectively. (b) Correlation between arteriolar mean blood flow and arteriolar diameter. (c), (d) Comparison of arteriolar diameter and mean blood flow between the two ATX groups. The young ATX mice had significantly larger arteriolar diameter and significantly higher arteriolar mean blood flow than the old ATX mice. (e), (f) Comparison of venular diameter and mean blood flow between the two groups of ATX mice. No significant difference was found for venules between the two groups. (g), (h) The diameter and the mean blood flow of descending arterioles at five different depths for the young and the old ATX mice. Arteriolar diameter and mean blood flow at all range of depths decreased significantly in the old ATX mice when compared with the young ATX mice. Data are shown as $\text{mean}\pm \mathrm{SEM}$. Unpaired $t$-test was performed: ****$p<0.0001$, ***$p<0.001$, and **$p<0.01$. Y, young and O, old.

3.3.

Reduced Blood Flow is Associated with Reduced Tissue PO₂ and Pockets of Hypoxia

Two-photon phosphorescence lifetime imaging was used to investigate whether reduced blood flow affected cerebral tissue oxygenation through impaired oxygen delivery from microvasculature. In each mouse, tissue ${\mathrm{PO}}_2$ data were acquired at six different depths ranging from 40- to $180\text{-}\mu \mathrm{m}$ beneath the cortical surface. Figure 3(b) shows the distribution of ${\mathrm{PO}}_2$ values for the young and the old ATX group. The distribution of tissue ${\mathrm{PO}}_2$ value for old ATX mice was shifted toward lower values when compared with young ATX mice. Focusing on the potential for hypoxia, we compared the frequency of samples with low ${\mathrm{PO}}_2$ value ($<15\mathrm{mmHg}$) between the two groups [inset of Fig. 3(b)]. The percentage of low ${\mathrm{PO}}_2$ points changed from 7.7% in young ATX mice to 12.6% in old ATX ones. Overall, the mean value of tissue ${\mathrm{PO}}_2$ in the young ATX mice was 15% higher than that in the old ATX mice [Fig. 3(c)]. Moreover, the old ATX group showed significantly higher spatial heterogeneity of tissue oxygenation, suggesting capillary delivery heterogeneity [Fig. 3(d)]. The analysis of cerebral tissue ${\mathrm{PO}}_2$ versus depth (limited to the depth that was accessible in this thin-skull preparation) showed a decreasing trend in both ATX groups, but the young ATX group had significantly higher tissue ${\mathrm{PO}}_2$ at all depths [Fig. 3(e)]. The decrease of cerebral tissue ${\mathrm{PO}}_2$ and the increase of its spatial heterogeneity could cause low ${\mathrm{PO}}_2$ sites to co-locate forming hypoxic micropockets. To look for the presence of hypoxic micropockets, we defined them as regions comprising at least 15 sampled points whose ${\mathrm{PO}}_2$ value is lower than 5 mmHg. Figure 3(a2) shows an example of an interpolated tissue ${\mathrm{PO}}_2$ image with the presence of a hypoxic micropocket. In our study, hypoxic micropockets were found in two old ATX mice out of seven, whereas none of the nine ATX young mice had hypoxic micropockets.

Fig. 3

Cerebral tissue ${\mathrm{PO}}_2$ measurement with two-photon phosphorescence lifetime imaging. (a1) A typical ${\mathrm{PO}}_2$ grid ($20\text{pixels}\times 20\text{pixels}$) covering an ROI of $400\mu \mathrm{m}\times 400\mu \mathrm{m}$. The scale bar is $100\mu \mathrm{m}$. (a2) The interpolated ${\mathrm{PO}}_2$ image of the ${\mathrm{PO}}_2$ grid (a1). A hypoxic micropocket was identified and delimited by the red lines. (b) The distribution of cerebral tissue ${\mathrm{PO}}_2$ for the two groups of ATX mice. The ${\mathrm{PO}}_2$ distribution of old ATX mice was shifted toward lower ${\mathrm{PO}}_2$ values when compared with the young ATX group. The inset shows that pixels with low ${\mathrm{PO}}_2$ values were more frequently seen in old ATX mice than in young ATX mice. (c) Cerebral tissue ${\mathrm{PO}}_2$ value in old ATX mice was significantly smaller than that in young ATX mice. (d) The old ATX mice showed a significantly higher spatial heterogeneity of tissue ${\mathrm{PO}}_2$ when compared with the young ATX group. The spatial heterogeneity was calculated for each ${\mathrm{PO}}_2$ grid and was defined as the coefficient of variation of each ${\mathrm{PO}}_2$ grid. (e) Tissue ${\mathrm{PO}}_2$ values at different depths were analyzed for the two groups of ATX mice. The decreasing trend of ${\mathrm{PO}}_2$ relative to depth was found in both ATX groups. The old ATX group had significantly lower cortical tissue ${\mathrm{PO}}_2$ at all investigated depths than the young ATX group. Data are shown as $\text{mean}\pm \mathrm{SEM}$. Unpaired $t$-test was performed: ****$p<0.0001$. Y, young and O, old.

3.4.

Cerebral Hypoperfusion and Decreased Tissue PO₂ is Associated with Capillary Dilation

To better understand how atherosclerotic disease affects cerebral microvasculature, we further investigated capillaries, which bridge between descending arterioles and cerebral tissue in terms of gas and substance exchange. Even though capillaries are not anticipated to be directly affected by atherosclerotic lesions because of their simple wall structure, high circulating plasma lipids and proinflammatory factors associated with atherosclerotic disease can still cause deleterious effects on capillary endothelium as discovered in a previous ex vivo study.²⁸ In the current work, we were interested in the structural and functional changes of cerebral capillaries in an in vivo pro-atherogenic environment. Using two-photon fluorescence intensity imaging, reference 2-D images were obtained from which multiple capillaries were selected for subsequent two-photon line scans [Figs. 4(a)–4(b)]. From the perpendicular and longitudinal spatiotemporal line-scan images, we were able to retrieve diameter, RBC flux, RBC velocity, RBC velocity heterogeneity, and hematocrit for each capillary. Comparison of these capillary parameters between the young and the old ATX group is shown in Figs. 4(c)–4(g). Changes in all parameters were statistically significant. RBC flux, RBC speed, and hematocrit of the old ATX mice decreased significantly, whereas the value of capillary diameter and RBC velocity heterogeneity were found significantly greater compared with young ATX mice.

Fig. 4

Two-photon fluorescence line scans to measure capillary parameters. (a1), (b1) A two-photon fluorescence intensity image in the $x-y$ plane with vessels appearing bright. The yellow line represents the laser scanning direction. Perpendicular line scans were perpendicular to capillaries, and longitudinal line scans were parallel to capillaries. The scale bar is $25\mu \mathrm{m}$. (a2) A spatiotemporal image composed of 200 consecutive perpendicular scans. A Gaussian function was fitted to the intensity profile averaged from the 200 perpendicular scans to estimate the capillary lumen diameter. (b2) A spatiotemporal image of longitudinal scans. The tilted dark streaks were due to the passage of RBCs in the capillary. RBC speed was calculated from the slope of the streaks, namely the tangent of the angle $\alpha$. (c)–(g) Comparison of the capillary parameters, including diameter, RBC flux, RBC velocity, RBC velocity heterogeneity, and hematocrit, between the young and the old ATX mice. RBC velocity heterogeneity is defined as the coefficient of variation of RBC velocity. RBC flux, RBC speed, and hematocrit of old ATX mice were significantly lower when compared with young ATX mice. The values of capillary diameter and RBC velocity heterogeneity of old ATX mice were significantly higher than those of young ATX mice. Data are shown as $\text{mean}\pm \mathrm{SEM}$. Unpaired $t$-test was performed: ****$p<0.0001$, ***$p<0.001$, and **$p<0.01$. Y, young and O, old.

4.

Discussion

As an organ consuming a high quantity of oxygen and energy substrates, the brain is vulnerable to hypoxia. The adequate oxygen supply to cerebral tissue and its temporal and spatial regulation relies on a well-functioning cerebral vasculature. In the current work, we investigated the detrimental effects of atherosclerotic disease on cerebral microvasculature and cerebral tissue oxygenation using ${\mathrm{LDLR}}^{-/-}{\mathrm{hApoB}}^{+/+}$ transgenic mice at 3 months of age, where no atherosclerotic lesions are found, and old mice at 12 months of age, where atherosclerosis is fully developed. Four optical imaging techniques were employed to characterize the microvascular environment: (1) ISOI for monitoring hemodynamic response in somatosensory cortex to whisker stimulation; (2) Doppler OCT for estimating arteriolar and venular size and blood flow; (3) two-photon phosphorescence lifetime imaging for measuring cerebral tissue ${\mathrm{PO}}_2$; and (4) two-photon fluorescence line scans to investigate capillaries. We used the thinned-skull window approach instead of open-skull technique to reduce inflammation and hemorrhage. As isoflurane, a commonly used anesthetic agent in animal studies for repeated experiments, modulates neural activity and has a vasodilator effect,^41,42 all our experiments were conducted with awake mice to avoid associated confounds. During imaging sessions, resting states (of the order of minutes) and short bouts of locomotion (of the order of seconds) of the mice were observed to be interleaved. We monitored animals during imaging acquisition. OCT and two-photon images were acquired while the mice stayed still to avoid locomotion. Their stress and attention levels were, however, not monitored. These factors could have a potential impact on the measured cerebral hemodynamic parameters. As for our ISOI acquisition, whisker stimulation under awake condition caused increased animal locomotion. We took multiple recordings and selected the ones where locomotion was minimized. Furthermore, its effect on hemodynamic response in the barrel cortex has previously been found to be negligible.⁴³

Data from ISOI showed that hemodynamic responses in terms of ${\mathrm{HbO}}_2$, HbR, and HbT were significantly lower in the old ATX group than in the young ATX group. Group-averaged time series of ${\mathrm{HbO}}_2$ and HbR concentration changes also confirmed that the old ATX mice had much weaker hemodynamic response to whisker deflection than the young group, whereas the difference in time delay between the beginning of stimulation and the response was found to be quite similar, as shown in Fig. 1(f). If we assume that whisker stimulation resulted in comparable increase of oxygen metabolism in the somatosensory cortex in both ATX groups, the smaller increase of ${\mathrm{HbO}}_2$ concentration and HbT concentration in the old ATX mice may indicate a less effective regulatory capacity for cerebral blood flow upon cortical activation. However, since neural activity was not measured in our study, we cannot conclude that the observed cerebral hemodynamic alteration in the 12-month-old ATX mice was completely due to atherosclerotic vascular disease. Since neurodegeneration and cerebrovascular disease are often intertwined,⁴⁴ reduced neural response could also contribute to the decreased hemodynamic response.

Using Doppler OCT, we found that arteriolar blood flow and arteriolar lumen area were highly correlated. The data obtained from the young and the old ATX mice also suggest that the decrease of arteriolar diameter and the deterioration of arteriolar blood flow were associated with the presence of atherosclerosis. As atherosclerosis developed in old ATX mice, their arterioles, the primary sites for blood pressure and blood flow regulation, were affected, reducing their diameter, which is in accordance with the observation from an ex vivo study.⁴⁵ Our analysis of arteriolar diameter versus depth also demonstrated that this reduction occurred not only in the proximal end branching off pial arteries but also in the deeper distal end with smaller lumen diameter. These diseased arterioles slowly lost the ability to supply an adequate quantity of oxygenated blood and to regulate the local blood flow. Drastically decreased oxygen supply to cerebral parenchyma by degenerated arterioles could eventually fail to meet the metabolic requirements of neurons. On the other hand, venules seemed not to be affected by atherosclerosis in terms of lumen size and mean blood flow. The fact that venules stayed intact may be explained by lack of smooth muscle cells in their tunica media and low local hemodynamic load.^46,47

Oxygen molecules diffuse from arterioles and capillaries to interstitial fluid, and neurons and glial cells extract oxygen directly from interstitial fluid.²³ The oxygen level in the surrounding media is thus crucial for these nerve and glial cells. Using two-photon microscopy with the phosphorescent dye PtP-C343, we found that the old ATX mice had significantly lower tissue ${\mathrm{PO}}_2$ value and significantly higher tissue ${\mathrm{PO}}_2$ heterogeneity compared with the young ATX mice. This finding indicates that atherosclerosis-related vascular disease modulates the balance between oxygen supply and oxygen consumption, which could lead to hypoxic environment in cerebral tissue and may cause irreversible damages to neurons.^22,48,49 Even without acute ischemic events, such chronic hypoxia in the brain can have adverse effects on cognition.^26,50,51 Hypoxic micropockets with extremely low ${\mathrm{PO}}_2$ values ($<5\mathrm{mmHg}$) were identified in two old ATX mice. However, the direct impact of these hypoxic micropockets on local neurons and glia remains unclear, and the possible causative role in hypoxic micropocket formation played by structural and functional alteration and thrombotic occlusions of nearby microvasculature still needs to be further studied.⁵²

Capillaries, as a critical component in oxygen delivery, were investigated using a two-photon fluorescence line-scan technique. The old ATX mice exhibited significant lower RBC flux, RBC speed, and hematocrit compared with the young ATX mice. The decrease of capillary RBC flux and RBC speed in the old ATX mice coincided with the decrease of their arteriolar blood flow. Meanwhile, the old ATX mice showed significantly higher RBC velocity heterogeneity than the young ATX group. A high RBC velocity heterogeneity value suggests a disturbed capillary flow pattern, which has been proven to limit the efficacy of oxygen extraction and impairs oxygen supply to brain tissue.^53,54 Thus alteration of these capillary properties (RBC flux, RBC velocity, RBC velocity heterogeneity, and hematocrit) along with degeneration of parenchymal arterioles probably resulted in the poor cerebral oxygenation in the old ATX mice.^23,55,56 Interestingly, capillary diameter of the old ATX group was found to be significantly larger than that of the young ATX group. Our in vivo finding is in concordance with a previous ex vivo study that associated larger capillary lumen diameter with atherosclerosis-susceptible transgenic mice with hyperlipidemia based on histological analysis.⁵⁷ It has been demonstrated that cerebral blood flow is not solely controlled by arterioles, and regulation can also occur at the capillary level by pericytes.^58–60 The observed capillary dilation in the old ATX mice could thus be a coping mechanism of cerebral microvasculature to partially compensate for oxygen supply decrease in the presence of atherosclerotic vascular disease to meet the metabolic requirements of the brain. In addition, capillary dilation also increases its permeability to solutes, which makes oxygen diffusion into surrounding tissue easier.⁶¹ Even though atherosclerosis-related capillary dilation may help mitigate cerebral hypoperfusion and hypoxia in the resting state, blood flow regulation capacity at the capillary level could decrease when a large number of neurons are activated by stimulations and a substantial amount of oxygen is needed. Furthermore, capillary dilation in old ATX mice may aggravate BBB disruption and impair its barrier functions, causing microhemorrhages in the cortex.²⁸

One of the limitations of our work is that the aging effect was not taken into account in our interpretation when we made comparisons between 3-month-old and 12-month-old ATX mouse groups. Previous studies of behavioral/cognitive tests (social exploration, open field, water maze, and novel object recognition) on WT mice indicated that most of the cognitive functions were preserved in old healthy mice (21 months) compared with young healthy ones (3 months) despite the aging process.⁶² However, not showing a significant drop in cognitive functions in old WT mice does not mean that the cerebral vasculature of aged mice remained intact. According to a recent publication, aging does modify capillary functions (RBC flux, RBC speed, and hematocrit) and tissue oxygenation.²² But the effects seen here at 12 months were larger and are hypothesized to be due to atherosclerosis. It should also be noted that the two aforementioned studies used two distinct substrains of mice (C57BL/6JRccHsd and C57BL/6JOlaHsd). As a result, we cannot conclude that the negative impact was solely attributed to atherosclerosis. Aging, as one of the dominant risk factors for atherosclerotic lesion formation,⁶³ may exacerbate cerebral vascular degeneration. Interestingly, the study by Moeini et al.²² reported that there were no significant changes on cerebral arteriolar diameter and hemodynamics among young, middle-aged, and old WT mice, which suggests that the arteriolar deterioration in 12-month-old ATX mice seen in our study were likely caused by atherosclerotic disease.

5.

Conclusion

In this paper, we demonstrate that atherosclerotic disease has detrimental effects on cerebral microvasculature and tissue oxygenation. Our data show that the decreased hemodynamic response to sensory stimulation is associated with the presence of atherosclerosis. When atherosclerotic disease develops, descending arterioles suffer from structural and functional degeneration, whereas ascending venules remain intact. During the progression of atherosclerosis, capillaries undergo RBC flux decrease and develop a more heterogeneous RBC speed pattern. Blood supply decrease from both arterioles and capillaries leads to cerebral tissue hypoxia. Meanwhile, capillary dilation occurs in old ATX mice, which partially compensates for blood flow decrease caused by atherosclerotic disease.