Impact of atherosclerotic disease on cerebral microvasculature and tissue oxygenation in awake LDLR−/−hApoB+/+ transgenic mice

Abstract. We explore cortical microvasculature changes during the progression of atherosclerosis using young and old transgenic atherosclerotic (ATX) mice with thinned-skull cranial window. In awake animals, exploiting intrinsic signal optical imaging, Doppler optical coherence tomography, and two-photon microscopy, we investigate how the progression of atherosclerotic disease affects the morphology and function of cortical microvasculature as well as baseline cerebral tissue oxygenation. Results show that aged ATX mice exhibited weaker hemodynamic response in the somatosensory cortex to whisker stimulation and that the diameter of their descending arterioles and associated mean blood flow decreased significantly compared with the young ATX group. Data from two-photon phosphorescence lifetime microscopy indicate that old ATX mice had lower and more heterogeneous partial pressure of oxygen (PO2) in cortical tissue than young ATX mice. In addition, hypoxic micropockets in cortical tissue were found in old, but not young, ATX mice. Capillary red blood cell (RBC) flux, RBC velocity, RBC velocity heterogeneity, hematocrit, and diameter were also measured using line scans with two-photon fluorescence microscopy. When compared with the young group, RBC flux, velocity, and hematocrit decreased and RBC velocity heterogeneity increased in old ATX mice, presumably due to disturbed blood supply from arterioles that were affected by atherosclerosis. Finally, dilation of capillaries in old ATX mice was observed, which suggests that capillaries play an active role in compensating for an oxygen deficit in brain tissue.


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][2][3][4] Studies have shown that atherosclerotic disease often plays a causative role in the onset of the ischemic CVDs. [5][6][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][9][10][11][12] The impact of atherosclerotic disease on cerebral small vessels, however, still remains to be fully elucidated. [13][14][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][17][18][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][21][22][23] In this work, we used LDLR −∕− hApoB þ∕þ 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][25][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 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.

Animal Preparation
The procedures and protocols were approved by the Animal Research Ethics Committee of the Montreal Heart Institute, and all animal experiments were performed in accordance with the Canadian Council on Animal Care recommendations. The animal model that we used in our study was the LDLR −∕− hApoB þ∕þ transgenic mice. They exhibit markedly high concentration of plasma LDL and spontaneously develop atherosclerotic lesions on a chow diet after the age of 6 months. 28,30-32 They were categorized into two age groups: young ATX mice (3-month old) and old ATX mice (12-month old). Our rationale in choosing the cut-off age of 12 months was as follows: ATX mice usually have shorter lifespans than wild type (WT) mice, thus making it difficult to study them at 24 months, which is typically considered to be old age in WT mice. Furthermore, confounding effects from other morbidity factors could become too important in 24-month-old ATX mice. In our previous work, 28 where both ATX and WT mice at 12 months of age were used, we observed cognitive differences in the Morris Maze and a decrease in brain perfusion. It was also shown that endothelial dysfunction was already present in ATX mice at 12 months of age when compared with 3-month-old ATX mice. 33 Thus the choice of 3-month-old and 12-month-old ATX mice allowed us to emphasize effects of atherosclerosis while minimizing the effects of aging (though present). At 8 to 10 days before measurements, a thinned-skull cranial window of 3-mm diameter was prepared over the left barrel cortex, and a titanium head bar was attached to the mouse skull. A detailed description of the surgical procedure can be found in our previous work. 22 During imaging sessions, the mouse head was fixed by the head bar onto a custom-built treadmill wheel, into which limbs were free to move to reduce stress. Four fixation training sessions were required prior to image acquisition to habituate the mice to head restraint. All measurements were made in awake mice to eliminate the influence of anesthesia on vascular and neural physiology. 34 ISOI was used to explore stimulus-evoked hemodynamic response. ISOI data were obtained from nine young ATX mice and eight old ATX mice using a home-built ISOI system. Mouse whiskers were deflected at a rate of 10 Hz by a piezo-based mechanical whisker stimulator to induce increased neural activity in the somatosensory cortex. Each stimulation period lasted 5 s followed by a 15-s interstimulus interval and a randomized jitter interval of an average of 3 s. Stimulation was repeated 10 times. The imaging system used green and red LEDs to illuminate the somatosensory cortex, and images were taken at a rate of 5 Hz for each color (10 Hz combined). Since oxygenated and deoxygenated haemoglobins have different extinction coefficients, relative changes in oxyhemoglobin (HbO 2 ), deoxyhemoglobin (HbR), and total hemoglobin (HbT) concentration in functional brain areas were calculated based on the images acquired with the two wavelengths following previous work. 37 Animals were monitored for locomotion and whisker activity during acquisitions. In some acquisitions, stimulations induced movement of the animal. Thus multiple acquisitions were acquired and the ones minimizing movement were analyzed.

Spectral-domain Doppler OCT
A spectral-domain OCT operating in Doppler mode was used to image descending arterioles and ascending venules for which the direction of blood flow is mostly parallel to the light beam. Images were acquired from five young ATX mice and five old ATX mice with a home-built spectral-domain OCT system. The critical parts of the OCT system included superluminescent diode illumination (LS2000C, Thorlabs) with a center wavelength at 1310 nm and a bandwidth of 200 nm, a custom-built spectrometer, a galvanometer scanner (GVS002, Thorlabs), and a 5× infinity corrected objective (Thorlabs). The axial and lateral resolution in biological tissue was ∼3.5 and ∼8.5 μm, respectively. 22 A 900 × 900 μm cortical area was scanned to get a 3-D Doppler OCT volume. The 3-D volume had 512 B-scans, and each B-scan was composed of 2048 A-lines. Adjacent A-lines were deliberately overlapped. The phase difference between two adjacent A-lines was computed to calculate the speed of blood flow in arterioles and venules. 38 The A-line acquisition rate was set to 50 kHz, which enabled the system to detect blood flow speed between −13 and 13 mm∕s along the direction of the incident beam. The scan was repeated 10 times in the same volume to enhance the signal-to-noise ratio (SNR). Following 3-D Doppler volume reconstruction, arterioles and venules were segmented on en face slices at depths between 140 and 290 μm beneath the cortical surface. Slices shallower than 140 μm or deeper than 290 μm were discarded to exclude the pial vessels and capillaries. Detected descending arterioles and ascending venules had an elliptical appearance on en face images. The vessel lumen diameter was estimated from the minor axis while the blood flow was calculated by multiplying the area of the ellipse by the blood flow velocity along the z axis.

Two-photon phosphorescence lifetime imaging
A custom-built two-photon microscope was used for phosphorescence lifetime imaging. A detailed description of the imaging system can be found in our previous publication. 22 Working with the PtP-C343 dye, phosphorescence lifetime imaging provided a way to quantify tissue oxygenation (PO 2 ). 39 Nine young ATX mice and seven old ATX mice were used in the measurement. Before PO 2 measurement, the PtP-C343 dye solution (∼150 μM in ACSF) was injected into the mouse brain tissue through the thinned skull using a glass micropipette slowly pushed by a microsyringe pump injector (UMP3, World Precision). Meanwhile, a FITC-Dextran solution (50 mg∕ml in saline) was administered through the tail vein for cerebral angiography, which was used for the selection of region of interest (ROI) in phosphorescence lifetime imaging. 2-D PO 2 grid measurements (20 pixels × 20 pixels) were taken in ROIs (400 μm × 400 μm) at different depths beneath the cortical surface. The phosphorescent dye at each location of the grid was excited by an 80-MHz femtosecond laser oscillator gated on for 25 μs, and a 275-μs photon collection window was applied right after to obtain a time-resolved decay curve of the phosphorescent molecules. Absolute tissue PO 2 was thus calculated based on the exponential decay rate, namely the lifetime. 22 This excitation/relaxation cycle was repeated 3000 times for each location to increase the SNR. A baby monitor operating in the IR was used to monitor animal behavior during experiments. Further details about the imaging system components and the animal handling procedure can be found in Ref. 22.

Two-photon fluorescence line scans
The same two-photon microscopy system was used for fluorescence line scans to investigate capillaries in seven young ATX mice and seven old ATX mice. In a separate session, FITC-Dextran solution (50 mg∕ml in saline) was administered by intravenous injection as a contrast agent to label blood plasma. We first scanned a cortical area of 100 μm × 100 μm to obtain 2-D en face fluorescence intensity images in the x − y plane.

Statistics
Data are presented as mean ± standard error of the mean (SEM) when results from the two groups of ATX mice are shown in the figures. Statistical comparisons between the two groups were performed using unpaired two-sample t-tests in MATLAB (MathWorks). The null hypothesis that there was no difference between the two groups is rejected if p-value was <0.01. The number of the young and the old ATX mice used for each imaging session is indicated above.

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 HbO 2 , HbR, and HbT concentration changes due to stimulation. Images are shown at 3 s after the beginning of a whisker stimulation. 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 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 ΔHbO 2 , ΔHbR, and ΔHbT than the young ATX group. Hemodynamic responses were averaged to obtain group-averaged time evolutions of 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 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.

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-μm 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.

Reduced Blood Flow is Associated with Reduced Tissue PO 2 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 PO 2 data were acquired at six different depths ranging from 40-to 180-μm beneath the cortical surface. Figure 3(b) shows the distribution of PO 2 values for the young and the old ATX group. The distribution of tissue 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 PO 2 value (<15 mmHg) between the two groups [inset of Fig. 3(b)]. The percentage of low PO 2 points changed from 7.7% in young ATX mice to 12.6% in old ATX ones. Overall, the mean value of tissue 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 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 PO 2 at all depths [ Fig. 3(e)]. The decrease of cerebral tissue PO 2 and the increase of its spatial heterogeneity could cause low PO 2 sites to colocate forming hypoxic micropockets. To look for the presence of hypoxic micropockets, we defined them as regions comprising at least 15 sampled points whose PO 2 value is lower than 5 mmHg. Figure 3(a2) shows an example of an interpolated tissue 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.

Cerebral Hypoperfusion and Decreased Tissue PO 2 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 proatherogenic 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.

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 LDLR −∕− hApoB þ∕þ transgenic mice at The PO 2 distribution of old ATX mice was shifted toward lower PO 2 values when compared with the young ATX group. The inset shows that pixels with low PO 2 values were more frequently seen in old ATX mice than in young ATX mice. (c) Cerebral tissue 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 PO 2 when compared with the young ATX group. The spatial heterogeneity was calculated for each PO 2 grid and was defined as the coefficient of variation of each PO 2 grid. (e) Tissue PO 2 values at different depths were analyzed for the two groups of ATX mice. The decreasing trend of PO 2 relative to depth was found in both ATX groups. The old ATX group had significantly lower cortical tissue PO 2 at all investigated depths than the young ATX group. Data are shown as mean AE SEM. Unpaired ttest was performed: ****p < 0.0001. Y, young and O, old.
Neurophotonics 045003-6 Oct-Dec 2019 • Vol. 6(4) 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 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 HbO 2 , HbR, and HbT were significantly lower in the old ATX group than in the young ATX group. Group-averaged time series of HbO 2 and HbR concentration changes also confirmed that the old ATX mice had much weaker hemodynamic (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 mean AE SEM. Unpaired t -test was performed: ****p < 0.0001, ***p < 0.001, and **p < 0.01. Y, young and O, old.
Neurophotonics 045003-7 Oct-Dec 2019 • Vol. 6 (4) 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 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 PO 2 value and significantly higher tissue 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 PO 2 values (<5 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][59][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, middleaged, 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.

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.

Disclosures
The authors declare no competing financial interests.