Animals and surgical preparation

All experiments were approved by the University Committee on Animal Resources of the University of Rochester Medical Center (Protocol No. 2011-023) and an effort was made to minimize the number of animals used. Male C57BL/6 mice, 8–12 weeks of age (Charles River), were anesthetized with ketamine/xylazine (100/10 mg kg−1, intraperitoneally). Body temperature was maintained at 37.5 °C with a rectal probe-controlled heated platform (Harvard Apparatus). A cranial window was prepared over the MCA vascular territory on the right anterolateral parietal bone leaving dura mater intact. The cranial window was sealed with agarose (0.8% at 37 °C) and a glass coverslip to prevent intracranial depressurization35. For a subset of experiments, instead of a cranial window, mice received a thin-skull preparation9,36. Afterwards, a cannula was placed in the cisterna magna and red fluorescent polystyrene microspheres (FluoSpheres™ 1.0 µm, 580/605 nm, 0.25% solids in artificial CSF (aCSF), Invitrogen) were briefly sonicated and infused at 2 µl min−1 for 5 min1.

Measurement of vital signs

ECG and respiratory rate were acquired at 1 kHz and 250 Hz, respectively, using a small animal physiological monitoring device (Harvard Apparatus). Arterial blood pressure was measured through an arterial catheter placed in the femoral artery and connected to a pressure transducer and monitor (World Precision Instruments). The signals were digitized and recorded with a DigiData 1550A digitizer and AxoScope software (Axon Instruments).

Acute arterial hypertension induction

Angiotensin-II (Tocris) was dissolved in NaCl 0.9% and infused into a femoral vein catheter at 5 ng g−1 min−1 at a volumetric rate of 1 µl min−1 using a syringe pump (Harvard Apparatus) for the duration of the experiment. Mice with less than a 20 mm Hg increase in blood pressure were excluded from the analysis. Control mice received an intravenous infusion of NaCl 0.9% at 1 µl min−1.

In vivo two-photon laser scanning microscopy

Two-photon imaging was performed using a resonant scanner B scope (Thorlabs) with a Mai Tai DeepSee HP Ti:Sapphire laser (Spectra Physics) or a Chameleon Ultra II laser (Coherent) attached to a galvo confocal scanning system (Fluoview 300, Olympus). A water-immersion 20× objective (1.0 NA, Olympus) was used on both systems. Intravascular fluorescein isothiocyanate–dextran (FITC–dextran, 2,000 kDa) and red microspheres were excited at an 820 nm wavelength. Images were acquired at 30 Hz (ThorSync software) simultaneously with physiological recordings (3 kHz, ThorSync software). Prior to infusion of the microspheres, a volumetric image of the vascular topology was acquired. Segments of the MCA were identified as large caliber surface vessels (50–60 µm diameter) that started at the anterolateral aspect of the window and traveled towards the midline. Direction of blood flow was confirmed before and after imaging using line scans.

Correlative imaging

To confirm the size of the PVS, a separate group of anesthetized mice received an intracisternal injection of Texas-Red dextran (2000 kDa, 0.5% in aCSF, Invitrogen) instead of microspheres. The dextran tracer was delivered using the same infusion paradigm as described above. After tracer reached the PVS of the cortical arteries, volumetric images were acquired for cross-sectional reconstruction. Mice were then perfused with a phosphate-buffered saline (PBS) solution with an Alexa Fluor 488 wheat germ agglutinin lectin (15 µg ml−1, Invitrogen) through an arterial catheter. Afterwards, mice were perfusion-fixed with 4% PFA. After fixation, the coverslip and the dura mater were removed due to tissue shrinking and images were acquired as described above.

BBB permeability assay

FITC-conjugated dextran (1%; 70 kDa; Sigma-Aldrich) in normal saline (4 ml kg−1) was injected via a femoral vein catheter. The dextran was allowed to circulate for 30 min while mice received either an Ang-II or NaCl 0.9% infusion (1 µl min−1). The brains were harvested, sectioned, and FITC extravasation was imaged and quantified (see below).

Tissue processing and imaging

For assessment of BBB permeability, mice were transcardially perfused with ice-cold 0.1 M PBS (pH 7.4, Sigma-Aldrich) followed by 4% PFA. Brain tissue was carefully dissected away from the skull and dura then post-fixed overnight in 4% PFA at 4 °C. For correlative imaging, mice were perfusion fixed as before but Alexa 488-conjugated lectin from wheat germ agglutinin (15 µg ml−1; Invitrogen) was added into the ice-cold PBS solution prior to the PFA perfusion step.

Ex vivo fluorescence imaging

Coronal slices (100 µm thickness) were obtained using a calibrated vibratome (VT1200S, Leica). Beginning at the anterior aspect of the corpus callosum, one section was collected every 5 sections until a total of 6 sections had been acquired for each animal. Brain sections were mounted with ProLong Gold Antifade with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Coronal sections were imaged at ×4 magnification using a montage epifluorescence microscope (BX51 Olympus and CellSens Software). Exposure times and magnifications were kept the same for all groups.

Image processing

The images obtained from two-photon microscopy are 16-bit with two channels (red and green), each with spatial dimensions of 512 × 512. The green channel captures the FITC–dextran in the vasculature, while the red channel captures the fluorescent microspheres flowing in the perivascular spaces. Note that in the figures presented in this study, we have interchanged the two channels to improve clarity (red vasculature is more intuitive), but the discussion here will refer to the two channels by their accurate, pre-interchanged colors. The first step of image processing is to perform image registration. A time series of rigid translations (no rotation or deformation) is calculated to an accuracy of 0.2 pixel using an efficient algorithm in MATLAB37. The translations are inspected for outliers (where the algorithm has failed due to an erroneous correlation) which are manually corrected via linear interpolation. Images from both the red and green channels are then sequentially read and rewritten with the rigid translations applied; the edges of each image are padded with zero-valued pixels such that all final images in each time series have the same spatial dimensions. One mouse was excluded because image registration could not be performed effectively. The second and final step of image processing was to mask regions with stagnant microspheres in the red channel. This was achieved by computing the average of the entire time series of registered red channel images, then applying a threshold to generate a binary mask. The binary mask was used as input to the particle tracking velocimetry software (discussed below) such that regions above the threshold were completely masked from any velocity measurements. This masking technique ensured that stagnant particles (typically stuck to the boundaries of the perivascular space) did not erroneously decrease the measurements of the flow speed. BBB permeability to FITC–dextran was quantified from ex vivo coronal sections by a blinded investigator. Each slice was analyzed for mean pixel intensity and the average was computed for all 6 sections taken from one brain. Full width at half maximum was quantified by measuring the intensity distribution across the center of 20 randomly selected particles from both types of cranial windows. All images were analyzed using ImageJ software (National Institutes of Health, imagej.nih.gov/ij/).

Particle tracking velocimetry

To obtain measurements of the speed of cerebrospinal fluid flow, we have quantified the motion of the microspheres in each registered time series of images using an automated PTV software in MATLAB25,26. This algorithm individually locates each particle with subpixel accuracy, tracks its location throughout the time series of images, and calculates its velocity in each frame. Hence, for each particle, a time series of positions and velocities is obtained. To further ensure that no stagnant particles were included in our measurements, we computed the average speed along each particle track by dividing the total displacement by the total number of frames in which that particle was tracked; particle tracks with average speeds lower than a given threshold (typically about 2 µm s−1) were excluded from the analysis. Supplementary Table 1 shows the total number of particle tracks and the total number of velocity measurements obtained for each of the 13 mice for which PTV has been performed. To compute the time-averaged flow velocity and speed, the domain was divided into approximately 70 × 70 boxes of resolution 7.5 × 7.5 pixels each. All individual velocity measurements for a given time interval were then binned and averaged according to their box positions. Average flow speeds were computed using only bins which had sufficient measurements (at least 20). Stagnant or nearly stagnant regions were excluded when calculating the changes in flow speed. Mice with fewer than 80 non-stagnant bins with sufficient measurements were excluded from the analysis. To calculate v rms , we computed the spatial root-mean-square of all speed measurements obtained at each instant of time.

Calculation of Reynolds and Péclet numbers

The Reynolds number is calculated as:

$${\mathrm{Re}} = \frac{{UL}}{

u },$$ (1)

where U is the spatial mean of the magnitude of the time-averaged flow velocity (e.g., the mean of measurements shown in Fig. 1d), L = 4.4 × 10−5 m is the average PVS width that has been measured (Fig. 2d), and ν = 0.697 × 10−6 m2 s−1 is the kinematic viscosity of water at 36.8 °C (a good approximation for cerebrospinal fluid). The Péclet number is calculated as:

$${\mathrm{Pe}} = \frac{{UL}}{D},$$ (2)

where D = 6.55 × 10−13 m2 s−1 is the diffusion coefficient for 1 µm spherical particles in a liquid at low Reynolds number. The diffusion coefficient is calculated using the Stokes–Einstein equation:

$$D = \frac{{kT}}{{6\pi \eta r}},$$ (3)

where k = 1.38 × 10−23 m2 kg s−2 K−1, T = 310 K is the body temperature of a mouse, η = 6.93 × 10−4 kg m−1 s−1 is the dynamic viscosity of water at 36.8 °C, and r = 0.5 × 10−6 m is the microsphere radius.

Artery diameter and wall velocity measurements

All line scans were recorded transverse to the MCA. The line scans were recorded at about 600 Hz with synchronized ECG measurements, from which we obtained about 120 artery diameter measurements per cardiac cycle (depending on the heart rate). Line scans at each location were recorded for at least 45 s. For each line scan time series, the outer edges of the artery were located with subpixel accuracy at each instant of time by performing a cubic interpolation of the pixel intensity profile onto a finer grid and identifying the two locations where the pixel intensity crossed a given threshold. For each time series, the threshold value was chosen to approximately coincide with the region of the steepest gradient in the pixel intensity profile. The artery diameter at each instant of time was calculated by measuring the distance between these two locations. Using the synchronized ECG measurements, the cardiac cycle was defined by sequential peaks of the R wave. The change in artery diameter was then calculated as the difference between the instantaneous artery diameter and a two-cardiac-cycle moving average of the artery diameter. The changes in artery diameter measurements were then binned and averaged according to the fraction of the cardiac cycle to obtain the mean change in the artery diameter Δd; this quantity was then normalized by dividing by the average artery diameter d. To measure the artery wall velocity, the mean change in artery diameter measurements (in dimensional distance versus time units) for each animal was fit with a high-order Fourier series in MATLAB and the derivative was calculated analytically.

Downstream velocity component

The downstream velocity component is calculated as \({\mathbf{u}} \cdot \widehat {\mathbf{u}}_{{\mathbf{avg}}}\), where u is the instantaneous particle velocity and \(\widehat {\mathbf{u}}_{{\mathbf{avg}}}\) is a field of unit vectors computed from the time-averaged velocity field (see, e.g., Fig. 1c).

PVS/artery area ratio

Each area used in computing an area ratio was obtained in MATLAB by generating a binary image of the relevant space (perivascular space or artery) and summing the binary image. To generate a binary image for a region which was visualized via fluorescent dye, a carefully chosen threshold was applied to each given image. To generate a binary image indicating the area of the perivascular spaces using microsphere trajectories, we followed the following procedure: all microsphere locations over the entire time series were superimposed onto one large set of coordinates. These (subpixel) coordinates were then rounded to the nearest pixel, and each microsphere was inflated to encompass a 3 × 3 pixel region. This set of coordinates was then used to construct the binary image. Before the area ratio was computed, a region of interest was manually chosen for the calculation such that we excluded the vasculature and perivascular spaces not associated with the middle cerebral artery. Furthermore, any region of overlap between the two binary images was subtracted from the perivascular binary image so that the area ratio would characterize the lateral extent of the perivascular space beyond the artery. Three-dimensional surface reconstructions and orthogonal views of the PVS were done using Imaris (Bitplane).

Statistical analysis

All statistical analyses were performed on GraphPad Prism 7 (GraphPad Software). No sample size calculations were done due to the lack of an existing effect size estimate. An effort was made to have n > 5 for each experiment. Statistical tests were selected after evaluating normality (D’Agostino–Pearson omnibus test) or after visualization of the distribution of data when the sample size did not allow for testing. In the latter cases, both parametric and nonparametric tests were performed and, in all cases, yielded the same result. All testing was two-tailed and exact P values were calculated at a 0.05 level of significance and stated in the figure legends. Animals were not randomized and blinding was done where possible, but was generally not practical for PTV given that the investigator required access to the arterial blood pressure data for analysis.

Code availability

All relevant codes are available from the authors.