Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension methods
Aim. Evidence-backed execution summary for Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension methods from Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Perivascular space size, shape, and change during fixation
reagent used in the protocol.
- Use
- By superimposing all particle tracks from each experiment (Fig. ), we can estimate the extent of the PVSs: each is ~40 µm wide, comparable to the adjacent artery. Volumetric images using a dextran CSF tracer confirmed this result (Fig. ), and orthogonal projections showed two non-connecting PV...
Correlative imaging
reagent used in the protocol.
- Use
- 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...
BBB permeability assay
reagent used in the protocol.
- Use
- 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...
Tissue processing and imaging
reagent used in the protocol.
- Use
- 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...
Flow pulsing with heartbeat and driven by artery walls
Both the cardiac and the respiratory cycles have been suggested as CSF flow drivers -,,. To identify the mechanisms driving flow in the PVS, we performed two-photon imaging while recording synchronized measurements of the electrocardiogram (ECG) and the respiratory cycle (Supplementary Movie ). After...
- Use
- Both the cardiac and the respiratory cycles have been suggested as CSF flow drivers -,,. To identify the mechanisms driving flow in the PVS, we performed two-photon imaging while recording synchronized measurements of the electrocardiogram (ECG) and the respiratory cycle (Supplementary Movie ). After...
Inducing acute hypertension to alter wall motion and flow
To test whether these changes in the vessel wall dynamics affect the efficiency of the perivascular pump, we imaged CSF flow and simultaneously recorded ECG, respiration, and arterial blood pressure before, during, and after administering Ang-II (Supplementary Movie ). The average flow speed (Fig. ) was...
- Use
- To test whether these changes in the vessel wall dynamics affect the efficiency of the perivascular pump, we imaged CSF flow and simultaneously recorded ECG, respiration, and arterial blood pressure before, during, and after administering Ang-II (Supplementary Movie ). The average flow speed (Fig. ) was...
Methods
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/...
- Use
- 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/...
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 Prec...
- Use
- 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 Prec...
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 mm̴...
- Use
- 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 mm̴...
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...
- Use
- 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...
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 An...
- Use
- 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 An...
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...
- Use
- 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...
Particle tracking velocimetry
Software used for acquisition, scoring, statistics, or reporting.
- Use
- 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 MATLAB,. This algorithm individually locates each particle with subpixel accuracy, tracks its location throughout the time s...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- 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&#...
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Inducing acute hypertension to alter wall motion and flow
To test whether these changes in the vessel wall dynamics affect the efficiency of the perivascular pump, we imaged CSF flow and simultaneously recorded ECG, respiration, and arterial blood pressure before, during, and after administering Ang-II (Supplementary Movie ). The average flow speed (Fig. ) was reduced by ~40% during Ang-II-induced hypertension (Fig. ). This decrease in flow speed was independent of the time after infusion (Supplementary Fig. ). Also, microsphere trajectories show more backflow during each cardiac period in acute hypertension (Fig., insets). To quantify changes in the instantaneous flow rate, we computed the downstream component of every velocity measurement (Fig. ). Hypertension increases backflow (negative downstream velocity) by 21% relative to controls (Fig. and Supplementary Fig. ). A small percentage of t...
Methods
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 depressurization. For a subset of experiments, instead of a cranial window, mice received a thin-skull preparation,. Afterwards, a cannula was placed in the...
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.
Calculation of Reynolds and Péclet numbers
The Reynolds number is calculated as: 1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{Re}} = \frac{{UL}}{\nu },$$\end{document} Re = U L ν, where U is the spatial mean of the magnitude of the time-averaged flow velocity (e.g., the mean of measurements shown in Fig. ), L = 4.4 × 10 -5 m is the average PVS width that has been measured (Fig. ), and ν = 0.697 × 10 -6 m 2 s -1 is the kinematic viscosity of water at 36.8 °C (a good approximation for cerebrospinal fluid). The Péclet number is calculated as: 2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepac...
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 synchroniz...
Measurement outputs
What raw and processed outputs should exist?
By superimposing all particle tracks from each experiment (Fig. ), we can estimate the extent of the PVSs: each is ~40 µm wide, comparable to the adjacent arter...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Both the cardiac and the respiratory cycles have been suggested as CSF flow drivers -,,. To identify the mechanisms driving flow in the PVS, we performed two-photon ima...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
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 &#...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
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 (Coheren...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
By superimposing all particle tracks from each experiment (Fig. ), we can estimate the extent of the PVSs: each is ~40 µm wide, comparable to the adjacent artery.
from paperScoring or quantification
Quantify the primary readouts for this experiment: By superimposing all particle tracks from each experiment (Fig. ), we can estimate the extent of the PVSs: each is ~40 µm wide, comparable to the adjacent arter...; Both the cardiac and the respiratory cycles have been suggested as CSF flow drivers -,,. To identify the mechanisms driving flow in the PVS, we performed two-photon ima...; 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 &#...; 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 (Coheren....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
By superimposing all particle tracks from each experiment (Fig. ), we can estimate the extent of the PVSs: each is ~40 µm wide, comparable to the adjacent arter...; Both the cardiac and the respiratory cycles have been suggested as CSF flow drivers -,,. To identify the mechanisms driving flow in the PVS, we performed two-photon ima...; Having determined that perivascular pumping is a principal driver of CSF flow in the PVS, we next investigated the effect of altering vascular wall dynamics. The amplitude and s...; To test whether these changes in the vessel wall dynamics affect the efficiency of the perivascular pump, we imaged CSF flow and simultaneously recorded ECG, respiration, and ar...
from paperReporting output
Report representative outputs alongside summary comparisons for By superimposing all particle tracks from each experiment (Fig. ), we can estimate the extent of the PVSs: each is ~40 µm wide, comparable to the adjacent arter..., Both the cardiac and the respiratory cycles have been suggested as CSF flow drivers -,,. To identify the mechanisms driving flow in the PVS, we performed two-photon ima..., 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 &#..., 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 (Coheren....
inferred from protocolStructured statistical methods
By superimposing all particle tracks from each experiment (Fig. ), we can estimate the extent of the PVSs: each is ~40 µm wide, comparable to the adjacent arter...; Both the cardiac and the respiratory cycles have been suggested as CSF flow drivers -,,. To identify the mechanisms driving flow in the PVS, we performed two-photon ima...; Having determined that perivascular pumping is a principal driver of CSF flow in the PVS, we next investigated the effect of altering vascular wall dynamics. The amplitude and s...; To test whether these changes in the vessel wall dynamics affect the efficiency of the perivascular pump, we imaged CSF flow and simultaneously recorded ECG, respiration, and ar...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
To test whether these changes in the vessel wall dynamics affect the efficiency of the perivascular pump, we imaged CSF flow and simultaneously recorded ECG, respiration, and arterial blood pressure before, during, and after administering Ang-II (Supplementary Movie ). The average flow speed (Fig. ) was reduced by ~40% during Ang-II-induced hypertension (Fig. ). This decrease in flow speed was independent of the time after infusion (Supplementary Fig. ). Also, microsphere trajectories show more backflow during each cardiac period in acute hypertension (Fig., insets). To quantify changes in the instantaneous flow rate, we computed the downstream component of every velocity measurement (Fig. ). Hypertension increases backflow (negative downstream velocity) by 21% relative to controls (Fig. and Supplementary Fig. ). A small percentage of the microspheres aggregate and adhere to the PVS wall, remaining in the imaging field for the duration of the experiment; however, hypertension did not increase the number of these stuck particles compared to the controls (Supplementary Fig. ). Fig. 5 Acute arterial hypertension reduces net CSF...
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 depressurization. For a subset of experiments, instead of a cranial window, mice received a thin-skull preparation,. 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 min.
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.
The Reynolds number is calculated as: 1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{Re}} = \frac{{UL}}{\nu },$$\end{document} Re = U L ν, where U is the spatial mean of the magnitude of the time-averaged flow velocity (e.g., the mean of measurements shown in Fig. ), L = 4.4 × 10 -5 m is the average PVS width that has been measured (Fig. ), and ν = 0.697 × 10 -6 m 2 s -1 is the kinematic viscosity of water at 36.8 °C (a good approximation for cerebrospinal fluid). The Péclet number is calculated as: 2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{Pe}} = \frac{{UL}}{D},$$\end{document} Pe = U L D, where D = 6.55 × 10 -13...
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 measuremen...
Machine-readable layer
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"text": "To test whether these changes in the vessel wall dynamics affect the efficiency of the perivascular pump, we imaged CSF flow and simultaneously recorded ECG, respiration, and arterial blood pressure before, during, and after administering Ang-II (Supplementary Movie ). The average flow speed (Fig. ) was reduced by ~40% during Ang-II-induced hypertension (Fig. ). This decrease in flow speed was independent of the time after infusion (Supplementary Fig. ). Also, microsphere trajectories show more backflow during each cardiac period in acute hypertension (Fig., insets). To quantify changes in the instantaneous flow rate, we computed the downstream component of every velocity measurement (Fig. ). Hypertension increases backflow (negative downstream velocity) by 21% relative to controls (Fig. and Supplementary Fig. ). A small percentage of t..."
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"text": "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 depressurization. For a subset of experiments, instead of a cranial window, mice received a thin-skull preparation,. Afterwards, a cannula was placed in the..."
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"text": "The Reynolds number is calculated as: 1 \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\mathrm{Re}} = \\frac{{UL}}{\\nu },$$\\end{document} Re = U L ν, where U is the spatial mean of the magnitude of the time-averaged flow velocity (e.g., the mean of measurements shown in Fig. ), L = 4.4 × 10 -5 m is the average PVS width that has been measured (Fig. ), and ν = 0.697 × 10 -6 m 2 s -1 is the kinematic viscosity of water at 36.8 °C (a good approximation for cerebrospinal fluid). The Péclet number is calculated as: 2 \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepac..."
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"text": "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 synchroniz..."
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