Aging exacerbates hypertension-induced cerebral microhemorrhages in mice: role of resveratrol treatment in vasoprotection methods
Aim. Evidence-backed execution summary for Aging exacerbates hypertension-induced cerebral microhemorrhages in mice: role of resveratrol treatment in vasoprotection methods from Aging exacerbates hypertension-induced cerebral microhemorrhages in mice: role of resveratrol treatment in vasoprotection.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Aging exacerbates hypertension-induced vascular oxidative stress: prevention by resveratrol treatment
reagent used in the protocol.
- Use
- We performed an implicit relationship analysis of entities (relevant vascular pathologies, genes, etc.) within the published literature to identify potential pressure-induced factors involved in cerebrovascular fragility and development of CMHs in aging using the IRIDESCENT text mining package (Wren & Garner, ). Thi...
Aging exacerbates hypertension-induced vascular oxidative stress: prevention by resveratrol treatment
reagent used in the protocol.
- Use
- Aging exacerbates hypertension-induced vascular oxidative stress. A: Representative confocal images showing stronger dihydroethidium (DHE) staining (red fluorescence) indicating increased O 2.- production in high pressure-exposed MCAs isolated from aged mice as compared to MCAs isolated from young mice. MCAs...
Aging exacerbates hypertension-induced MMP activation: prevention by resveratrol treatment
reagent used in the protocol.
- Use
- Redox-sensitive activation of MMPs is thought to play a central role in the pathogenesis of CMHs (Wakisaka et al.,, ). Because previous studies showed that high intraluminal pressure is the primary stimulus for upregulation of vascular MMPs (Lehoux et al., ), we compared pressure-induced MMP activation...
Aging exacerbates hypertension-induced MMP activation: prevention by resveratrol treatment
reagent used in the protocol.
- Use
- Aging exacerbates hypertension-induced redox-sensitive matrix metalloproteinases (MMP) activation. Representative compressed Z stacks of confocal images of MCAs showing stronger MMPsense 645 FAST fluorescence (red) in high pressure-exposed MCAs isolated from aged mice as compared to MCAs isolated from young mice, in...
Induction of spontaneous intracerebral microhemorrhages and resveratrol treatment
reagent used in the protocol.
- Use
- To study spontaneous intracerebral microhemorrhages, we have used a mouse model previously characterized by the Heistad laboratory (Wakisaka et al.,, ): mice with hypertension induced by combined treatment with angiotensin II (Ang II) and ω-nitro- l -arginine methyl ester (L-NAME). As aging is associated...
Detection of high pressure-induced production of ROS in isolated cerebral arteries
reagent used in the protocol.
- Use
- Two segments of the middle cerebral arteries were isolated from the brains of young and aged mice, as reported (Toth et al.,, ). The vessels were mounted onto two glass micropipettes in an organ chamber in oxygenated (21% O 2, 5% CO 2, 75% N 2 ) Krebs' buffer (composed of (in mmol L -1 ): 110.0 N...
Quantitative real-time RT-PCR
reagent used in the protocol.
- Use
- A quantitative real-time RT-PCR technique was used to analyze mRNA expression for following genes in cortical samples of mice from each experimental group: Nox1, Nox2, Nox4, and Ncf1 (p47phox) using a Strategene MX3000 platform, as previously reported (Toth et al., ). In brief, total RNA was isolated wit...
Aging exacerbates hypertension-induced MMP activation: prevention by resveratrol treatment
To substantiate our findings, vascular MMP activity was also compared in hypertensive young and aged mice by in vivo administration of the MMPsense 645 FAST substrate. Activity of MMP-9 was barely detectable by confocal microscopy in normotensive young mice and was not increased significantly by hypertension. Repres...
- Use
- To substantiate our findings, vascular MMP activity was also compared in hypertensive young and aged mice by in vivo administration of the MMPsense 645 FAST substrate. Activity of MMP-9 was barely detectable by confocal microscopy in normotensive young mice and was not increased significantly by hypertension. Repres...
Induction of spontaneous intracerebral microhemorrhages and resveratrol treatment
To study spontaneous intracerebral microhemorrhages, we have used a mouse model previously characterized by the Heistad laboratory (Wakisaka et al.,, ): mice with hypertension induced by combined treatment with angiotensin II (Ang II) and ω-nitro- l -arginine methyl ester (L-NAME). As aging is associated...
- Use
- To study spontaneous intracerebral microhemorrhages, we have used a mouse model previously characterized by the Heistad laboratory (Wakisaka et al.,, ): mice with hypertension induced by combined treatment with angiotensin II (Ang II) and ω-nitro- l -arginine methyl ester (L-NAME). As aging is associated...
Analysis of gait function
Gait coordination was examined by the CatWalk System (Noldus Information Technology Inc. Leesburg, VA, USA). Using the CatWalk system, the detection of paw prints' size, pressure, and pattern during volunteer running on an illuminated glass walkway by a camera placed under the glass surface provides an automated com...
- Use
- Gait coordination was examined by the CatWalk System (Noldus Information Technology Inc. Leesburg, VA, USA). Using the CatWalk system, the detection of paw prints' size, pressure, and pattern during volunteer running on an illuminated glass walkway by a camera placed under the glass surface provides an automated com...
Histological analysis of intracerebral hemorrhages
Mice were euthanized and transcardially perfused with heparinized PBS for 5 min and decapitated. Subsequently, the brains were isolated and fixed in 10% formalin at room temperature for 1 day. The next day, the brains were placed in fresh 10% formalin (at 4 °C, for 2 days), then in 70% ethan...
- Use
- Mice were euthanized and transcardially perfused with heparinized PBS for 5 min and decapitated. Subsequently, the brains were isolated and fixed in 10% formalin at room temperature for 1 day. The next day, the brains were placed in fresh 10% formalin (at 4 °C, for 2 days), then in 70% ethan...
Detection of high pressure-induced production of ROS in isolated cerebral arteries
Two segments of the middle cerebral arteries were isolated from the brains of young and aged mice, as reported (Toth et al.,, ). The vessels were mounted onto two glass micropipettes in an organ chamber in oxygenated (21% O 2, 5% CO 2, 75% N 2 ) Krebs' buffer (composed of (in mmol L -1 ): 110.0 N...
- Use
- Two segments of the middle cerebral arteries were isolated from the brains of young and aged mice, as reported (Toth et al.,, ). The vessels were mounted onto two glass micropipettes in an organ chamber in oxygenated (21% O 2, 5% CO 2, 75% N 2 ) Krebs' buffer (composed of (in mmol L -1 ): 110.0 N...
Detection of high pressure-induced activation of MMPs in isolated cerebral arteries
In separate experiments, pressure-induced MMP activity was measured in cannulated segments of the middle cerebral arteries. The arteries from young and aged animals were pressurized to 60 or 160 mmHg in the presence of MMPsense 645 FAST substrate (PerkinElmer Inc, Boston MA, USA; 3 µmol L -...
- Use
- In separate experiments, pressure-induced MMP activity was measured in cannulated segments of the middle cerebral arteries. The arteries from young and aged animals were pressurized to 60 or 160 mmHg in the presence of MMPsense 645 FAST substrate (PerkinElmer Inc, Boston MA, USA; 3 µmol L -...
Detection of hypertension-induced MMP activation in the cerebral vessels in situ
Mice from each experimental group were temporarily anesthetized with ketamine/xylazine and injected retro-orbitally with a 100 µL dose of 40 nmol L -1 MMPsense 645 FAST substrate. After 12 h of circulation of the substrate, animals were transcardially perfused with PBS containing 1...
- Use
- Mice from each experimental group were temporarily anesthetized with ketamine/xylazine and injected retro-orbitally with a 100 µL dose of 40 nmol L -1 MMPsense 645 FAST substrate. After 12 h of circulation of the substrate, animals were transcardially perfused with PBS containing 1...
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Increased incidence of CMHs is associated with early gait dysfunction in hypertensive aged mice: prevention by resver...
Increased incidence of cerebral microhemorrhages (CMHs) is associated with early gait dysfunction in hypertensive aged mice: prevention by resveratrol treatment. Panel A shows that dysfunction of gait coordination (indicated by changes in the regulatory index) is manifested before the onset of signs of intracerebral hemorrhage detectable by standard neurological examination. Regularity index is a measure of interpaw coordination. In healthy, fully coordinated animals, its value is 100%. Inset: significant decline in the regularity index between the day before induction of hypertension and the day before the onset of clinically detectable hemorrhage (S-1) in hypertensive aged mice. Data are mean ± SEM. * P < 0.05 vs. Day baseline. ( n = 20). B: Data from cross-sectional studies. Shown are changes in regularity index induced by treatment with angiote...
Analysis of gait function
Gait coordination was examined by the CatWalk System (Noldus Information Technology Inc. Leesburg, VA, USA). Using the CatWalk system, the detection of paw prints' size, pressure, and pattern during volunteer running on an illuminated glass walkway by a camera placed under the glass surface provides an automated computerized way to assess gait function and the spatial and temporal aspects of interlimb coordination (Hamers et al., ). Briefly, animals were trained to cross the walkway and then, in a dark room, had 3 consecutive runs every day in the instrument throughout the experimental period. Data were averaged across 3 runs in which the animal maintained a constant speed across the walkway. After manual identification and labeling of each footprint, the regularity index was calculated. The regularity index (%) is a fractional measure of interpaw coordination, which expresses t...
Histological analysis of intracerebral hemorrhages
Mice were euthanized and transcardially perfused with heparinized PBS for 5 min and decapitated. Subsequently, the brains were isolated and fixed in 10% formalin at room temperature for 1 day. The next day, the brains were placed in fresh 10% formalin (at 4 °C, for 2 days), then in 70% ethanol (at 4 °C, for 2 days), followed by embedding in paraffin. The brains were serially sectioned at 8 µm thickness yielding approximately 1500 sections per brain. The first two sections of every five section were stained with hematoxylin to reveal the brain structure and diaminobenzidine (DAB) to highlight the presence of hemorrhages. DAB turns into dark brown when it undergoes a reaction with peroxidases present in red blood cells therefore allowing precise detection of extravasated blood cells in the parenchyma of the brain. All stained sections we...
Detection of hypertension-induced MMP activation in the cerebral vessels in situ
Mice from each experimental group were temporarily anesthetized with ketamine/xylazine and injected retro-orbitally with a 100 µL dose of 40 nmol L -1 MMPsense 645 FAST substrate. After 12 h of circulation of the substrate, animals were transcardially perfused with PBS containing 1 × heparin and FITC-dextran (to highlight the vascular lumen). Then, the mice were decapitated and the brains were removed and cut in half. From the left hemisphere, the frontal cortex containing the photoactive substrate was isolated and homogenized. To quantify MMP activity, the background corrected fluorescence (Ex: 649 nm, Em: 666 nm) was measured spectrophotofluorometrically using a microplate reader and normalized to tissue weight. The right hemisphere was embedded in OCT media, and cryosectioned and confocal images of brain areas containing cross sections o...
Quantitative real-time RT-PCR
A quantitative real-time RT-PCR technique was used to analyze mRNA expression for following genes in cortical samples of mice from each experimental group: Nox1, Nox2, Nox4, and Ncf1 (p47phox) using a Strategene MX3000 platform, as previously reported (Toth et al., ). In brief, total RNA was isolated with a Mini RNA Isolation Kit (Zymo Research, Orange, CA, USA) and was reverse transcribed using Superscript III RT (Invitrogen). Amplification efficiencies were determined using a dilution series of a standard vascular sample. Quantification was performed using the efficiency-corrected ΔΔCq method. The relative quantities of the reference genes Hprt, Ywhaz, B2 m, and Actb were determined, and a normalization factor was calculated based on the geometric mean for internal normalization. Fidelity of the PCR reaction was determined by melting temperature analysis...
Measurement outputs
What raw and processed outputs should exist?
Figure shows that early and progressive dysfunction of gait coordination in hypertensive aged mice can be detected before the onset of signs of intracerebral hemorrhage detectab...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
We performed an implicit relationship analysis of entities (relevant vascular pathologies, genes, etc.) within the published literature to identify potential pressure-induced fa...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Aging exacerbates hypertension-induced vascular oxidative stress. A: Representative confocal images showing stronger dihydroethidium (DHE) staining (red fluorescence) indicating...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
NADPH oxidases are important sources of ROS in the cerebral vasculature, whose increased expression and activity contribute to hypertension-induced vascular oxidative stress (Gi...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
Figure shows that early and progressive dysfunction of gait coordination in hypertensive aged mice can be detected before the onset of signs of intracerebral hemorrhage detectable by standardized neurological examination.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Figure shows that early and progressive dysfunction of gait coordination in hypertensive aged mice can be detected before the onset of signs of intracerebral hemorrhage detectab...; We performed an implicit relationship analysis of entities (relevant vascular pathologies, genes, etc.) within the published literature to identify potential pressure-induced fa...; Aging exacerbates hypertension-induced vascular oxidative stress. A: Representative confocal images showing stronger dihydroethidium (DHE) staining (red fluorescence) indicating...; NADPH oxidases are important sources of ROS in the cerebral vasculature, whose increased expression and activity contribute to hypertension-induced vascular oxidative stress (Gi....
from paperStatistical comparison
Figure shows that early and progressive dysfunction of gait coordination in hypertensive aged mice can be detected before the onset of signs of intracerebral hemorrhage detectab...; Increased incidence of cerebral microhemorrhages (CMHs) is associated with early gait dysfunction in hypertensive aged mice: prevention by resveratrol treatment. Panel A shows t...; Aging exacerbates hypertension-induced vascular oxidative stress. A: Representative confocal images showing stronger dihydroethidium (DHE) staining (red fluorescence) indicating...; NADPH oxidases are important sources of ROS in the cerebral vasculature, whose increased expression and activity contribute to hypertension-induced vascular oxidative stress (Gi...
from paperReporting output
Report representative outputs alongside summary comparisons for Figure shows that early and progressive dysfunction of gait coordination in hypertensive aged mice can be detected before the onset of signs of intracerebral hemorrhage detectab..., We performed an implicit relationship analysis of entities (relevant vascular pathologies, genes, etc.) within the published literature to identify potential pressure-induced fa..., Aging exacerbates hypertension-induced vascular oxidative stress. A: Representative confocal images showing stronger dihydroethidium (DHE) staining (red fluorescence) indicating..., NADPH oxidases are important sources of ROS in the cerebral vasculature, whose increased expression and activity contribute to hypertension-induced vascular oxidative stress (Gi....
inferred from protocolStructured statistical methods
Figure shows that early and progressive dysfunction of gait coordination in hypertensive aged mice can be detected before the onset of signs of intracerebral hemorrhage detectab...; Increased incidence of cerebral microhemorrhages (CMHs) is associated with early gait dysfunction in hypertensive aged mice: prevention by resveratrol treatment. Panel A shows t...; Aging exacerbates hypertension-induced vascular oxidative stress. A: Representative confocal images showing stronger dihydroethidium (DHE) staining (red fluorescence) indicating...; NADPH oxidases are important sources of ROS in the cerebral vasculature, whose increased expression and activity contribute to hypertension-induced vascular oxidative stress (Gi...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
Increased incidence of cerebral microhemorrhages (CMHs) is associated with early gait dysfunction in hypertensive aged mice: prevention by resveratrol treatment. Panel A shows that dysfunction of gait coordination (indicated by changes in the regulatory index) is manifested before the onset of signs of intracerebral hemorrhage detectable by standard neurological examination. Regularity index is a measure of interpaw coordination. In healthy, fully coordinated animals, its value is 100%. Inset: significant decline in the regularity index between the day before induction of hypertension and the day before the onset of clinically detectable hemorrhage (S-1) in hypertensive aged mice. Data are mean ± SEM. * P < 0.05 vs. Day baseline. ( n = 20). B: Data from cross-sectional studies. Shown are changes in regularity index induced by treatment with angiotensin II plus L-NAME (HT groups) in young (3 months, n = 20), aged (24 months, n = 20), and resveratrol-treated aged ( n = 20) C57BL/6 mice on day 5 postinduction of hypertension. Note that aging exacerbates hypertension-induced decline in the regulatory, wh...
Gait coordination was examined by the CatWalk System (Noldus Information Technology Inc. Leesburg, VA, USA). Using the CatWalk system, the detection of paw prints' size, pressure, and pattern during volunteer running on an illuminated glass walkway by a camera placed under the glass surface provides an automated computerized way to assess gait function and the spatial and temporal aspects of interlimb coordination (Hamers et al., ). Briefly, animals were trained to cross the walkway and then, in a dark room, had 3 consecutive runs every day in the instrument throughout the experimental period. Data were averaged across 3 runs in which the animal maintained a constant speed across the walkway. After manual identification and labeling of each footprint, the regularity index was calculated. The regularity index (%) is a fractional measure of interpaw coordination, which expresses the number of normal step sequence patterns relative to the total number of paw placements. The formula of regularity index is as follows: NSSPx4/PP × 100 (%), where NSSP represents the number of normal step sequence patterns and PP the total number of paw placements. In healthy, fully...
Mice were euthanized and transcardially perfused with heparinized PBS for 5 min and decapitated. Subsequently, the brains were isolated and fixed in 10% formalin at room temperature for 1 day. The next day, the brains were placed in fresh 10% formalin (at 4 °C, for 2 days), then in 70% ethanol (at 4 °C, for 2 days), followed by embedding in paraffin. The brains were serially sectioned at 8 µm thickness yielding approximately 1500 sections per brain. The first two sections of every five section were stained with hematoxylin to reveal the brain structure and diaminobenzidine (DAB) to highlight the presence of hemorrhages. DAB turns into dark brown when it undergoes a reaction with peroxidases present in red blood cells therefore allowing precise detection of extravasated blood cells in the parenchyma of the brain. All stained sections were screened, and images were acquired in the evidence of a positive DAB reaction. Digital images were analyzed with imagej software (NIH) to identify the location and quantify the number and size of hemorrhages. The size of hemorrhages was estimated as follows: (area (mm 2 ) of CMHs on each section)...
Mice from each experimental group were temporarily anesthetized with ketamine/xylazine and injected retro-orbitally with a 100 µL dose of 40 nmol L -1 MMPsense 645 FAST substrate. After 12 h of circulation of the substrate, animals were transcardially perfused with PBS containing 1 × heparin and FITC-dextran (to highlight the vascular lumen). Then, the mice were decapitated and the brains were removed and cut in half. From the left hemisphere, the frontal cortex containing the photoactive substrate was isolated and homogenized. To quantify MMP activity, the background corrected fluorescence (Ex: 649 nm, Em: 666 nm) was measured spectrophotofluorometrically using a microplate reader and normalized to tissue weight. The right hemisphere was embedded in OCT media, and cryosectioned and confocal images of brain areas containing cross sections of penetrating small arteries were captured.
A quantitative real-time RT-PCR technique was used to analyze mRNA expression for following genes in cortical samples of mice from each experimental group: Nox1, Nox2, Nox4, and Ncf1 (p47phox) using a Strategene MX3000 platform, as previously reported (Toth et al., ). In brief, total RNA was isolated with a Mini RNA Isolation Kit (Zymo Research, Orange, CA, USA) and was reverse transcribed using Superscript III RT (Invitrogen). Amplification efficiencies were determined using a dilution series of a standard vascular sample. Quantification was performed using the efficiency-corrected ΔΔCq method. The relative quantities of the reference genes Hprt, Ywhaz, B2 m, and Actb were determined, and a normalization factor was calculated based on the geometric mean for internal normalization. Fidelity of the PCR reaction was determined by melting temperature analysis and visualization of the product on a 2% agarose gel.
Machine-readable layer
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