Source Paper
Stefano Tarantini, Noa M. Valcarcel‐Ares, Andriy Yabluchanskiy, Gabor A. Fulop, Peter Hertelendy et al.
Aging Cell • 2018
Summary Moment‐to‐moment adjustment of cerebral blood flow ( CBF ) via neurovascular coupling has an essential role in maintenance of healthy cognitive function. In advanced age, increased oxidative stress and cerebromicrovascular endothelial dysfunction impair neurovascular coupling, likely contributing to age‐related decline of higher cortical functions. There is increasing evidence showing that mitochondrial oxidative stress plays a critical role in a range of age‐related cellular impairments, but its role in neurovascular uncoupling remains unexplored. This study was designed to test the hypothesis that attenuation of mitochondrial oxidative stress may exert beneficial effects on neurovascular coupling responses in aging. To test this hypothesis, 24‐month‐old C57 BL /6 mice were treated with a cell‐permeable, mitochondria‐targeted antioxidant peptide ( SS ‐31; 10 mg kg −1 day −1, i.p.) or vehicle for 2 weeks. Neurovascular coupling was assessed by measuring CBF responses (laser speckle contrast imaging) evoked by contralateral whisker stimulation. We found that neurovascular coupling responses were significantly impaired in aged mice. Treatment with SS –31 significantly improved neurovascular coupling responses by increasing NO ‐mediated cerebromicrovascular dilation, which was associated with significantly improved spatial working memory, motor skill learning, and gait coordination. These findings are paralleled by the protective effects of SS –31 on mitochondrial production of reactive oxygen species and mitochondrial respiration in cultured cerebromicrovascular endothelial cells derived from aged animals. Thus, mitochondrial oxidative stress contributes to age‐related cerebromicrovascular dysfunction, exacerbating cognitive decline. We propose that mitochondria‐targeted antioxidants may be considered for pharmacological microvascular protection for the prevention/treatment of age‐related vascular cognitive impairment ( VCI ).
Objective: Assessment of gait coordination as a measure of motor function in aged mice treated with mitochondrial-targeted antioxidant peptide SS-31
This is a Gait Coordination Test protocol using mouse as the model organism. The procedure involves 19 procedural steps, 8 equipment items, 7 materials. Extracted from a 2018 paper published in Aging Cell.
Model and subjects
mouse • C57BL/6 • unknown • 24 months old (aged); 3 months old (young controls) • Not specified • 6
Study window
~2 week study window | ~2 hours hands-on
Core workflow
Animal treatment initiation • SS-31 or vehicle administration • Neurovascular coupling assessment via laser speckle contrast imaging
Primary readouts
Key equipment and reagents
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24-month-old C57BL/6 aged mice were assigned to treatment groups
Note: Young 3-month-old mice served as controls
“24-month-old C57 BL/6 mice were treated with a cell-permeable, mitochondria-targeted antioxidant peptide (SS-31; 10 mg kg−1 day−1, i.p.) or vehicle”
Intraperitoneal injection of SS-31 peptide or vehicle control
Note: Dose: 10 mg/kg/day via intraperitoneal injection
“treated with a cell-permeable, mitochondria-targeted antioxidant peptide (SS-31; 10 mg kg−1 day−1, i.p.) or vehicle for 2 weeks”
Measure cerebral blood flow responses in whisker barrel cortex evoked by contralateral whisker stimulation using laser speckle contrast imaging
Note: Baseline CBF recorded before stimulation; CBF changes measured as percent change from baseline
“Neurovascular coupling was assessed by measuring CBF responses (laser speckle contrast imaging) evoked by contralateral whisker stimulation”
Surgical preparation of open cranial window for direct measurement of cerebral blood flow and pharmacological agent administration
Note: Used for L-NAME administration studies
“Studies using an open cranial window preparation showed that in young animals administration of the NO synthase inhibitor L-NAME”
Measure cerebral blood flow above whisker barrel cortex during contralateral whisker stimulation in presence and absence of L-NAME
Note: Performed in young, aged, and SS-31-treated aged mice
“Representative traces of cerebral blood flow (CBF; measured with a laser Doppler probe above the whisker barrel cortex) during contralateral whisker stimulation (30 s, 5 Hz)”
Administer NO synthase inhibitor L-NAME and measure resulting changes in CBF response to whisker stimulation
Note: Assesses NO-mediated component of neurovascular coupling
“in the absence and presence of the NO synthase inhibitor L-NAME (3 × 10−4 M)”
Isolate and cannulate branches of the middle cerebral artery from young, aged, and SS-31-treated aged mice
Note: Vessels maintained under pressure to develop spontaneous myogenic tone (~30%)
“endothelium-dependent vasodilator responses were tested in isolated, cannulated branches of the MCA. Pressurized MCAs developed spontaneous myogenic tone (~30%)”
Administer acetylcholine to isolated MCA vessels and measure vasodilator response in presence and absence of L-NAME
Note: Tests endothelium-dependent vasodilation and NO-mediated component
“In young vessels, administration of acetylcholine resulted in significant dilation, whereas these responses were significantly attenuated in vessels derived from aging mice”
Administer ATP to isolated MCA vessels and measure vasodilator response in presence and absence of L-NAME
Note: Tests endothelium-dependent vasodilation via purinergic receptor activation
“administration of acetylcholine and ATP resulted in significant dilation, whereas these responses were significantly attenuated in vessels derived from aging mice”
Administer NO donor sodium nitroprusside to isolated MCA vessels and measure vasodilator response
Note: Tests endothelium-independent vasodilation and smooth muscle cell function
“Vasodilator responses elicited by administration of the NO donor sodium nitroprusside (SNP) did not differ significantly among the experimental groups”
Isolate and culture cerebromicrovascular endothelial cells (CMVECs) from young and aged animals
Note: Cells used for in vitro assessment of mitochondrial ROS and respiration
“cultured CMVECs derived from aged animals”
Measure mitochondrial ROS production in cultured CMVECs using MitoSox fluorescence method
Note: Baseline mtROS measured in aged CMVECs; changes measured after SS-31 treatment
“we assessed the effects of SS-31 on cellular mtROS production in cultured CMVECs derived from aged animals using the MitoSox fluorescence method”
Treat cultured CMVECs with SS-31 peptide and measure resulting changes in mtROS production
Note: Concentration: 10−5 M; rapid antioxidative effects observed
“SS-31 treatment elicited significant decreases in mtROS production in aged CMVECs, eliminating the difference between the two age groups. The antioxidative effects of SS-31 were manifested rapidly, with maximal reduction in mtROS being evident after 2 hr post-treatment”
Measure cellular oxygen consumption rate (OCR) in CMVECs as marker of oxidative phosphorylation and mitochondrial respiration
Note: Performed after SS-31 treatment to assess mitochondrial function improvement
“Attenuation of mtROS production in aged CMVECs was associated with significant improvement in cellular oxygen consumption rate (OCR; a marker of oxidative phosphorylation)”
Collect cerebral cortex tissue from young, aged, and SS-31-treated aged mice for gene expression analysis
Note: Tissue used for qPCR analysis of genes regulating NO mediation
“Markers of oxidative stress and expression of genes regulating neurovascular coupling responses in the cerebral cortex were assessed”
Quantify mRNA expression of genes regulating nitric oxide mediation and oxidative stress in cerebral cortex
Note: Genes analyzed: Nos1, Nos3, Arg1, Arg2, Nox1, Nox2, Sod1, Sod2
“qPCR data showing cortical mRNA expression of nitric oxide synthases Nos3 and Nos1, arginases (Arg1, Arg2), NADPH oxidases (Nox1, Nox2), and superoxide dismutases (Sod1, Sod2)”
Conduct battery of behavioral tests to characterize cognitive function and motor coordination
Note: Includes assessment of gait coordination, spatial working memory, and motor skill learning
“Mice were behaviorally evaluated on a battery of tests for characterization of cognitive function and motor coordination”
Evaluate gait coordination as measure of motor function in young, aged, and SS-31-treated aged mice
Note: Part of behavioral testing battery; improved by SS-31 treatment
“Treatment with SS-31 significantly improved neurovascular coupling responses by increasing NO-mediated cerebromicrovascular dilation, which was associated with significantly improved spatial working memory, motor skill learning, and gait coordination”
Analyze data using one-way ANOVA with post hoc Tukey's tests
Note: Significance threshold: p < 0.05
“one-way ANOVA with post hoc Tukey's tests”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
Assessment of gait coordination as a measure of motor function in aged mice treated with mitochondrial-targeted antioxidant peptide SS-31
Objective
Assessment of gait coordination as a measure of motor function in aged mice treated with mitochondrial-targeted antioxidant peptide SS-31
Subjects
From papermouse • C57BL/6 • unknown • 24 months old (aged); 3 months old (young controls) • Not specified
Sample count
From paper6
Cohort notes
From paperAged mice compared to young animals as controls
Animal treatment initiation (Not specified)
SS-31 or vehicle administration (2 weeks)
Neurovascular coupling assessment via laser speckle contrast imaging (30 seconds stimulation at 5 Hz)
Open cranial window preparation (Not specified)
Cerebral blood flow (CBF) responses to whisker stimulation measured by laser speckle contrast imaging
From paperData analyzed using one-way ANOVA with post hoc Tukey's tests.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
CBF changes expressed as percent change from baseline
From paperData analyzed using one-way ANOVA with post hoc Tukey's tests.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
NO-mediated component of neurovascular coupling assessed via L-NAME inhibition
From paperData analyzed using one-way ANOVA with post hoc Tukey's tests.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Endothelium-dependent vasodilation to acetylcholine and ATP in isolated MCA vessels
From paperData analyzed using one-way ANOVA with post hoc Tukey's tests.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Cerebral blood flow (CBF) responses to whisker stimulation measured by laser speckle contrast imaging
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
CBF changes expressed as percent change from baseline
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
NO-mediated component of neurovascular coupling assessed via L-NAME inhibition
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Endothelium-dependent vasodilation to acetylcholine and ATP in isolated MCA vessels
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
Preprocessing / cleaning
Data analyzed using one-way ANOVA with post hoc Tukey's tests.
Scoring or quantification
Quantify the primary readouts for this experiment: Cerebral blood flow (CBF) responses to whisker stimulation measured by laser speckle contrast imaging; CBF changes expressed as percent change from baseline; NO-mediated component of neurovascular coupling assessed via L-NAME inhibition; Endothelium-dependent vasodilation to acetylcholine and ATP in isolated MCA vessels.
Normalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Cerebral blood flow (CBF) responses to whisker stimulation measured by laser speckle contrast imaging, CBF changes expressed as percent change from baseline, NO-mediated component of neurovascular coupling assessed via L-NAME inhibition, Endothelium-dependent vasodilation to acetylcholine and ATP in isolated MCA vessels.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Stefano Tarantini et al. (2018). Treatment with the mitochondrial‐targeted antioxidant peptide <scp>SS</scp>‐31 rescues neurovascular coupling responses and cerebrovascular endothelial function and improves cognition in aged mice. Aging Cell
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