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 spatial working memory as a measure of cognitive function in aged mice treated with mitochondrial-targeted antioxidant peptide SS-31
This is a Spatial Working Memory Test protocol using mouse as the model organism. The procedure involves 15 procedural steps, 8 equipment items, 6 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
Core workflow
Animal treatment initiation • Neurovascular coupling assessment via laser speckle contrast imaging • Open cranial window preparation for laser Doppler measurements
Primary readouts
Key equipment and reagents
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24-month-old C57BL/6 mice were treated with SS-31 peptide or vehicle control
Note: Intraperitoneal (i.p.) administration route
“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”
Measure cerebral blood flow (CBF) responses in the whisker barrel cortex evoked by contralateral whisker stimulation using laser speckle contrast imaging
Note: Baseline CBF measured before stimulation; CBF changes recorded relative to baseline
“CBF responses in the whisker barrel cortex elicited by contralateral whisker stimulation were significantly decreased in aged mice compared to young animals”
Prepare open cranial window to allow direct measurement of cerebral blood flow using laser Doppler probe positioned above whisker barrel cortex
Note: Surgical preparation required; allows assessment of NO mediation
“Studies using an open cranial window preparation showed that in young animals administration of the NO synthase inhibitor L-NAME significantly decreased neurovascular coupling responses”
Measure cerebral blood flow using laser Doppler probe during contralateral whisker stimulation (30 seconds, 5 Hz) in absence and presence of L-NAME
Note: Measurements taken in young, aged, and SS-31-treated aged mice; L-NAME administered to assess NO contribution
“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) in the absence and presence of the NO synthase inhibitor L-NAME”
Isolate branches of the middle cerebral artery (MCA) from young, aged, and SS-31-treated aged mice and cannulate for vascular function testing
Note: Pressurized vessel myograph used for measurements
“Dilation of cannulated branches of the middle cerebral artery, isolated from young, aged, and SS-31-treated aged mice”
Measure dilation responses of isolated MCA branches to acetylcholine administration in absence and presence of L-NAME
Note: Pressurized MCAs developed spontaneous myogenic tone (~30%); responses compared across age groups and treatment conditions
“In young vessels, administration of acetylcholine resulted in significant dilation, whereas these responses were significantly attenuated in vessels derived from aging mice”
Measure dilation responses of isolated MCA branches to ATP administration in absence and presence of L-NAME
Note: Responses compared across age groups and treatment conditions
“administration of acetylcholine and ATP resulted in significant dilation, whereas these responses were significantly attenuated in vessels derived from aging mice”
Measure dilation responses of isolated MCA branches to sodium nitroprusside (NO donor) to assess smooth muscle cell function
Note: Responses should not differ significantly among experimental groups if smooth muscle function is preserved
“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 from aged and young animals for in vitro assessment of mitochondrial function
Note: Cells derived from aged animals used for SS-31 treatment studies
“cultured CMVECs derived from aged animals”
Assess mitochondrial ROS (mtROS) production in cultured CMVECs using MitoSox fluorescence method before and after SS-31 treatment
Note: Time course measurements taken; SS-31 concentration 10−5 M
“we assessed the effects of SS-31 on cellular mtROS production in cultured CMVECs derived from aged animals using the MitoSox fluorescence method. 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) as a marker of oxidative phosphorylation and mitochondrial respiration in CMVECs after SS-31 treatment
Note: OCR improvement associated with mtROS reduction
“Attenuation of mtROS production in aged CMVECs was associated with significant improvement in mitochondrial respiration (Figure 2 g), which is compatible with the idea that SS-31 energizes mitochondria”
Assess mRNA expression of genes regulating NO mediation and oxidative stress in cerebral cortex tissue from young, aged, and SS-31-treated aged mice
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)”
Measure markers of oxidative stress in cerebral cortex tissue from experimental groups
Note: Specific markers not detailed in provided text
“Markers of oxidative stress and expression of genes regulating neurovascular coupling responses in the cerebral cortex were assessed”
Conduct behavioral tests to characterize cognitive function and motor coordination in young, aged, and SS-31-treated aged mice
Note: Tests include spatial working memory, motor skill learning, and gait coordination assessments
“Mice were behaviorally evaluated on a battery of tests for characterization of cognitive function and motor coordination”
Analyze all quantitative data using one-way ANOVA with post hoc Tukey's tests for multiple comparisons
Note: Significance threshold p < 0.05; data presented as mean ± SEM
“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 spatial working memory as a measure of cognitive function in aged mice treated with mitochondrial-targeted antioxidant peptide SS-31
Objective
Assessment of spatial working memory as a measure of cognitive 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 (24-month-old) compared to young animals (3-month-old)
Animal treatment initiation (2 weeks)
Neurovascular coupling assessment via laser speckle contrast imaging (30 seconds stimulation at 5 Hz)
Open cranial window preparation for laser Doppler measurements (Not specified)
Laser Doppler measurement of CBF during whisker stimulation (30 seconds stimulation)
Cerebral blood flow (CBF) responses to whisker stimulation measured by laser speckle contrast imaging
From paperOne-way ANOVA with post hoc Tukey's tests for multiple comparisons between young, aged, and SS-31-treated aged groups.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
CBF responses measured by laser Doppler flowmetry
From paperOne-way ANOVA with post hoc Tukey's tests for multiple comparisons between young, aged, and SS-31-treated aged groups.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
NO-mediated neurovascular coupling responses (assessed via L-NAME inhibition)
From paperOne-way ANOVA with post hoc Tukey's tests for multiple comparisons between young, aged, and SS-31-treated aged groups.
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
From paperOne-way ANOVA with post hoc Tukey's tests for multiple comparisons between young, aged, and SS-31-treated aged groups.
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 responses measured by laser Doppler flowmetry
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 neurovascular coupling responses (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
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
One-way ANOVA with post hoc Tukey's tests for multiple comparisons between young, aged, and SS-31-treated aged groups.
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 responses measured by laser Doppler flowmetry; NO-mediated neurovascular coupling responses (assessed via L-NAME inhibition); Endothelium-dependent vasodilation to acetylcholine.
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 responses measured by laser Doppler flowmetry, NO-mediated neurovascular coupling responses (assessed via L-NAME inhibition), Endothelium-dependent vasodilation to acetylcholine.
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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