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: Evaluation of motor skill learning ability in aged mice to assess the effects of SS-31 treatment on cognitive and motor function
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24-month-old C57BL/6 mice were treated with SS-31 or vehicle control
Note: Intraperitoneal injection route; dose 10 mg/kg/day
“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 responses in whisker barrel cortex evoked by contralateral whisker stimulation
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”
Surgical preparation of open cranial window for direct measurement of cerebral blood flow

Double Cannula System Pack
Matches: Cannulated middle cerebral artery (MCA) preparation apparatus
Note: Used for measuring CBF responses with laser Doppler probe
“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 above whisker barrel cortex during contralateral whisker stimulation
Note: Measurements taken in absence and presence of L-NAME
“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)”
Apply nitric oxide synthase inhibitor L-NAME to assess NO-mediated neurovascular coupling
Note: Concentration: 3 × 10−4 M; administered during open cranial window preparation
“In untreated aged animals, administration of L-NAME was without effect (Figure 2 a,b). In contrast, in SS-31-treated aged mice L-NAME significantly decreased CBF responses”
Isolate branches of middle cerebral artery and cannulate for vascular function testing

Double Cannula System Pack
Matches: Cannulated middle cerebral artery (MCA) preparation apparatus
Note: Vessels allowed 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 dilation response
Note: Endothelium-dependent vasodilator response; tested in absence and presence of L-NAME
“In young vessels, administration of acetylcholine (Figure 2 c) and ATP (Figure 2 d) resulted in significant dilation, whereas these responses were significantly attenuated in vessels derived from aging mice”
Administer ATP to isolated MCA vessels and measure dilation response
Note: Endothelium-dependent vasodilator response; tested in absence and presence of L-NAME
“In young vessels, administration of acetylcholine (Figure 2 c) and ATP (Figure 2 d) resulted in significant dilation”
Administer sodium nitroprusside (NO donor) to isolated MCA vessels to assess smooth muscle cell dilator capacity
Note: Endothelium-independent vasodilator; used to assess smooth muscle 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 from aged and young animals
Note: Cells used for in vitro assessment of mitochondrial function and ROS production
“we assessed the effects of SS-31 on cellular mtROS production in cultured CMVECs derived from aged animals using the MitoSox fluorescence method”
Measure mitochondrial reactive oxygen species production in cultured CMVECs using MitoSox fluorescence
Note: SS-31 treatment concentration: 10−5 M; measurements taken at multiple time points
“SS-31 treatment elicited significant decreases in mtROS production in aged CMVECs, eliminating the difference between the two age groups (Figure 2 f). 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 as a marker of oxidative phosphorylation in CMVECs
Note: Performed on aged CMVECs treated with SS-31 to assess mitochondrial respiration improvement
“Attenuation of mtROS production in aged CMVECs was associated with significant improvement in mitochondrial respiration (Figure 2 g)”
Measure cortical mRNA expression of genes regulating NO mediation and oxidative stress
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)”
Aged mice (24-month-old) were treated with SS-31 (10 mg/kg/day, i.p.) or vehicle for 2 weeks
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