Nicotinamide mononucleotide (NMN) supplementation rescues cerebromicrovascular endothelial function and neurovascular coupling responses and improves cognitive function in aged mice methods
Aim. Evidence-backed execution summary for Nicotinamide mononucleotide (NMN) supplementation rescues cerebromicrovascular endothelial function and neurovascular coupling responses and improves cognitive function in aged mice methods from Nicotinamide mononucleotide (NMN) supplementation rescues cerebromicrovascular endothelial function and neurovascular coupling responses and improves cognitive function in aged mice.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Quantitative real-time RT-PCR
reagent used in the protocol.
- Use
- A quantitative real time RT-PCR technique was used to analyze mRNA expression of genes relevant for neurovascular impairment and age-related mitochondrial dysfunction in cortical, cerebrovascular, aortic and endothelial samples using validated TaqMan probes (Applied Biosystems) and a Strategen MX3000 platform, as pr...
Results
reagent used in the protocol.
- Use
- CBF responses in the whisker barrel cortex elicited by contralateral whisker stimulation were significantly decreased in aged mice compared to young animals indicating impaired NVC in aging (representative CBF tracings are shown in A, summary data are shown in B) [ ]. We found that a 14-day treatment with NMN signif...
NMN attenuates mitochondrial oxidative stress and improves mitochondrial bioenergetics in aged cerebromicrovascular e...
reagent used in the protocol.
- Use
- To substantiate the endothelial protective effects of NMN in vitro, we assessed the effects of NMN on cellular mtROS production in cultured primary cerebromicrovascular endothelial cells (CMVECs) derived from aged animals using the MitoSox fluorescence method. First we demonstrated that in aged CMVECs NAD + content...
Analysis of gait function
To determine how aging and NMN treatment affect gait coordination, we tested the animals using an automated computer assisted method (CatWalk; Noldus Information Technology Inc. Leesburg, VA) as described [,, ]. Using the CatWalk system the detection of paw print size, pressure and pattern during volunteer running...
- Use
- To determine how aging and NMN treatment affect gait coordination, we tested the animals using an automated computer assisted method (CatWalk; Noldus Information Technology Inc. Leesburg, VA) as described [,, ]. Using the CatWalk system the detection of paw print size, pressure and pattern during volunteer running...
Electron microscopy
Brains (n = 5 animals in each group) were perfusion fixed under 100 mmHg pressure using Karnowsky's method [ ]. Thin sections were obtained with an ultramicrotome, stained with osmium tetroxide, and examined with a transmission electron microscope as previously described [ ]. Mitochondrial volume d...
- Use
- Brains (n = 5 animals in each group) were perfusion fixed under 100 mmHg pressure using Karnowsky's method [ ]. Thin sections were obtained with an ultramicrotome, stained with osmium tetroxide, and examined with a transmission electron microscope as previously described [ ]. Mitochondrial volume d...
NMN attenuates mitochondrial oxidative stress and improves mitochondrial bioenergetics in aged cerebromicrovascular e...
To substantiate the endothelial protective effects of NMN in vitro, we assessed the effects of NMN on cellular mtROS production in cultured primary cerebromicrovascular endothelial cells (CMVECs) derived from aged animals using the MitoSox fluorescence method. First we demonstrated that in aged CMVECs NAD + content...
- Use
- To substantiate the endothelial protective effects of NMN in vitro, we assessed the effects of NMN on cellular mtROS production in cultured primary cerebromicrovascular endothelial cells (CMVECs) derived from aged animals using the MitoSox fluorescence method. First we demonstrated that in aged CMVECs NAD + content...
Restoration of cerebromicrovascular function is associated with improved cognitive function in aged mice treated with...
We also evaluated hippocampal-dependent learning and memory employing the elevated plus-maze. For young mice, transfer latency on day 2 was significantly decreased (by ∼49%) compared to day 1 ( A), indicating an intact learning effect. In contrast, for aged mice the transfer latency on day 1 and day 2 were sim...
- Use
- We also evaluated hippocampal-dependent learning and memory employing the elevated plus-maze. For young mice, transfer latency on day 2 was significantly decreased (by ∼49%) compared to day 1 ( A), indicating an intact learning effect. In contrast, for aged mice the transfer latency on day 1 and day 2 were sim...
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Analysis of gait function
To determine how aging and NMN treatment affect gait coordination, we tested the animals using an automated computer assisted method (CatWalk; Noldus Information Technology Inc. Leesburg, VA) as described [,, ]. Using the CatWalk system the detection of paw print size, pressure and pattern during volunteer running on an illuminated glass walkway by a camera placed under the glass surface provides an automated analysis of gait function and the spatial and temporal aspects of interlimb coordination [, ]. Briefly, animals were trained to cross the walkway and then, in a dark and silent room (<20 lux of illumination), animals were tested in three consecutive runs. Data were averaged across ten runs in which the animal maintained a constant speed across the walkway. After manual identification and labeling of each footprint, spatial and temporal indices of gait were calculated (includin...
Quantitative real-time RT-PCR
A quantitative real time RT-PCR technique was used to analyze mRNA expression of genes relevant for neurovascular impairment and age-related mitochondrial dysfunction in cortical, cerebrovascular, aortic and endothelial samples using validated TaqMan probes (Applied Biosystems) and a Strategen MX3000 platform, as previously reported [,, ]. Targets included the nitric oxide synthases eNOS and nNOS ( Nos3 and Nos1, respectively), arginases ( Arg1, Arg2; which regulate NO synthase activity and were proposed to contribute to endothelial dysfunction in aging [ ]), antioxidant enzymes, enzymes involved in NAD metabolism and nuclear- and mitochondrion-encoded subunits of the electron transport chain. In brief, total RNA was isolated with a Mini RNA Isolation Kit (Zymo Research, Orange, CA) and was reverse transcribed using Superscript III RT (Invitrogen) as described previously [ ]. Quan...
NMN attenuates mitochondrial oxidative stress and improves mitochondrial bioenergetics in aged cerebromicrovascular e...
To substantiate the endothelial protective effects of NMN in vitro, we assessed the effects of NMN on cellular mtROS production in cultured primary cerebromicrovascular endothelial cells (CMVECs) derived from aged animals using the MitoSox fluorescence method. First we demonstrated that in aged CMVECs NAD + content was significantly decreased, whereas it was normalized by treatment with NMN ( A). We found that in aged CMVECs mtROS production was significantly increased as compared to that in CMVECs derived from young animals ( B and C), which associated with a decreased production of NO (DAF fluorescence; D) as well as impaired mitochondrial membrane potential ( E), and decreased ATP levels ( F). NMN treatment attenuated mtROS generation ( B and C), increased NO production ( D), rescued mitochondrial membrane potential ( E) and restored cellular ATP content ( E) in aged CMVECs, elimi...
NMN reverses age-related decline in mitochondrially encoded genes without promoting mitochondrial biogenesis
We could exclude that mitochondrial protective effects of NMN are linked to promotion of mitochondrial biogenesis. Using electron microscopy and unbiased morphometric methods we found that mitochondrial volume density in endothelial cells in the cerebral microcirculation was unaffected by NMN treatment ( A-D). NMN treatment of aged mice also does not affect mtDNA content in cerebral arteries ( E). Findings obtained in cultured CMVECs showing unaltered mtDNA content after NMN treatment extend the in vivo data ( F). Fig. 3 Treatment with NMN rescues age-related downregulation of mitochondrially encoded subunits of the electron transport chain without promoting mitochondrial biogenesis. A-C) Representative electronmicrographs showing mitochondria in cerebromicrovascular endothelial cells in young (A), aged (B) and NMN treated aged mice (C); (arrowheads, mitochondria; nu, nucleus; b...
Measurement outputs
What raw and processed outputs should exist?
To determine how aging and NMN treatment affect gait coordination, we tested the animals using an automated computer assisted method (CatWalk; Noldus Information Technology Inc....
- 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
A quantitative real time RT-PCR technique was used to analyze mRNA expression of genes relevant for neurovascular impairment and age-related mitochondrial dysfunction in cortica...
- 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
To substantiate the endothelial protective effects of NMN in vitro, we assessed the effects of NMN on cellular mtROS production in cultured primary cerebromicrovascular endothe...
- 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 could exclude that mitochondrial protective effects of NMN are linked to promotion of mitochondrial biogenesis. Using electron microscopy and unbiased morphometric methods we...
- 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
CBF responses in the whisker barrel cortex elicited by contralateral whisker stimulation were significantly decreased in aged mice compared to young animals indicating impaired NVC in aging (representative CBF tracings are shown in A, summary data are shown in B) [ ].
from paperScoring or quantification
Quantify the primary readouts for this experiment: To determine how aging and NMN treatment affect gait coordination, we tested the animals using an automated computer assisted method (CatWalk; Noldus Information Technology Inc....; A quantitative real time RT-PCR technique was used to analyze mRNA expression of genes relevant for neurovascular impairment and age-related mitochondrial dysfunction in cortica...; To substantiate the endothelial protective effects of NMN in vitro, we assessed the effects of NMN on cellular mtROS production in cultured primary cerebromicrovascular endothe...; We could exclude that mitochondrial protective effects of NMN are linked to promotion of mitochondrial biogenesis. Using electron microscopy and unbiased morphometric methods we....
from paperStatistical comparison
CBF responses in the whisker barrel cortex elicited by contralateral whisker stimulation were significantly decreased in aged mice compared to young animals indicating impaired...; To substantiate the endothelial protective effects of NMN in vitro, we assessed the effects of NMN on cellular mtROS production in cultured primary cerebromicrovascular endothe...; We could exclude that mitochondrial protective effects of NMN are linked to promotion of mitochondrial biogenesis. Using electron microscopy and unbiased morphometric methods we...; We also evaluated hippocampal-dependent learning and memory employing the elevated plus-maze. For young mice, transfer latency on day 2 was significantly decreased (by ∼49...
from paperReporting output
Report representative outputs alongside summary comparisons for To determine how aging and NMN treatment affect gait coordination, we tested the animals using an automated computer assisted method (CatWalk; Noldus Information Technology Inc...., A quantitative real time RT-PCR technique was used to analyze mRNA expression of genes relevant for neurovascular impairment and age-related mitochondrial dysfunction in cortica..., To substantiate the endothelial protective effects of NMN in vitro, we assessed the effects of NMN on cellular mtROS production in cultured primary cerebromicrovascular endothe..., We could exclude that mitochondrial protective effects of NMN are linked to promotion of mitochondrial biogenesis. Using electron microscopy and unbiased morphometric methods we....
inferred from protocolStructured statistical methods
CBF responses in the whisker barrel cortex elicited by contralateral whisker stimulation were significantly decreased in aged mice compared to young animals indicating impaired...; To substantiate the endothelial protective effects of NMN in vitro, we assessed the effects of NMN on cellular mtROS production in cultured primary cerebromicrovascular endothe...; We could exclude that mitochondrial protective effects of NMN are linked to promotion of mitochondrial biogenesis. Using electron microscopy and unbiased morphometric methods we...; We also evaluated hippocampal-dependent learning and memory employing the elevated plus-maze. For young mice, transfer latency on day 2 was significantly decreased (by ∼49...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (4)
To determine how aging and NMN treatment affect gait coordination, we tested the animals using an automated computer assisted method (CatWalk; Noldus Information Technology Inc. Leesburg, VA) as described [,, ]. Using the CatWalk system the detection of paw print size, pressure and pattern during volunteer running on an illuminated glass walkway by a camera placed under the glass surface provides an automated analysis of gait function and the spatial and temporal aspects of interlimb coordination [, ]. Briefly, animals were trained to cross the walkway and then, in a dark and silent room (<20 lux of illumination), animals were tested in three consecutive runs. Data were averaged across ten runs in which the animal maintained a constant speed across the walkway. After manual identification and labeling of each footprint, spatial and temporal indices of gait were calculated (including swing speed, cadence, regularity index, brake and propulsion phase duration, stand index, duty cycle; size-adjusted base of support, stride length and distance between ipsilateral prints; stride length and stride time coefficient of variance, interlimb couplings).
A quantitative real time RT-PCR technique was used to analyze mRNA expression of genes relevant for neurovascular impairment and age-related mitochondrial dysfunction in cortical, cerebrovascular, aortic and endothelial samples using validated TaqMan probes (Applied Biosystems) and a Strategen MX3000 platform, as previously reported [,, ]. Targets included the nitric oxide synthases eNOS and nNOS ( Nos3 and Nos1, respectively), arginases ( Arg1, Arg2; which regulate NO synthase activity and were proposed to contribute to endothelial dysfunction in aging [ ]), antioxidant enzymes, enzymes involved in NAD metabolism and nuclear- and mitochondrion-encoded subunits of the electron transport chain. In brief, total RNA was isolated with a Mini RNA Isolation Kit (Zymo Research, Orange, CA) and was reverse transcribed using Superscript III RT (Invitrogen) as described previously [ ]. Quantification was performed using the efficiency-corrected ΔΔCq method. The relative quantities of the reference genes Hprt, Ywhaz, B2m, Actb and S18 were determined and a normalization factor was calculated based on the geometric mean for internal normalization. Fidelity of the PCR reaction...
To substantiate the endothelial protective effects of NMN in vitro, we assessed the effects of NMN on cellular mtROS production in cultured primary cerebromicrovascular endothelial cells (CMVECs) derived from aged animals using the MitoSox fluorescence method. First we demonstrated that in aged CMVECs NAD + content was significantly decreased, whereas it was normalized by treatment with NMN ( A). We found that in aged CMVECs mtROS production was significantly increased as compared to that in CMVECs derived from young animals ( B and C), which associated with a decreased production of NO (DAF fluorescence; D) as well as impaired mitochondrial membrane potential ( E), and decreased ATP levels ( F). NMN treatment attenuated mtROS generation ( B and C), increased NO production ( D), rescued mitochondrial membrane potential ( E) and restored cellular ATP content ( E) in aged CMVECs, eliminating the difference between the two age groups. NMN treatment also attenuated increased H 2 O 2 release from aged CMVECs as measured by the Amplex Red assay ( ). Attenuation of mtROS production in NMN-treated aged CMVECs was associated with significant improvement of both basal and maximal mitocho...
We could exclude that mitochondrial protective effects of NMN are linked to promotion of mitochondrial biogenesis. Using electron microscopy and unbiased morphometric methods we found that mitochondrial volume density in endothelial cells in the cerebral microcirculation was unaffected by NMN treatment ( A-D). NMN treatment of aged mice also does not affect mtDNA content in cerebral arteries ( E). Findings obtained in cultured CMVECs showing unaltered mtDNA content after NMN treatment extend the in vivo data ( F). Fig. 3 Treatment with NMN rescues age-related downregulation of mitochondrially encoded subunits of the electron transport chain without promoting mitochondrial biogenesis. A-C) Representative electronmicrographs showing mitochondria in cerebromicrovascular endothelial cells in young (A), aged (B) and NMN treated aged mice (C); (arrowheads, mitochondria; nu, nucleus; bm, basal lamina; AC, astrocyte; scale bar: 500 nm). D) Summary data showing that NMN treatment does not affect mitochondrial volume density in aged cerebromicrovascular endothelial cells. E) NMN treatment of aged mice does affect mtDNA content in cerebral arteries. F) NMN treatment (5 &#...
Machine-readable layer
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