NAD⁺ depletion drives age-related monocyte hyperinflammation after stroke and is reversed by nicotinamide riboside

NAD⁺ depletion drives age-related monocyte hyperinflammation after stroke and is reversed by nicotinamide riboside | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article NAD⁺ depletion drives age-related monocyte hyperinflammation after stroke and is reversed by nicotinamide riboside Mica Cabrera, Hannah Ennerfelt, Abrar I. Alsaadi, Brent Eastman, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7483662/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Nov, 2025 Read the published version in Journal of Neuroinflammation → Version 1 posted 9 You are reading this latest preprint version Abstract BACKGROUND: Aging exacerbates post-stroke inflammation, contributing to worse neurological outcomes. However, the mechanisms underlying this age-dependent immune dysregulation remain unclear. Because immune cell metabolism critically shapes inflammatory responses, we investigated whether the metabolic state of circulating monocytes, key immune cells that traffic to the ischemic brain, is altered by age after stroke. We further examined whether enhancing cellular NAD⁺ availability with nicotinamide riboside (NR) could mitigate age-associated neuroinflammatory responses and improve stroke outcome. METHODS: Ischemic stroke was induced in young and aged mice using the distal middle cerebral artery occlusion model. We assessed monocyte metabolic profiles via untargeted metabolomics, mitochondrial function assays, multi-analyte cytokine/chemokine profiling, and flow cytometry. Given the contribution of monocyte-derived intestinal macrophages to gut barrier disruption after stroke, we evaluated gut barrier integrity, immune cell composition, and systemic inflammation. Stroke outcomes were also determined by infarct size, motor function, and brain inflammatory status. RESULTS: Our findings show that the availability of the essential energy co-factor, NAD + , is a key age-dependent factor that regulates monocyte and intestinal macrophage immune responses after stroke. Aged monocytes showed decreased NAD + levels and increased inflammatory responses compared to young monocytes. Pretreatment with NR elevated cellular NAD + levels in aged monocytes, normalized intestinal macrophage numbers and activation states, preserved gut barrier integrity, reduced systemic and brain inflammation, and improved stroke outcomes in aged mice. CONCLUSION: These findings highlight the importance of NAD + in mitigating the post-stroke response in aging and the potential of NAD + supplementation as a preventive strategy for patients at risk for cerebrovascular disease. Stroke Aging Inflammation Metabolism NAD+ Nicotinamide riboside Quinolinic acid Monocytes Gut Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION An important consequence of acute ischemic stroke is secondary injury, which occurs as a result of robust and sustained immune cell infiltration into the ischemic brain, driven initially by monocytes and neutrophils. ( 1 ) This inflammatory response is significantly exacerbated in aged individuals compared to young. ( 2 , 3 ) The underlying cause of this aggravated inflammatory response in aging remains unclear. Several studies have established that the metabolic state of immune cells plays a pivotal role in regulating immune responses, as metabolic reprogramming ensures sufficient energy for host defense and tissue homeostasis. ( 4 ) This metabolic homeostasis is disrupted with aging, impairing the ability to mount appropriate immune responses to stimuli. ( 5 ) We hypothesized that energy metabolism could play a critical role in the immune responses to stroke in aged individuals. To study this, we investigated the metabolism of blood monocytes that along with neutrophils, are the first immune cells to infiltrate the ischemic brain where they differentiate into macrophages. In the ischemic area, macrophages clear cellular debris, promote repair, regeneration, and inflammation resolution; processes that depend on robust and adaptable energy metabolism. ( 4 ) The balance between glycolytic and oxidative metabolism can determine whether immune cells adopt an activated, pro-inflammatory state or maintain homeostatic regulatory functions. ( 6 , 7 ) Homeostatic immune cells depend primarily on oxidative phosphorylation and fatty acid β-oxidation to generate ATP. However, upon activation, they shift toward aerobic glycolysis, also known as the Warburg effect. ( 7 , 8 ) This metabolic shift fuels immune activation, with increased glucose utilization driving a pro-inflammatory phenotype in macrophages. ( 8 ) As glycolysis serves as a primary energy source for activated immune cells during inflammation, nicotinamide adenine dinucleotide (NAD + ), a key energy co-factor, is rapidly consumed to sustain this glycolytic activity and can potentially become limiting. ( 9 ) NAD + is critical for maintaining energy homeostasis and modulating immune function. ( 5 ) For example, inhibiting the de novo biosynthetic pathway for NAD + (downstream of the Kynurenine pathway) in aged, but not young macrophages, disrupts oxidative phosphorylation, impairs phagocytic function and the immune response to lipopolysaccharide (LPS). ( 5 ) NAD + levels decline significantly with age, impairing immune cell responses, while increasing cellular NAD + can enhance energy metabolism and reduce inflammation. ( 5 , 10 , 11 ) In addition to trafficking to the ischemic brain, monocytes continually migrate to selected organs where they replenish macrophage populations, including the intestine, lung and spleen. Monocytes that traffic to the intestinal lamina propria function critically in maintaining intestinal homeostasis and gut barrier integrity. ( 12 , 13 ) Following stroke, a burst of increased adrenergic input leads to transient opening of the gut barrier, translocation of microbial components, and hyper-activation of lamina propria macrophages, which in turn, further disrupts the gut epithelial barrier, leading to systemic bacterial dissemination. ( 14 , 15 ) In this study, we investigated the metabolic status of blood monocytes from young and aged mice at several time points after stroke. We also examined monocyte-derived lamina propria macrophages and the integrity of the gut barrier after stroke in young and aged mice. Our results show that NAD + is critically dysregulated in blood monocytes from aged mice and is associated with disrupted immune responses and an accumulation of quinolinic acid (QA), a neurotoxic metabolite of the Kynurenine pathway. Prolonged pretreatment of aged mice with nicotinamide riboside (NR) to elevate cellular NAD⁺ levels significantly improved monocyte energy metabolism, reduced inflammation, rescued the loss of lamina propria macrophages and gut barrier function after stroke. NR supplementation also significantly improved motor function and reduced infarct size following stroke. MATERIALS AND METHODS Animals All experiments and procedures were performed in accordance with the National Institutes of Health (NIH) guidelines, and all protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Case Western Reserve University and Stanford University. All animals were socially housed in a pathogen-free barrier facility environmentally controlled for temperature and humidity, on a 12-hour light–dark cycle, with food and water available ad libitum. C57BL/6J mice were purchased from Jackson laboratories or obtained from the NIH aged rodent colony. Distal middle cerebral artery occlusion surgery and collection of all tissue samples were performed between ZT2 and ZT5 ( zeitgebers time ). This time interval was maintained to ensure consistency of any possible circadian effects and reduce variability. Distal middle cerebral artery occlusion Distal middle cerebral artery occlusion (dMCAo), a permanent coagulation of the middle cerebral artery via electrocoagulation was performed as described previously. ( 16 ) Young (3 − 6 months) and aged (18 − 20 months) C57BL/6J male mice were anesthetized with 3% isoflurane and then with 70% N 2 O + 30% O 2 maintained throughout the surgical procedure, and injected intramuscularly with 0.3 mg/kg buprenorphine (BERLAB0.5-232305; ZooPharm, Laramie, WY). The mice were placed on a heating blanket at 37˚C to maintain body temperature. Eye ointment (Artificial Tears Ophthalmic Lubricant, Akorn, Inc. Lake Forest, IL) was applied to both eyes to prevent dryness during the procedure. The mice were randomized and subjected to either dMCAo or sham surgery which involved thinning of the skull without coagulation of the middle cerebral artery. Nicotinamide Riboside (NR) treatment Mice were administered with either water (vehicle) or 400 mg/kg of nicotinamide riboside (NR; ASB-000114315-101; Niagen; Chromadex, Irvine CA) daily via oral gavage at the same time of day (between ZT 2–3) for 4 weeks. Mice were weighed and values recorded every week and no adverse reactions to NR was observed. Certificates of Analysis provided by the manufacturer and performed on separate lots reported ~ 99% purity of the NR preparation. Immunofluorescence Mice were euthanized in CO 2 chamber and transcardially perfused with 10 mM PBS. Brain and gut tissue were isolated and immersed in 4% paraformaldehyde (PFA; 15714-1L; Electron Microscopy Sciences, Hatfield, PA) overnight and then serially cryopreserved in 15% sucrose and then 30% sucrose solution. 1 cm gut tissue sections were collected from the duodenum, jejunum and ileum and embedded in optimal cutting temperature compound (O.C.T, 23-730-571; Fisher Scientific, Waltham, MA) and then longitudinally sectioned at 10 µm per section. Four sections per mouse were mounted on Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA) and immunostained. For each brain, five 40 µm brain sections were coronally sectioned using a sliding microtome (Microm HM430, Thermo Fisher Scientific, Waltham, MA) at 500 µm intervals between approximately 1.5 mm rostral and 1.5 mm caudal to bregma. Brain tissue slices were placed in freezing media and stored at − 20˚C. Briefly, both brain and gut tissue samples were washed 3× in PBS for 5 minutes and permeabilized in 0.2% Triton-X100 in PBS for 20 minutes at room temperature. Samples were then washed in 10 mM PBS and incubated for 1 hour in blocking buffer (0.2% Triton-X100 supplemented with 10% normal donkey serum in 10 mM PBS) and then in primary antibody (see appendix for list of antibodies) overnight at 4˚C. Next, the samples were washed 3× in PBS for 5 minutes and then incubated in secondary antibody (see appendix for the list of antibodies) for 2 hours. Samples were washed 3× in 10 mM PBS for 5 minutes and then incubated in Hoechst stain for 10 minutes in 10 mM PBS. Samples were washed in 10 mM PBS and then mounted with Prolong™ Gold antifade reagent (P36930; Invitrogen, Eugene, OR), and tissue samples mounted onto Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA). Gut tissue sections were acquired on Zeiss 780 LSM confocal microscope (Carl Zeiss, Thornwood, NY) using either 20×/0.45 NA dry objective lens or 40×/0.95 NA oil immersion objective lens. Mean fluorescence intensity was quantified using ImageJ ( http://imagej.nih.gov/ij ). Quantification of stroke infarct area Measurement of infarct area was carried out by an investigator blinded to treatment. 40 µm brain slices were immunostained with MAP2 to delineate infarcted area which had a relatively high fluorescent signal due to dead and dying neurons, and IBA1 and CD68 to quantify microglia (see appendix for antibody details). Images were acquired using a 10× objective lens with a 0.40 NA on a Leica SP8 confocal microscope (Leica Microsystems Inc, Deerfield, IL). Infarct area was quantified using ImageJ ( http://imagej.nih.gov/ij ). Blood monocyte isolation Blood was collected by transcardiac puncture from mice with 0.25 mM EDTA (anticoagulant) and mixed with ACK lysing buffer (A1049201; Thermo Fisher Scientific, Waltham, MA). Samples were incubated on ice for 15 minutes and centrifuged at 300 ×g for 6 minutes. Samples were then resuspended in 10 mM PBS containing 2% FBS and 1 mM EDTA. EasySep™ mouse monocyte isolation kit (19861; STEMCELL Technologies, Vancouver, BC, Canada) was used to isolate and enrich blood monocytes by immunomagnetic negative selection. Chemokine and cytokine multiplex assay – Luminex Briefly, blood was collected by transcardiac puncture from mice with 0.25 mM EDTA, and either centrifuged at 2000 ×g at 4˚C for 10 minutes and blood plasma collected, or blood monocytes isolated using the EasySep™ mouse monocyte isolation kit (19861; STEMCELL Technologies, Vancouver, BC, Canada). Mice were perfused with 10 mM PBS and brain tissue was collected from each mouse. Cells and tissue samples were lysed with cell lysis buffer (RIPA buffer; 89901; Thermo Fisher Scientific, Waltham, MA) and protein quantified by BCA (A55864, Thermo Fisher Scientific, Waltham, MA). Equal concentrations of samples were stored at − 80°C and cytokine analysis was carried out at the Human Immune Monitoring Core (Stanford University, CA) using magnetic bead-based multiplex Luminex assays (LXSAMSM; R&D Systems, Inc., Minneapolis, MN). Plates were read using a Luminex LabMap200 instrument. Mean fluorescence intensity (MFI) was averaged over duplicate wells for each cytokine per sample on each plate. Endotoxin Lipopolysaccharide Assay Briefly, blood was collected by transcardiac puncture from mice with 0.25 mM EDTA. Samples were centrifuged at room temperature for 10 minutes at 2500 rpm. Blood plasma was removed and tested for bacterial lipopolysaccharide (LPS) using the LPS ELISA kit (MBS261904; Mouse Lipopolysaccharide ELISA Kit; MyBioSource, Inc. San Diego, CA). FITC-Dextran Mice were fasted for 4 hours with ad libitum access to water at the beginning of the light cycle at ZT 0, 24 hours post stroke. Blood samples were collected via tail snip, centrifuged at 5000 rpm for 10 minutes at room temperature and kept on ice. Then 80 mg/mL of 4 kDa FITC-dextran (46944-500MG-F; Millipore Sigma, St. Louis, MO) diluted in sterile 10 mM PBS (per mouse) was administered via oral gavage. 4 hours post-administration, blood samples were collected via transcardiac puncture with 0.25 mM EDTA, centrifuged at 5000 rpm at room temperature for 10 minutes. Blood plasma was removed and the levels of FITC-dextran measured with a fluorescent plate reader. Real-Time Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) Blood monocytes were isolated using the EasySep™ mouse monocyte isolation kit (19861; STEMCELL Technologies, Vancouver, BC, Canada) from each mouse as described previously and seeded in a seahorse XFe24 cell culture microplate (102340-100; Agilent, Santa Clara, CA) and allowed to adhere for 1 hour at 5% CO 2 at 37°C. Cells were washed twice with Agilent seahorse XF Media (103680-100; Agilent, Santa Clara, CA) supplemented with 1 mM pyruvate (Gibco™ 11360070; Fisher Scientific, Waltham, MA), 2 mM L-glutamine (400-106-100; GeminiBio, West Sacramento, CA) and 2 mM D-glucose (G7021-1KG; Sigma-Aldrich, St. Louis, MO) in a final volume of 500 µL. Cells were then incubated in a 0% CO 2 chamber at 37°C for 1 hour before being placed into a seahorse XFe24 analyzer (US421134; Agilent, Santa Clara, CA). Cells were treated with 2.5 µM oligomycin, 2 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and 0.5 µM rotenone/antimycin (103015-100; Seahorse XF Cell Mito Stress Test Kit; Agilent, Santa Clara, CA). 1 µM of Hoescht (H3570; Invitrogen, Waltham, MA) was injected at the end of the assay for cell counting. A total of three OCR and pH measurements were taken after each compound was administered from which basal respiration, maximal respiration, ATP production, spare respiratory capacity and extracellular acidification rates were calculated. All values were normalized by Hoescht-positive cell counting in the Cytation1 imaging platform (20081913; Agilent, Santa Clara, CA). Flow cytometry sample preparation The small intestine (SI) was removed following perfusion and placed in cold 10 mM PBS. The SI was then cut into segments that underwent cleaning by flushing with cold sterile 10 mM PBS. Gut segments were then cut open and into 2 cm pieces before being placed in dissociation buffer: HBSS (without calcium/magnesium) containing 1% 0.5M EDTA and 1% HEPES and placed on a shaker at 180 rpm at 37˚C for 40 minutes. Following incubation, samples were poured through a 70 µm strainer and the flow-through was discarded. The tissue on the strainer was then moved to a new tube and dissociated mechanically with scissors for ~ 30 seconds. Samples were resuspended in HBSS (without calcium/magnesium) containing 2% FBS, 0.5 mg/mL collagenase E and 0.5 mg/mL DNase I and placed on the shaker at 180 rpm at 37˚C for 45 minutes. Samples were again filtered through a 70 µm strainer and washed with 10 mL of cold HBSS (without calcium/magnesium). Samples were then centrifuged at 1400 rpm for 8 minutes at 4˚C and the supernatant discarded. Samples were washed again with 5 mL of HBSS (without calcium/magnesium) and centrifuged at 1400 rpm for 8 minutes at 4˚C. Supernatants were discarded and cell pellets resuspended in FACS buffer before staining for flow cytometry. Flow cytometry procedure Samples were transferred to a 96-well v bottom plate (277143; Thermo Fisher Scientific, Waltham, MA), and washed 1× with FACS buffer (500 mL of 10 mM PBS containing 1 mL of 0.5M EDTA, and 1 g of Bovine Serum Albumin; BSA). Gut cells were stained at a dilution of 1:200 with CD11b (BV421), Ly6G (BV605), Ly6C (V450), CD45 (AF 700), MHCII (PerCP-efluor710), EGR2 (PE-Cy7), Live Dead Aqua, and with 1:1000 FC block in in FACS buffer for 25 minutes at 4°C. The cell pellets were centrifuged at 500 ×g for 3 minutes and then washed with FACS buffer then resuspended in 200 µL of FACS buffer and run on Cytek Aurora flow cytometry instrument (Fremont, CA). Analysis was performed using FlowJo software v10. Metabolomics Water Soluble Metabolites: A solvent mixture of 80:20 methanol/water (500 µL) containing seven internal standards was used to resuspend the pellets that were composed of blood monocytes isolated from mice. Cell suspensions were vortexed for 30 seconds, sonicated in a water bath (30 seconds sonication, 30 seconds on ice, repeated 3 times), vortexed for 30 seconds and incubated for 2 hours at − 20°C to allow for protein precipitation. The supernatant was collected after centrifugation at 10,000 rpm for 10 minutes at 4°C and then evaporated to dryness under nitrogen. The dry extracts were then reconstituted with 100 µL of 50:50 methanol/water before analysis. Supernatants were centrifuged at 16,000 ×g for 20 minutes to remove any residual debris before analysis. Extracts were analyzed within 36 hours by liquid chromatography coupled to a mass spectrometer (LC − MS). The LC–MS method involved hydrophilic interaction chromatography (HILIC) coupled to the Q Exactive PLUS mass spectrometer (Thermo Fisher Scientific, Waltham, MA) as previously described. ( 17 ) The LC separation was performed on a XBridge BEH Amide column (150 mm 3 2.1 mm, 2.5 mm particle size; Waters, Milford, MA). Solvent A was 95%:5% H 2 O: acetonitrile with 20 mM ammonium bicarbonate, and solvent B was acetonitrile. The gradient was 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B; 30 min, 85% B. Other LC parameters were: flow rate, 150 mL/min; column temperature, 25˚C; injection volume, 10 µL; and autosampler temperature, 5˚C. The mass spectrometer was operated in both negative and positive ion mode for the detection of metabolites. Other MS parameters were: resolution of 140,000 at m/z 200, automatic gain control (AGC) target at 3 6 , maximum injection time of 30 ms and scan range of m/z 75-1000. Raw LC/MS data were converted to mzXML format using the command line “msconvert” utility. ( 18 ) Data was processed and analyzed using MAVEN software (Princeton University, Princeton, NJ). Significant metabolites were formally identified by matching fragmentation spectra to public spectral libraries or by matching retention time and fragmentation spectra to authentic standards when possible. Determination of the NAD + , Kynurenine pathway and related metabolites Analyte standards for NAD + metabolites The following standards (see appendix for list of reagents catalogue numbers) Nicotinamide (NAM); Nicotinamide mononucleotide (NMN); nicotinamide adenine dinucleotide (NAD + ); nicotinic acid mononucleotide (NaMN); nicotinic acid adenine dinucleotide (NaAD); adenosine monophosphate (AMP); adenosine diphosphate ribose (ADPr); NAM-d4; AMP 15 N 5 were purchased from either Medical Isotopes, Inc, or Sigma-Aldrich (St. Louis, MO). For NaMN-d4 standard, the M + 4 D/molecule was significantly less than the M + 3 isotopomer and significantly less than the unlabeled molecular ion peak at identical concentrations in solution. Therefore, the actual incorporation of deuterium in NaMN-d4 was determined by mass spectrometry analysis where M + 3 D/molecule was the major isotopomer and NaMN-d3 was used as an internal standard for the quantification. Analyte standards for the Kynurenine pathway metabolites The following standards (see appendix for list of reagents catalogue numbers) L-Tryptophan (TRP); Kynurenine (KYN); 3 Hydroxy DL-Kynurenine (3HK); Kynurenic acid (KA); 3 Hydroxyanthranilic acid (3HANA); Quinolinic acid (QA); TRP-d5; and KA-d5 – were purchased from Sigma-Aldrich (St. Louis, MO). KYN-d4; 3HK-d3; 3HANA-d3 and QA-d3 were purchased from Medical Isotopes, Inc. Calibration curve preparation Individual analytes and internal standard (IS) primary stock solutions include: 10 mM (NAM; NMN; NaMN, NaAD; ADPr): 20 mM (NAD+; AMP); 2 mM (NAD + -d4); 5 mM (NaMN-d3) and 10 mM (NAM-d4; NMN-d4; AMP-15N5), were prepared individually in water corrected for lot purity and salt. For the Kynurenine pathway metabolites, individual analytes and corresponding internal standards (IS) 10 mM primary stock solutions were prepared separately in water containing 0.1% formic acid and 0.02% ascorbic acid corrected for lot purity. Intermediate stock solutions were prepared by mixing individual stock solutions of each analyte followed by dilution. These intermediate stock solutions were serially diluted with water to obtain a series of standard spiking solutions, which were used to generate the calibration curve. Calibration curves were prepared by spiking 10 µL of each standard working solution into 25 µL of homogenization buffer (0.5N perchloric acid in water) followed by addition of 10 µL internal standard solutions. For NAD + metabolites: 4 µM for NAD + -d4 and AMP-15N5; 2 µM for NAM-d4; NMN-d4 and NaMN-d3. A calibration curve was prepared fresh with each set of samples. Using 25 µL aliquots, the calibration curve range for NAD + and AMP was 0.016–160 µM; for NAM; NMN; NaMN; NaAD; and ADPr was 0.008–80 µM. Calibration curves for the Kynurenine pathway metabolites were prepared likewise. Final concentration of IS in the sample was 4000nM for TRP-d5; 400nM for QA-d3, 3HANA-d3, 3HK-d3 and 200nM for KYN-d4 and KA-d5. A calibration curve was prepared fresh with each set of samples. Using 25 µL aliquot the calibration curve range for TRP 4 nM – 20 uM; KYN 0.4–4000 nM; KA 0.4–1000 nM; 3HK 2–4000 nM; 3HANA and QA 10–4000 nM. Sample collection and extraction procedure The extraction procedure was modified from that of Liang X. et al. ( 19 ) Samples isolated from mice include blood monocytes, infarcted cortex, and gut. Samples were weighed and placed into lysing matrix D vials containing 1.4 mm zirconium-silicate spheres (1169130-CF; MP Biomedicals, Solon, OH); snap-frozen with liquid nitrogen and kept frozen at -80°C until analysis. Before LC-MS/MS analysis, samples were removed from the − 80°C freezer and placed on ice. A cold solution of 0.5N perchloric acid was added to frozen samples in the following amounts: Monocytes – 100 µL; cortex and gut – 200 µL. Samples were homogenized using Bead Mill-4 (15-340-164; Thermo Fisher Scientific, Waltham, MA) twice at 30 seconds at a speed of 5 m/s and then centrifuged. 25 µL of supernatant was used for analysis. 10 µL internal standard solution and 10 µL water solution was added to 25 µL aliquots of supernatant and vortexed. For the NAD + metabolites, samples were diluted with 60 µL 20mM ammonium formate buffer with 0.1% formic acid in water, vortexed, centrifuged, transferred to injection vial and analyzed by LC-MS/MS. For the Kynurenine pathway metabolites, samples were diluted with 40 µL 20mM ammonium formate buffer with 0.1% formic acid in water, vortexed, centrifuged, transferred to injection vial and analyzed by LC-MS/MS. LC-MS/MS Since some of the analytes namely NMN/NaMN and NAD + /NaAD have a mass difference of 1 amu, chromatographic separation is critical. At least two selective reaction monitoring (SRM) transitions – one quantifier and one qualifier – were carefully selected for each analyte. Distinctive qualifier to quantifier ion intensity ratios and retention times were essential to authenticate the target analytes. All analyses were carried out by positive electrospray LC-MS/MS using a Waters Acquity I-class LC system with Waters Xevo TQ-XS triple quadrupole mass spectrometer (RRID:SCR_018510). Chromatographic conditions include: Atlantis® T3, 2.1x100 mm, 3 µm particle size column (186003718; Waters Corp. Milford, MA) was operated at 30°C with a flow rate of 0.2 mL/min. Mobile phases consisted of A: 20 mM ammonium formate/0.1% formic acid in water and B: 20 mM ammonium formate/0.1% formic acid in acetonitrile/methanol/water (45:45:10). Elution profile include: initial hold at 0% B for 3 minutes, followed by a gradient of 0%-30% in 6 minutes, then 30%-95% in 2 minutes, hold at 95% for 1 minute; total run time was 15 minutes. Injection volume was 10 µL. SRM was used for quantification. The mass transitions were as follows: NAM: m/z 122.58 → m/z 105.42 (quantifier); m/z 122.58 → m/z 79.35 (qualifier); NMN: m/z 335.03 → m/z 122.49 (quantifier); m/z 335.03 → m/z 96.37 (qualifier); NaMN: m/z 336.03 → m/z 123.46 (quantifier); m/z 336.03 → m/z 96.36 (qualifier); ADPr: m/z 560.03 → m/z 135.59 (quantifier) and m/z 560.03 → m/z 347.96 (qualifier); NAD + : m/z 664.16 → m/z 135.55 (quantifier) and m/z 664.16 → m/z 428.09 (qualifier); NaAD: m/z 665.16 → m/z 135.56(quantifier) and m/z 665.16 → m/z 428.04 (qualifier); AMP: m/z 347.93 → m/z 135.56 (quantifier) and m/z 347.93 → m/z 118.49 (qualifier); NAM-d4: m/z 126.58 → m/z 83.16 ; NMN-d4: m/z 339.03 → m/z 126.53; NaMN-d3: m/z 338.5 → m/z 126.54; NAD + -d4: m/z 668.16 → m/z 135.56; and AMP 15N5: m/z 353.19 → m/z 140.60. Dwell time was 25 ms. For the Kynurenine pathway metabolites, the mass transitions were as follows: TRP: m/z 204.776 → m/z 131.594 (quantifier); m/z 204.776 → m/z 117.525 (qualifier); KYN: m/z 208.72 → m/z 93.427 (quantifier); m/z 208.72 → m/z 117.516 (qualifier); 3HK: m/z 224.776 → m/z 109.528 (quantifier); m/z 224.776 → m/z 165.654 (qualifier); KA: m/z 189.656 → m/z 143.548 (quantifier) and m/z 189.656 → m/z 115.546 (qualifier); 3HANA: m/z 153.584 → m/z 107.504 (quantifier) and m/z 153.584 → m/z 135.576 (qualifier); QA: m/z 167.584 → m/z 105.479 (quantifier) and m/z 167.584 → m/z 77.314 (qualifier); TRP-d5: m/z 209.84 → m/z 149.711 ; KYN-d4: m/z 212.84 → m/z 97.492; 3HK-d3: m/z 227.784 → m/z 110.708; KA-d5: m/z 194.72 → m/z 93.245; and 3HANA-d3: m/z 156.648 → m/z 138.591; QA-d3 m/z 170.648 → m/z 108.497. Dwell time was also 25 ms. Quantification Quantitative analysis was done with TargetLynx quantification software (Waters Corp. Milford, MA) using an internal standard approach. NAM-d4; NMN-d4; NaMN-d3 were the internal standards used for quantification of NAM; NMN; NaMN, respectively; NAD + -d4 for NAD + and NaAD; and AMP15N5 for AMP and ADPr. TRP-d5; KYN-d4; 3HK-d3; KA-d5; 3HANA-d3 and QA-d3 were the internal standards for quantification of TRP; KYN; 3HK; KA; 3HANA and QA, respectively. Calibration curves were linear (R > 0.98) over the concentration range using a weighting factor of 1/X2 where X is the concentration. The back-calculated standard concentrations were ± 15% from nominal values, and ± 20% at the lower limit of quantitation (LLOQ). Rotarod The rotarod test was used to evaluate motor function and coordination. Mice were pretrained on the rotarod for one week before stroke was induced, with 3 trials separated by 15-minute inter-trial intervals. The mice were kept on the rotarod until they learned to stay on for 300 sec. Training began with a constant low-speed rotation (5 rpm) for 5 minutes on the first day of training. On all subsequent days, each mouse was placed on the rod with the rotation speed increasing from 4–40 rpm (ramping) over 5 minutes. Testing was performed on the day before stroke and the day after stroke and involved placing the mice on the rotarod for a maximum of 300 sec and measuring the length of time they remained on the rotarod with ramping, and reverse ramping speeds before falling on the soft pad below. The rotarod (Ugo Basile, Italy) consisted of a striated rod (consisting of 5 lanes with diameter: 3 cm; rod width: 5.8 cm; fall height: 16 cm). Statistical analysis For all experiments, mice were randomly assigned to either sham, stroke, vehicle-treated or NR-treated groups. Investigators were blinded to group allocation during data collection and analysis. No power analysis was performed to determine sample sizes; prior literature using similar experimental paradigms that yielded interpretable results and the laboratory’s previous experience was used. ( 14 ) All analyses were performed using either GraphPad Prism software version 10 (GraphPad Software, LaJolla, CA), R statistical package software version R 4.4.0, or Metaboanalyst 6.0. Unless otherwise specified, values represent the means ± SEM of at least three independent experiments. Data were analyzed by unpaired Student’s t -test with Welch’s correction, one-way ANOVA, two-way ANOVA or repeated measures two-way ANOVA with Tukey’s posthoc or Bonferroni multiple comparison test to determine significance. Normality of the distribution of the data was tested with Kolmogorov–Smirnov normality tests using the column statistics function of GraphPad Software. Metabolomics data analysis was carried out using MetaboAnalyst 6.0. Metabolomic data were log-transformed and scaled according to the auto-scaling feature (mean-centered and divided by the standard deviation of each variable). Metabolites that were significantly different by ANOVA (with FDR correction) were subjected to hierarchical clustering analysis using the Euclidean distance measure and Ward clustering algorithm. Differentially expressed metabolites underwent pathway-based enrichment analysis using 84 metabolite sets based on KEGG metabolic pathways in MetaboAnalyst 6.0. RESULTS Aging impairs blood monocyte energy metabolism after stroke. To evaluate acute age-dependent changes in the energy metabolism of blood monocytes in response to stroke, we subjected young (3–6 month) and aged (18–20 month) male mice to distal middle cerebral artery occlusion (dMCAo), and isolated blood monocytes at 4.5 hours, 24 hours and 72 hours post stroke (Fig. 1 A). We assessed the metabolic function of blood monocytes using a Mitostress test that induces mitochondrial stress to measure basal respiration (BR) and ATP production, indicators of oxidative phosphorylation, and extracellular acidification rates (ECAR), an indicator of glycolytic activity. At 4.5 hours after stroke, there were no differences in BR, ATP production or ECAR in the blood monocytes from young stroke compared to sham mice (Fig. 1 B and 1 C). Significant increases in BR and ATP production but no changes in ECAR were observed in aged stroked compared to sham mice (Fig. 1 B and 1 C). At 24 hours post stroke, BR was moderately reduced and ATP production and ECAR were significantly lower in young stroked mice compared to sham (Fig. 1 B and 1 C). In aged stroked mice, while previously increased at 4.5 hours, BR, ATP production and ECAR were significantly reduced (Fig. 1 B and 1 C), suggesting metabolic exhaustion in the blood monocytes compared to aged sham. By 72 hours after stroke, all metabolic parameters rebounded to sham levels in young mice, while in aged stroked mice, the decrease in BR and ATP production persisted with ECAR unchanged, suggesting that the blood monocytes remained metabolically depleted (Fig. 1 B and 1 C). Together, these results indicate that blood monocytes in young stroked mice experience a brief depletion of metabolic reserves under stress at 24 hours post stroke, followed by recovery. In contrast, blood monocytes in aged stroked mice initially exhibit an elevated metabolic response, engaging both oxidative phosphorylation and glycolysis to fuel their activation followed by a prolonged phase of metabolic exhaustion. Age-dependent changes in blood monocyte NAD + and quinolinic acid levels following stroke. To further explore the metabolic response of blood monocytes following stroke, we conducted metabolomic analyses to identify stroke-induced alterations in metabolic pathways and assess how these changes are affected by age (Figure S1 –S3). Enrichment pathway analyses revealed changes in the nicotinamide adenine dinucleotide (NAD + ) metabolic pathway at all time-points in aged stroked mice that were absent in young mice (Fig. 1 D and Figure S4A–C). NAD + plays a critical role in specifying immune responses in aged mice, ( 5 , 20 ) and is synthesized via three major pathways: the Preiss-Handler, Salvage and the Kynurenine pathways (KP), also known as the NAD + de novo biosynthetic pathway. ( 21 ) The KP is a significant source of NAD + during an immune challenge, ( 5 ) and has been shown to be activated in stroke patients. ( 22 , 23 ) Activation of the KP has been linked to stroke severity, with higher kynurenine/tryptophan ratios observed in the blood plasma of patients who died from stroke. ( 22 ) Quinolinic acid (QA), a metabolite of the KP, which is converted to the NAD + precursor nicotinamide adenine mononucleotide (NaMN), is a neurotoxic molecule that binds N -methyl- D -aspartate (NMDA) receptors to induce excitotoxicity in neurons, and is a potent mediator of inflammation. ( 24 , 25 ) Therefore, we investigated if stroke regulates NAD + and QA levels in blood monocytes in an age-dependent manner. At 4.5 hours after stroke, we found no differences in NAD + levels in the blood monocytes from young mice (Fig. 1 E), however, NAD + was significantly increased in blood monocytes from aged stroked mice compared to sham (Fig. 1 F). At 24 hours post-stroke, NAD + levels in blood monocytes from young mice were significantly elevated (Fig. 1 E). In contrast, while aged mice exhibited increased NAD + at 4.5 hours post-stroke, levels were significantly reduced by 24 hours, indicating NAD + depletion (Fig. 1 F). At 72 hours post stroke, NAD + levels of blood monocytes from young and aged stroked mice had rebounded to sham levels (Fig. 1 E and 1 F). QA levels were increased in blood monocytes from young and aged mice at 4.5 hours post stroke (Fig. 1 E and 1 F). The increase in QA persisted at 24 hours post stroke in both young and aged mice (Fig. 1 E and 1 F). However, by 72 hours post stroke, QA levels rebounded to sham levels in young mice but remained elevated in aged stroked mice (Fig. 1 E and 1 F). Overall, these results suggest that the Kynurenine pathway (KP) is activated to boost NAD⁺ production in blood monocytes after stroke in both young and aged mice. However, in aged mice, the conversion of quinolinic acid (QA) to the NAD⁺ precursor NaMN appears to be impaired, resulting in QA accumulation (Figure S4D). Replenishing NAD + levels increases oxygen consumption rates in blood monocytes from aged mice after stroke. Nicotinamide riboside (NR) increases NAD + levels, improves metabolism, reduces inflammation in human monocyte-derived macrophages, and attenuates neuronal loss in a mouse model of acute ischemia. ( 10 , 11 , 26 – 28 ) These findings are based on studies in young mouse models and not aged mice, which are more representative of human stroke populations. Here, we tested whether NAD + regulates blood monocyte function during stroke in an age-dependent manner. Therefore, we administered either vehicle (water) or NR to young and aged mice via oral gavage every day for 4 weeks and then subjected the mice to stroke (Figure S5A). Blood monocytes were isolated at 24 hours post stroke, given that we observed the most distinct age-related metabolic differences at this time-point. A Mitostress test revealed no differences in basal respiration (BR) and ATP production between vehicle and NR-treated young stroked mice, however, there was a significant decrease in ECAR, suggesting reduced glycolytic rates (Fig. 2 A). In aged stroked mice, BR, ATP production and ECAR were all significantly increased with NR treatment (Fig. 2 A), consistent with improved oxidative phosphorylation and glycolysis. To determine if NR increased NAD + levels via the KP, we measured NaMN as a readout for increased flux through the Priess-Handler and KP, as demonstrated in previous studies. ( 26 , 29 ) This approach was chosen given the limitations of directly measuring NAD⁺ in small sample sets such as blood monocytes. In the blood monocytes from young mice, there were no differences in NaMN levels between vehicle and NR-treated stroked mice (Fig. 2 B). However, in the aged stroked mice, NaMN levels were significantly increased (Fig. 2 B), suggesting NR may have improved QA conversion to NaMN. To confirm that QA was converted to NaMN, we measured QA levels. Indeed, NR modestly decreased QA levels in blood monocytes from young stroked mice, and significantly reduced QA levels in aged stroked mice compared to vehicle-treated mice (Fig. 2 B). Together, these results suggest that prolonged pretreatment with NR decreased the stroke-induced increase in QA levels in blood monocytes from aged mice. Nicotinamide riboside reduces the inflammatory response of blood monocytes after stroke in aged mice. To determine if the inflammatory response of blood monocytes correlates with changes in their metabolic state, we measured immune factors in isolated blood monocytes using a chemokine and cytokine multiplex assay. At 4.5 hours post stroke, macrophage chemoattractant protein/C-C ligand 7 (MCP3/CCL7), a chemokine that plays a role in monocyte/macrophage/neutrophil recruitment through activation of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway to induce cell proliferation and survival, ( 30 ) was the only immune factor increased in the blood monocytes from young stroked mice compared to sham including IL-7Rα (Fig. 2 C). In aged stroked mice, several immune factors were increased, including MCP3/CCL7, indicating increased monocyte cell proliferation, monocyte differentiation, and migration. At 24 hours post stroke, in blood monocytes from young stroked mice, we detected a decrease in macrophage colony-stimulating factor (MCSF1), a cytokine required for monocytic differentiation into macrophages. ( 31 ) However, in aged stroked mice, there was an increase in the expression of several immune factors that play roles in monocytic activation, proliferation, migration and differentiation (Fig. 2 C). By 72 hours post stroke, we detected no differences in young mice, while the increase in IL-7Rα persisted in aged stroked mice (Fig. 2 C). IL-7Rα expression promotes antimicrobial activity in monocytes, ( 32 ) indicating a potential response to elevated bacterial load resulting from increased gut barrier permeability after stroke. ( 15 , 33 ) Next, we measured immune factors in the blood plasma as indicators of systemic, whole-body inflammation. We observed changes in inflammation over time and age-related differences that were exacerbated by stroke (Figure S5B–D). A majority of these immune factors converge on the JAK/STAT pathway to mediate immune cell proliferation, activation, differentiation and migration as well as downstream signaling to promote inflammation. While young mice exhibited only a mild systemic inflammatory response to stroke within 72 hours, aged mice showed an initially modest response that intensified by 24 hours post stroke before subsiding by 72 hours (Figure S5B–D). Next, to determine whether NAD + replenishment from pretreatment with NR could influence stroke-induced inflammation, we measured immune factors in blood monocytes from vehicle and NR-treated young and aged mice 24 hours after stroke. No significant differences in any immune factors were observed due to NR treatment in young stroked mice (Fig. 2 D). However, NR reduced levels of several inflammatory factors that were increased in the blood monocytes from aged stroked mice (Fig. 2 E). We also investigated the effect of NR on systemic inflammation by measuring the levels of immune factors in the blood plasma from sham, vehicle and NR-treated young and aged stroked mice. We observed no differences between all groups of young mice (Figure S6B), however there was a significant reduction in stroke-induced inflammation in aged stroked mice following NR treatment (Figure S6C). Altogether, these results show a marginal effect of NR on blood monocyte and systemic inflammation in young mice, while in aged mice, NR has significant suppressive effect on stroke-mediated inflammatory responses. Nicotinamide riboside alters stroke-induced inflammation in the ipsilateral and contralateral cortex. Next, we tested whether NR might also regulate inflammation in the brain of stroked mice. We isolated the ipsilateral infarct core and a corresponding area in the contralateral brain at 24 hours post stroke in vehicle and NR-treated young and aged stroked mice and performed a chemokine and cytokine multiplex assay. We determined that NR reduced all detected immune factors in the ipsilateral cortex of both young and aged stroked mice, with the exception of IL-1α and MDC/CCL22 in the aged stroked mice (Fig. 3 A), both of which were increased and play a role in immune cell recruitment and regulation. In the contralateral cortex of young stroked mice, we observed no effect of NR on the expression of immune factors (Fig. 3 B). In aged mice, however, several cytokines and chemokines were decreased while others were elevated, indicating an altered inflammatory response in the contralateral cortex (Fig. 3 B). We then assessed the effect of NR pretreatment on brain NAD + levels. NR increased NAD⁺ levels in the contralateral cortex of young mice but did not affect NAD⁺ levels in the ipsilateral cortex, while in aged stroked mice, NR significantly elevated NAD + levels in both hemispheres (Fig. 3 C and 3 D). These results suggest that NR alters the immune response in both the ipsilateral and contralateral hemispheres in aged mice following stroke, and these changes may be due to the levels of NAD + availability in the brain. Nicotinamide riboside changes the composition of macrophage cell populations in the gut. Monocytes traffic to the gut lamina propria where they critically maintain the gut barrier, ( 12 ) which becomes compromised following stroke. ( 15 , 33 ) Therefore, we investigated effects of NR pretreatment on lamina propria macrophage cell composition using flow cytometry (Figure S6A). The number of gut monocyte-derived macrophages (MDMs) identified as CD45 + CD11b + , Ly6C + , F4/80 + cells (Figure S6A) and tissue resident macrophages (TRMs), identified as CD45 + CD11b + , Ly6C + , CD62L + cells (Figure S6A) did not change with stroke at 24 hours but was significantly increased with NR treatment in young mice (Fig. 4 A). In aged mice, stroke significantly reduced the populations of gut MDMs and TRMs, however this was completely prevented by NR pretreatment (Fig. 4 A), suggesting a beneficial effect of increased cellular NAD + in gut macrophages and/or broadly across the gut epithelium. Next, we investigated the populations of P1 (newly infiltrating inflammatory monocytes – Ly6C Hi MHCII Low ), P2 (inflammatory monocytes – Ly6C Int MHCII Hi ) and P3 (new tissue resident macrophages – Ly6C low MHCII Hi ) cells in the gut that consists of both MDMs and TRMs. ( 34 ) The populations of P1, P2 and P3 macrophages were decreased with stroke in aged but not young stroked mice, and NR prevented loss of the P1 populations in aged stroked mice (Fig. 4 B). To further characterize the polarization states of gut macrophages, we assessed the number of cells positive for CD71 (marker for proliferating macrophages and required for phagocytosis), ( 35 ) and MHCII (marker for activated macrophages and antigen presentation). ( 36 ) In the gut of both young and aged stroked mice, NR increased the populations of CD71 + and MHCII + MDMs (Fig. 4 C and 4 D), and restored stroke-induced changes in the population of CD71 + and MHCII + TRMs in the gut of both young and aged stroked mice (Fig. 4 C and 4 D). We also assessed CD71 + , MHCII + P1, P2 and P3 macrophages, and while we did not detect CD71 + , MHCII + P1 and P3 macrophages in both young and aged mice, NR increased the populations of CD71 + and MHCII + P2 macrophages in young stroked mice and restored these populations in aged stroked mice (Fig. 4 E and 4 F). Finally, we evaluated overall intestinal inflammation and determined that NR had no effect on general inflammation in the gut of young mice 24 hours after stroke (Figure S6D). However, in the gut of aged stroked mice, NR restored the expression of several immune factors to sham levels 24 hours after stroke, thus reducing inflammation in the gut (Figure S6E). Together, these results suggest that NR administration prior to stroke protects against stroke-induced decline in intestinal macrophages and reduces gut inflammation. Nicotinamide riboside reduces stroke-induced gut permeability in aged mice. Stroke compromises gut barrier integrity by triggering adrenergic signals to the intestine, causing a transient but consequential disruption of the gut epithelium. This allows the translocation of microbial components across the gut epithelium and hyperactivation of lamina propria macrophages, and this persistent gut barrier damage increases the risk of bacterial infection and worsens stroke outcomes. ( 14 , 15 , 33 ) Given the effect of NR on monocyte/macrophage cell populations in the gut, we next tested whether gut permeability might be improved following NR pretreatment. Fluorescein isothiocyanate-labeled dextran (FITC-Dextran) was orally administered to sham, vehicle and NR-treated young and aged stroked at 24 hours post stroke, and levels measured in the blood plasma 4 hours later. NR treatment did not alter plasma FITC-Dextran levels in young stroked mice. However, in aged stroked mice, NR administration resulted in FITC-Dextran levels comparable to sham mice, indicating reduced gut barrier permeability relative to vehicle-treated stroked mice (Fig. 5 A). To confirm these beneficial effects of NR on gut barrier integrity, we measured endotoxin lipopolysaccharide (LPS) levels in plasma 24 hours after stroke. No differences were observed in young mice, however, endotoxin levels were significantly reduced in NR-treated aged compared to vehicle-treated stroked mice (Fig. 5 B), confirming that NR rescued stroke-induced increases in gut barrier permeability. To further probe the integrity of the gut epithelium, we immunostained for tight junction proteins, zona occludens-1 (ZO-1) and epithelial cell adhesion molecule (EpCAM), and probed for CD11b, a marker for resident macrophages. NR had no effect on levels of ZO-1 and EpCAM but restored expression of CD11b to sham levels in the young stroked mice (Fig. 5 C and 5 D). In aged stroked mice, NR restored expression of ZO-1 and CD11b to sham levels, and marginally increased EpCAM (Fig. 5 C and 5 D). To assess whether improved gut integrity was linked to changes in NAD⁺, we measured levels of NAD + and nicotinic acid (NA), which studies have shown increases as a result of NR metabolism by the gut microbiome ( 29 ) (Fig. 5 E). In the gut from young mice, no differences were observed in NA or NAD + levels between sham, vehicle and NR-treated stroked mice, however, both NA and NAD + levels were significantly increased in the gut of aged NR-treated stroked mice compared to sham and vehicle-treated stroked mice (Fig. 5 F). These findings support highly beneficial effects of NR pretreatment in aged stroked mice with protection of gut barrier function. Nicotinamide riboside improves stroke induced motor deficits. Given the improved metabolism and reduced inflammation in aged stroked mice from NR pretreatment, we then assessed other measures of stroke severity. We evaluated motor coordination and balance by subjecting sham, vehicle and NR-treated young and aged mice to a rotarod test, which measures the latency to fall from an accelerating/decelerating rotating rod, 24 hours after stroke. The latency to fall from the rotarod was improved in NR-treated young stroked mice in the first trial, however by the third trial, there were no significant differences between sham, vehicle and NR-treated young stroked mice (Fig. 6 A). Likewise, NR-treated aged stroked mice performed better in the first trial compared to sham and vehicle-treated stroked mice (Fig. 6 B). Next, we reversed the direction of motion of the rotarod to challenge motor coordination and determine if the mice would self-correct and remain on the rotarod. For both young and aged mice, NR-treated mice had a higher latency to fall compared to sham and vehicle-treated stroked mice (Fig. 6 A and 6 B). In line with this improved motor control, we also observed that infarct area, measured at 24 hours after stroke, was reduced in NR-treated young and aged stroked mice compared with sham and vehicle-treated stroked mice (Fig. 6 C and 6 D). We also investigated the number of IBA1-positive cells expressing CD68 to measure activation of microglia in the infarct area. In the young, NR marginally decreased expression of IBA1 and CD68 (Fig. 6 E), and in aged NR-treated stroked mice, both IBA1 and CD68 expression were significantly reduced compared to vehicle-treated stroked mice (Fig. 6 F). Interestingly, the ratio of CD68/IBA1 expression was unchanged in both young and aged NR-treated stroked mice, suggesting that NR had no effect on overall microglial activation. Altogether, NR pretreatment protects against stroke-related motor deficits and reduces stroke infarct size in both young and aged mice. DISCUSSION The precise cause of the robust and maladaptive inflammatory response to stroke in aged individuals is unclear. This is the first study to investigate the metabolism of blood monocytes and evaluate the effect of boosting NAD + levels on the response of peripheral blood monocytes to stroke. Nicotinamide riboside (NR) has been shown to enhance NAD + levels and alter the metabolism and inflammatory status of human blood monocytes by increasing purine levels required for nucleotide synthesis. ( 28 ) Here, we find that prolonged pretreatment with NR prior to stroke reduces detrimental age-dependent metabolic and inflammatory changes in blood monocytes and prevents post-stroke increases in gut barrier permeability. Our studies demonstrate that stroke triggers age-dependent metabolic changes in blood monocytes by reducing NAD + levels and increasing levels of the neurotoxic quinolinic acid (QA). NR supplementation improves stroke outcomes by broadly replenishing NAD⁺ levels in blood monocytes, intestine, and brain, while mitigating inflammation and endotoxemia caused by gut barrier disruption. Several studies have investigated acute and chronic transcriptomic and inflammatory changes of immune cells in the blood and brain-infarct areas of young and aged mice after stroke, ( 3 , 14 , 37 ) however, the metabolic status of immune cells after stroke has not been explored. In this study, we focused on peripheral blood monocytes because these cells infiltrate the brain in response to stroke, alongside neutrophils, to drive secondary stroke injury. Because of the link between metabolism and immune function, we investigated the metabolic state of blood monocytes immediately post-stroke during the period in which they traffic to the ischemic brain. Our results reveal age-dependent metabolic and inflammatory changes over time, with the most pronounced changes occurring 24 hours post stroke. While several metabolites changed over time in both young and aged blood monocytes, the NAD + metabolome was altered across all time points only in aged stroked mice, leading us to investigate NAD + biosynthetic pathways, including the Kynurenine pathway (KP). Activation of the KP correlates with increased stroke severity. ( 22 ) QA is generated by the KP and the elevated levels of QA in aged blood monocytes observed in our study support both the activation of the KP in monocytes after stroke as well as the failure to convert to the NAD + precursor, nicotinic acid mononucleotide (NaMN). In the brain, QA induces excitotoxicity in neurons by activating NMDA receptors. ( 24 ) QA can also reduce nucleotide levels including GTP, ATP and NAD + , leading to mitochondrial dysfunction in synapses and neuronal damage. ( 38 ) The increased monocytic QA level in aged mice could potentially worsen excitatory injury following trafficking to the ischemic brain. Replenishing NAD + levels is neuroprotective in young mouse models of stroke and in aging. ( 39 – 43 ) We investigated the effects of NR supplementation on stroke outcome in young and aged mice, focusing on the effects on blood monocytes and gut barrier integrity. The choice of NR compared to nicotinamide (NAM) and nicotinamide mononucleotide (NMN) was made due to its robust enhancement of mitochondrial function, the feasibility of daily oral administration, avoidance of the side effects associated with NAM, and the instability of NMN. ( 26 , 44 ) Prolonged daily NR pretreatment in aged mice enhanced basal respiration, ATP production, and glycolytic activity and reduced expression of multiple pro-inflammatory molecules in blood monocytes following stroke. Additionally, increased NaMN and decreased QA levels in aged blood monocytes suggested enhanced KP metabolic flux. Overall, NR had a more potent effect on aged mice compared to young mice as the elevated levels of inflammatory mediators in both blood monocytes and plasma were most reduced in aged mice. Increased gut permeability and dysbiosis are significant contributors to neuroinflammation and worse stroke outcomes in aging. ( 14 , 15 , 33 , 45 ) While NR did not show any measurable effects in young mice, it reduced gut permeability in aged stroked mice. Indeed, NR has been shown to improve ethanol-induced gut barrier damage via mitochondrial sirtuins in intestinal epithelial cells. ( 46 ) Moreover, in aged mice, pretreatment with NR completely prevented age-associated losses of MDMs and TRMs in the intestinal lamina propria which are critical to gut barrier function. The improvement in gut barrier function observed in aged NR-pretreated stroked mice may be the result of improved overall epithelial health from increased absorption of NR and NMN, as well as improved MDM and TRM immune states, as reflected by reversal of inflammatory markers CD71 + and MHCII + . Alongside the overall improved immune state of monocytes and intestinal macrophages, NR pretreatment also improved motor coordination in both young and aged stroked mice, reduced infarct size and mitigated inflammation in the brain. A recent study performed in young mice demonstrated reduced stroke infarct volume with a single post-ischemic NR treatment, but poorer stroke outcomes with three days of pre-ischemic NR treatment. ( 42 ) In that study, it is possible that the timing was not sufficient for cells to metabolically adapt to such high levels of NAD + prior to the ischemic event. The authors also observed improved outcome with a single post-ischemic dose of Nam, however, it is important to note that several other studies have demonstrated Nam inhibition of sirtuins, essential mediators of metabolic homeostasis, as well as adverse effects of Nam on liver function. ( 44 , 47 ) A limitation of the current study is that only male mice were examined. There are well established mechanistic differences between male and female responses to stroke, ( 48 ) and potential sex-specific metabolic differences will be explored in future studies. Regarding the metabolism of NR, further studies are required to understand how NR is metabolized in the gut by the gut microbiome after stroke. It is known that NR is converted to nicotinamide mononucleotide (NMN) by nicotinamide riboside kinase (NRK1) and then to NAD + , however, oral administration of NR leads to an increase in nicotinic acid (NA) which is metabolized to nicotinic acid mononucleotide (NaMN) by the gut microbiome, ( 26 , 29 , 49 ) a common metabolite of the Kynurenine (KP) and Preiss-Handler pathways, suggesting activation of either pathway by NR through a mechanism that is not fully understood. Another limitation is the technical challenge in measuring NAD + levels in very small samples such as blood monocytes due to its rapid turnover, low analyte concentration and instability. To address this limitation, we measured NaMN as a readout for increased NAD + levels when undetected, given that orally administered NR has been shown to significantly increase NaMN. ( 26 , 29 ) In conclusion, we identified age-related metabolic alterations in monocytes following stroke, characterized by reduced NAD⁺ and elevated QA levels, which were associated with changes in inflammatory factors. Additionally, we showed that prolonged NR pretreatment effectively replenishes NAD⁺, enhances the metabolic and inflammatory responses of blood monocytes to stroke, and broadly improves stroke outcomes by strengthening gut barrier function and reducing inflammation. Our findings highlight the potential of NR as a preventive therapeutic strategy for patients at high risk of cardiovascular and cerebrovascular disease. Declarations Competing Interests The authors declare that they have no competing interests. Authors and Affiliations Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH, USA (C.A.I., B.A.E), Department of Neurology and Neurological Science, Stanford University School of Medicine, Stanford, CA, USA (C.A.I., K.I.A., M.C., J.H., H.E.E., Y.A.A., Q.W.), Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA (K.I.A.), Stanford Neurosciences Graduate Program, Stanford University, Stanford, CA, USA (J.H.), Mass Spectrometry Center, Stanford University, Stanford, CA, USA (L.A.), Department of Biochemistry and Molecular Biology, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA (A.I.A., M.R.M.), Department of Molecular Biosciences, The University of Texas at Austin, TX, USA (M.C.), Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel (E.B.), Chan Zuckerberg Biohub, San Francisco, CA, USA (K.A.I). Funding sources This work was supported by Burroughs Wellcome Postdoctoral Diversity Enrichment Program grant no. G-1022372 (C.A.I). The Howard Hughes Medical Institute Hanna H. Gray Fellows Program Faculty Phase grant no. GT15655 (M.R.M). The Burroughs Welcome Fund PDEP Transition to Faculty grant no. 1022604 (M.R.M). NIH Grants T32GM108563 (A.I.A.), RO1NS100180 (K.I.A.). The American Heart Foundation grant no. 19PABH1345800 (K.I.A.). K.I.A. is a Chan Zuckerberg–San Francisco Biohub Investigator. Author Contribution C.A.I and K.I.A. conceived and planned this study. C.A.I., M.R.M., and K.I.A. contributed to supervising the experimental design. C.A.I., H.E., M.C., A.I.A., L.A., Y.A.A., Q.W., and E.B. conducted the experiments. C.A.I., H.E., M.C., A.I.A., L.A., J.H., B.A.E, and M.R.M performed collection of the data and statistical analysis. C.A.I. and K.I.A. wrote the manuscript. All authors reviewed and approved of the manuscript. Acknowledgments We would like to acknowledge the Stanford University Mass Spectrometry core facility for the use of the Xevo TQ-XS mass spectrometer system (RRID:SCR_018510) that was purchased with funding from National Institutes of Health Shared Instrumentation Grant S10OD026962. We thank the Stanford University Cell Sciences Imaging Core Facility for the use of the Leica SP8 confocal microscope (RRID: SCR_017787), supported by the Award Number 1S10OD010580-01A1 from the National Center for Research Resources (NCRR). We would also like to acknowledge the Stanford University Human Immune Monitoring Center (HIMC) and the Case Western Reserve University Bioanalyte core facility for the Luminex experiments. We would also like to acknowledge the Huck Institutes’ Metabolomics core facility (RRID:SCR_023864) for use of the OE 240 LC-MS and Sergei Koshkin for helpful discussions on sample preparation and analysis. 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Oral supplementation of nicotinamide riboside alters intestinal microbial composition in rats and mice, but not humans. NPJ Aging. 2023;9(1):7. PubMed PMID: 37012386. PMCID: PMC10070358. Epub 20230403. Additional Declarations No competing interests reported. Supplementary Files NADdepletiondrivesagerelatedmonocytehyperinflammationafterstrokeandisreversedbynicotinamideribosidesupplementarymaterials.docx Cite Share Download PDF Status: Published Journal Publication published 27 Nov, 2025 Read the published version in Journal of Neuroinflammation → Version 1 posted Editorial decision: Revision requested 23 Oct, 2025 Reviews received at journal 21 Oct, 2025 Reviewers agreed at journal 09 Oct, 2025 Reviews received at journal 26 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers invited by journal 06 Sep, 2025 Editor assigned by journal 03 Sep, 2025 Submission checks completed at journal 29 Aug, 2025 First submitted to journal 28 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Alsaadi","email":"","orcid":"","institution":"Huck Institutes of the Life Sciences, Pennsylvania State University","correspondingAuthor":false,"prefix":"","firstName":"Abrar","middleName":"I.","lastName":"Alsaadi","suffix":""},{"id":512521947,"identity":"6baa0f69-8bbb-4572-b2c5-4474aedcf9dd","order_by":3,"name":"Brent Eastman","email":"","orcid":"","institution":"Case Western Reserve University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Brent","middleName":"","lastName":"Eastman","suffix":""},{"id":512521948,"identity":"2bad51dd-4c98-43a6-820a-85f8073858c6","order_by":4,"name":"Ludmila Alexandrova","email":"","orcid":"","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Ludmila","middleName":"","lastName":"Alexandrova","suffix":""},{"id":512521949,"identity":"5c16ff99-8b4f-4f6e-b51a-3ab051f0235e","order_by":5,"name":"Yeonglong Albert Ay","email":"","orcid":"","institution":"Stanford University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yeonglong","middleName":"Albert","lastName":"Ay","suffix":""},{"id":512521950,"identity":"9091dea6-0cff-43b0-a052-4ee9d04d3f91","order_by":6,"name":"Jolie Huang","email":"","orcid":"","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Jolie","middleName":"","lastName":"Huang","suffix":""},{"id":512521952,"identity":"220103de-0ce2-4088-aa10-34e485ee8a32","order_by":7,"name":"Qian Wang","email":"","orcid":"","institution":"Stanford University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Wang","suffix":""},{"id":512521954,"identity":"8ac30c7f-edbb-4acf-8767-cea339c2afd0","order_by":8,"name":"Eran Blacher","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"prefix":"","firstName":"Eran","middleName":"","lastName":"Blacher","suffix":""},{"id":512521955,"identity":"2d0b6025-c545-4cfd-8daa-d0d258206be1","order_by":9,"name":"Melanie R. 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Andreasson","email":"","orcid":"","institution":"Stanford University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Katrin","middleName":"I.","lastName":"Andreasson","suffix":""},{"id":512521957,"identity":"bba81598-3dfe-4529-abbe-81931b868d4f","order_by":11,"name":"Chinyere Agbaegbu Iweka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYFACxsYHCRVQNg+RWpoNPpwhTQsDm+DMNlK08M9IbmPmnWcnL9/ewPjgbRthHQwSZw62Pebdlmy44cwBZsO5xGgxYG9sN+bddoBxg0QCmzQvUVqYGdukeeccsJ8//wH7b+K0sDe2Sc5sOJDYcIOBjZkoLUC/AAP5WHLyhjOJzZJzzhGhhX9G+sMHCTV2tvPbDx/88KaMCC1IgLGBNPWjYBSMglEwCnADAOlVNvOZX9wsAAAAAElFTkSuQmCC","orcid":"","institution":"Case Western Reserve University","correspondingAuthor":true,"prefix":"","firstName":"Chinyere","middleName":"Agbaegbu","lastName":"Iweka","suffix":""}],"badges":[],"createdAt":"2025-08-28 23:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7483662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7483662/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12974-025-03638-6","type":"published","date":"2025-11-27T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91196295,"identity":"9f9c39c7-9de8-4863-a38b-e8c8857a375e","added_by":"auto","created_at":"2025-09-12 15:02:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":481576,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime course of bioenergetic responses of blood monocytes from young and aged mice after stroke. (A)\u003c/strong\u003eSchematic illustrating the timeline of stroke induction using the dMCAo (distal middle cerebral artery occlusion) model, and collection of blood monocytes from young (3–6 mo) and aged (18–20 mo) mice. \u003cstrong\u003e(B) \u003c/strong\u003eSeahorse (Mitostress test) analysis of basal respiration, ATP production and \u003cstrong\u003e(C)\u003c/strong\u003e extracellular acidification rates (ECAR) of blood monocytes isolated from young and aged mice at 4.5 hours, 24 hours, and 72 hours after stroke. n=3–5 male mice per group. Data are represented as the mean ± SEM. p-values were calculated using two-tailed Student’s t-test. \u003cstrong\u003e(D)\u003c/strong\u003e Analysis of enriched metabolic pathways in blood monocytes isolated from aged mice at 4.5, 24 and 72 hours after stroke. Data were analyzed using MetaboAnalyst 6.0. Red arrows indicate changes in nicotinamide adenine dinucleotide (NAD+) metabolic pathways. Negative and positive values indicate decreased and increased pathways respectively. LC-MS/MS measurement of NAD+ and quinolinic acid (QA) levels in blood monocytes isolated from \u003cstrong\u003e(E) \u003c/strong\u003eyoung and \u003cstrong\u003e(F) \u003c/strong\u003eaged male mice at 4.5 hours, 24 hours and 72 hours post stroke. n=4–5 male mice per group. Data are represented as the mean ± SEM. p-values were calculated using two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7483662/v1/589c89debb22a0d77bc1e0ae.png"},{"id":91197748,"identity":"f5055ff9-f699-44ab-b9ca-b99433d1cb60","added_by":"auto","created_at":"2025-09-12 15:10:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":459424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNAD\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e replenishment improves metabolism of blood monocytes and reduces inflammation in aged mice after stroke.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eSeahorse (Mitostress test) analysis of basal respiration, ATP production and extracellular acidification rates (ECAR) from blood monocytes isolated 24 hours after stroke from\u003cstrong\u003e \u003c/strong\u003eyoung (3–6 mo) and aged (18–20 mo) mice treated with either vehicle (water; control) or nicotinamide riboside (NR) via oral gavage every day for 4 weeks. n=3–5 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test.\u003cstrong\u003e (B)\u003c/strong\u003e LC-MS/MS measurement levels of nicotinic acid mononucleotide (NaMN; left) and quinolinic acid (QA; right) in blood monocytes isolated from control and NR-treated young and aged mice at 24 hours after stroke. n=4–5 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test.\u003cstrong\u003e (C)\u003c/strong\u003e Multi-analyte cytokine/chemokine (Luminex) quantification of differences in the expression of immune factors in blood monocytes from young and aged sham and stroke mice at 4.5 hours (left), 24 hours (middle) and 72 hours (right) after stroke. n=4–5 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Multi-analyte Luminex quantification of differences in the expression of immune factors in blood monocytes isolated from sham control (vehicle; water), stroke control (vehicle; water) and stroke NR-treated \u003cstrong\u003e(D)\u003c/strong\u003e young and \u003cstrong\u003e(E)\u003c/strong\u003e aged mice 24 hours after stroke. n=4–6 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using one-way ANOVA with Bonferroni correction.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7483662/v1/467a751d79edf851e530019d.png"},{"id":91198907,"identity":"641dcb85-d38b-41f7-904e-bd8ab1344c8e","added_by":"auto","created_at":"2025-09-12 15:18:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":439040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNicotinamide riboside alters inflammation and increases NAD\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e levels in the brain after stroke. \u003c/strong\u003eMulti-analyte Luminex quantification of differences in the expression of immune factors in the \u003cstrong\u003e(A)\u003c/strong\u003e ipsilateral and \u003cstrong\u003e(B)\u003c/strong\u003e contralateral brain isolated from vehicle (water; control) and NR-treated young (3–6 mo) and aged (18–20 mo) mice at 24 hours after stroke. n=4–5 male mice per group. Data are represented as the mean ± SEM. p-values were calculated using two-tailed Student’s t-test. LC-MS/MS measurement of NAD+ levels in \u003cstrong\u003e(C)\u003c/strong\u003e ipsilateral and \u003cstrong\u003e(D)\u003c/strong\u003e contralateral brain from sham, vehicle and NR-treated young and aged mice at 24 hours post stroke. n=5 male mice per group. Data are represented as the mean ± SEM. Values were calculated using one-way ANOVA with Bonferroni correction.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7483662/v1/b6ee242b3299802507136365.png"},{"id":91197749,"identity":"6494285e-e6c2-4296-8e14-ce1049b78dba","added_by":"auto","created_at":"2025-09-12 15:10:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":355245,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNicotinamide riboside alters the composition of gut macrophages in aged mice.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Number of CD45\u003csup\u003e+\u003c/sup\u003e monocyte-derived macrophages (MDMs) and tissue resident macrophages (TRMs) isolated from the gut of sham (vehicle; water), stroke control (vehicle; water) and stroke NR-treated young (3–6 mo; top) mice and aged (18–20 mo; bottom) mice at 24 hours after stroke. \u003cstrong\u003e(B) \u003c/strong\u003eNumber of P1 (Ly6C\u003csup\u003eHi \u003c/sup\u003eMHCII\u003csup\u003eLow\u003c/sup\u003e), P2 (Ly6C\u003csup\u003eInt\u003c/sup\u003e MHCII\u003csup\u003eHi\u003c/sup\u003e) and P3 (Ly6C\u003csup\u003elow\u003c/sup\u003e MHCII\u003csup\u003eHi\u003c/sup\u003e) macrophages in the gut of sham control, stroke control and stroke NR-treated young and\u003cstrong\u003e \u003c/strong\u003eaged mice. \u003cstrong\u003e(C) \u003c/strong\u003eNumber of CD71\u003csup\u003e+\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e MDMs and TRMs in the gut of sham control, stroke control and stroke NR-treated young mice. \u003cstrong\u003e(D) \u003c/strong\u003eNumber of CD71\u003csup\u003e+\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e MDMs and TRMs in the gut of sham control, stroke control and stroke NR-treated aged mice. \u003cstrong\u003e(E) \u003c/strong\u003eNumber of CD71\u003csup\u003e+\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e P2 macrophages in the gut of\u003cstrong\u003e \u003c/strong\u003esham control, stroke control and stroke NR-treated young mice. \u003cstrong\u003e(F) \u003c/strong\u003eNumber of CD71\u003csup\u003e+\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e P2 macrophages in the gut of\u003cstrong\u003e \u003c/strong\u003esham control, stroke control and stroke NR-treated aged mice. n=4–5 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using one-way ANOVA with Bonferroni correction.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7483662/v1/f160d9c718ef780c4af3edfd.png"},{"id":91196298,"identity":"e39052d5-460e-4307-b45e-e99bee2bded0","added_by":"auto","created_at":"2025-09-12 15:02:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1904391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNicotinamide riboside restores gut integrity in aged stroke mice. (A)\u003c/strong\u003e FITC-Dextran was measured in the blood plasma from sham control (vehicle; water), stroke control (vehicle; water), and stroke NR-treated young (3–6 mo) and aged (18–20 mo) mice 24 hours after stroke. n=4–5 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using one-way ANOVA with Bonferroni correction. \u003cstrong\u003e(B)\u003c/strong\u003e Blood plasma concentration levels of the bacterial endotoxin lipopolysaccharide (LPS) measured 24 hours after stroke in sham control, stroke control and stroke NR-treated young and aged stroke mice. n=4–5 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using one-way ANOVA with Bonferroni correction. \u003cstrong\u003e(C)\u003c/strong\u003e Representative confocal images of ZO-1, EpCAM and CD11b in the lamina propria in sham control, stroke control and stroke NR-treated young (top) and aged (bottom) male mice 24 hours post stroke. Scale bar, 50 µm.\u003cstrong\u003e (D)\u003c/strong\u003e Mean fluorescent intensity quantification of ZO-1, EpCAM and CD11b expression 24 hours after stroke in sham control, stroke control and stroke NR-treated\u003cstrong\u003e \u003c/strong\u003eyoung (top) and aged (bottom) mice. n=4–5 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using one-way ANOVA with Bonferroni correction. \u003cstrong\u003e(E)\u003c/strong\u003e Schematic illustrating metabolism of NR in the gut to NAD\u003csup\u003e+\u003c/sup\u003e (left; created in Biorender). \u003cstrong\u003e(F)\u003c/strong\u003e LC-MS/MS measurement of nicotinic acid (NA) and NAD\u003csup\u003e+\u003c/sup\u003e levels in sham control, stroke control and stroke NR-treated young (left) and aged (right) mice. n=5–6 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using one-way ANOVA with Bonferroni correction.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7483662/v1/b6467bd0abec1577e993aa32.png"},{"id":91196297,"identity":"440d3950-6a74-43cf-bc1c-2e85cd56dbc6","added_by":"auto","created_at":"2025-09-12 15:02:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1915800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNicotinamide riboside improves motor coordination and reduces stroke infarct size. \u003c/strong\u003eMotor coordination using the rotarod test shows latency to fall in 3 trials (left graph) before stroke in sham control (vehicle; water), stroke control (vehicle; water), and stroke NR-treated \u003cstrong\u003e(A)\u003c/strong\u003e young (3–6 mo) and \u003cstrong\u003e(B)\u003c/strong\u003e aged (18–20 mo) mice 24 hours after stroke. Direction of rotarod was reversed to challenge motor coordination 24 hours after stroke (right graph). n=5 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using one-way ANOVA with Bonferroni correction. \u003cstrong\u003e(C) \u003c/strong\u003eRepresentative confocal images of the brain isolated from stroke control and stroke NR-treated young and aged mice 24 hours after stroke. Brain slices were immunostained for DAPI (blue), MAP2 (neuronal marker in green), IBA1 (microglial marker, magenta) and CD68 (microglial lysosomal marker, red). \u003cstrong\u003e(D)\u003c/strong\u003e Stroke infarct size was measured as a percentage of corrected infarct area (see Methods) in young and aged stroke control and NR-treated mice at 24 hours post stroke. n=5 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Dotted lines indicate stroke infarct area. Scale bar, 500 µm. Dotted lines show infarct area. Mean fluorescent intensity quantification of the expression and ratio of CD68 and IBA1 expression 24 hours after stroke in NR-treated \u003cstrong\u003e(E)\u003c/strong\u003e young and \u003cstrong\u003e(F)\u003c/strong\u003eaged male mice. n=5 male mice per group. Data are represented as the mean ± SEM. \u003cem\u003ep\u003c/em\u003e-values were calculated using two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7483662/v1/cd15c5c2793c5021f216a804.png"},{"id":97178617,"identity":"07a63b1a-3f84-4fe8-bc2a-7ea6a6a97237","added_by":"auto","created_at":"2025-12-01 16:11:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6415481,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7483662/v1/9d53e621-5610-4c85-bc06-89b4ff53d988.pdf"},{"id":91196306,"identity":"2ca8d7b2-14e0-495f-9c8c-6ad631778dbc","added_by":"auto","created_at":"2025-09-12 15:02:06","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4744323,"visible":true,"origin":"","legend":"","description":"","filename":"NADdepletiondrivesagerelatedmonocytehyperinflammationafterstrokeandisreversedbynicotinamideribosidesupplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7483662/v1/2aae876d91b2c65b06b93e4a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"NAD⁺ depletion drives age-related monocyte hyperinflammation after stroke and is reversed by nicotinamide riboside","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAn important consequence of acute ischemic stroke is secondary injury, which occurs as a result of robust and sustained immune cell infiltration into the ischemic brain, driven initially by monocytes and neutrophils.\u003csup\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/sup\u003e This inflammatory response is significantly exacerbated in aged individuals compared to young.\u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/sup\u003e The underlying cause of this aggravated inflammatory response in aging remains unclear. Several studies have established that the metabolic state of immune cells plays a pivotal role in regulating immune responses, as metabolic reprogramming ensures sufficient energy for host defense and tissue homeostasis.\u003csup\u003e(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/sup\u003e This metabolic homeostasis is disrupted with aging, impairing the ability to mount appropriate immune responses to stimuli.\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eWe hypothesized that energy metabolism could play a critical role in the immune responses to stroke in aged individuals. To study this, we investigated the metabolism of blood monocytes that along with neutrophils, are the first immune cells to infiltrate the ischemic brain where they differentiate into macrophages. In the ischemic area, macrophages clear cellular debris, promote repair, regeneration, and inflammation resolution; processes that depend on robust and adaptable energy metabolism.\u003csup\u003e(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/sup\u003e The balance between glycolytic and oxidative metabolism can determine whether immune cells adopt an activated, pro-inflammatory state or maintain homeostatic regulatory functions.\u003csup\u003e(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/sup\u003e Homeostatic immune cells depend primarily on oxidative phosphorylation and fatty acid β-oxidation to generate ATP. However, upon activation, they shift toward aerobic glycolysis, also known as the Warburg effect.\u003csup\u003e(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/sup\u003e This metabolic shift fuels immune activation, with increased glucose utilization driving a pro-inflammatory phenotype in macrophages.\u003csup\u003e(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/sup\u003e As glycolysis serves as a primary energy source for activated immune cells during inflammation, nicotinamide adenine dinucleotide (NAD\u003csup\u003e+\u003c/sup\u003e), a key energy co-factor, is rapidly consumed to sustain this glycolytic activity and can potentially become limiting.\u003csup\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/sup\u003e NAD\u003csup\u003e+\u003c/sup\u003e is critical for maintaining energy homeostasis and modulating immune function.\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/sup\u003e For example, inhibiting the \u003cem\u003ede novo\u003c/em\u003e biosynthetic pathway for NAD\u003csup\u003e+\u003c/sup\u003e (downstream of the Kynurenine pathway) in aged, but not young macrophages, disrupts oxidative phosphorylation, impairs phagocytic function and the immune response to lipopolysaccharide (LPS).\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/sup\u003e NAD\u003csup\u003e+\u003c/sup\u003e levels decline significantly with age, impairing immune cell responses, while increasing cellular NAD\u003csup\u003e+\u003c/sup\u003e can enhance energy metabolism and reduce inflammation.\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn addition to trafficking to the ischemic brain, monocytes continually migrate to selected organs where they replenish macrophage populations, including the intestine, lung and spleen. Monocytes that traffic to the intestinal lamina propria function critically in maintaining intestinal homeostasis and gut barrier integrity.\u003csup\u003e(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/sup\u003e Following stroke, a burst of increased adrenergic input leads to transient opening of the gut barrier, translocation of microbial components, and hyper-activation of lamina propria macrophages, which in turn, further disrupts the gut epithelial barrier, leading to systemic bacterial dissemination.\u003csup\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn this study, we investigated the metabolic status of blood monocytes from young and aged mice at several time points after stroke. We also examined monocyte-derived lamina propria macrophages and the integrity of the gut barrier after stroke in young and aged mice. Our results show that NAD\u003csup\u003e+\u003c/sup\u003e is critically dysregulated in blood monocytes from aged mice and is associated with disrupted immune responses and an accumulation of quinolinic acid (QA), a neurotoxic metabolite of the Kynurenine pathway. Prolonged pretreatment of aged mice with nicotinamide riboside (NR) to elevate cellular NAD⁺ levels significantly improved monocyte energy metabolism, reduced inflammation, rescued the loss of lamina propria macrophages and gut barrier function after stroke. NR supplementation also significantly improved motor function and reduced infarct size following stroke.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e All experiments and procedures were performed in accordance with the National Institutes of Health (NIH) guidelines, and all protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Case Western Reserve University and Stanford University. All animals were socially housed in a pathogen-free barrier facility environmentally controlled for temperature and humidity, on a 12-hour light\u0026ndash;dark cycle, with food and water available ad libitum. C57BL/6J mice were purchased from Jackson laboratories or obtained from the NIH aged rodent colony. Distal middle cerebral artery occlusion surgery and collection of all tissue samples were performed between \u003cem\u003eZT2\u003c/em\u003e and \u003cem\u003eZT5\u003c/em\u003e (\u003cem\u003ezeitgebers time\u003c/em\u003e). This time interval was maintained to ensure consistency of any possible circadian effects and reduce variability.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDistal middle cerebral artery occlusion\u003c/h3\u003e\n\u003cp\u003eDistal middle cerebral artery occlusion (dMCAo), a permanent coagulation of the middle cerebral artery via electrocoagulation was performed as described previously.\u003csup\u003e(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/sup\u003e Young (3\u0026thinsp;\u0026minus;\u0026thinsp;6 months) and aged (18\u0026thinsp;\u0026minus;\u0026thinsp;20 months) C57BL/6J male mice were anesthetized with 3% isoflurane and then with 70% N\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;30% O\u003csub\u003e2\u003c/sub\u003e maintained throughout the surgical procedure, and injected intramuscularly with 0.3 mg/kg buprenorphine (BERLAB0.5-232305; ZooPharm, Laramie, WY). The mice were placed on a heating blanket at 37˚C to maintain body temperature. Eye ointment (Artificial Tears Ophthalmic Lubricant, Akorn, Inc. Lake Forest, IL) was applied to both eyes to prevent dryness during the procedure. The mice were randomized and subjected to either dMCAo or sham surgery which involved thinning of the skull without coagulation of the middle cerebral artery.\u003c/p\u003e\n\u003ch3\u003eNicotinamide Riboside (NR) treatment\u003c/h3\u003e\n\u003cp\u003eMice were administered with either water (vehicle) or 400 mg/kg of nicotinamide riboside (NR; ASB-000114315-101; Niagen; Chromadex, Irvine CA) daily via oral gavage at the same time of day (between \u003cem\u003eZT\u003c/em\u003e2\u0026ndash;3) for 4 weeks. Mice were weighed and values recorded every week and no adverse reactions to NR was observed. Certificates of Analysis provided by the manufacturer and performed on separate lots reported\u0026thinsp;~\u0026thinsp;99% purity of the NR preparation.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eMice were euthanized in CO\u003csub\u003e2\u003c/sub\u003e chamber and transcardially perfused with 10 mM PBS. Brain and gut tissue were isolated and immersed in 4% paraformaldehyde (PFA; 15714-1L; Electron Microscopy Sciences, Hatfield, PA) overnight and then serially cryopreserved in 15% sucrose and then 30% sucrose solution. 1 cm gut tissue sections were collected from the duodenum, jejunum and ileum and embedded in optimal cutting temperature compound (O.C.T, 23-730-571; Fisher Scientific, Waltham, MA) and then longitudinally sectioned at 10 \u0026micro;m per section. Four sections per mouse were mounted on Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA) and immunostained. For each brain, five 40 \u0026micro;m brain sections were coronally sectioned using a sliding microtome (Microm HM430, Thermo Fisher Scientific, Waltham, MA) at 500 \u0026micro;m intervals between approximately 1.5 mm rostral and 1.5 mm caudal to bregma. Brain tissue slices were placed in freezing media and stored at \u0026minus;\u0026thinsp;20˚C. Briefly, both brain and gut tissue samples were washed 3\u0026times; in PBS for 5 minutes and permeabilized in 0.2% Triton-X100 in PBS for 20 minutes at room temperature. Samples were then washed in 10 mM PBS and incubated for 1 hour in blocking buffer (0.2% Triton-X100 supplemented with 10% normal donkey serum in 10 mM PBS) and then in primary antibody (see appendix for list of antibodies) overnight at 4˚C. Next, the samples were washed 3\u0026times; in PBS for 5 minutes and then incubated in secondary antibody (see appendix for the list of antibodies) for 2 hours. Samples were washed 3\u0026times; in 10 mM PBS for 5 minutes and then incubated in Hoechst stain for 10 minutes in 10 mM PBS. Samples were washed in 10 mM PBS and then mounted with Prolong\u0026trade; Gold antifade reagent (P36930; Invitrogen, Eugene, OR), and tissue samples mounted onto Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA). Gut tissue sections were acquired on Zeiss 780 LSM confocal microscope (Carl Zeiss, Thornwood, NY) using either 20\u0026times;/0.45 NA dry objective lens or 40\u0026times;/0.95 NA oil immersion objective lens. Mean fluorescence intensity was quantified using ImageJ (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://imagej.nih.gov/ij\u003c/span\u003e\u003cspan address=\"http://imagej.nih.gov/ij\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eQuantification of stroke infarct area\u003c/h3\u003e\n\u003cp\u003eMeasurement of infarct area was carried out by an investigator blinded to treatment. 40 \u0026micro;m brain slices were immunostained with MAP2 to delineate infarcted area which had a relatively high fluorescent signal due to dead and dying neurons, and IBA1 and CD68 to quantify microglia (see appendix for antibody details). Images were acquired using a 10\u0026times; objective lens with a 0.40 NA on a Leica SP8 confocal microscope (Leica Microsystems Inc, Deerfield, IL). Infarct area was quantified using ImageJ (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://imagej.nih.gov/ij\u003c/span\u003e\u003cspan address=\"http://imagej.nih.gov/ij\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eBlood monocyte isolation\u003c/h2\u003e\u003cp\u003eBlood was collected by transcardiac puncture from mice with 0.25 mM EDTA (anticoagulant) and mixed with ACK lysing buffer (A1049201; Thermo Fisher Scientific, Waltham, MA). Samples were incubated on ice for 15 minutes and centrifuged at 300 \u0026times;g for 6 minutes. Samples were then resuspended in 10 mM PBS containing 2% FBS and 1 mM EDTA. EasySep\u0026trade; mouse monocyte isolation kit (19861; STEMCELL Technologies, Vancouver, BC, Canada) was used to isolate and enrich blood monocytes by immunomagnetic negative selection.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eChemokine and cytokine multiplex assay – Luminex\u003c/h3\u003e\n\u003cp\u003eBriefly, blood was collected by transcardiac puncture from mice with 0.25 mM EDTA, and either centrifuged at 2000 \u0026times;g at 4˚C for 10 minutes and blood plasma collected, or blood monocytes isolated using the EasySep\u0026trade; mouse monocyte isolation kit (19861; STEMCELL Technologies, Vancouver, BC, Canada). Mice were perfused with 10 mM PBS and brain tissue was collected from each mouse. Cells and tissue samples were lysed with cell lysis buffer (RIPA buffer; 89901; Thermo Fisher Scientific, Waltham, MA) and protein quantified by BCA (A55864, Thermo Fisher Scientific, Waltham, MA). Equal concentrations of samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C and cytokine analysis was carried out at the Human Immune Monitoring Core (Stanford University, CA) using magnetic bead-based multiplex Luminex assays (LXSAMSM; R\u0026amp;D Systems, Inc., Minneapolis, MN). Plates were read using a Luminex LabMap200 instrument. Mean fluorescence intensity (MFI) was averaged over duplicate wells for each cytokine per sample on each plate.\u003c/p\u003e\n\u003ch3\u003eEndotoxin Lipopolysaccharide Assay\u003c/h3\u003e\n\u003cp\u003eBriefly, blood was collected by transcardiac puncture from mice with 0.25 mM EDTA. Samples were centrifuged at room temperature for 10 minutes at 2500 rpm. Blood plasma was removed and tested for bacterial lipopolysaccharide (LPS) using the LPS ELISA kit (MBS261904; Mouse Lipopolysaccharide ELISA Kit; MyBioSource, Inc. San Diego, CA).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eFITC-Dextran\u003c/h2\u003e\u003cp\u003eMice were fasted for 4 hours with \u003cem\u003ead libitum\u003c/em\u003e access to water at the beginning of the light cycle at \u003cem\u003eZT\u003c/em\u003e0, 24 hours post stroke. Blood samples were collected via tail snip, centrifuged at 5000 rpm for 10 minutes at room temperature and kept on ice. Then 80 mg/mL of 4 kDa FITC-dextran (46944-500MG-F; Millipore Sigma, St. Louis, MO) diluted in sterile 10 mM PBS (per mouse) was administered via oral gavage. 4 hours post-administration, blood samples were collected via transcardiac puncture with 0.25 mM EDTA, centrifuged at 5000 rpm at room temperature for 10 minutes. Blood plasma was removed and the levels of FITC-dextran measured with a fluorescent plate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eReal-Time Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR)\u003c/h2\u003e\u003cp\u003eBlood monocytes were isolated using the EasySep\u0026trade; mouse monocyte isolation kit (19861; STEMCELL Technologies, Vancouver, BC, Canada) from each mouse as described previously and seeded in a seahorse XFe24 cell culture microplate (102340-100; Agilent, Santa Clara, CA) and allowed to adhere for 1 hour at 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Cells were washed twice with Agilent seahorse XF Media (103680-100; Agilent, Santa Clara, CA) supplemented with 1 mM pyruvate (Gibco\u0026trade; 11360070; Fisher Scientific, Waltham, MA), 2 mM L-glutamine (400-106-100; GeminiBio, West Sacramento, CA) and 2 mM D-glucose (G7021-1KG; Sigma-Aldrich, St. Louis, MO) in a final volume of 500 \u0026micro;L. Cells were then incubated in a 0% CO\u003csub\u003e2\u003c/sub\u003e chamber at 37\u0026deg;C for 1 hour before being placed into a seahorse XFe24 analyzer (US421134; Agilent, Santa Clara, CA). Cells were treated with 2.5 \u0026micro;M oligomycin, 2 \u0026micro;M carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and 0.5 \u0026micro;M rotenone/antimycin (103015-100; Seahorse XF Cell Mito Stress Test Kit; Agilent, Santa Clara, CA). 1 \u0026micro;M of Hoescht (H3570; Invitrogen, Waltham, MA) was injected at the end of the assay for cell counting. A total of three OCR and pH measurements were taken after each compound was administered from which basal respiration, maximal respiration, ATP production, spare respiratory capacity and extracellular acidification rates were calculated. All values were normalized by Hoescht-positive cell counting in the Cytation1 imaging platform (20081913; Agilent, Santa Clara, CA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry sample preparation\u003c/h2\u003e\u003cp\u003eThe small intestine (SI) was removed following perfusion and placed in cold 10 mM PBS. The SI was then cut into segments that underwent cleaning by flushing with cold sterile 10 mM PBS. Gut segments were then cut open and into 2 cm pieces before being placed in dissociation buffer: HBSS (without calcium/magnesium) containing 1% 0.5M EDTA and 1% HEPES and placed on a shaker at 180 rpm at 37˚C for 40 minutes. Following incubation, samples were poured through a 70 \u0026micro;m strainer and the flow-through was discarded. The tissue on the strainer was then moved to a new tube and dissociated mechanically with scissors for ~\u0026thinsp;30 seconds. Samples were resuspended in HBSS (without calcium/magnesium) containing 2% FBS, 0.5 mg/mL collagenase E and 0.5 mg/mL DNase I and placed on the shaker at 180 rpm at 37˚C for 45 minutes. Samples were again filtered through a 70 \u0026micro;m strainer and washed with 10 mL of cold HBSS (without calcium/magnesium). Samples were then centrifuged at 1400 rpm for 8 minutes at 4˚C and the supernatant discarded. Samples were washed again with 5 mL of HBSS (without calcium/magnesium) and centrifuged at 1400 rpm for 8 minutes at 4˚C. Supernatants were discarded and cell pellets resuspended in FACS buffer before staining for flow cytometry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry procedure\u003c/h2\u003e\u003cp\u003eSamples were transferred to a 96-well v bottom plate (277143; Thermo Fisher Scientific, Waltham, MA), and washed 1\u0026times; with FACS buffer (500 mL of 10 mM PBS containing 1 mL of 0.5M EDTA, and 1 g of Bovine Serum Albumin; BSA). Gut cells were stained at a dilution of 1:200 with CD11b (BV421), Ly6G (BV605), Ly6C (V450), CD45 (AF 700), MHCII (PerCP-efluor710), EGR2 (PE-Cy7), Live Dead Aqua, and with 1:1000 FC block in in FACS buffer for 25 minutes at 4\u0026deg;C. The cell pellets were centrifuged at 500 \u0026times;g for 3 minutes and then washed with FACS buffer then resuspended in 200 \u0026micro;L of FACS buffer and run on Cytek Aurora flow cytometry instrument (Fremont, CA). Analysis was performed using FlowJo software v10.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMetabolomics\u003c/h2\u003e\u003cp\u003eWater Soluble Metabolites: A solvent mixture of 80:20 methanol/water (500 \u0026micro;L) containing seven internal standards was used to resuspend the pellets that were composed of blood monocytes isolated from mice. Cell suspensions were vortexed for 30 seconds, sonicated in a water bath (30 seconds sonication, 30 seconds on ice, repeated 3 times), vortexed for 30 seconds and incubated for 2 hours at \u0026minus;\u0026thinsp;20\u0026deg;C to allow for protein precipitation. The supernatant was collected after centrifugation at 10,000 rpm for 10 minutes at 4\u0026deg;C and then evaporated to dryness under nitrogen. The dry extracts were then reconstituted with 100 \u0026micro;L of 50:50 methanol/water before analysis. Supernatants were centrifuged at 16,000 \u0026times;g for 20 minutes to remove any residual debris before analysis. Extracts were analyzed within 36 hours by liquid chromatography coupled to a mass spectrometer (LC\u0026thinsp;\u0026minus;\u0026thinsp;MS). The LC\u0026ndash;MS method involved hydrophilic interaction chromatography (HILIC) coupled to the Q Exactive PLUS mass spectrometer (Thermo Fisher Scientific, Waltham, MA) as previously described.\u003csup\u003e(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e The LC separation was performed on a XBridge BEH Amide column (150 mm 3 2.1 mm, 2.5 mm particle size; Waters, Milford, MA). Solvent A was 95%:5% H\u003csub\u003e2\u003c/sub\u003eO: acetonitrile with 20 mM ammonium bicarbonate, and solvent B was acetonitrile. The gradient was 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B; 30 min, 85% B. Other LC parameters were: flow rate, 150 mL/min; column temperature, 25˚C; injection volume, 10 \u0026micro;L; and autosampler temperature, 5˚C. The mass spectrometer was operated in both negative and positive ion mode for the detection of metabolites. Other MS parameters were: resolution of 140,000 at m/z 200, automatic gain control (AGC) target at 3\u003csup\u003e6\u003c/sup\u003e, maximum injection time of 30 ms and scan range of m/z 75-1000. Raw LC/MS data were converted to mzXML format using the command line \u0026ldquo;msconvert\u0026rdquo; utility.\u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/sup\u003e Data was processed and analyzed using MAVEN software (Princeton University, Princeton, NJ). Significant metabolites were formally identified by matching fragmentation spectra to public spectral libraries or by matching retention time and fragmentation spectra to authentic standards when possible.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of the NAD\u003csup\u003e+\u003c/sup\u003e, Kynurenine pathway and related metabolites\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003eAnalyte standards for NAD\u003csup\u003e+\u003c/sup\u003e metabolites\u003c/h2\u003e\u003cp\u003eThe following standards (see appendix for list of reagents catalogue numbers) Nicotinamide (NAM); Nicotinamide mononucleotide (NMN); nicotinamide adenine dinucleotide (NAD\u003csup\u003e+\u003c/sup\u003e); nicotinic acid mononucleotide (NaMN); nicotinic acid adenine dinucleotide (NaAD); adenosine monophosphate (AMP); adenosine diphosphate ribose (ADPr); NAM-d4; AMP\u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e5\u003c/sub\u003e were purchased from either Medical Isotopes, Inc, or Sigma-Aldrich (St. Louis, MO). For NaMN-d4 standard, the M\u0026thinsp;+\u0026thinsp;4 D/molecule was significantly less than the M\u0026thinsp;+\u0026thinsp;3 isotopomer and significantly less than the unlabeled molecular ion peak at identical concentrations in solution. Therefore, the actual incorporation of deuterium in NaMN-d4 was determined by mass spectrometry analysis where M\u0026thinsp;+\u0026thinsp;3 D/molecule was the major isotopomer and NaMN-d3 was used as an internal standard for the quantification.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eAnalyte standards for the Kynurenine pathway metabolites\u003c/h2\u003e\u003cp\u003eThe following standards (see appendix for list of reagents catalogue numbers) L-Tryptophan (TRP); Kynurenine (KYN); 3 Hydroxy DL-Kynurenine (3HK); Kynurenic acid (KA); 3 Hydroxyanthranilic acid (3HANA); Quinolinic acid (QA); TRP-d5; and KA-d5 \u0026ndash; were purchased from Sigma-Aldrich (St. Louis, MO). KYN-d4; 3HK-d3; 3HANA-d3 and QA-d3 were purchased from Medical Isotopes, Inc.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eCalibration curve preparation\u003c/h2\u003e\u003cp\u003eIndividual analytes and internal standard (IS) primary stock solutions include: 10 mM (NAM; NMN; NaMN, NaAD; ADPr): 20 mM (NAD+; AMP); 2 mM (NAD\u003csup\u003e+\u003c/sup\u003e-d4); 5 mM (NaMN-d3) and 10 mM (NAM-d4; NMN-d4; AMP-15N5), were prepared individually in water corrected for lot purity and salt. For the Kynurenine pathway metabolites, individual analytes and corresponding internal standards (IS) 10 mM primary stock solutions were prepared separately in water containing 0.1% formic acid and 0.02% ascorbic acid corrected for lot purity. Intermediate stock solutions were prepared by mixing individual stock solutions of each analyte followed by dilution. These intermediate stock solutions were serially diluted with water to obtain a series of standard spiking solutions, which were used to generate the calibration curve. Calibration curves were prepared by spiking 10 \u0026micro;L of each standard working solution into 25 \u0026micro;L of homogenization buffer (0.5N perchloric acid in water) followed by addition of 10 \u0026micro;L internal standard solutions. For NAD\u003csup\u003e+\u003c/sup\u003e metabolites: 4 \u0026micro;M for NAD\u003csup\u003e+\u003c/sup\u003e-d4 and AMP-15N5; 2 \u0026micro;M for NAM-d4; NMN-d4 and NaMN-d3. A calibration curve was prepared fresh with each set of samples. Using 25 \u0026micro;L aliquots, the calibration curve range for NAD\u003csup\u003e+\u003c/sup\u003e and AMP was 0.016\u0026ndash;160 \u0026micro;M; for NAM; NMN; NaMN; NaAD; and ADPr was 0.008\u0026ndash;80 \u0026micro;M.\u003c/p\u003e\u003cp\u003eCalibration curves for the Kynurenine pathway metabolites were prepared likewise. Final concentration of IS in the sample was 4000nM for TRP-d5; 400nM for QA-d3, 3HANA-d3, 3HK-d3 and 200nM for KYN-d4 and KA-d5. A calibration curve was prepared fresh with each set of samples. Using 25 \u0026micro;L aliquot the calibration curve range for TRP 4 nM \u0026ndash; 20 uM; KYN 0.4\u0026ndash;4000 nM; KA 0.4\u0026ndash;1000 nM; 3HK 2\u0026ndash;4000 nM; 3HANA and QA 10\u0026ndash;4000 nM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSample collection and extraction procedure\u003c/h2\u003e\u003cp\u003eThe extraction procedure was modified from that of Liang X. et al.\u003csup\u003e(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/sup\u003e Samples isolated from mice include blood monocytes, infarcted cortex, and gut. Samples were weighed and placed into lysing matrix D vials containing 1.4 mm zirconium-silicate spheres (1169130-CF; MP Biomedicals, Solon, OH); snap-frozen with liquid nitrogen and kept frozen at -80\u0026deg;C until analysis. Before LC-MS/MS analysis, samples were removed from the \u0026minus;\u0026thinsp;80\u0026deg;C freezer and placed on ice. A cold solution of 0.5N perchloric acid was added to frozen samples in the following amounts: Monocytes \u0026ndash; 100 \u0026micro;L; cortex and gut \u0026ndash; 200 \u0026micro;L. Samples were homogenized using Bead Mill-4 (15-340-164; Thermo Fisher Scientific, Waltham, MA) twice at 30 seconds at a speed of 5 m/s and then centrifuged. 25 \u0026micro;L of supernatant was used for analysis. 10 \u0026micro;L internal standard solution and 10 \u0026micro;L water solution was added to 25 \u0026micro;L aliquots of supernatant and vortexed. For the NAD\u003csup\u003e+\u003c/sup\u003e metabolites, samples were diluted with 60 \u0026micro;L 20mM ammonium formate buffer with 0.1% formic acid in water, vortexed, centrifuged, transferred to injection vial and analyzed by LC-MS/MS. For the Kynurenine pathway metabolites, samples were diluted with 40 \u0026micro;L 20mM ammonium formate buffer with 0.1% formic acid in water, vortexed, centrifuged, transferred to injection vial and analyzed by LC-MS/MS.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eLC-MS/MS\u003c/h2\u003e\u003cp\u003eSince some of the analytes namely NMN/NaMN and NAD\u003csup\u003e+\u003c/sup\u003e/NaAD have a mass difference of 1 amu, chromatographic separation is critical. At least two selective reaction monitoring (SRM) transitions \u0026ndash; one quantifier and one qualifier \u0026ndash; were carefully selected for each analyte. Distinctive qualifier to quantifier ion intensity ratios and retention times were essential to authenticate the target analytes. All analyses were carried out by positive electrospray LC-MS/MS using a Waters Acquity I-class LC system with Waters Xevo TQ-XS triple quadrupole mass spectrometer (RRID:SCR_018510). Chromatographic conditions include: Atlantis\u0026reg; T3, 2.1x100 mm, 3 \u0026micro;m particle size column (186003718; Waters Corp. Milford, MA) was operated at 30\u0026deg;C with a flow rate of 0.2 mL/min. Mobile phases consisted of A: 20 mM ammonium formate/0.1% formic acid in water and B: 20 mM ammonium formate/0.1% formic acid in acetonitrile/methanol/water (45:45:10). Elution profile include: initial hold at 0% B for 3 minutes, followed by a gradient of 0%-30% in 6 minutes, then 30%-95% in 2 minutes, hold at 95% for 1 minute; total run time was 15 minutes. Injection volume was 10 \u0026micro;L.\u003c/p\u003e\u003cp\u003eSRM was used for quantification. The mass transitions were as follows: NAM: m/z 122.58 \u0026rarr; m/z 105.42 (quantifier); m/z 122.58 \u0026rarr; m/z 79.35 (qualifier); NMN: m/z 335.03 \u0026rarr; m/z 122.49 (quantifier); m/z 335.03 \u0026rarr; m/z 96.37 (qualifier); NaMN: m/z 336.03 \u0026rarr; m/z 123.46 (quantifier); m/z 336.03 \u0026rarr; m/z 96.36 (qualifier); ADPr: m/z 560.03 \u0026rarr; m/z 135.59 (quantifier) and m/z 560.03 \u0026rarr; m/z 347.96 (qualifier); NAD\u003csup\u003e+\u003c/sup\u003e: m/z 664.16 \u0026rarr; m/z 135.55 (quantifier) and m/z 664.16 \u0026rarr; m/z 428.09 (qualifier); NaAD: m/z 665.16 \u0026rarr; m/z 135.56(quantifier) and m/z 665.16 \u0026rarr; m/z 428.04 (qualifier); AMP: m/z 347.93 \u0026rarr; m/z 135.56 (quantifier) and m/z 347.93 \u0026rarr; m/z 118.49 (qualifier); NAM-d4: m/z 126.58 \u0026rarr; m/z 83.16 ; NMN-d4: m/z 339.03 \u0026rarr; m/z 126.53; NaMN-d3: m/z 338.5 \u0026rarr; m/z 126.54; NAD\u003csup\u003e+\u003c/sup\u003e-d4: m/z 668.16 \u0026rarr; m/z 135.56; and AMP 15N5: m/z 353.19 \u0026rarr; m/z 140.60. Dwell time was 25 ms.\u003c/p\u003e\u003cp\u003eFor the Kynurenine pathway metabolites, the mass transitions were as follows: TRP: m/z 204.776 \u0026rarr; m/z 131.594 (quantifier); m/z 204.776 \u0026rarr; m/z 117.525 (qualifier); KYN: m/z 208.72 \u0026rarr; m/z 93.427 (quantifier); m/z 208.72 \u0026rarr; m/z 117.516 (qualifier); 3HK: m/z 224.776 \u0026rarr; m/z 109.528 (quantifier); m/z 224.776 \u0026rarr; m/z 165.654 (qualifier); KA: m/z 189.656 \u0026rarr; m/z 143.548 (quantifier) and m/z 189.656 \u0026rarr; m/z 115.546 (qualifier); 3HANA: m/z 153.584 \u0026rarr; m/z 107.504 (quantifier) and m/z 153.584 \u0026rarr; m/z 135.576 (qualifier); QA: m/z 167.584 \u0026rarr; m/z 105.479 (quantifier) and m/z 167.584 \u0026rarr; m/z 77.314 (qualifier); TRP-d5: m/z 209.84 \u0026rarr; m/z 149.711 ; KYN-d4: m/z 212.84 \u0026rarr; m/z 97.492; 3HK-d3: m/z 227.784 \u0026rarr; m/z 110.708; KA-d5: m/z 194.72 \u0026rarr; m/z 93.245; and 3HANA-d3: m/z 156.648 \u0026rarr; m/z 138.591; QA-d3 m/z 170.648 \u0026rarr; m/z 108.497. Dwell time was also 25 ms.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eQuantification\u003c/h2\u003e\u003cp\u003eQuantitative analysis was done with TargetLynx quantification software (Waters Corp. Milford, MA) using an internal standard approach. NAM-d4; NMN-d4; NaMN-d3 were the internal standards used for quantification of NAM; NMN; NaMN, respectively; NAD\u003csup\u003e+\u003c/sup\u003e-d4 for NAD\u003csup\u003e+\u003c/sup\u003e and NaAD; and AMP15N5 for AMP and ADPr. TRP-d5; KYN-d4; 3HK-d3; KA-d5; 3HANA-d3 and QA-d3 were the internal standards for quantification of TRP; KYN; 3HK; KA; 3HANA and QA, respectively. Calibration curves were linear (R\u0026thinsp;\u0026gt;\u0026thinsp;0.98) over the concentration range using a weighting factor of 1/X2 where X is the concentration. The back-calculated standard concentrations were \u0026plusmn;\u0026thinsp;15% from nominal values, and \u0026plusmn;\u0026thinsp;20% at the lower limit of quantitation (LLOQ).\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eRotarod\u003c/h2\u003e\u003cp\u003eThe rotarod test was used to evaluate motor function and coordination. Mice were pretrained on the rotarod for one week before stroke was induced, with 3 trials separated by 15-minute inter-trial intervals. The mice were kept on the rotarod until they learned to stay on for 300 sec. Training began with a constant low-speed rotation (5 rpm) for 5 minutes on the first day of training. On all subsequent days, each mouse was placed on the rod with the rotation speed increasing from 4\u0026ndash;40 rpm (ramping) over 5 minutes. Testing was performed on the day before stroke and the day after stroke and involved placing the mice on the rotarod for a maximum of 300 sec and measuring the length of time they remained on the rotarod with ramping, and reverse ramping speeds before falling on the soft pad below. The rotarod (Ugo Basile, Italy) consisted of a striated rod (consisting of 5 lanes with diameter: 3 cm; rod width: 5.8 cm; fall height: 16 cm).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eFor all experiments, mice were randomly assigned to either sham, stroke, vehicle-treated or NR-treated groups. Investigators were blinded to group allocation during data collection and analysis. No power analysis was performed to determine sample sizes; prior literature using similar experimental paradigms that yielded interpretable results and the laboratory\u0026rsquo;s previous experience was used.\u003csup\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/sup\u003e All analyses were performed using either GraphPad Prism software version 10 (GraphPad Software, LaJolla, CA), R statistical package software version R 4.4.0, or Metaboanalyst 6.0. Unless otherwise specified, values represent the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of at least three independent experiments. Data were analyzed by unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test with Welch\u0026rsquo;s correction, one-way ANOVA, two-way ANOVA or repeated measures two-way ANOVA with Tukey\u0026rsquo;s posthoc or Bonferroni multiple comparison test to determine significance. Normality of the distribution of the data was tested with Kolmogorov\u0026ndash;Smirnov normality tests using the column statistics function of GraphPad Software. Metabolomics data analysis was carried out using MetaboAnalyst 6.0. Metabolomic data were log-transformed and scaled according to the auto-scaling feature (mean-centered and divided by the standard deviation of each variable). Metabolites that were significantly different by ANOVA (with FDR correction) were subjected to hierarchical clustering analysis using the Euclidean distance measure and Ward clustering algorithm. Differentially expressed metabolites underwent pathway-based enrichment analysis using 84 metabolite sets based on KEGG metabolic pathways in MetaboAnalyst 6.0.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eAging impairs blood monocyte energy metabolism after stroke.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate acute age-dependent changes in the energy metabolism of blood monocytes in response to stroke, we subjected young (3\u0026ndash;6 month) and aged (18\u0026ndash;20 month) male mice to distal middle cerebral artery occlusion (dMCAo), and isolated blood monocytes at 4.5 hours, 24 hours and 72 hours post stroke (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We assessed the metabolic function of blood monocytes using a Mitostress test that induces mitochondrial stress to measure basal respiration (BR) and ATP production, indicators of oxidative phosphorylation, and extracellular acidification rates (ECAR), an indicator of glycolytic activity. At 4.5 hours after stroke, there were no differences in BR, ATP production or ECAR in the blood monocytes from young stroke compared to sham mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Significant increases in BR and ATP production but no changes in ECAR were observed in aged stroked compared to sham mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). At 24 hours post stroke, BR was moderately reduced and ATP production and ECAR were significantly lower in young stroked mice compared to sham (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In aged stroked mice, while previously increased at 4.5 hours, BR, ATP production and ECAR were significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), suggesting metabolic exhaustion in the blood monocytes compared to aged sham. By 72 hours after stroke, all metabolic parameters rebounded to sham levels in young mice, while in aged stroked mice, the decrease in BR and ATP production persisted with ECAR unchanged, suggesting that the blood monocytes remained metabolically depleted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTogether, these results indicate that blood monocytes in young stroked mice experience a brief depletion of metabolic reserves under stress at 24 hours post stroke, followed by recovery. In contrast, blood monocytes in aged stroked mice initially exhibit an elevated metabolic response, engaging both oxidative phosphorylation and glycolysis to fuel their activation followed by a prolonged phase of metabolic exhaustion.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAge-dependent changes in blood monocyte NAD\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eand quinolinic acid levels following stroke.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further explore the metabolic response of blood monocytes following stroke, we conducted metabolomic analyses to identify stroke-induced alterations in metabolic pathways and assess how these changes are affected by age (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u0026ndash;S3). Enrichment pathway analyses revealed changes in the nicotinamide adenine dinucleotide (NAD\u003csup\u003e+\u003c/sup\u003e) metabolic pathway at all time-points in aged stroked mice that were absent in young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Figure S4A\u0026ndash;C). NAD\u003csup\u003e+\u003c/sup\u003e plays a critical role in specifying immune responses in aged mice,\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/sup\u003e and is synthesized via three major pathways: the Preiss-Handler, Salvage and the Kynurenine pathways (KP), also known as the NAD\u003csup\u003e+\u003c/sup\u003e \u003cem\u003ede novo\u003c/em\u003e biosynthetic pathway.\u003csup\u003e(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e The KP is a significant source of NAD\u003csup\u003e+\u003c/sup\u003e during an immune challenge,\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/sup\u003e and has been shown to be activated in stroke patients.\u003csup\u003e(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e)\u003c/sup\u003e Activation of the KP has been linked to stroke severity, with higher kynurenine/tryptophan ratios observed in the blood plasma of patients who died from stroke.\u003csup\u003e(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e Quinolinic acid (QA), a metabolite of the KP, which is converted to the NAD\u003csup\u003e+\u003c/sup\u003e precursor nicotinamide adenine mononucleotide (NaMN), is a neurotoxic molecule that binds \u003cem\u003eN\u003c/em\u003e-methyl-\u003cem\u003eD\u003c/em\u003e-aspartate (NMDA) receptors to induce excitotoxicity in neurons, and is a potent mediator of inflammation.\u003csup\u003e(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eTherefore, we investigated if stroke regulates NAD\u003csup\u003e+\u003c/sup\u003e and QA levels in blood monocytes in an age-dependent manner. At 4.5 hours after stroke, we found no differences in NAD\u003csup\u003e+\u003c/sup\u003e levels in the blood monocytes from young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), however, NAD\u003csup\u003e+\u003c/sup\u003e was significantly increased in blood monocytes from aged stroked mice compared to sham (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). At 24 hours post-stroke, NAD\u003csup\u003e+\u003c/sup\u003e levels in blood monocytes from young mice were significantly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In contrast, while aged mice exhibited increased NAD\u003csup\u003e+\u003c/sup\u003e at 4.5 hours post-stroke, levels were significantly reduced by 24 hours, indicating NAD\u003csup\u003e+\u003c/sup\u003e depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). At 72 hours post stroke, NAD\u003csup\u003e+\u003c/sup\u003e levels of blood monocytes from young and aged stroked mice had rebounded to sham levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eQA levels were increased in blood monocytes from young and aged mice at 4.5 hours post stroke (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The increase in QA persisted at 24 hours post stroke in both young and aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). However, by 72 hours post stroke, QA levels rebounded to sham levels in young mice but remained elevated in aged stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Overall, these results suggest that the Kynurenine pathway (KP) is activated to boost NAD⁺ production in blood monocytes after stroke in both young and aged mice. However, in aged mice, the conversion of quinolinic acid (QA) to the NAD⁺ precursor NaMN appears to be impaired, resulting in QA accumulation (Figure S4D).\u003c/p\u003e\u003cp\u003e\u003cb\u003eReplenishing NAD\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003elevels increases oxygen consumption rates in blood monocytes from aged mice after stroke.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNicotinamide riboside (NR) increases NAD\u003csup\u003e+\u003c/sup\u003e levels, improves metabolism, reduces inflammation in human monocyte-derived macrophages, and attenuates neuronal loss in a mouse model of acute ischemia.\u003csup\u003e(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/sup\u003e These findings are based on studies in young mouse models and not aged mice, which are more representative of human stroke populations. Here, we tested whether NAD\u003csup\u003e+\u003c/sup\u003e regulates blood monocyte function during stroke in an age-dependent manner. Therefore, we administered either vehicle (water) or NR to young and aged mice via oral gavage every day for 4 weeks and then subjected the mice to stroke (Figure S5A). Blood monocytes were isolated at 24 hours post stroke, given that we observed the most distinct age-related metabolic differences at this time-point. A Mitostress test revealed no differences in basal respiration (BR) and ATP production between vehicle and NR-treated young stroked mice, however, there was a significant decrease in ECAR, suggesting reduced glycolytic rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In aged stroked mice, BR, ATP production and ECAR were all significantly increased with NR treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), consistent with improved oxidative phosphorylation and glycolysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine if NR increased NAD\u003csup\u003e+\u003c/sup\u003e levels via the KP, we measured NaMN as a readout for increased flux through the Priess-Handler and KP, as demonstrated in previous studies.\u003csup\u003e(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/sup\u003e This approach was chosen given the limitations of directly measuring NAD⁺ in small sample sets such as blood monocytes. In the blood monocytes from young mice, there were no differences in NaMN levels between vehicle and NR-treated stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, in the aged stroked mice, NaMN levels were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting NR may have improved QA conversion to NaMN. To confirm that QA was converted to NaMN, we measured QA levels. Indeed, NR modestly decreased QA levels in blood monocytes from young stroked mice, and significantly reduced QA levels in aged stroked mice compared to vehicle-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Together, these results suggest that prolonged pretreatment with NR decreased the stroke-induced increase in QA levels in blood monocytes from aged mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNicotinamide riboside reduces the inflammatory response of blood monocytes after stroke in aged mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine if the inflammatory response of blood monocytes correlates with changes in their metabolic state, we measured immune factors in isolated blood monocytes using a chemokine and cytokine multiplex assay. At 4.5 hours post stroke, macrophage chemoattractant protein/C-C ligand 7 (MCP3/CCL7), a chemokine that plays a role in monocyte/macrophage/neutrophil recruitment through activation of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway to induce cell proliferation and survival,\u003csup\u003e(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/sup\u003e was the only immune factor increased in the blood monocytes from young stroked mice compared to sham including IL-7Rα (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In aged stroked mice, several immune factors were increased, including MCP3/CCL7, indicating increased monocyte cell proliferation, monocyte differentiation, and migration. At 24 hours post stroke, in blood monocytes from young stroked mice, we detected a decrease in macrophage colony-stimulating factor (MCSF1), a cytokine required for monocytic differentiation into macrophages.\u003csup\u003e(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003c/sup\u003e However, in aged stroked mice, there was an increase in the expression of several immune factors that play roles in monocytic activation, proliferation, migration and differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). By 72 hours post stroke, we detected no differences in young mice, while the increase in IL-7Rα persisted in aged stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). IL-7Rα expression promotes antimicrobial activity in monocytes,\u003csup\u003e(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u003c/sup\u003e indicating a potential response to elevated bacterial load resulting from increased gut barrier permeability after stroke.\u003csup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eNext, we measured immune factors in the blood plasma as indicators of systemic, whole-body inflammation. We observed changes in inflammation over time and age-related differences that were exacerbated by stroke (Figure S5B\u0026ndash;D). A majority of these immune factors converge on the JAK/STAT pathway to mediate immune cell proliferation, activation, differentiation and migration as well as downstream signaling to promote inflammation. While young mice exhibited only a mild systemic inflammatory response to stroke within 72 hours, aged mice showed an initially modest response that intensified by 24 hours post stroke before subsiding by 72 hours (Figure S5B\u0026ndash;D).\u003c/p\u003e\u003cp\u003eNext, to determine whether NAD\u003csup\u003e+\u003c/sup\u003e replenishment from pretreatment with NR could influence stroke-induced inflammation, we measured immune factors in blood monocytes from vehicle and NR-treated young and aged mice 24 hours after stroke. No significant differences in any immune factors were observed due to NR treatment in young stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, NR reduced levels of several inflammatory factors that were increased in the blood monocytes from aged stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). We also investigated the effect of NR on systemic inflammation by measuring the levels of immune factors in the blood plasma from sham, vehicle and NR-treated young and aged stroked mice. We observed no differences between all groups of young mice (Figure S6B), however there was a significant reduction in stroke-induced inflammation in aged stroked mice following NR treatment (Figure S6C).\u003c/p\u003e\u003cp\u003eAltogether, these results show a marginal effect of NR on blood monocyte and systemic inflammation in young mice, while in aged mice, NR has significant suppressive effect on stroke-mediated inflammatory responses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNicotinamide riboside alters stroke-induced inflammation in the ipsilateral and contralateral cortex.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, we tested whether NR might also regulate inflammation in the brain of stroked mice. We isolated the ipsilateral infarct core and a corresponding area in the contralateral brain at 24 hours post stroke in vehicle and NR-treated young and aged stroked mice and performed a chemokine and cytokine multiplex assay. We determined that NR reduced all detected immune factors in the ipsilateral cortex of both young and aged stroked mice, with the exception of IL-1α and MDC/CCL22 in the aged stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), both of which were increased and play a role in immune cell recruitment and regulation. In the contralateral cortex of young stroked mice, we observed no effect of NR on the expression of immune factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In aged mice, however, several cytokines and chemokines were decreased while others were elevated, indicating an altered inflammatory response in the contralateral cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe then assessed the effect of NR pretreatment on brain NAD\u003csup\u003e+\u003c/sup\u003e levels. NR increased NAD⁺ levels in the contralateral cortex of young mice but did not affect NAD⁺ levels in the ipsilateral cortex, while in aged stroked mice, NR significantly elevated NAD\u003csup\u003e+\u003c/sup\u003e levels in both hemispheres (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results suggest that NR alters the immune response in both the ipsilateral and contralateral hemispheres in aged mice following stroke, and these changes may be due to the levels of NAD\u003csup\u003e+\u003c/sup\u003e availability in the brain.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNicotinamide riboside changes the composition of macrophage cell populations in the gut.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMonocytes traffic to the gut lamina propria where they critically maintain the gut barrier,\u003csup\u003e(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/sup\u003e which becomes compromised following stroke.\u003csup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/sup\u003e Therefore, we investigated effects of NR pretreatment on lamina propria macrophage cell composition using flow cytometry (Figure S6A). The number of gut monocyte-derived macrophages (MDMs) identified as CD45\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e, Ly6C\u003csup\u003e+\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003e cells (Figure S6A) and tissue resident macrophages (TRMs), identified as CD45\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e, Ly6C\u003csup\u003e+\u003c/sup\u003e, CD62L\u003csup\u003e+\u003c/sup\u003e cells (Figure S6A) did not change with stroke at 24 hours but was significantly increased with NR treatment in young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In aged mice, stroke significantly reduced the populations of gut MDMs and TRMs, however this was completely prevented by NR pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), suggesting a beneficial effect of increased cellular NAD\u003csup\u003e+\u003c/sup\u003e in gut macrophages and/or broadly across the gut epithelium.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we investigated the populations of P1 (newly infiltrating inflammatory monocytes \u0026ndash; Ly6C\u003csup\u003eHi\u003c/sup\u003e MHCII\u003csup\u003eLow\u003c/sup\u003e), P2 (inflammatory monocytes \u0026ndash; Ly6C\u003csup\u003eInt\u003c/sup\u003e MHCII\u003csup\u003eHi\u003c/sup\u003e) and P3 (new tissue resident macrophages \u0026ndash; Ly6C\u003csup\u003elow\u003c/sup\u003e MHCII\u003csup\u003eHi\u003c/sup\u003e) cells in the gut that consists of both MDMs and TRMs.\u003csup\u003e(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e)\u003c/sup\u003e The populations of P1, P2 and P3 macrophages were decreased with stroke in aged but not young stroked mice, and NR prevented loss of the P1 populations in aged stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To further characterize the polarization states of gut macrophages, we assessed the number of cells positive for CD71 (marker for proliferating macrophages and required for phagocytosis),\u003csup\u003e(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e)\u003c/sup\u003e and MHCII (marker for activated macrophages and antigen presentation).\u003csup\u003e(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)\u003c/sup\u003e In the gut of both young and aged stroked mice, NR increased the populations of CD71\u003csup\u003e+\u003c/sup\u003e and MHCII\u003csup\u003e+\u003c/sup\u003e MDMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), and restored stroke-induced changes in the population of CD71\u003csup\u003e+\u003c/sup\u003e and MHCII\u003csup\u003e+\u003c/sup\u003e TRMs in the gut of both young and aged stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). We also assessed CD71\u003csup\u003e+\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e P1, P2 and P3 macrophages, and while we did not detect CD71\u003csup\u003e+\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e P1 and P3 macrophages in both young and aged mice, NR increased the populations of CD71\u003csup\u003e+\u003c/sup\u003e and MHCII\u003csup\u003e+\u003c/sup\u003e P2 macrophages in young stroked mice and restored these populations in aged stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eFinally, we evaluated overall intestinal inflammation and determined that NR had no effect on general inflammation in the gut of young mice 24 hours after stroke (Figure S6D). However, in the gut of aged stroked mice, NR restored the expression of several immune factors to sham levels 24 hours after stroke, thus reducing inflammation in the gut (Figure S6E). Together, these results suggest that NR administration prior to stroke protects against stroke-induced decline in intestinal macrophages and reduces gut inflammation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNicotinamide riboside reduces stroke-induced gut permeability in aged mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStroke compromises gut barrier integrity by triggering adrenergic signals to the intestine, causing a transient but consequential disruption of the gut epithelium. This allows the translocation of microbial components across the gut epithelium and hyperactivation of lamina propria macrophages, and this persistent gut barrier damage increases the risk of bacterial infection and worsens stroke outcomes.\u003csup\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/sup\u003e Given the effect of NR on monocyte/macrophage cell populations in the gut, we next tested whether gut permeability might be improved following NR pretreatment. Fluorescein isothiocyanate-labeled dextran (FITC-Dextran) was orally administered to sham, vehicle and NR-treated young and aged stroked at 24 hours post stroke, and levels measured in the blood plasma 4 hours later. NR treatment did not alter plasma FITC-Dextran levels in young stroked mice. However, in aged stroked mice, NR administration resulted in FITC-Dextran levels comparable to sham mice, indicating reduced gut barrier permeability relative to vehicle-treated stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To confirm these beneficial effects of NR on gut barrier integrity, we measured endotoxin lipopolysaccharide (LPS) levels in plasma 24 hours after stroke. No differences were observed in young mice, however, endotoxin levels were significantly reduced in NR-treated aged compared to vehicle-treated stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), confirming that NR rescued stroke-induced increases in gut barrier permeability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further probe the integrity of the gut epithelium, we immunostained for tight junction proteins, zona occludens-1 (ZO-1) and epithelial cell adhesion molecule (EpCAM), and probed for CD11b, a marker for resident macrophages. NR had no effect on levels of ZO-1 and EpCAM but restored expression of CD11b to sham levels in the young stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In aged stroked mice, NR restored expression of ZO-1 and CD11b to sham levels, and marginally increased EpCAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eTo assess whether improved gut integrity was linked to changes in NAD⁺, we measured levels of NAD\u003csup\u003e+\u003c/sup\u003e and nicotinic acid (NA), which studies have shown increases as a result of NR metabolism by the gut microbiome\u003csup\u003e(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In the gut from young mice, no differences were observed in NA or NAD\u003csup\u003e+\u003c/sup\u003e levels between sham, vehicle and NR-treated stroked mice, however, both NA and NAD\u003csup\u003e+\u003c/sup\u003e levels were significantly increased in the gut of aged NR-treated stroked mice compared to sham and vehicle-treated stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These findings support highly beneficial effects of NR pretreatment in aged stroked mice with protection of gut barrier function.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNicotinamide riboside improves stroke induced motor deficits.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the improved metabolism and reduced inflammation in aged stroked mice from NR pretreatment, we then assessed other measures of stroke severity. We evaluated motor coordination and balance by subjecting sham, vehicle and NR-treated young and aged mice to a rotarod test, which measures the latency to fall from an accelerating/decelerating rotating rod, 24 hours after stroke. The latency to fall from the rotarod was improved in NR-treated young stroked mice in the first trial, however by the third trial, there were no significant differences between sham, vehicle and NR-treated young stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Likewise, NR-treated aged stroked mice performed better in the first trial compared to sham and vehicle-treated stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Next, we reversed the direction of motion of the rotarod to challenge motor coordination and determine if the mice would self-correct and remain on the rotarod. For both young and aged mice, NR-treated mice had a higher latency to fall compared to sham and vehicle-treated stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn line with this improved motor control, we also observed that infarct area, measured at 24 hours after stroke, was reduced in NR-treated young and aged stroked mice compared with sham and vehicle-treated stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). We also investigated the number of IBA1-positive cells expressing CD68 to measure activation of microglia in the infarct area. In the young, NR marginally decreased expression of IBA1 and CD68 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), and in aged NR-treated stroked mice, both IBA1 and CD68 expression were significantly reduced compared to vehicle-treated stroked mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Interestingly, the ratio of CD68/IBA1 expression was unchanged in both young and aged NR-treated stroked mice, suggesting that NR had no effect on overall microglial activation. Altogether, NR pretreatment protects against stroke-related motor deficits and reduces stroke infarct size in both young and aged mice.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe precise cause of the robust and maladaptive inflammatory response to stroke in aged individuals is unclear. This is the first study to investigate the metabolism of blood monocytes and evaluate the effect of boosting NAD\u003csup\u003e+\u003c/sup\u003e levels on the response of peripheral blood monocytes to stroke. Nicotinamide riboside (NR) has been shown to enhance NAD\u003csup\u003e+\u003c/sup\u003e levels and alter the metabolism and inflammatory status of human blood monocytes by increasing purine levels required for nucleotide synthesis.\u003csup\u003e(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/sup\u003e Here, we find that prolonged pretreatment with NR prior to stroke reduces detrimental age-dependent metabolic and inflammatory changes in blood monocytes and prevents post-stroke increases in gut barrier permeability. Our studies demonstrate that stroke triggers age-dependent metabolic changes in blood monocytes by reducing NAD\u003csup\u003e+\u003c/sup\u003e levels and increasing levels of the neurotoxic quinolinic acid (QA). NR supplementation improves stroke outcomes by broadly replenishing NAD⁺ levels in blood monocytes, intestine, and brain, while mitigating inflammation and endotoxemia caused by gut barrier disruption.\u003c/p\u003e\u003cp\u003eSeveral studies have investigated acute and chronic transcriptomic and inflammatory changes of immune cells in the blood and brain-infarct areas of young and aged mice after stroke,\u003csup\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/sup\u003e however, the metabolic status of immune cells after stroke has not been explored. In this study, we focused on peripheral blood monocytes because these cells infiltrate the brain in response to stroke, alongside neutrophils, to drive secondary stroke injury. Because of the link between metabolism and immune function, we investigated the metabolic state of blood monocytes immediately post-stroke during the period in which they traffic to the ischemic brain. Our results reveal age-dependent metabolic and inflammatory changes over time, with the most pronounced changes occurring 24 hours post stroke.\u003c/p\u003e\u003cp\u003eWhile several metabolites changed over time in both young and aged blood monocytes, the NAD\u003csup\u003e+\u003c/sup\u003e metabolome was altered across all time points only in aged stroked mice, leading us to investigate NAD\u003csup\u003e+\u003c/sup\u003e biosynthetic pathways, including the Kynurenine pathway (KP). Activation of the KP correlates with increased stroke severity.\u003csup\u003e(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e QA is generated by the KP and the elevated levels of QA in aged blood monocytes observed in our study support both the activation of the KP in monocytes after stroke as well as the failure to convert to the NAD\u003csup\u003e+\u003c/sup\u003e precursor, nicotinic acid mononucleotide (NaMN). In the brain, QA induces excitotoxicity in neurons by activating NMDA receptors.\u003csup\u003e(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)\u003c/sup\u003e QA can also reduce nucleotide levels including GTP, ATP and NAD\u003csup\u003e+\u003c/sup\u003e, leading to mitochondrial dysfunction in synapses and neuronal damage.\u003csup\u003e(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003c/sup\u003e The increased monocytic QA level in aged mice could potentially worsen excitatory injury following trafficking to the ischemic brain.\u003c/p\u003e\u003cp\u003eReplenishing NAD\u003csup\u003e+\u003c/sup\u003e levels is neuroprotective in young mouse models of stroke and in aging.\u003csup\u003e(\u003cspan additionalcitationids=\"CR40 CR41 CR42\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e)\u003c/sup\u003e We investigated the effects of NR supplementation on stroke outcome in young and aged mice, focusing on the effects on blood monocytes and gut barrier integrity. The choice of NR compared to nicotinamide (NAM) and nicotinamide mononucleotide (NMN) was made due to its robust enhancement of mitochondrial function, the feasibility of daily oral administration, avoidance of the side effects associated with NAM, and the instability of NMN.\u003csup\u003e(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e)\u003c/sup\u003e Prolonged daily NR pretreatment in aged mice enhanced basal respiration, ATP production, and glycolytic activity and reduced expression of multiple pro-inflammatory molecules in blood monocytes following stroke. Additionally, increased NaMN and decreased QA levels in aged blood monocytes suggested enhanced KP metabolic flux. Overall, NR had a more potent effect on aged mice compared to young mice as the elevated levels of inflammatory mediators in both blood monocytes and plasma were most reduced in aged mice.\u003c/p\u003e\u003cp\u003eIncreased gut permeability and dysbiosis are significant contributors to neuroinflammation and worse stroke outcomes in aging.\u003csup\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e)\u003c/sup\u003e While NR did not show any measurable effects in young mice, it reduced gut permeability in aged stroked mice. Indeed, NR has been shown to improve ethanol-induced gut barrier damage via mitochondrial sirtuins in intestinal epithelial cells.\u003csup\u003e(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e)\u003c/sup\u003e Moreover, in aged mice, pretreatment with NR completely prevented age-associated losses of MDMs and TRMs in the intestinal lamina propria which are critical to gut barrier function. The improvement in gut barrier function observed in aged NR-pretreated stroked mice may be the result of improved overall epithelial health from increased absorption of NR and NMN, as well as improved MDM and TRM immune states, as reflected by reversal of inflammatory markers CD71\u003csup\u003e+\u003c/sup\u003e and MHCII\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlongside the overall improved immune state of monocytes and intestinal macrophages, NR pretreatment also improved motor coordination in both young and aged stroked mice, reduced infarct size and mitigated inflammation in the brain. A recent study performed in young mice demonstrated reduced stroke infarct volume with a single post-ischemic NR treatment, but poorer stroke outcomes with three days of pre-ischemic NR treatment.\u003csup\u003e(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e)\u003c/sup\u003e In that study, it is possible that the timing was not sufficient for cells to metabolically adapt to such high levels of NAD\u003csup\u003e+\u003c/sup\u003e prior to the ischemic event. The authors also observed improved outcome with a single post-ischemic dose of Nam, however, it is important to note that several other studies have demonstrated Nam inhibition of sirtuins, essential mediators of metabolic homeostasis, as well as adverse effects of Nam on liver function.\u003csup\u003e(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eA limitation of the current study is that only male mice were examined. There are well established mechanistic differences between male and female responses to stroke,\u003csup\u003e(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e)\u003c/sup\u003e and potential sex-specific metabolic differences will be explored in future studies. Regarding the metabolism of NR, further studies are required to understand how NR is metabolized in the gut by the gut microbiome after stroke. It is known that NR is converted to nicotinamide mononucleotide (NMN) by nicotinamide riboside kinase (NRK1) and then to NAD\u003csup\u003e+\u003c/sup\u003e, however, oral administration of NR leads to an increase in nicotinic acid (NA) which is metabolized to nicotinic acid mononucleotide (NaMN) by the gut microbiome,\u003csup\u003e(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e)\u003c/sup\u003e a common metabolite of the Kynurenine (KP) and Preiss-Handler pathways, suggesting activation of either pathway by NR through a mechanism that is not fully understood. Another limitation is the technical challenge in measuring NAD\u003csup\u003e+\u003c/sup\u003e levels in very small samples such as blood monocytes due to its rapid turnover, low analyte concentration and instability. To address this limitation, we measured NaMN as a readout for increased NAD\u003csup\u003e+\u003c/sup\u003e levels when undetected, given that orally administered NR has been shown to significantly increase NaMN.\u003csup\u003e(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn conclusion, we identified age-related metabolic alterations in monocytes following stroke, characterized by reduced NAD⁺ and elevated QA levels, which were associated with changes in inflammatory factors. Additionally, we showed that prolonged NR pretreatment effectively replenishes NAD⁺, enhances the metabolic and inflammatory responses of blood monocytes to stroke, and broadly improves stroke outcomes by strengthening gut barrier function and reducing inflammation. Our findings highlight the potential of NR as a preventive therapeutic strategy for patients at high risk of cardiovascular and cerebrovascular disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eAuthors and Affiliations\u003c/h2\u003e\u003cp\u003eDepartment of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH, USA (C.A.I., B.A.E), Department of Neurology and Neurological Science, Stanford University School of Medicine, Stanford, CA, USA (C.A.I., K.I.A., M.C., J.H., H.E.E., Y.A.A., Q.W.), Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA (K.I.A.), Stanford Neurosciences Graduate Program, Stanford University, Stanford, CA, USA (J.H.), Mass Spectrometry Center, Stanford University, Stanford, CA, USA (L.A.), Department of Biochemistry and Molecular Biology, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA (A.I.A., M.R.M.), Department of Molecular Biosciences, The University of Texas at Austin, TX, USA (M.C.), Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel (E.B.), Chan Zuckerberg Biohub, San Francisco, CA, USA (K.A.I).\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding sources\u003c/h2\u003e\u003cp\u003eThis work was supported by Burroughs Wellcome Postdoctoral Diversity Enrichment Program grant no. G-1022372 (C.A.I). The Howard Hughes Medical Institute Hanna H. Gray Fellows Program Faculty Phase grant no. GT15655 (M.R.M). The Burroughs Welcome Fund PDEP Transition to Faculty grant no. 1022604 (M.R.M). NIH Grants T32GM108563 (A.I.A.), RO1NS100180 (K.I.A.). The American Heart Foundation grant no. 19PABH1345800 (K.I.A.). K.I.A. is a Chan Zuckerberg\u0026ndash;San Francisco Biohub Investigator.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.A.I and K.I.A. conceived and planned this study. C.A.I., M.R.M., and K.I.A. contributed to supervising the experimental design. C.A.I., H.E., M.C., A.I.A., L.A., Y.A.A., Q.W., and E.B. conducted the experiments. C.A.I., H.E., M.C., A.I.A., L.A., J.H., B.A.E, and M.R.M performed collection of the data and statistical analysis. C.A.I. and K.I.A. wrote the manuscript. All authors reviewed and approved of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eWe would like to acknowledge the Stanford University Mass Spectrometry core facility for the use of the Xevo TQ-XS mass spectrometer system (RRID:SCR_018510) that was purchased with funding from National Institutes of Health Shared Instrumentation Grant S10OD026962. We thank the Stanford University Cell Sciences Imaging Core Facility for the use of the Leica SP8 confocal microscope (RRID: SCR_017787), supported by the Award Number 1S10OD010580-01A1 from the National Center for Research Resources (NCRR). We would also like to acknowledge the Stanford University Human Immune Monitoring Center (HIMC) and the Case Western Reserve University Bioanalyte core facility for the Luminex experiments. We would also like to acknowledge the Huck Institutes\u0026rsquo; Metabolomics core facility (RRID:SCR_023864) for use of the OE 240 LC-MS and Sergei Koshkin for helpful discussions on sample preparation and analysis. Finally, we thank Chromadex for providing the Nicotinamide Riboside used in this study.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eThe datasets used and analyzed during the current study are included in this published article and are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOffner H, Subramanian S, Parker SM, Afentoulis ME, Vandenbark AA, Hurn PD. Experimental stroke induces massive, rapid activation of the peripheral immune system. J Cereb Blood Flow Metab. 2006;26(5):654\u0026ndash;65. PubMed PMID: 16121126. 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Epub 20230403.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Stroke, Aging, Inflammation, Metabolism, NAD+, Nicotinamide riboside, Quinolinic acid, Monocytes, Gut","lastPublishedDoi":"10.21203/rs.3.rs-7483662/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7483662/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND:\u003c/strong\u003e Aging exacerbates post-stroke inflammation, contributing to worse neurological outcomes. However, the mechanisms underlying this age-dependent immune dysregulation remain unclear. Because immune cell metabolism critically shapes inflammatory responses, we investigated whether the metabolic state of circulating monocytes, key immune cells that traffic to the ischemic brain, is altered by age after stroke. We further examined whether enhancing cellular NAD⁺ availability with nicotinamide riboside (NR) could mitigate age-associated neuroinflammatory responses and improve stroke outcome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS:\u003c/strong\u003e Ischemic stroke was induced in young and aged mice using the distal middle cerebral artery occlusion model. We assessed monocyte metabolic profiles via untargeted metabolomics, mitochondrial function assays, multi-analyte cytokine/chemokine profiling, and flow cytometry. Given the contribution of monocyte-derived intestinal macrophages to gut barrier disruption after stroke, we evaluated gut barrier integrity, immune cell composition, and systemic inflammation. Stroke outcomes were also determined by infarct size, motor function, and brain inflammatory status.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS:\u003c/strong\u003e Our findings show that the availability of the essential energy co-factor, NAD\u003csup\u003e+\u003c/sup\u003e, is a key age-dependent factor that regulates monocyte and intestinal macrophage immune responses after stroke. Aged monocytes showed decreased NAD\u003csup\u003e+\u003c/sup\u003e levels and increased inflammatory responses compared to young monocytes. Pretreatment with NR elevated cellular NAD\u003csup\u003e+\u003c/sup\u003e levels in aged monocytes, normalized intestinal macrophage numbers and activation states, preserved gut barrier integrity, reduced systemic and brain inflammation, and improved stroke outcomes in aged mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSION: \u003c/strong\u003eThese findings highlight the importance of NAD\u003csup\u003e+\u003c/sup\u003e in mitigating the post-stroke response in aging and the potential of NAD\u003csup\u003e+\u003c/sup\u003e supplementation as a preventive strategy for patients at risk for cerebrovascular disease.\u003c/p\u003e","manuscriptTitle":"NAD⁺ depletion drives age-related monocyte hyperinflammation after stroke and is reversed by nicotinamide riboside","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 15:02:01","doi":"10.21203/rs.3.rs-7483662/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-23T15:55:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T21:56:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279604299915224291580813267863713631588","date":"2025-10-09T20:47:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-26T13:34:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69434971987702765584907840752103644381","date":"2025-09-07T07:43:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-07T00:39:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-03T06:17:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-29T09:59:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2025-08-28T23:42:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"32b0290f-0cd8-4899-b927-d6376e4cebda","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:04:29+00:00","versionOfRecord":{"articleIdentity":"rs-7483662","link":"https://doi.org/10.1186/s12974-025-03638-6","journal":{"identity":"journal-of-neuroinflammation","isVorOnly":false,"title":"Journal of Neuroinflammation"},"publishedOn":"2025-11-27 15:57:02","publishedOnDateReadable":"November 27th, 2025"},"versionCreatedAt":"2025-09-12 15:02:01","video":"","vorDoi":"10.1186/s12974-025-03638-6","vorDoiUrl":"https://doi.org/10.1186/s12974-025-03638-6","workflowStages":[]},"version":"v1","identity":"rs-7483662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7483662","identity":"rs-7483662","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}