After determining that the gut microbiome influences stroke outcomes, we subsequently tested the hypothesis that a heightened inflammatory response, as occurs with “inflammaging,” accompanies the aged microbiome when transplanted into young mice. We further assessed short‐chain fatty acid (SCFA) changes, because these compounds are primarily produced by gut microbiota. Previous studies suggest that these SCFAs decline with age, 26 and SCFAs have important homeostatic and anti‐inflammatory effects. 14 In these studies, we show that aged microbiome leads to an exaggerated systemic inflammatory response and reduced levels of SCFAs in young mice.

Little is known about how the gut‐brain axis changes with aging. This lack of knowledge is noteworthy given that aging alone alters the gut microbiota (dysbiosis) and likely contributes to age‐related inflammation, 4 , 3 - 15 a condition often referred to as “inflammaging.” 16 - 18 Importantly, age‐related dysbiosis may be a major contributor to the increasing prevalence of many age‐related diseases and the poorer outcomes observed in the elderly after acute injury. Stroke is predominately a disease associated with aging, and outcomes after experimental stroke are poorer in aged mice compared to young. 19 - 25 In this work, we first found that the microbiota shifted after stroke in young mice and resembled that observed in uninjured aged mice. We then determined whether aged mice would benefit from manipulation of the gut microbiota to create a more youthful bacterial population. In this study, we first tested the hypothesis that a youthful microbiota, when established in aged mice, produces more positive outcomes following stroke. Conversely, an aged microbiota, when established in young mice, produces more negative outcomes following stroke. We demonstrate that a “youthful” microbiota in aged mice is beneficial both at baseline and after an injury.

Differences in Phyla in the gut microbiota of young and aged mice were analyzed using the unweighted UniFrac distance and plotted in a principal coordinates analysis (PCoA). The UniFrac distance is a measure that takes into account the branch length shared by the young and aged microbiota when placed on a common phylogenetic tree. 38 Survival following MCAO was analyzed using the Mantel‐Cox and Gehan‐Breslow‐Wilcoxon tests.

Parametric analysis consisted of a Student's t test or two‐way analysis of variance (ANOVA) followed by Holm‐Sidak, Bonferroni, or Tukey post‐hoc analysis, when appropriate. Because the NDS consists of categorical data, the Kruskal‐Wallace ANOVA on ranks was used followed by a Dunn's post‐hoc analysis, when appropriate. The Mantel‐Cox and the Gehan‐Breslow‐Wilcoxon tests were used to analyze survival after MCAO.

Values are presented as mean ± standard error of the mean with the exception of categorical data, which are expressed using a box and whisker plot. Statistical significance was set at p ≤ 0.05. For the box and whisker plot, the box boundary closest to zero represents the 25th percentile; the box boundary furthest from zero represents the 75th percentile; the horizontal line within the box represents the median, and the error bars above and below the box represent the 90th and 10th percentile, respectively. When the statistical package could not calculate the 90th and 10th percentile the error bars were omitted. Individual outliers are depicted as circular black symbols.

In the initial study, we determined whether the fecal microbiota, representing bacteria primarily residing in the colon, differed between young (8–12 weeks) and aged (16–18 months) mice. In study 2, we determined whether transient MCAO (60 minutes) altered the gut microbiota. Fecal pellets were collected from young and aged mice before MCAO and 7 days following reperfusion in MCAO mice or an equivalent time for sham‐operated mice. In study 3, we determined whether fecal microbiota could be altered in young mice to resemble that of aged mice using supernatant gavages from fecal suspensions of the aged mice (see above). Conversely, we determined whether fecal microbiota could be altered in aged mice to resemble that in young mice using supernatant gavages from fecal suspensions of the young mice. In study 4, we determined the effect of fecal transplants between young and aged age mice on infarct size and recovery from MCAO. In study 5, adult mice (24 weeks) received either microbiota from aged (18–20 months) or young donors (8–12 weeks) to assess their impact on circulating cytokines following MCAO or sham MCAO. In study 6, young mice (8–12 weeks) and aged mice (16–18 months) received either microbiota from aged (18–20 months) or young donors (8–12 weeks) to assess the changes in fecal SCFAs concentrations following FTG, and recovery was evaluated.

SCFAs were analyzed in fecal samples as previously described. 37 In short, fecal samples were diluted 1:10 (w/v) in 50% aqueous acetonitrile and homogenized. SCFAs in the supernatant were derivatized using a mixture of 12C6‐3‐nitrophenlhydrazine (200mM) and (N‐ethyl‐N'‐(dimethylaminopropyl) carbodiimide hydrolyzed urea derivative (120mM). Samples was further spiked with derivatized 13C6‐3NPH‐HCl and 5‐ul aliquots were analyzed by liquid chromatography/mass spectrometry (electrospray ionization negative mode) using an Acquity UPLC HSS T3 1.8 µm, 2.1 × 100mm high‐performance liquid chromatography column.

Following reperfusion mice were deeply anesthetized using an Avertin overdose; mice were perfused transcardially using ice‐cold PBS solution followed by 4% paraformaldehyde solution to fix tissues. Brains were harvested and cryoprotected in 30% sucrose for 72 hours. Each brain was frozen and sectioned (30 μm) in a coronal plane using a frozen slicing microtome. Sections were stained with cresyl violet and imaged for further analyzing of infarct volumes, expressed as a percentage of the contralateral hemisphere using SigmaScan Pro5 (Systat Software Inc., Chicago, IL). 35 , 36

Motor strength was measured using the hang wire test at 3 and 7 days post‐MCAO or post‐sham MCAO. Mice were placed in the center of a wire‐cage top (18 × 9in) that was slowly inverted and placed at a height of 36in above a cage containing regular bedding. The time that elapsed between inversion to the moment the mouse fell from the wire‐cage top was recorded. Results from three trials for each mouse (45‐minute gap between each trial) was averaged to obtain a single observation for a given mouse.

Mice were placed in the front right corner of a clear, acrylic box (16 × 16in), and activity was assessed for 10 minutes. Activity was measured as the total number of laser beam breaks using a computer‐operated PAS open field system (San Diego Instruments, San Diego, CA). Tests were administered before MCAO and at 3 and 7 days post‐MCAO for 20‐minute intervals using Noldus/EthoVision behavior tracking software.

The Neurological Deficit Score (NDS) was performed to assess MCAO outcomes as previously described. 34 The NDS was scored at reperfusion, 1 and 3 days post‐MCAO using a 5‐point scale where: 0 = no deficit; 1 = forelimb weakness and torso turning to the ipsilateral side when held by tail; 2 = circling to affected side; 3 = unable to bear weight on affected side; and 4 = no spontaneous locomotor activity, or barrel rolling.

Behavioral testing was conducted at the same time of day for each testing time point. Before testing, all mice were acclimated for 1 hour in the testing room in their home cages. All equipment was cleaned with 70% ethanol between trials. Animals were pretested on some of these tests to obtain baseline scores. Mice that died during the experimental timeline were excluded from all the behavioral analysis, including the data for the previous days, to avoid any bias and from infarct‐size analysis. Animal mortality is shown for survival curves.

Transient cerebral ischemia was induced by occluding the right middle cerebral artery (MCA) with silicon sutures, 0.21 and 0.23mm in diameter for young and aged mice, respectively, under isoflurane anesthesia. 32 , 33 After 60 minutes of MCA occlusion (MCAO), the suture was removed to allow for reperfusion. Rectal muscle temperature was monitored and maintained at 37 °C by a Monotherm temperature control system. Sham mice underwent the same procedure; however, the suture was not advanced to the middle cerebral artery. Following ischemic stroke, all animals were given 0.5ml of saline subcutaneously daily for 1 week in addition to access to soft food. In studies where the microbiota was altered by antibiotic and fecal gavages, mice were allowed 1 month to establish a new microbiota before MCAO or sham MCAO was conducted.

Microbiota in fecal samples, representative of that in the distal colon, were collected from mice and stored in sterile tubes at –80 °C until analyzed. Bacteria taxa in each fecal sample were analyzed by amplifying the V4 to V5 hypervariable regions of the 16S ribosomal RNA (rRNA) gene using high‐throughput sequence analysis (Illumina MisSeq platform; Illumina, San Diego, CA). 27 Quality filtered 16S rRNA sequences were clustered into operational taxonomic units (OTUs), with 97% similarity, by closed reference OTU‐picking using the UCLUST algorithm and GreenGenes reference database (v13.5) as implemented in Quantitative Insights Into Microbial Ecology (QIIME versions 1.6 and 1.7). 28 - 30 Sequences were checked for chimeras using ChimeraSlayer with standard options as implemented in QIIME. Sequences not clustered were identified using the Ribosomal Database Project to the lowest possible taxonomic level. 31 The data were randomly rarefied to 10,000 sequences per sample before any downstream analysis.

Recipient mice were treated with 50‐ul antibiotic gavages consisting of 500mg of streptomycin HCl/ml of sterile water for 2 consecutive days. The premise of the antibiotic treatment was to decrease the bacterial load in the recipient mice to reduce competition for the repopulating microbiota from the donor mice. Twenty‐four hours after the second antibiotic gavage, donor supernatant was orally gavaged (50 μl) to recipient mice daily for 5 days. Recipient mice were maintained for up to 2 months after oral gavage. Continued presence of donor biome was confirmed by 16S before ischemic stroke was induced.

Feces from 3 aged mice residing in different cages were collected and pooled; similarly, feces from 3 young mice were collected and pooled. Each pooled sample was diluted in chilled phosphate‐buffered saline solution (PBS; 120 mg feces/1ml buffer) and homogenized for 5 minutes until a paste‐like consistency was achieved. The suspension was vortexed for 1 minute and centrifuged 800 g for 3 minutes. The supernatant was collected, aliquoted, for gavaging into recipient mice and a frozen sample was used for further analysis at a later date.

Mice were ordered from commercial vendors and verified to be free of exclusionary pathogens. For this study, both young and aged C57Bl6 mice were from Charles River Laboratories (Wilmington, MA), mice were aged in‐house, and were all fed a presterilized (irradiated) diet (Picolab rodent diet 20 (#5053) LabDiet, St Louis, MO, USA) for 2 or more months before fecal transplant gavage (FTG) studies. Additional studies were conducted to determine whether age‐related changes in the microbiome were similar in cohorts born and raised in our facility to those mice ordered from commercial vendors and maintained in our facility for a minimum of 2 months to control for dietary and other environmental variables.

This study was conducted in accord with the National Institutes of Health Guidelines for the Care and Use of Animals in research and under protocols approved by the Center for Lab Animal Care at the University of Connecticut Health Center and the University of Texas McGovern Medical School. Young male C57BL/6 male mice (8–12 weeks) and aged in‐house C57BL/6 male mice (18–20 months) were used in this set of studies. One additional cohort of 24‐week‐old male mice was used in study 5 to assess the systemic cytokine response (see below). Mice were housed 4 to 5 per cage in standard facilities with a 12‐hour light/dark schedule in a temperature‐ (21.7–22.7 °C) and humidity‐controlled (40–60 RH) controlled vivarium, with ad libitum access to food and water. It is important to note that the vivarium ambient temperature is set to conform to Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) guidelines, though the temperature inside cages may reach 26 to 28 °C attributed to thermal contribution of animals and that the animals huddle together. An initial cohort of mice were housed in cages with wire mesh bottoms to prevent coprophagy. A second cohort of mice was housed on corncob bedding, and differences between fecal microbiome between the two cohorts were analyzed in order to determine whether housing on mesh affected the gut microbiota. Mice in the facility are housed in individually ventilated cages on irradiated 1/8‐in corncob bedding and receive irradiated, nutritionally balanced, pelleted rodent diet (Picolab rodent diet 20 (#5053) LabDiet, St Louis, MO, USA) and filtered tap water. Cages are serviced under high‐efficiency particulate air filtered animal‐transfer stations. This is a barrier facility that requires investigators to wear personal protective equipment for entry—including disposable gown, shoe covers, hair cover, mask, and gloves.

Results

Differences Exist Between Aged and Young Gut Microbiota Populations We determined whether gut microbiota, represented by the bacteria residing in feces, differed between young (8–12 weeks) and aged (18–20 months) male mice. Figure 1A shows the relative abundance of phyla in the young and aged microbiota. The vast majority of the gut microbiota are represented by bacteria from the phyla, Bacteroidetes and Firmicutes, with the remaining phyla combined representing less than 5% of the total bacteria. Note that aging increases the relative abundance of the Firmicutes while decreasing that of the Bacteroidetes. This shift in these phyla are graphically represented in Figure 1B as the ratio of Firmicutes/Bacteroidetes (F:B). The F:B ratio increased ∼9‐fold with aging (from 0.26 ± 0.04 to 2.31 ± 0.41; p < 0.001; n = 17 and 15 for young and aged, respectively). Furthermore, we confirmed similar F:B ratios in mice born and raised in our facility to that observed in animals purchased from an outside vendor and housed for 4 weeks in our facility. There were no significant differences in microbiome composition between mice born and raised in‐house compared to those purchased for either young mice or aged mice. There was a significant difference between young and aged mice (***p < 0.001 and *p < 0.05 compared to young and aged mice born at different locations). These data are consistent with our results in Figure 1. Thus, the increase in F:B ratio was a function of age and not the vendor or facilities where mice were born. Although there were no significant differences between young or aged mice that were purchased or born in‐house, there were significant differences in the gut microbiome between young and aged animals. Figure 1 Open in figure viewer PowerPoint (A) Pie chart of phyla in the young and age microbiota obtained from 16S rRNA gene analysis in feces. (B) Firmicutes/Bacteroidetes (F:B) ratio in the young and aged microbiota. Firmicutes and Bacteroidetes comprise most of the bacteria in the gut. An increase in the F:B ratio is indicative of dysbiosis. *p < 0.001 (C) Principal coordinates analysis showing bacteria from the microbiota of young (N = 16) and aged mice (N = 11). p = 0.009 between young and aged microbiota using analysis of similarity of UniFrac distance. PC = principal component; rRNA = ribosomal RNA. In these studies, we will use the term “dysbiosis” to describe the increase in F:B ratio attributed to the fact that it represents a detrimental imbalance of the microbiota, and the definition of dysbiosis can differ in the literature.11 We demonstrate in subsequent studies that the aged microbiota fits our definition of dysbiosis. A number of indices have been developed to describe the degree of similarity or distance between microbiota communities. One of these indices, termed UniFrac distance, reduces the microbiota from the community in a single mouse to a coordinate, based on the phylogenetic distances between bacteria determined from the 16S rRNA sequencing.38 A PCoA plot (Fig 1C) demonstrates a clear separation in the UniFrac distances of the gut microbiota in young (circled in the lower right) and aged mice (circled in the upper left). Analysis of Similarity (ANOSIM) using the unweighted UniFrac distance matrix demonstrated that the aged microbiota was statistically significant from the young microbiota (p = 0.009). The PCoA analysis complements the F:B ratios (Fig 1B) in demonstrating that aging has a profound effect on the gut microbiota.

MCAO Causes Significant Shifts in the Microbiota Figure 2A shows relative phyla abundance of the gut microbiota in young and aged mice before and 1 week after a 60‐minute MCAO. As observed with aging, MCAO decreased the relative abundance of the Bacteroidetes while increasing the relative abundance of the Firmicutes in young and aged mice. Note the appearance of other phyla following MCAO especially in the aged mice; the F:B ratios for data described in Figure 2A are shown in Figure 2B. Note that stroke produced dysbiosis in the young mice and produced further dysbiosis in the aged mice as indicated by increased F:B ratios. Seven days poststroke, the F:B ratio increased more than 3‐fold in young mice (p < 0.001; n = 16) compared to that in prestroke young mice. In aged mice, the poststroke F:B ratio increase more than 40% (p = 0.014; n = 14 and 15) compared to prestroke. Although the primary comparisons for these studies is between the F:B ratios pre‐ and poststroke in young mice and the F:B ratios pre‐ and post‐stroke in aged mice, we do note that the F:B ratio in young mice poststroke (0.72 ± 0.13) was still less than the F:B ratio in aged mice preceding stroke (1.37 ± 0.08). Figure 2 Open in figure viewer PowerPoint (A) Stacked bar chart of relative abundance of phyla in aged and young mice before and 1 week following MCAO. (B) Firmicutes/Bacteroidetes (F:B) ratio pre‐ and post‐MCAO in young and aged mice. MCAO significantly increased the F:B ratio in young (*p < 0.012) and aged mice (**p = 0.014). (C) F:B ratio in young and aged recipient mice receiving contents from young and aged donor mice. Two‐way ANOVA revealed a significant recipient effect (p = 0.03) and a significant donor effect (p < 0.001). *p < 0.001 compared to young mice with young microbiota (YY) and **p ≤ 0.001 compared to all other groups using Holm‐Sidak post‐hoc analysis. ANOVA = analysis of variance; MCAO = middle cerebral artery occlusion.

Microbiota Manipulation by Fecal Transplants We determined that after antibiotic treatment, the gut microbiota could be manipulated by gavaging supernatant from fecal suspensions of young mice into aged mice and, conversely, gavaging supernatant of aged mice into young mice. The study consisted of four groups of mice: (1) YY, young mice inoculated with the young microbiota (young control); (2) YA, young mice inoculated with aged microbiota; (3) AA, aged mice inoculated with the aged microbiota (aged control); and (4) AY, aged mice inoculated with young microbiota. One month after gavage from the donor mice, the F:B ratios of the recipient mice were similar to that of the donors (Fig 2C). Two‐way ANOVA revealed both significant recipient (p = 0.026) and donor (p < 0.001) effects (n = 14–16 per group). Mice transplanted with young microbiota showed the lowest F:B ratios regardless of the age of the recipient mice. Also, note that the F:B ratio in YY was similar to that in young mice without transplant (compare F:B ratios in Fig 2C to that in Fig 1B). On the other hand, mice transplanted with aged microbiota showed an increased F:B ratio regardless of the age of the recipient (Fig 2C). Thus, we show that we cannot only successfully manipulate the microbiota to resemble that of the donor mice regardless of the age of the recipient mice, but also that these changes were sustained in the recipient.

Behavioral Outcomes Following MCAO Are Influenced by the Microbiota In the next study, we determined the effect of young or aged microbiota on stroke outcome in cohorts of young and aged mice. The young cohort consisted of the sham‐operated and MCAO groups in that were inoculated with either young microbiota (YY) or aged microbiota (YA). Similarly, studies were conducted in aged mice that were inoculated with aged microbiota (AA) or young microbiota (AY). Mice were assessed using the NDS upon reperfusion, and 1 and 3 weeks following reperfusion (Fig 3A; n = 10–14 per group) or at equivalent time points for sham‐operated mice. After reperfusion, there were no significant differences in the NDS score of young mice with different gut microbiomes (Fig 3A, top panel) or in aged mice having different gut microbiomes (Fig 3A, bottom panel). NDS scores were increased at reperfusion (greater deficit) compared to subsequent trials at 1 and 3 weeks for both young (p < 0.001) and aged mice (p < 0.001, Kruskal‐Wallace one‐way ANOVA on ranks). At 1 week following reperfusion, AY mice showed an improved NDS score compared to AA mice (p = 0.036), indicating an improved outcome with a young microbiome (Fig 3A, bottom panel). Although the NDS score was similarly improved in YY mice compared to YA mice, statistical significance was not achieved. At 3 weeks post‐MCAO, a similar pattern was observed in aged mice with the young microbiota trending toward an improvement. There were no significant differences in NDS among any of the sham groups (data not shown). Figure 3 Open in figure viewer PowerPoint (A) Neurological Deficit Score (NDS) at the time of reperfusion and 1 and 3 weeks postreperfusion in young recipient (top panel) and aged recipient mice (bottom panel). Kruskal‐Wallis ANOVA on ranks showed statistical significance in both young (p < 0.001) and aged (p < 0.001) mice (n = 10–14 per group). *p < 0.036 compared to AY. (B) Length of time mice could hang on a wire mesh once it was inverted. Studies were conducted 3 and 7 days post‐MCAO or post‐sham MCAO in young mice (left panel) or aged mice (right panel) having different microbiota (n = 7 mice per group of young and aged recipients). In young recipient mice (left panel), there was a significant main effect of the microbiome at both 3 (p = 0.004) and 7 days poststroke (p = 0.018, two‐way ANOVA). *p < 0.006 and **p = 0.037 compared to corresponding group with young microbiome at the same time (Holm‐Sidak post‐hoc test). In aged mice (right panel), there was a significant main effect at 3 days (p = 0.036). ***p = 0.034 compared to AY at 7 days. (C) Percent time out of 5 minutes that mice spend hanging for hang‐wire behavior test. Note that mice with aged microbiome, regardless of chronological age, performed worse. There was a significant main effect of the microbiome in young mice (left) and aged mice (right; p < 0.001 for both chronological age groups) and with time (p < 0.001 for left and right panels). Pre‐FTG = prefecal transfer gavage. *p ≤ 0.01 compared to corresponding time point; **p = 0.036 compared to young biome pre‐FTG. ANOVA = analysis of variance; FMT = fecal microbiota transplantation; MCAO = middle cerebral artery occlusion. Figure 3B shows the results of the hang‐wire test, a measure of motor strength, 3 and 7 days post‐MCAO or post sham‐MCAO in young (left panel) and aged (right, panel, n = 7 mice per group of young and aged) mice. In young mice (left panel), there was a main effect of the microbiome at both 3 (p = 0.004) and 7 days poststroke (p = 0.018, two‐way ANOVA, n = 7 for all groups of young and aged mice). In addition to the main effect of microbiome at 3 and 7 days, there was also a significant effect of the microbiome at 3 days (p = 0.037) and 7 days in in young MCAO (p = 0.037, Holm‐Sidak post‐hoc test; Fig 3B, left panel). Thus, mice with an aged microbiome had reduced motor strength overall (including sham and MCAO groups), but most pronounced at 3 and 7 days following MCAO compared to mice with a young microbiome. Overall, aged mice (n = 7 per group) were unable to hang on to the wire mesh top as long as the young mice regardless of the microbiome or treatment group (compare left and right panels in Fig 3B). At 3 days, there was a significant main effect of MCAO (p = 0.036, two‐way ANOVA; Fig 3B, right panel). Aged mice repopulated with young microbiota (AY) had an increased hang time after MCAO at 7 days (25 ± 8 seconds) compared to the aged mice with aged microbiota (AA) after MCAO (8 ± 8 seconds), which was significant (p = 0.034, Mann‐Whitney rank‐sum test with Bonferroni correction). Some of the differences in hang time between young and aged mice could be attributed to the differences in body weight of young and aged mice. Regardless of a potential confounding effect of the greater weights observed in aged mice, the data are clear that young mice with an aged microbiota (YA) had decreased hang times following MCAO compared to young mice with a young microbiota (YY). Open field activity was used to assess locomotor function and anxiety (n = 10 per group). In sham MCAO mice, there were no differences in open field activity at any time point regardless of the young or aged gut microbiota (Fig 4A, top two panels). However, open field activity in MCAO mice showed significant main effects for the microbiome and with time in young MCAO mice (p < 0.001 and p < 0.001, respectively, two‐way repeated‐measures ANOVA; Fig 4, left bottom panel) and in aged MCAO mice (p < 0.001 and p = 0.007, respectively, bottom right panel). Three days after MCAO, activities decreased in all groups compared to pre‐MCAO levels regardless of age or status of the microbiome. At 7 days post‐MCAO, activities rebounded to or toward that of the pre‐MCAO mice in all groups. However, YA was significantly less than YY at 7 days (P < 0.001; Fig 4A, bottom left panel) and AA was significantly less the AY at 7 days (P < 0.001; Fig 4A, bottom right panel). Additionally, activity levels in mice with aged microbiomes did not fully recover to the pre‐MCAO activity levels for young mice (p < 0.001 for YA at 7 days compared to pre‐MCAO) or aged mice (p < 0.001 for AA at 7 days compared to pre‐MCAO). Figure 4 Open in figure viewer PowerPoint (A) Open field test to assess locomotor function and anxiety before MCAO (Pre‐) and 3 and 7 days postreperfusion in sham mice (top panels in A; n = 10/group). In sham MCAO mice, there were no differences in activity at any time point regardless of the young or aged gut microbiota (A, top two panels). Open field activity in MCAO mice showed significant main effects for the microbiome and for time in young MCAO mice (p < 0.001 and p < 0.001, respectively, two‐way repeated‐measures ANOVA, left bottom panel) and in aged MCAO mice (p < 0.001 and p = 0.007 right bottom panel). Activity significantly decreased from pre‐MCAO levels at 3 days regardless of microbiome (*p < 0.001 compared to pre‐MCAO activity in the corresponding recipient age group). YA was significantly less than YY at 7 days (**p < 0.001, bottom left panel) and AA was significantly less than AY at 7 days (#p < 0.001, bottom right panel). Additionally, activity levels in mice with aged microbiomes did not fully recover to the pre‐MCAO activity levels for young mice (p < 0.001 for YA at 7 days compared to pre‐MCAO) or aged mice (p < 0.001 for AA at 7 days compared to pre‐MCAO). (B) Survival in the various groups after MCAO. The increased mortality in the young mice having an aged microbiota was statistically significant by the Mantel‐Cox test (p = 0.04) and the Gehan‐Breslow‐Wilcoxon test (p = 0.03). ANOVA = analysis of variance; MCAO = middle cerebral artery occlusion.

Mice With an Aged Microbiota Have Increased Mortality Following MCAO Mortality over 21 days in young and aged mice following MCAO is shown in Figure 4B. Young mice with a young microbiota (YY) had no mortality 21 days after MCAO (n = 13). However, mortality in young mice reconstituted with aged microbiota (YA) increased to 36% (5 deaths in 14 mice) after MCAO. Aged mice with young microbiota (AY) had a 7% mortality rate (1 death in 14 mice) and aged mice with an aged microbiota (AA) had a 14% mortality rate (2 deaths in 14 mice) after MCAO. The increased mortality in the young recipients with aged microbiota was statistically significant by the Mantel‐Cox test (p = 0.04) and the Gehan‐Breslow‐Wilcoxon test (p = 0.03).

Effects of Altered Microbiota on Infarct Volume in Young and Aged Mice To examine whether microbiota transplants influenced histological injury from stroke, cohorts of mice receiving fecal transplants were perfused and infarct size was analyzed using cresyl violet staining (Fig 5A). Regardless of the biome received, aged mice had significantly less cerebral atrophy 30 days after stroke compared to young mice (p < 0.001), consistent with previous studies.54 Significant differences were observed in percent of tissue loss with biome transfers in aged mice that received young microbiota (9.9 ± 0.5%) compared to aged mice that received aged microbiota (13.1 ± 1.0%; p < 0.05), whereas no significant differences were found between young mice that received young microbiota (23.0 ± 2.6%) compared to young mice that received aged microbiota (25.8 ± 3.3%; p < 0.05). Figure 5 Open in figure viewer PowerPoint (A) Relative infarct volume 30 days after stroke in young and aged mice with either a young microbiota (biome) or aged biome. Regardless of the biome received, aged mice (N = 10) had significantly smaller levels of cerebral atrophy after stroke compared to young mice (N = 10; p = 0.004 Kruskal‐Wallis ANOVA on ranks). *p < 0.05 compared to corresponding species in the young. (B, left side of panels) Fecal propionate and acetate compositions were significantly less in aged mice when compared to young at baseline p < 0.001 for each SCFA, n = 8/group, left side of the upper and lower panels). (B, right side of panels) Changes in acetate and propionate after fecal transplant gavage but immediately before MCAO (time 0) and several time points over 28 days post‐MCAO. As with the naïve mice, both acetate and propionate were decreased in mice with aged microbiomes. For acetate in both the young and aged mice (B, top panel), there was a significant main effect of microbiome (young or aged, p < 0.001; N = 8/group), a significant effect of time after MCAO (p = 0.018), and a significant interaction between time after MCAO and microbiome (p = 0.005). Similarly, propionate showed a significant main effect of the microbiome for both young and aged mice (p < 0.001; N = 8/group) and a significant interaction between microbiome and time (p = 0.012). *p ≤ 0.01 compared to corresponding group with an aged microbiome (Holm‐Sidak post‐hoc test). ANOVA = analysis of variance; MCAO = middle cerebral artery occlusion; SCFA = short‐chain fatty acid.

Reduced SCFAs Were Observed in Mice Harboring Aged Microbiota Given that bacterial‐derived SCFAs have a prominent role in gut and brain health,39-44 we measured SCFAs in fecal pellets. Figure 5B shows the levels of two important SCFAs in pellets from naïve young and aged mice (ie, no gavages or MCAO). Note that fecal levels for both acetate and propionate were decreased by ∼68% for each SCFA in aged mice compared to that measured from young mice (p < 0.001 for each SCFA; n = 8/group; left side of the upper and lower panels in Fig 5B). Butyrate decreased by almost 80%, but did not reach statistical significance (Fig 7). Fecal levels of butyrate and four other SCFAs in naïve young and aged mice are shown in Figure 7. Figure 5B (right side of the upper and lower panels) show the change in acetate and propionate after fecal transplant gavage, but immediately before MCAO (time 0) and several time points over 28 days post‐MCAO. Note that the level of acetate and propionate was determined by the microbiome (aged or young biome) and not by the age of the recipient mouse. As with the naïve mice, both acetate and propionate were decreased in mice with aged microbiomes. For acetate in both the young and aged mice (5B, top panel), there was a significant main effect of microbiome (young or aged, p < 0.001; N = 8/group), a significant effect of time after MCAO (p = 0.018), and a significant interaction between time after MCAO and microbiome (p = 0.005). Similarly, propionate showed a significant main effect of the microbiome for both young and aged mice (p < 0.001; N = 8 per group) and a significant interaction between microbiome and time (p = 0.012).

Impact of Microbiota and MCAO on Circulating Immune Markers Adult mice (24 weeks of age) were inoculated with microbiota from young or aged donors 1 month before MCAO or sham surgery. Cytokine levels measured from plasma 28 days after MCAO or sham MCAO demonstrated that the microbiota (young versus aged) had significant effects on the systemic cytokine response to stroke observed even a month after injury (Fig 6). Protective cytokines had a more pronounced increase following MCAO in mice with a young microbiota compared to mice having aged microbiota as shown in Figure 6. Interleukin (IL)‐4 and granulocyte colony stimulating factor (G‐CSF) increased more in mice with young microbiota than mice having an aged microbiota. In Figure 6, bars having the same alphabet letter are significantly different from one another. Although IL‐10 did not reach statistical significance, it trended similar to IL‐4 and G‐CSF. Figure 6 Open in figure viewer PowerPoint Plasma cytokines in adult MCAO or sham mice reconstituted with a young or aged microbiota. Plasma was sampled 7 days following the MCAO or sham MCAO. Two‐way ANOVA revealed significant effects of MCAO on IL‐4, G‐CSF (proinflammatory), IL‐6, TNFα, Eotaxin, and RANTES (anti‐inflammatory; p ≤ 0.04). Bars with the same letter are significantly different (p < 0.05) using the Holm‐Sidak post‐hoc analysis. n = 4 to 5 per group except Eotaxin and RANTES, which had 2 to 4 and 3 to 5 per group. ANOVA = analysis of variance; G‐CSF = granulocyte‐colony stimulating factor; IL = interleukin; MCAO = middle cerebral artery occlusion; RANTES = regulated on activation, normal T‐cell expressed and secreted; TNFα = tumor necrosis factor alpha. Altering the microbiota had the opposite effects on several proinflammatory cytokines (bottom two rows in Fig 6). Young mice with aged microbiota had a greater increase in proinflammatory cytokines following stroke compared to adult mice with young microbiota. Taking the data as a whole, it is clear that the cytokine response is more proinflammatory in animals with aged microbiota compared to animals hosting young microbiota.