Old age decreases long-term survival and gait performance after TBI
To determine whether old age impacts long-term recovery after TBI we assessed young and old male C57BL/6 mice using a battery of behavioral tests over the course of a 12-week period (Fig. 1a). Old mice showed significantly higher mortality beginning at 9 weeks as determined by the Mantel-Cox test (Fig. 1b). Body weights gradually increased in young mice and decreased in old mice in a normal age-related fashion, however, TBI-induced weight loss was evident early during the first week in the older group (Fig. 1c). To rule out any confounding effects in motor performance caused by differences in pain sensitivity we subjected mice to the hot plate test. Both age groups displayed similar increases in pain sensitivity to heat stimuli between 3-6 weeks after injury (Fig. 1d).
Motor function was measured using the open field (OF) for spontaneous locomotor activity, Catwalk for gait analysis, rotarod for balance and stamina, and grip testing for forelimb strength. Baseline (i.e., pre-TBI) motor function was significantly reduced with age in all tests. A significant group effect of injury was seen in rotarod performance, especially in young mice which showed decreased latency to fall at every timepoint after TBI (Fig. 1e). Forelimb grip strength was significantly reduced during the first week for both age groups but rebounded back to baseline or higher by 3 weeks (Fig. 1f). Together these data show that the effects of normal aging are more pronounced than TBI. Motor recovery was evident in both age groups for OF behavior and grip strength, while long-term deficits were seen in gait dynamics and rotarod performance.
OF results showed an injury effect in the total distance traveled and mean speed, marked by an early increase in each metric during the first week (Fig. 1g-i). This finding could not be attributed to increased anxiety-like behavior as no change in inner/outer zone preference was seen for either age (Fig. 1j). Gait analysis showed significant and lasting changes in all four limbs for all mice following injury (Fig. S1) but given the unilateral nature of the injury we focused on the contralateral, or right-side limb dynamics. Our results revealed long-term deficits in step sequence regularity and pattern for both age groups at 12 weeks post-injury (Fig. 1k, l). The duration of contact with the walkway of the right forepaw, or stand time, was chronically increased in old, but not young TBI mice (Fig. 1m). Stride length was impeded early at 3 weeks in old mice and persisted up to 12 weeks (Fig. 1n). No injury effect was seen in the swing time for the right forelimb (Fig. 1o); however, swing speed was significantly increased at 12 weeks in old mice (Fig. 1p). Chronic alterations in body speed variation were evident in both age groups (Fig. 1q), whereas only young mice exhibited significantly decreased body speed late after TBI (Fig. 1r).
Old age exacerbates long-term cognitive dysfunction after TBI
To investigate the role of age on cognitive decline following TBI we examined mice using Y-maze and novel object recognition (NOR) testing, to assess short-term spatial working memory and long-term recognition memory, respectively. Significant group effects of age and injury were seen in the percentage of spontaneous alternations in the Y-maze test (Fig. 2a); however, no difference was seen between pre-TBI and 12 weeks post-injury for either group. An injury-induced decrease in the number of arm entries was also seen in both age groups, with aged mice showing relatively fewer arm entries in general (Fig. 2b). NOR testing showed no clear preference in time spent between left and right objects during the sample phase, except for the young sham group (Fig. 2c). During the choice phase, a statistical preference was found for the novel object in all groups, except for the old TBI group which displayed late impairment in recognition memory (Fig. 2d). These findings indicate that old age is associated with a more pronounced loss of recognition rather than spatial memory during the chronic phase of TBI.
To assess depressive-like phenotype we subjected mice to novelty suppressed feeding (NSF) and social interaction testing late after TBI. Given the high degree of mortality seen in the aged TBI group, we chose tests that were less stressful than those that typically measure behavioral despair. In the NSF test, a significant effect of injury, but not age, was seen in the ratio of time required for food-deprived mice to travel to the center zone of a novel versus familiar environment to obtain food (Fig. 2e). We then evaluated social behavior in chronically injured mice. No preference in time spent between left and right cups was seen in any group (Fig. 2f). However, when one cup was replaced with a stranger mouse, all groups spent significantly more time exploring the mouse (Fig. 2g). Moreover, when the other empty cup was replaced with a second stranger mouse, all groups showed a preference for the novel mouse (Fig. 2h). While an effect of age was found in time spent exploring the second stranger mouse (purple bars) in sham mice by Tukey’s post-hoc test for multiple comparisons (p<0.05), no statistical differences were seen between the injury groups. These data suggest that despite modest differences at baseline, depression-like behavior is prevalent in all age groups following TBI.
Old age increases lesion volume, white matter loss, and microglial activation at 16 weeks following TBI
We determined the effect of old age on chronic neurodegeneration after TBI. Quantification of cresyl violet stained tissue sections revealed significantly larger lesion volumes in old mice compared to their younger counterparts (Fig. 3a-b). IHC confirmed there were fewer NeuN-positive neurons in the ipsilateral cortex of old mice at 16 weeks post-injury (Fig. 3c-d). Concomitantly, the number of Iba1-positive microglia was found to be statistically increased in the old TBI cortex, but not young, as determined by two-way ANOVA with Tukey’s multiple comparison’s test (Fig. 3e-f). Age-related pathological abnormalities were also seen in myelination of the medial corpus collosum as evidenced by Fluoromyelin stain intensity (Fig. 3g-h). Together, we demonstrate a positive association between microglia/macrophage number, severity of neuronal degeneration, and white matter degradation after chronic TBI, which is exacerbated in older mice.
Gene pathways associated with microglia activation, phagocytosis, and autophagy are elevated with age in both the injured cortex and hippocampus
To address age-related differences in the transcriptional response to TBI during the chronic phase, ipsilateral cortical and hippocampal tissues were sampled from young and old injured mice at 16 weeks post-injury. The Neuroinflammation panel tested a total of 757 genes within three themes of Immunity & Inflammation, Neurobiology & Neuropathology and Metabolism & Stress [28, 29]. PCA of all normalized gene counts in the cortex and hippocampus revealed a distinct separation of samples into individual groups across the first two principal coordinates (Fig. 4a-b). In the cortex, the first component accounted for the majority of the variation (20.2%) across samples and separated the groups by injury, while the second principal coordinate (12.9%) separated the groups by age (Fig. 4a). To further analyze the connection between aging, TBI, and the behaviors reflective of cognitive decline and depression that we observed in tested mice, hippocampal tissue was also analyzed. In the hippocampus, injury accounted for 19.2% of the variation compared to 15.3% by age (Fig. 4b). In both regions, the old TBI samples clustered further away on the x-axis compared to the young TBI and two sham groups, perhaps reflecting the higher number of inflammatory genes that were increased in the brain of old mice at 16 weeks post-TBI.
Four pairwise comparisons of perilesional cortices and hippocampi were performed and outlined in Fig. S2: (1) young sham vs. old sham – (Set 1); (2) young TBI vs. young sham – (Set 2); (3) old TBI vs. old sham — (Set 3); and (4) old TBI vs. young TBI — (Set 4). TBI resulted in increased expression of genes in both young and old mice (Set 2 and 3), while age-related differences were seen in both baseline (Set 1) and TBI conditions (Set 4). At baseline, 130 genes (17.1%) were differentially expressed (DE) between young and old sham groups, indicating that age affected the homeostatic regulation of genes related to the inflammatory process in a fundamental way (Fig. S2a). In the hippocampus, 121 genes were found to be DE with age (Fig. S2b). The number genes found to be down-regulated was greater in the cortex than hippocampus for both Sets 3 and 4. Venn diagrams illustrate the number of DE genes that overlap between Sets 1, 2, 3, and 4 (Fig. S2c-d). With further analysis we were able to identify 83 genes in the cortex that were uniquely related to age and do not overlap with any genes from Set 2 and Set 3 (Fig. S2c). In addition, a total of 130 genes were found to be related to TBI injury effects (Fig. S2c). In the hippocampus, a total of 74 genes were downregulated and 47 genes were upregulated when comparing samples in Old Sham to Young Sham (Fig. S2b). At 16 weeks after injury, Set 2 had as many as 126 genes within the inflammation panel downregulated while only 21 genes were upregulated. Similarly, the comparison between Old TBI versus Old Sham showed a total of 110 DE genes, with 83 being downregulated in the injury group while a mere 27 genes were upregulated. As demonstrated in the adjacent Venn diagrams, we identified 100 age-related genes and 101 injury-related genes (Fig. S2d).
An initial analysis of the DE genes in each set showed several genes with large fold changes in the cortex, as illustrated in a volcano plot for each gene set (Fig. 4c-j). To further investigate the effects of normal aging on transcriptional activation, we compared the percentage of genes modified by age (Set 1 and 4) for each pathway annotation. All pathways were affected with advanced age, but genes that facilitate oligodendrocyte function, neurons and neurotransmission, autophagy, astrocyte function, innate immune response, epigenetic regulation, and microglia function were far more likely to be activated in the cortex of old sham mice compared to young sham control. Alternatively, pathway analysis of genes related to injury (Set 2 and 3) showed that genes associated with oligodendrocyte function, angiogenesis, neurons and neurotransmission, matrix remodeling, astrocyte function, microglia function, and cellular stress were predominantly activated after injury (Fig. 4c-d). Pathway analysis of DE genes in the cortex is shown (Fig. 5). A heatmap of DE genes implicated in each notated pathway generally show an age-related up-regulation in the chronic phase of TBI (Fig. 5a-g). The complement pathway, which has been previously implicated in age-related neurodegenerative disease and synaptic maintenance, was found to be increased both with age and injury (Fig. 5h). Genes associated with microglia function, the complement pathway, and autophagy regulation were also up-regulated with age and/or injury in the hippocampus (Fig. 6a-c). Genes associated with epigenetic regulation were also increased with age and injury (Fig. 6d). A heatmap of DE genes implicated in astrocyte function, oligodendrocyte function, and neurons & neurotransmission showed a more mixed expression pattern after injury (Fig. 6e-g). More specifically, the age- and chronic injury-related upregulation of Axl, CD14, CD68, CD74, Ctss, Clec7a, Trem2, and Tyrobp genes in both the cortex and hippocampus is consistent with that seen in the multiple models of age-related neurodegenerative diseases, and is representative of a disease-activated microglia (DAM) signature that reflects a pathological state of the CNS [33].
As an additional measure for examining transcriptional changes, we validated several genes that showed significant changes via real-time quantitative PCR (Fig. S3 and S4). For example, the gene Clec7a that encodes a glycoprotein linked to the modulation of the natural killer gene complex region and connected to the innate immune response functions. Real-time qPCR validation of this gene revealed significantly higher expression levels of Clec7a in the cerebral cortex of Old TBI mice compared to the Old Sham group, but barely any change in the Young TBI versus Young Sham group. Another gene, ncf1 (neutrophil cytosolic factor 1), encodes a subunit of neutrophil NADPH oxidase, which is activated to produce superoxide anions. Taken together, our results show a diverse transcriptional response of inflammatory genes in old mice compared to young mice after TBI, with several genes showing differential expression between the Old TBI and Young TBI groups.
To better illustrate the relationship between neuropathology and expression of autophagic markers, we confirmed immuno-reactivity of LC3 and p62 at the protein level in the perilesional cortex (Fig. 7a-d). Significant effects of age- and TBI were seen in both the number of LC3-positive and p62-positive cells at 16 weeks post-injury, consistent with our Nanostring findings. In sum, these data suggest autophagic changes in the aged and injured brain may precipitate chronic microglial activation and neuronal degeneration.
Old age increases infiltration of lymphocytes and exaggerates microglial responses to TBI
Next, we investigated the cellular inflammatory response at early (48 h) and late (16 weeks) timepoints after TBI. Brain leukocyte identification and characterization was performed using flow cytometry. Old age was associated with fewer microglia (CD45intCD11b+Ly6C-) and less robust microglial accumulation in terms of absolute number (Fig. 8a-b). Normal age-related reductions in microglia number have been reported previously by our group and others and likely reflects age-related dystrophy and proliferative senescence [34]. The number of infiltrating myeloid cells (CD45hiCD11b+), however, was comparable between young and old groups and showed two-way ANOVA group effects of injury and age, including a significant interaction between them (Fig. 8c). Delayed infiltration and accumulation of putative lymphocyte populations (CD45hiCD11b-) was dramatically in old mice at all timepoints post-injury (Fig. 8d). These findings highlight the profound effects of aging on the central and peripheral immune response to TBI and suggests that blood-brain barrier integrity may be uniquely compromised in the aged brain long after injury.
To address the rapidly shifting dynamics of microglia proliferation that occurs during the first week of injury, we examined two commonly used nuclear protein markers of cellular proliferation, Ki67 and proliferating cell nuclear antigen (PCNA). Both markers were significantly upregulated in microglia at 48 h after TBI, but no statistical difference between injury groups was found (Fig. 8e-f). Despite being fewer in number, microglia in old sham mice showed significantly higher basal expression of Ki67, indicating active cell cycling. The percentage of Ki67-positive microglia remained elevated late after TBI in young but not old microglia as determined by Dunnett’s test (p=0.05). These data do not indicate age-related difference in the kinetic progression of microglial proliferation at 48 h, however, during the chronic stages of injury microglia from old TBI mice showed reduced proliferative capacity relative to sham compared to those from the young TBI group.
We then examined the activation state of microglia based on light scatter properties and ex vivo functional analysis. Significant effects of age and injury were seen microglia size and granularity at all timepoints, as measured by forward and side scatter intensity, respectively (Fig. S5a-b). Old microglia became markedly more granular after 48 h post-injury compared to young microglia, suggesting increased inflammatory activation. Reactive oxygen species (ROS) production is associated with oxidative stress which can exacerbate neurodegeneration [18, 35]. Old microglia had dramatically higher ROS production at baseline and at all timepoints post-injury, whereas young microglia exhibited a more robust increase in ROS relative to its respective sham control (Fig. 8g). The percentage of microglia producing detectable ROS remained elevated at 16 weeks in young mice as determined by Dunnett’s test. Lastly, we measured cytokine production in microglia which showed significant effects of both age and injury in expression level of IL1β and TNF protein (Fig. 8h-i). Both cytokines showed an age-related increase during the acute phase of TBI, which largely abated by 16 weeks (p<0.05). Taken together, our data imply that old age primes microglia to become more reactive to acute TBI, consistent with previous work. Independent of any change in cell number, basic features of microglial activation during the chronic phase of TBI appear to be more subtle on a per cell basis.
Microglial phagocytosis of neurons, expression of autophagy markers, and lipofuscin content are increased with age and injury
The emergence of a DAM signature led us to surmise that age-related deficits in autophagy function underlie chronic microglial activation and dysfunction following TBI. To better understand the temporal effects of TBI on microglia function, we performed an extensive characterization of phagocytosis and autophagy informed largely by our NanoString results indicating age-related increases in these specific pathways late after injury. Phagocytosis was measured by intracellular detection of neuronal antigens, myelin content, and the lysosomal/endosomal membrane marker CD68. Significant group effects of both age and injury were seen in the percentage of NeuN-positive microglia (Fig. 9a). The frequency of NeuN-positive microglia increased sharply at 48 h in both age groups and remained high at 16 weeks in old mice. Similar changes were seen in intracellular myelin content; however, while levels trended higher in old mice at 16 weeks (p=0.06) it did not meet statistical significance in the two-way ANOVA multiple comparisons test (Fig. 9b). Intracellular detection of vesicular glutamate transporter 1 (Vglut1), a synaptic protein in neurons, was also increased acutely in microglia after TBI (Fig. 9c). A group effect of age was found, indicating a higher presence of this synaptic marker in old microglia. The phagocytosis marker CD68 was significantly higher in old microglia both at baseline and at 16 weeks but was elevated to a similar extent at 48 h in both age groups (Fig. 9d). These findings suggest that microglial phagocytosis of dead or dying neurons is robust in the early stages of TBI, and modestly increased with both age and time post-injury, consistent with our gene expression data.
Lysosome and autophagosome content in microglia were significantly increased with both age and injury and remained elevated for up to 16 weeks (Fig. 9e-g). Protein expression of Sqstm1/p62 and Lamp2 were increased at 48 h, but no effect of age was seen (Fig. 9h-i). The ubiquitin–proteasome system degrades intracellular proteins into peptide fragments that can be presented by major histocompatibility complex (MHC) class I molecules. Significant group effects of age and injury were found in surface expression of MHCI with the highest expression seen at 48 h (Fig. 9j). Defects in the autophagy pathway can result in the cytosolic and lysosomal accumulation of an autofluorescent amalgam of oxidized lipids, proteins, and metals known as lipofuscin. Group effects of age and injury were seen in intracellular lipid content, iron levels, peptide/protein aggregation, and autofluorescence (Fig. S5c-f). Neutral lipid and iron content increased sharply at 48 h, consistent with the increase in phagocytic activity. Protein aggregation and autofluorescence were largely unperturbed at 48 h, however, both were found to be elevated above sham levels at 16 weeks in young microglia as determined by Dunnett’s test (p<0.05 and p<0.05, respectively). Collectively, these data imply that phagocytosis and autophagic function are exacerbated with age and TBI, consistent with our DAM gene signature. Although these processes are more active during the acute stages of injury, many markers, including the accumulation of lipofuscin material, show a sustained increase during the chronic phase.
Age-related changes in microglial histone acetylation patterns, metabolic activity levels, and expression of senescence markers are altered after TBI
Given the widespread effects of aging on microglial function, we also evaluated changes in epigenetic regulation, metabolic activity, and senescence-like phenotype following TBI. Microglia activation is associated with increased histone deacetylase (HDAC) and sirtuin (SIRT) activity resulting in hypoacetylation of histones and non-histone proteins and increased expression of inflammatory markers [36]. Our results show an age-related loss of histone 3 (H3) within the general microglia population, however, H3 expression levels were dramatically increased at 48 h in both age groups (Fig. 10a). The percentage of microglia expressing acetylated lysine residues was significantly reduced with age and injury indirectly suggesting increased HDAC and SIRT activity (Fig. 10b). To further probe the impact of TBI on histone modification we examined acetylation patterns on H3. Significant effects of age and injury, or injury alone, were seen in H3 hypoacetylation of the lysine residue at N-terminal positions 9, 18, 27, and 36 (Fig. 10c-f). These data suggest that aging and TBI result in the removal of acetyl groups from H3 which is known to increase chromatin condensation and decrease gene transcription.
TBI acutely increased mitochondrial activity, glucose uptake, and ATP production in microglia (Fig. 11a-c). At 16 weeks post-injury, the cellular demand for glucose remained higher than sham control in young mice as determined by Dunnett’s test (p<0.01). These findings suggest that TBI causes a metabolic crisis in microglia that is associated with a chronic increase in glucose uptake in young mice.
We have previously demonstrated that a small population of microglia upregulate markers of cellular senescence in an age- and TBI-dependent manner [37]. However, the sustained presence of a senescent-like phenotype has yet to be reported in microglia at chronic timepoints after injury. Expression of the DNA damage sensor, p-H2AX, showed a basal elevation with age (Fig. 11d). Interestingly, p-H2AX levels were significantly decreased at 48 h, likely due to a stress-mediated increase in DNA repair mechanisms. Although expression was further increased from 48 h to 16 weeks, levels did not surpass that seen in sham controls. Expression of the cell cycle arrest markers p16 and p21 were increased with normal aging (Fig. 11e-f). The percent of p16-positive microglia trended higher in young TBI mice at 16 weeks compared to sham, but significance was not achieved by two-way ANOVA multiple comparison test. It should be noted that the absolute number of p16-positive microglia may be significantly increased in the absence of a statistical change in percentage. In contrast, a robust increase in the relative expression of p21 was seen at 48 h in both age groups, however, levels were higher in old microglia at both timepoints post-injury (Fig. 11f). Together these results indicate that aging interacts with TBI to modify key aspects of microglial homeostasis, including post-translational epigenetic regulation, metabolic function, and development of a senescence-like phenotype.
Trehalose treatment enhances functional recovery in motor and cognitive tasks and decreases depressive-like behavior
Old mice were administered trehalose or sucrose control in their drinking water beginning on d1 for up to 8 weeks after surgery. A battery of behavioral tests was performed to monitor changes in motor performance, cognitive function, and anxiety/depression (Fig. 12a). Trehalose treatment resulted in a delayed decrease in body weight (Fig. 12b). No statistical deficits were seen in rotarod performance at any timepoint in mice treated with trehalose, whereas mice treated with sucrose control exhibited lasting impairment for up to 8 weeks after TBI (Fig. 12c). OF activity was then assessed (Fig. 12d). Interestingly, sucrose-treated mice traveled significant shorter distances in the OF during the first week of injury (Fig. 12e). This was also accompanied by a decrease in speed, not seen in trehalose-treated mice (Fig. 12f). A statistical increase in time spent in the center zone of the OF late after injury was recorded in mice administered trehalose, suggesting that enhancing autophagy during TBI may result in delayed anxiolytic effects (Fig. 12g). These data suggest continued administration of trehalose may attenuate motor deficits in aged mice after TBI.
To address the effect of trehalose on long-term cognitive outcomes in old mice, we evaluated mice using the Y-maze and NOR tests. While both treatment groups showed a temporary reduction in the number of arm entries early after TBI, no therapeutic effects were seen (Fig. 12h-i). However, trehalose-treated mice did show improvement in recognition memory at 8 weeks post-TBI compared to sucrose control (Fig. 12j-k). Lastly, NSF and social interaction tests were employed to evaluate anxiety/depressive-like phenotype. Trehalose-treated mice displayed shorter latency times to reach food at the center of the OF compared to sucrose control, suggesting less depression-related behavior, consistent with our OF findings (Fig. 12l). Finally, social interaction testing showed that trehalose increased the amount of time spent interacting with a novel stranger mouse in the social discrimination phase (i.e., sequence 3) of the test (Fig. 12m-o). Together these findings show that continued trehalose treatment can preserve recognition memory and reduce anxiety/depression in old mice late after injury.
Trehalose treatment reduces chronic brain inflammation and phagocytosis of neuronal synapses by modulating multiple steps of the autophagy pathway in microglia
To better understand the impact of trehalose on chronic microglia activation and neuroinflammation in old mice after TBI we examined autophagic function at 9 weeks post-injury. Trehalose treatment caused a clear reduction in the number of proliferated microglia (CD45intCD11b+Ly6C-) present in the ipsilateral hemisphere and significantly prevented the delayed recruitment or accumulation of peripherally-derived myeloid (CD45hiCD11b+) and lymphocytes (CD45hiCD11b-) (Fig. 13a-d). Next, we examined the effect of trehalose on phagocytosis. A significant injury-related increase was found in microglial expression of CD68, but no effect of treatment was seen (Fig. 13e). Lipid content trended higher in sucrose-treated mice (p=0.05), while trehalose treatment caused no change (Fig. 13f). The percentage of microglia that engulfed neuronal Vglut1 and the relative level of intracellular NeuN protein were both significantly lower in trehalose-treated mice (Fig. 13g-h).
A comprehensive analysis of autophagy-related markers showed no effect of treatment or injury at 8 weeks as measured by LysoTracker dye, lamp1, lamp2, and Sqstm1/p62 (Fig. S6a-d). Although increased formation of LC3II-positive autophagosomes was found in sucrose- but not trehalose-treated mice, this difference was not statistically significant (p=0.05; Fig. 14a). A group effect of TBI was seen in ATG5 and ATG7 expression in microglia; trehalose treatment prevented a significant reduction in ATG7 (Fig. 14b-c). Examination of lysosomal enzymatic activity in microglia revealed a robust increase in cathepsin D and lysosomal lipase activity in mice treated with trehalose (Fig. 14d-e). There was a trend towards increased ubiquitin expression in microglia in sucrose-treated mice, but changes did not reach significance (p=0.05; Fig. 14f). Whereas antigen presentation by MHCI was more augmented after trehalose treatment, it did not alter injury-induced increases in intracellular protein aggregation (Fig. 14g-h). Lastly, trehalose-mediated protection was associated with attenuated lysine acetylation in microglia (Fig. S6e). Taken together, our results indicate that long-term trehalose treatment reduces chronic brain inflammation via modulation of several key proteins involved in the autophagy pathway, including the upstream regulation of phagocytosis.