Neuroinflammation (i.e., expression of inflammatory response factors and glial activation), is a hallmark of brain aging [46, 47] and is implicated in the cognitive deficits that accompany neurodegenerative conditions and profound brain insults (e.g., TBI and sepsis) [[48–50]]. Markers of neuroinflammation (e.g., CD74, GFAP) have been reported to increase in the hippocampus with age but the manner in which these changes may contribute to cognitive decline with normative aging remain to be determined. Here, we demonstrate that a coordinated induction of MHC II immune response-associated genes and concomitant astrocyte/microglial activation in the hippocampus occurs with advanced aging in both cognitively intact and impaired animals but does not correlate with age-related deficits of cognitive function.
In this work, bioinformatic analysis of the hippocampal synaptosomal transcriptomes of adult and aged rats behaviorally assessed for spatial learning and memory identified 21 genes as components of an age-induced MHC II-associated antigen presentation and response pathway. Upregulation of these genes with advanced aging was confirmed by qPCR in whole-hippocampus synaptosome fractions and in hippocampal subregion (CA1, CA3 and DG) dissections. In agreement with our findings, examination of primary microarray datasets from previous transcriptomic studies of age-related changes in hippocampal gene expression from humans , non-human primates , and rodents [53, 54] reveals upregulation of a number of immune response factors, including induction of MHC II-associated genes, that have not previously been systematically pursued in follow-up studies. For example, transcriptomic analyses of Fischer 344 rat hippocampal gene expression reveals age-related upregulation of MHC II alpha chain, Cd74, Fcgr3, and C3 , as well as induction of MHC II beta and invariant chains and multiple complement components . Importantly, no statistical relationships between age-related increases in these specific inflammation-response genes and cognitive performance were identified in these studies. Increased Cd74, C4a and C3 mRNA expression, which we observed in aged rats compared to 12-month old adults, has also been observed in the hippocampal transcriptome between young (4-6 months) and aged (24 months) Fischer 344 rats .
Our findings also share commonalities with previous targeted gene expression studies of hippocampal aging. Increased hippocampal expression of Hla-dra has been reported in aged (24 months) versus young (3 months) Fischer 344 × Brown Norway rats . We have expanded on this work by demonstrating that increased expression of multiple MHC II components [i.e., MHC II alpha (Hla-dra), beta (Hla-drb1) and invariant (Cd74) chains, and the MHC II antigen-loading cofactor (Hla-dmb)], and multiple MHC II pathway-associated genes occurs between mature adult (12 months) and aged (26-28 months) rats. Similarly, the present study builds upon a previous finding of increased toll-like receptor (TLR) expression with aging in mouse whole-brain preparations , by demonstrating that increased expression of TLRs 3, 4, and 7 occurs primarily in hippocampal synapses and in the hippocampal CA3 subregion, and that TLR expression does not correlate with cognitive impairment.
Our findings extend these previous reports by characterizing a large set of MHC II components and associated immune/inflammation response factors and by demonstrating that increased expression of these genes occurs in both cognitively intact and impaired animals but that the extent of induction does not correlate with cognitive deficits. Interestingly, the magnitude of induction of these MHC II-mediated immune response genes was greater in CA1 and CA3 than in DG in nearly all cases, suggesting that pyramidal cell-containing subregions may undergo age-related neuroinflammation to a greater extent than granule cell-containing regions. Further, age-related increases in immune/inflammation response gene expression were, in many cases, larger in hippocampal synaptosome fractions than in dissected hippocampal subregions. MHC II pathway gene expression in hippocampal synaptosomes likely stems largely from astrocytic and microglial processes closely associated with synaptic compartments, while downstream inflammatory signaling factors may derive from the synaptic terminals themselves. This suggests that heightened antigen processing/immune response within the synaptic milieu may contribute to age-related dysfunction of hippocampal synapses and potentially plays a role in synapse loss [[60–62]].
Immunohistochemical studies of both human and nonhuman primate aging have demonstrated upregulation of MHC II alpha/beta chains in hippocampal microglia [[18, 21, 63, 64]]. We have observed, in both aged cognitively intact and aged cognitively impaired rats, widespread mild and moderate microglial activation indicated by increased numbers of microglia expressing CD74 (i.e., the MHC II invariant chain), and altered morphological and immunoreactive phenotypes. While the percentage of activated microglia (reflecting the ratio of CD74+/Iba1+ to total Iba1+ microglia) was consistently increased by several fold in all aged rats compared to adults, these glia maintained a ramified morphology, rather than the ameboid, morphology typical of a phagocytic phenotype. This suggests that the neuroinflammatory state in the aged hippocampus, while elevated compared to adults, is not severe enough to induce a transition from a "surveilling" microglial phenotype [65, 66] to the reactive phenotype associated with dramatic CNS insults and neurodegeneration [[66–68]]. In agreement with previous work, the total number of microglia (Iba1+ cells) did not increase with aging , indicating that increases in Iba1 protein expression and numbers of CD74+ microglia reflect age-related activation rather than microglial proliferation or infiltration. Iba1/CD74 co-labeling extends previous findings of increased hippocampal microglial activation (CD74+ only) in comparisons of young (3-6 months) and aged (24+ months) [70, 71] by demonstrating that an increased percentage of total hippocampal microglia are activated with advanced aging compared to mature adults. Additionally, previous studies [[70–72]] have generally compared young (3-6 months) and old (24+ months) rats, while our results demonstrate that microglial activation occurs between mature adulthood (12 months) and advanced age (24-26 months). Furthermore, we extend a previous finding of qualitative differences in populations of activated microglia in the aged Wistar rat hippocampus  by demonstrating that increased activation is evident across CA1, CA3, and DG, and that, within each subregion, the percentages of qualitatively distinct activated microglia (mild and moderate) are equivalent. Similar to the work by Gavilan and colleagues , fewer activated microglia were observed in close proximity to pyramidal and granule cell layers than associated with synapse-containing hippocampal layers. While adding to the evidence of hippocampal microglial activation with advanced aging, our findings also clearly demonstrate that the examined marker of microglial activation (CD74) is not directly associated with deficits of spatial learning and maze performance, in agreement with previous studies in the Long Evans rat .
We also observed increased astrocyte activation, as indicated by increased GFAP expression and morphological alterations, which occurred in the absence of astrocyte proliferation. Studies of humans, nonhuman primates, and rodent models of human aging have demonstrated increased glial fibrillary acidic protein (GFAP) gene and protein expression indicative of astrocyte activation and age-related astrocyte hypertrophy, in agreement with our findings [[73–79]]. Our observations agree with one of the first investigations of age-related hippocampal astrocyte activation, which demonstrated that these dystrophic astrocyte processes are often oriented in the same direction . Our work also revealed that while astrocyte activation increases throughout the hippocampus with advanced aging, no cognitive decline-associated differences in GFAP protein content or numbers of GFAP+ astrocytes are apparent between aged intact and aged impaired rats.
Our finding that MHC II pathway induction and glial activation occur in aged rats regardless of cognitive status raises the important question of whether neuroinflammation plays a role in the pathogenesis of cognitive decline . Pathological gliosis and inflammation are associated with severe cognitive dysfunction in neurodegenerative/advanced disease states, traumatic brain injury, and direct inflammatory stimulation [[17–24]]. Hippocampal neuroinflammation has also been suggested to play a functional role in the etiology of age-related cognitive impairment [80, 81]. A lesser degree of neuroinflammation occurs with normative aging than with disease or injury, and ranges from increased oxidative stress to induction of inflammatory signal transducing factors including cytokines, chemokines, complement, and stress response proteins which we and others have previously described in studies of hippocampal aging [[12, 25, 27, 55, 82–84]]. Age-related alterations in these factors suggest an allostatic shift toward a heightened basal neuroinflammatory state in the aging hippocampus. Establishment and maintenance of such a state has been termed "para-inflammation" and is thought to represent an adaptive response to persistent sub-threshold stimuli such as cellular/tissue stress .
The present work demonstrates that, while neuroinflammation at the level of MHC II pathway expression and glial activation may represent an aspect of age-related hippocampal dysregulation necessary for development of cognitive deficits, these measures do not correlate to cognitive performance deficits. It is likely that multiple age-related processes, including decreased neurotransmission, glial dysfunction, and increased neuroinflammation, combine with cognitive decline-related dysregulation of plasticity- and myelin-associated proteins and genes [27, 55], to form an additive array of insults to impede healthy neuronal function and learning and memory . Thus far, the triggering event(s) that cause the transition from an aged, cognitively intact state to an aged, cognitively impaired state have remained elusive. Additionally, the potential remains that other measures of neuroinflammation, including cytokines in both the CNS and systemically could directly correlate to impaired spatial learning and memory. The importance of the interplay between systemic inflammation, neuroinflammation, and brain function with aging was recently underscored by a report demonstrating inhibition of hippocampal neurogenesis by the systemic cytokine CCL11 . Ultimately, our understanding of the functional roles of neuroinflammation in both protective and harmful CNS processes must advance in order to identify whether there are appropriate points for interventions seeking modulate age-related neuroinflammation with the goal of preventing or reversing age-related hippocampal dysfunction.