Aging is associated with glial senescence in the brainstem - implications for age-related sympathetic overactivity

Abstract

Accumulating evidence suggests that the sympathetic nervous system (SNS) overactivity plays a crucial role in age-related increase in the risk for cardiovascular diseases such as hypertension, myocardial infarction, stroke and heart diseases. Previous studies indicate that neuroinflammation in key brainstem regions that regulate sympathetic outflow plays a pathogenic role in aging-mediated sympathoexcitation. However, the molecular mechanisms underlying this phenomenon are not clear. While senescent cells and their secretory phenotype (SASP) have been implicated in the pathogenesis of several age-related diseases, their role in age-related neuroinflammation in the brainstem and SNS overactivity has not been investigated. To test this, we isolated brainstems from young (2-4 months) and aged (24 months) male C57BL/6J mice and assessed senescence using a combination of RNA-in situ hybridization, PCR analysis, multiplex assay and SA-β gal staining. Our results show significant increases in p16^Ink4a expression, increased activity of SA-β gal and increases in SASP levels in the aged brainstem, suggesting age-induced senescence in the brainstem. Further, analysis of senescence markers in glial cells enriched fraction from fresh brainstem samples demonstrated that glial cells are more susceptible to senesce with age in the brainstem. In conclusion, our study suggests that aging induces glial senescence in the brainstem which likely causes inflammation and SNS overactivity.

Keywords: aging; brainstem; glial cells; senescence; sympathetic nervous system.

Figure 1

Age-induced changes in serum NE and senescence markers in the brainstem. (A) Changes in serum NE levels in young and aged animals measured using a commercial ELISA kit (mean±SE, n=4-6/group). (B) Real-time PCR analysis of gene expression levels of senescence markers p16, p21 and p53 (n=4/group) (C) Representative images of RNA-ISH showing p16-positive cells in the RVLM of the brainstem in young and aged animals. (D) Semi-quantitative analysis measuring the number of p16-positive cells in the RVLM by RNA-ISH. (E) Representative images of SA-β gal staining in the brainstem and (F) Quantification of cells positive for SA-β gal staining in the RVLM of the brainstem. *denotes a significant difference (p < 0.05) from young animals.

Figure 2

Age-induced changes in NF-κB activity and mRNA levels of SASP factors in the brainstem. (A) NF-κB DNA-binding capacity measured by ELISA in nuclear protein extracted from the brainstem of young and aged animals. (B) Gene expression levels of SASP factors in the brainstem measured by real-time PCR analysis. Data are expressed as mean±SE, n=4-5/group. *denotes a significant difference (p < 0.05) from young animals.

Figure 3

Aging induces glial senescence in the brainstem. (A) Purity of glial cell-enriched fraction assessed by real-time PCR analysis. (B–H) Gene expression analysis of senescence markers in the glial cell-enriched fraction from young and aged brainstem. Data are expressed as mean±SE, n=4-5/group. *denotes a significant difference (p < 0.05) from young animals.