Metabolic Profiling in the Plasma of Rats in Response to Aging and Ischemic Stroke by Nuclear Magnetic Resonance Spectroscopy

Introduction: Metabolic dysfunction is a common hallmark of aging, and it influences the neurodegenerative diseases including stroke incidence and outcome. Several metabolomics-based studies have revealed metabolite biomarkers in stroke patients and yet metabolic changes as a result of aging and focal ischemia/reperfusion (I/R) time are not completely elucidated. In this study, we examined the dysregulation of metabolites in the plasma as a function of age and cerebral ischemic injury. Methods: We compared the metabolic pattern in young (3 months) and older rats (12 months) before, and 2 days after I/R injury. Stroke was induced by middle cerebral artery occlusion. For time course study, plasma samples from younger animals were collected at day 1, 2, 3, 7, 14 and 21 of I/R (n=3). We used Nuclear Magnetic Resonance spectroscopy and compared the metabolite peaks using spectral binning and targeted profiling. The data was analyzed using one way analysis of variance, principal component analysis (PCA) and hierarchical clustering. Pathway enrichment and topological analysis tools were used to identify the age and stroke responsive metabolic pathways. Results: PCA revealed global differences in plasma of naive young and old rats, with old rats showing significantly lower glucose levels, and higher formate levels. Also, older animals showed reduced levels of acetate and formate and very high levels of 3-hydroxybutyrate 2 days after I/R. Younger rats showed significant differences starting from day 3 onwards with lower glucose concentration and increased 3-hydroxybutyrate, creatine, formate and glycine concentrations, which remained at a higher level on day 7 of I/R. Interestingly, the metabolic profile of plasma at day 21 of I/R showed similar pattern as that of control naive animals. Pathway topology analysis revealed enrichment of citric acid cycle and glycine, serine and threonine metabolic pathways in older animals as early as 2 days after I/R. However, younger animals showed a delayed metabolic response after day 3 of I/R. Conclusions: We have demonstrated that the metabolic profile of plasma is altered as a function of age, ischemic stroke and time of reperfusion. These results suggests an important role for imbalance in metabolites in stroke pathogenesis.