Development of a cyclophosphamide stress test to predict resilience to aging in mice.

The concept of resilience, defined as the ability to recover from stress, is a potential platform to predict healthy aging. However, specific stress tests for resilience have not yet been fully established in humans so investigations in animal models are of interest. The chemotherapeutic drug cyclophosphamide (Cyp) was selected as a chemical stressor to investigate resilience response in C57Bl/6 male mice at 4, 15, and 28 months of age. Following a single intraperitoneal injection of Cyp (100 mg/kg), tail blood was collected for counting white blood cells (WBC) every other day for 25 days, and physiological performance tests performed. Cyp induced a consistent pattern in neutrophil count in all three age groups, with a nadir at day 5 and a rebound at day 7 with different rates in each group. The neutrophil to lymphocyte ratio (NLR) showed an age-dependent rebound response 7 days after Cyp injection, with a similar pattern of decline back toward baseline. Mice in the 15-month age group with high pre-injection Cyp NLR had significantly higher total WBC counts after Cyp injection compared with mice with low pre-injection Cyp NLR, indicating a correlation between NLR and Cyp-altered WBC counts. In addition, mice with high pre-injection Cyp NLR showed significant learning impairment compared with mice with low pre-injection Cyp NLR, suggesting low NRL intensity can predict resilience to age-related cognitive decline. These observations provide the rationale to translate findings from the mouse to humans in developing in vitro Cyp stress tests.

Neutrophil response to cyclophosphamide predicts resilience to age-related learning impairment.

The ability to respond to stress, defined as resilience, was measured by white blood cell counts in C57BL/6 mice of various ages receiving a nonlethal dose of cyclophosphamide (CYP). Neutrophil counts dipped and then rebounded in a consistent and age-dependent manner. Low neutrophil rebound correlated with improved learning in middle-age mice suggesting CYP-nduced neutrophil response may predict resilience to aging.

A minimally invasive, low-stress method for serial blood collection in aging mice.

Hematologic analysis is an efficient and valuable tool for real-time health monitoring and immune analysis in mouse aging studies. However, many frequently used blood sampling techniques in mice are incompatible with continuous monitoring with increasing age, as they may involve anesthesia, cause severe stress, or require a high volume of blood. This technical report describes a convenient relatively noninvasive procedure for counting white blood cells in C57Bl/6 mice by an optimized tail blood collection method followed by Wright-Giemsa and Türk staining. This technique can be performed on unanesthetized mice in less than 1 min with minimal stress using only a few microliters of blood. White blood cell analysis can include but is not limited to total and differential white blood cell count and cell morphology. The low blood volume collected is ideal for aging mice in longer-term experiments requiring regular and continuous monitoring.