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

Abstract

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.

Keywords: Aging mice; Cyclophosphamide; Neutrophil lymphocyte ratio; Resilience to aging; Stress test; WBC count.

Fig. 1

a Counts for WBC, lymphocyte, and neutrophils before Cyp injection in C57BL/6 mice varied in the different age groups. b The NLR in the same mice increased with increasing age. Results are presented as mean ± SD. *p < 0.05 compared with other two age groups

Fig. 2

The WBC count as well as the lymphocyte and neutrophil counts decreased rapidly following a single IP injection of 100 mg/kg Cyp. Results were normalized to baseline levels (dotted line). a The WBC count more quickly returned to baseline levels with increasing age. b The lymphocyte count showed a similar pattern as the WBC count. c The neutrophil count significantly increased to above baseline levels at day 7. The 4-month-old mice had a lower nadir at day 5 and sharper rebound at day 7, while the other age groups had a more delayed rebound response. Error bars represent SEM. *p < 0.05 compared with other ages

Fig. 3

The NLR showed an age-dependent rebound response at day 7 after Cyp injection, but the gradual decline back toward baseline was similar in all age groups. The NLR was calculated as the neutrophil count divided by the lymphocyte count multiplied by 100. Results were normalized to baseline levels (dotted line). Error bars represent SEM. *p < 0.05 compared with other ages

Fig. 4

The WBC and neutrophil responses after Cyp injection were found to correlate with pre-injection NLR. a 15-month-old mice were separated into high and low NLR baseline (pre-injection) groups using 0.15 (dash line) as the separation boundary. b Mice with high NLR baseline had higher total WBC counts from day 1 to day 7 (red) with a higher rebound at day 7 after Cyp injection. *p < 0.05 between high and low responders. c Mice with high NLR baseline had higher neutrophil counts from day 1 to day 7 (red) with a higher rebound on day 7. *p < 0.05 between high and low responders

Fig. 5

Pre-injection NLR was related to learning impairment induced by Cyp. a Pre-Cyp injection NLR aligned with learning curves of 15-month and 28-month mice injected with Cyp. The Pearson correlation coefficients of 15-month and 28-month age groups were 0.655 and 0.784 respectively with p < 0.05, indicating a significant positive relationship. b Escape times in the box maze navigation task were significantly faster in Cyp-injected mice with low NLR baseline in both 15-month and 28-month age groups