Figure 2. Examples of preliminary data collected in our aging sled dog study. (A) Use of the treadmill test to assess physical fitness of dogs. Left panel: example of a dog being tested on the treadmill by Drs. Huson and Wakshlag. Right panel: two independent parameters measured in the treadmill test show a gradual decline in physical performance of aged sled dogs over 12 months of observation. Mean distance (miles covered by dog running for 20 min at maximum speed adjusted for each dog) and Δ lactate (difference in blood lactate levels before and after performance) are shown. N=65-60 each timepoint. Error bars indicate standard deviation. Groups were compared by one-way ANOVA. (B) Overview of workflow for immunologic assays. Blood is collected from dogs into the appropriate tubes (EDTA, CPT) for corresponding downstream assays. Some assays, such as phagocytosis, T cell, MDSC staining, and EliSpot, are conducted immediately. Others, such as determination of cytokine levels and antibody titers elicited by leptospirosis vaccination, use serum and PBMC stored until analysis. (C) Age-dependence of T cell composition. The ratio of T cells with TH1 vs. TH2 phenotypes was determined by IFNγ/IL-4 EliSpot assays in groups of aged (8-12 years old; N=60) and young (2–4-year-old; N=10) sled dogs. Groups were compared by Mann-Whitney test. (D) Age-dependence of PMN-MDSC levels in peripheral blood. The percentage of PBMC expressing MDSC markers (CADO48A+CD14+) was determined by flow cytometry for the same groups of dogs as in C. Groups were compared by Mann-Whitney test. (E–G) Cognitive and neural marker tests (cross-validation of results). N=91 (E) Dogs’ performance in problem-solving V-tests correlates with CDS scores obtained from questionnaires completed by handlers. V-test performance is indicated by color as shown on the right y-axis; questionnaire scores reflecting the number of signs of cognitive dysfunction are shown on the x-axis. (F) Dogs that fail the V-test tend to have higher plasma levels of NfL (pg/ml). (G) Dogs that fail the V-test tend to interact less with the human in the “novel person” open field test. The “successful” group here includes dogs who were successful at least one attempt. For all panels, * - p < 0.05, **- p< 0.01, ****- p< 0.0001.