PSVI-31 - The Effect of Aging on Peripheral Blood Mononuclear Cell Proliferation in Labrador Retrievers

It is well documented that companion animals are living longer and as animals age their cells undergo immuno-aging or senescence, an age associated process of deterioration. In an effort to see how aging plays a role in immune cell proliferation, twelve healthy Labrador Retrievers (6-Young, 3Male/3Female, 6-Senior, 3Male/3Female) were selected to participate in a trial to determine what effect aging might have on the proliferative rate of peripheral blood mononuclear cells (PBMC). Mean age for Senior group was 8.74yrs, Young 2.38yrs (P.< 0.0001). Peripheral blood mononuclear cells (PBMC) were isolated, counted, seeded at a density of 5x104 in 96-well MP, cultured in RPMI-1640 complete medium (5% FBS), and mitogenic stimulation via 2-fold serial dilutions of Phytolacca Americana lectin (pokeweed mitogen-PWM) ranging from 0.8 -0.025 µg/mL. Following 48hr incubation in 5% CO2/95% humidity, 100µM Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU; a synthetic analog nucleoside of thymidine), was added to each culture well and further incubated 24hrs to allow DNA incorporation. Cells were harvested, denatured, and BrdU incorporated in newly synthesized DNA was bound by monoclonal antibodies (anti-BrdU-POD). The immune complexes were detected by tetramethyl-benzidine (TMB), reaction stopped with 1M Sulfuric acid (H2SO4), and quantified by measuring absorbance at 450nm. Proliferation was higher in Young dogs than Senior dogs at every concentration, signifying overall statistical significance (Mean: Young-0.972, Senior-0.697±0.057 SEM, P = 0.001). Rate difference by concentration was as follows: PWM 0.00 µg/mL: Young-0.109, Senior-0.194±0.031 (P = 0.099); 0.025 µg/mL: Young-0.788, Senior-0.481±0.201 (P = 0.321); 0.05 µg/mL: Young-1.002, Senior-0.610±0.207 (P = 0.227); 0.1 µg/mL: Young-1.113, Senior-0.776±0.195 (P = 0.244); 0.2 µg/mL: Young-1.249, Senior-0.915±0.172 (P = 0.217); 0.4 µg/mL: Young-1.271, Senior-0.965±0.148 (P = 0.191); 0.8 µg/mL: Young-1.252, Senior-0.936±0.117 (P = 0.101). These results coincide with other published literature, and these data suggest age plays a critical role in immunity and proliferative ability of PBMCs in the Labrador Retriever.