2017 ACVIM Forum Research Abstract Program

We studied the pharmacological effect of TX on equine neutrophils obtained by jugular venipuncture from 6 healthy horses, and purified by Ficoll gradient centrifugation. Respiratory burst was evaluated by chemoluminescence induced by opsonized zymosan in neutrophils. Luminol-emitted luminescence was measured at 37°C for 3 hours using a luminometer. The Different concentrations of TX and its combination with selective estrogen receptor modulators (SERM) were evaluated (TX 0.5; 1; 2.5; 5; 7.5; 10; 15 and 20 lM; ICI 182,780 [high affinity estrogen receptor antagonist] 1, 10 and 100 nM; G15 [selective G-protein estrogen receptor antagonist] 1, 10 and 100 nM; b-estradiol 1, 10 and 100 nM; G1 [selective G-protein estrogen receptor agonist] 1, 10 and 100 nM; ICI 100 nM + TX 11.5 lM; G15 100 nM + TX 11.5 lM; b-estradiol 100 nM + TX 11.5 lM; and G1 100 nM + TX 11.5 lM). Phosphatidylserine expression was quantified by flow cytometry using a commercial Annexin V-FITC kit (BD Biosciences). Cells were incubated at 37°C in a humidity chamber (CO 2 5%) for 20 minutes with: DMSO 0.1% (vehicle), TX 11.5 lM,; b-estradiol 100 nM; ICI 100 nM; G15 100 nM and G1 100 nM. Results showed that TX inhibits neutrophil respiratory burst induced by opsonized zymosan in a dose dependent manner. Nuclear (17-b-Estradiol) and GPR30 cell membrane (G1) estrogen receptor agonists and their antagonists (ICI 182,780 and G15 respectively) do not block nor reproduce TX 0 s effect. Thus, TX does not inhibit respiratory burst through estrogen receptors. TX 11.5 lM also produced an increase in phosphatidylserine translocation, which was not observed with any of the SERM used. This shows a direct pro-apoptotic effect on neutrophils. In conclusion, TX 0 s inhibitory and pro-apoptotic effect on neutrophils seems to be independent of estrogen receptors. We hypothesized that magnesium sulfate (MgS0 4 ) has bronchodilator effects in severe asthmatic horses and potentiates the effects of inhaled salbutamol.