Melatonin Modulates Leukocytes Immune Responses in Freshwater Snakes, Natrix piscator
The role of melatonin in altering cell-mediated nonspecific immune responses has been documented in mammals, but there is no report available in reptiles. We designed the present study to evaluate the role of melatonin in altering innate immune responses of leukocytes in freshwater snakes. We administered melatonin injections (dose: 5 and 10 μg/g body weight) during evening hours. Animals receiving saline served as controls. Snakes were sacrificed after 10 and 20 days. We studied the alteration in total and differential leukocyte counts, blood neutrophil phagocytosis, nitric oxide production, superoxide production, and lymphoproliferation. We did not observe a consistent and significant change in total leukocyte count, whereas monocyte, eosinophil, and basophil counts were increased in response to melatonin. Interestingly, the phagocytic response of neutrophils was inhibited when treated with melatonin. Nitrite release and superoxide production by leukocytes were significantly higher in snakes receiving melatonin injections. Exogenous melatonin also enhanced the mitogen-induced lymphocyte proliferation in a manner dependent on dose and duration of melatonin treatment.Abstract
Effects of in vivo melatonin (5 μg and 10 μg/g body weight) on Total Leukocyte Count (TLC), Lymphocyte, Monocyte, Heterophil, Basophil, and Eosinophil count in freshwater snakes, Natrix piscator (*P < 0.05). Control animals received saline injection.
Effects of in vivo melatonin (5 μg and 10 μg/g body weight) on NBT reduction assay, leukocyte phagocytosis, and nitrite release in freshwater snakes, Natrix piscator (*P < 0.05, **P < 0.01, ***P < 0.001). NBT reduction results in the formation of blue formazan deposit in leukocytes. Upper left panel shows number of blue cells scored randomly in 20 optic fields at 4 × 10 X magnification. NBT assay result is shown in lower left panel. Phagocytosis (Upper right panel) is shown as phagocytic index and percentage phagocytosis. Nitrite assay result is shown in lower right panel.
Effects of in vivo administration of melatonin (5μg and 10μg/g body weight) for 10 days (A) and 20 days (B) on Con A, PHA, and LPS induced lymphocyte proliferation in freshwater snakes, Natrix piscator. Error bars bearing the same superscript do not differ significantly (P < 0.05). ConA5, ConA10, and ConA20 = concanavalin A 5, 10, and 20 μg/mL, respectively; PHA5 and PHA10 = phytohemagglutinin 5 and 10 μg/mL, respectively; LPS10 and 20 = lipopolysaccharide 10 and 20 μg/mL, respectively. Stimulation index was calculated by the formula: Stimulation Index = Optical Density of stimulated culture / Optical Density of unstimulated culture.
Effects of in vitro melatonin (aMT0, aMT100, aMT200, and aMT500 - Melatonin 0, 100, 200 and 500 pg/mL, respectively) on mitogen induced lymphocyte proliferation. Lymphocytes were obtained from freshwater snakes treated with in vivo melatonin for 10 days (A) and 20 days (B). The error bars bearing the same superscript do not differ significantly (P < 0.05). ConA5, ConA10, and ConA20 = concanavalin A 5, 10, and 20 μg/mL, respectively; PHA5 and PHA10 = phytohemagglutinin 5 and 10 μg/mL, respectively; LPS10 and 20 = lipopolysaccharide 10 and 20 μg/mL, respectively.
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