IMIBIO-SL   20937
INSTITUTO MULTIDISCIPLINARIO DE INVESTIGACIONES BIOLOGICAS DE SAN LUIS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
NOREPINEPHRINE MODULATES DAILY RHYTHMS IN EX VIVO SPLENIC MACROPHAGES
Autor/es:
ANZULOVICH AC; DELGADO SM; RAMIREZ M; CARGNELLUTTI E
Lugar:
SAN LUIS
Reunión:
Congreso; XXXVII REUNION CIENTIFICA DE LA SOCIEDAD DE BIOLOGIA DE CUYO; 2019
Resumen:
The splenic macrophages (MΦ) phagocytes and eliminates circulating pathogens, and orchestrate the development of the specific acquired immune response. Even though circadian effects on the immune system have been documented, the circadian regulation in the spleen has not been completely elucidated yet. In mammals, the central clock in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus synchronizes cell-autonomous clocks. Communication between SCN and spleen occurs by the sympathetic nervous system (SNS), through nerves that release norepinephrine (NE) in areas of T and MΦ cells. Previously, other authors reported daily oscillation of NE in spleen. Our focus was to determine whether rhythmic expression of the molecular clock in the splenic MΦ is regulate by SNS innervation. For this purpose, we disrupted the autonomic innervation to the spleen of 4-month old male Holtzman rats, by splenic injections of guanethidine. Guanethidine has several effects on peripheral sympathetic neurons including blockade of neural transmission, depletion of neuronal NE stores, and blockade of reuptake of NE into the neurons. Animals were maintain under 12h-light: 12h-dark conditions and ad-libitum food/water intake until the experiment. First, we evaluated the effects of local guanethidine injection on spleen macroscopic appearance, size, weight, and lymphocyte number. Control animals received intrasplenic saline solution injection. The Student t test was used for comparison of data between both groups. Second, to study the NE temporal impact on the molecular clock of splenic MΦ, ten days after injection of saline solution or guanethidine, control (N=4/ZT) and sympathectomized rats (N=3/ZT) were euthanized at different times during a 24 h period (ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22) and spleen was aseptically removed for ex vivo cultures. The clock transcription factor, BMAL1 and ACTIN, were analysed by Western blot from splenic adherent cells. Time point data were compute by 1-way analysis of variance (ANOVA) and followed by Tukey post hoc test. Further, chronobiologic statistics were used for validating temporal changes as rhythms. Thus, each series of data were analysed by Cosinor method. We found no significant differences in the spleen macroscopic appearance, size, weight, and lymphocyte number between both treated groups, suggesting local guanethidine injections did not have any toxicity on spleen cells. The splenic MΦ from control rats showed a diary oscillation of BMAL1 (% rhythm: 71.8), with its acrophase occurring at the middle of the light period. In contrast, the ex vivo splenic MΦ from guanethidine-treated animals lost the diary oscillation of BMAL1. Our results would indicate a regulation of the molecular clock in splenic adherent cells by the SCN, through the NE sympathetic pathway.