Chronic stress adaptation of the nitric oxide synthases and IL-1β levels in brain structures and hypothalamic-pituitary-adrenal axis activity induced by homotypic stress.

The aim of this study was to determine the effect of repeated restraint stress (RS) on a single restraint (homotypic) stress-induced expression of interleukin-1β (IL-1β), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in the prefrontal cortex (PFC), hippocampus and hypothalamus and their adaptational changes in chronic stress. Also the involvement of plasma IL-1β in adrenocorticotropic hormone (ACTH) and corticosterone secretion during chronic stress was investigated. Rats were subjected to a single restraint for 10 minutes or restrained twice a day for 3, 7 and 14 consecutive days and 24 hours after the last stress period rats were restrained for 10 min and rapidly decapitated 0, 1, 2 and 3 hours later. The IL-1β, nNOS and iNOS protein levels in brain structures samples were analyzed by Western blot procedure and IL-1β, ACTH and corticosterone levels were determined in plasma. Single restraint induced a strongest decrease of iNOS protein levels (1-3 h) in the PFC and a weaker decline in the hippocampus and hypothalamus (3 h) after stress cessation. Single restraint markedly increased IL-1β protein level in PFC and hippocampus. In the PFC repeated restraint for 3 days significantly increased the homotypic stress induced iNOS and IL-1β protein levels and this increase gradually declined after 7 and 14 days of repeated restraint. Much weaker yet a parallel changes appeared with neuronal NOS level. In the hippocampus prior stress for 3, 7 and 14 days significantly increased the homotypic stress induced iNOS protein level parallel with IL-1β level which gradually declined with prolonged period of repeated restraint. In the hippocampus a longer restraint period, 7 and 14 days markedly decreased nNOS protein level evoked by homotypic stress. In the hypothalamus prior stress for 3 days strongly enhanced the homotypic stress-induced iNOS level and repeated stress for 7 and 14 days blunted this effect. Repeated stress increased IL-1β level in response to homotypic stress after 3 days and after 14 days. The present results indicate time-related similarities in the potent alterations in IL-1β and iNOS protein levels in brain structures. Single restraint induced a significant increase of plasma IL-1β level which was abolished by pretreatment with IL-1 receptor antagonist (IL-1Ra). A parallel strong increase of plasma ACTH and corticosterone levels were significantly impaired by IL-1Ra suggesting a marked involvement of stress-induced stimulation of ACTH and corticosterone by IL-1β in single restraint. In repeatedly restrained rats IL-1Ra significantly blunted plasma IL-1β level induced by homotypic stress. A parallel strong increase in plasma ACTH level by homotypic stress was not substantially altered by pretreatment with IL-1Ra in repeatedly stressed rats. Plasma a corticosterone level increased by homotypic stress in rats restrained for 3 and 14 days was not affected by pretreatment with IL-1Ra, but after for 7 days its level was significantly enhanced. These results suggest that repeated stress desensitizes IL-1β-induced stimulatory component in a single restraint stress-induced hypothalamic-pituitary-adrenal (HPA) axis stimulation. A sensitization by homotypic stress of corticosterone response after restraint for 7 days may depend on other stimulatory systems acting within adrenal glands during prolonged stress. Comparative data from the same model of rather mild psychological stress allows for the comparison of functional adaptive changes of NO synthases and IL-1β in brain structures involved in stress regulation. In general, the iNOS system is strongly sensitized by repeated stress for 3 days in prefrontal cortex and hippocampus. Increased plasma IL-1β level by a single restraint stress is significantly involved in ACTH and corticosterone secretion. Repeated stress for 3-14 days reduces this participation of IL-1β in pituitary-adrenal stimulation.