Research ReportEffects of chronic alcohol consumption and withdrawal on the response of the male and female hypothalamic–pituitary–adrenal axis to acute immune stress
Highlights
► Chronic ethanol consumption and withdrawal impair HPA axis functioning. ► Excess alcohol and withdrawal have sex-specific effects on HPA axis activity. ► The response of the HPA axis to ethanol withdrawal is gender-specific. ► Chronic ethanol consumption disrupts the HPA axis response to a novel stress. ► Sex steroid hormones influence the response of the HPA axis to acute stress.
Introduction
It is widely known, from both human research and studies with experimental animals, that the acute exposure to ethanol leads to a transient, but powerful activation of the main components of the hypothalamic–pituitary–adrenal (HPA) axis (for a review, see Madeira and Paula-Barbosa, 1999). Specifically, a single alcohol administration increases the blood levels of adrenocorticotrophic hormone (ACTH) and corticosterone, and enhances the synthesis of corticotrophin-releasing hormone (CRH) and vasopressin (VP) by neurons of the medial parvocellular division of the hypothalamic paraventricular nucleus (PVNmp), the central site that regulates the activity of the HPA axis (Whitnall, 1993). There are also data indicating that more prolonged exposures to ethanol lead to the development of tolerance in laboratory animals. In particular, it has been shown that when the exposure is maintained for a few days or weeks, the plasma concentrations of corticosterone are lower than those detected after acute exposures, and the mRNA levels of CRH are either increased or unchanged, and those of VP are decreased (reviewed in Madeira and Paula-Barbosa, 1999). In addition, studies using experimental models of alcohol consumption over months have shown that corticosterone concentrations return to normal levels, but the synthesis, and consequent expression, of CRH and VP in the PVNmp is reduced (Silva et al., 2002, Silva et al., 2009). In theory, these changes might merely reflect the adaptation of the HPA axis to alcohol exposure (Ogilvie et al., 1998, Spencer and McEwen, 1990). However, the observation that withdrawal does not abrogate the effects of chronic alcohol consumption on the number of CRH- and VP-producing neurons in the PVNmp (Silva et al., 2002, Silva et al., 2009) suggests that the down-regulation of these neuropeptides might actually result from ethanol effects on cellular function.
To explore this possibility, we have tested the hypothesis that rats submitted to prolonged alcohol consumption and to subsequent withdrawal are unable to mount an adequate response of the HPA axis to stressors. It is well known that acute stress activates the HPA axis by increasing the synthesis of CRH and VP in the parvocellular neurons of the PVN and the release of ACTH by the pituitary, leading to the production of glucocorticoids (Harbuz and Lightman, 1992, Itoi et al., 2004, Kovács and Sawchenko, 1996, Ma et al., 1997, Tsigos and Chrousos, 2002). The endotoxin lipopolysaccharide (LPS) is a systemic stressor that induces the production of endogenous cytokines, which, in turn, stimulate the synthesis and the release of CRH and VP into the hypophysial portal blood, with subsequent pituitary-adrenal activation and increased glucocorticoid secretion (Beishuizen and Thijs, 2003, Rivest et al., 1995). In the present experiment, we have administered a single intraperitoneal injection of LPS to rats consuming alcohol for 6 months and to rats withdrawn from chronic alcohol consumption for 2 months, and measured the impact of the immune stimulation on the mRNA levels of CRH and VP in the PVNmp and on the circulating levels of corticosterone.
There is ample evidence that the activity of the HPA axis is sexually dimorphic in basal conditions and after stimulation, with females producing more corticosterone and having more CRH and VP neurons in the PVNmp than males (Figueiredo et al., 2007, Kudielka and Kirschbaum, 2005, Silva et al., 2009). The vulnerability of the HPA axis to excess alcohol and to ethanol withdrawal also differs between the sexes. Females secrete more corticosterone than males in response to acute and short-term alcohol exposures (Ogilvie and Rivier, 1997) and, unlike males, females have reduced circulating levels of corticosterone after withdrawal from chronic alcohol consumption together with a further depression in CRH and VP expression by PVNmp neurons (Silva et al., 2009). There are also studies showing that, in rodents, the HPA response to endotoxin and to cytokines is enhanced by gonadectomy and attenuated by estradiol and testosterone replacement (Kudielka and Kirschbaum, 2005, Seale et al., 2004a, Seale et al., 2004b, Spinedi et al., 1992). Therefore, in this study we have analyzed in parallel the influence of LPS on the functioning of the HPA axis during chronic alcohol consumption and after withdrawal in males and in females. To examine the degree to which the sexual dimorphism of the HPA axis response to LPS is influenced by the estrous cycle, and specifically whether circulating estrogens are responsible for the sexually dimorphic response, females were ovariectomized and injected 2 weeks later, before LPS administration, with either estradiol benzoate or vehicle.
Section snippets
Ethanol consumption and blood alcohol levels
The variations in the amount of liquid diet ingested over the period of alcohol consumption were very similar to those described in an earlier study (Silva et al., 2009): the volume of fluid ingested by ethanol-treated rats decreased to about half the amount of water drunk by controls during the first month of alcohol consumption and, then, remained relatively steady. The mean daily ethanol consumption over the entire experimental period, expressed in g/day/ kg bw, was significantly higher (p <
Discussion
Data of the present study show that the response of the HPA axis to an acute immune challenge during prolonged alcohol consumption and ethanol withdrawal is dependent on gender and, in females, relies on the circulating levels of estrogens. In males, corticosterone release in response to a single intraperitoneal injection of LPS is attenuated during prolonged alcohol consumption and within control levels after withdrawal. Females do not increase corticosterone concentrations in response to LPS
Animals and treatments
Male and female Wistar rats were housed three per cage and maintained throughout the experiments under 12 h light/dark cycles (lights on at 07:00) and temperature of 22 °C. Solid diet (4RF21/C Mucedola, Milan, Italy) and water were freely available until rats were 2-month-old. At this age, rats were randomly assigned to the following experimental groups: (1) Ethanol-treated rats were given an aqueous ethanol solution as their only available liquid source for 6 months, i.e., until the age of 8
Acknowledgments
This work is supported by National Funds through FCT — Fundação para a Ciência e a Tecnologia within the scope of the Strategic Project Centro de Morfologia Experimental (CME/FM/UP) — 2011–2012 and Project PEst-OE/SAU/UI0121/2011. We are grateful to Dr. Conceição Gonçalves for help with biochemical measurements. We also thank Professors M. M. Paula-Barbosa for helpful comments on the manuscript and Armando Pinto for statistical advise. The authors have no conflict of interests to declare.
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