Major depressive disorder patients present chronic stress and decreased immunity. The Wistar-Kyoto rat (WKY) is a strain in which the hypothalamic-pituitary-adrenal axis is overactivated. To determine whether chronic stress induces changes in corticosterone levels and splenic lymphoid tissue, 9-week-old male rats were subject to restraint stress (3 h daily), chemical stress (hydrocortisone treatment, 50 mg/Kg weight), mixed stress (restraint plus hydrocortisone), or control treatment (without stress) for 1, 4, and 7 weeks. The serum corticosterone levels by RIA and spleens morphology were analyzed. Corticosterone levels as did the structure, size of the follicles and morphology of the parenchyma (increase in red pulp) in the spleen, varied depending on time and type of stressor. These changes indicate that chronic stress alters the immune response in the spleen in WKY rats by inducing morphological changes, explaining in part the impaired immunity that develops in organisms that are exposed to chronic stress.
Chronic stress induces immunosuppressive effects and increases susceptibility to various diseases, such as infection, autoimmune disease, and neoplasia [
To study the function of stress in depression, several animal models have been developed, permitting researchers to examine aspects of neurobiology of this disease. One of the most important systems that controlled the stress response is the hypothalamus-pituitary-adrenal axis (HPA), or stress axis. Wistar-Kyoto rats (WKY) are hypersensitive to stress and display dysregulation of the HPA axis, in association with anhedonic behavior [
To maintain homeostasis, glucocorticoids establish a negative feedback loop through specific receptors [
During chronic stress, glucocorticoid levels rise significantly and induce a negative immune response [
Male Wistar-Kyoto rats (180–200 g) were purchased from Harlan (Indianapolis, IN, USA) and group-housed in clear plastic containers (450 × 240 × 200 mm) with food and water
WKY rats were assigned to experimental groups (
Hydrocortisone (50 mg/kg), prepared daily, was dissolved in ethanol (2.2 p. 100) and added to drinking water.
Rats were sacrificed by rapid decapitation in the morning (10:00 h) or immediately after the restraint stress test. Trunk blood was collected into Vacutainer tubes and centrifuged at 2500 rpm for 15 min at 4°C, and the serum was stored at −70°C until analysis. Serum CORT was analyzed using an RIA kit (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA). The Coat-A-Count Rat Corticosterone method is a solid-phase 125I-radioimmunoassay, the detection limit of which is 5.7 ng/mL. The samples (50
Spleens were fixed in paraformaldehyde 4 p. 100 for 24 h, paraffin embedded, sectioned serially (4 to 5
Data are presented as mean ± SD, calculated from each experimental group (six rats per group). Statistical significance was determined by one-way ANOVA, followed by Bonferroni’s
Figures
Effect of stressors on body and adrenal gland weight and corticosterone levels in Wistar-Kyoto rats. Wistar-Kyoto rats (
Corticosterone levels (Figure
The histological architecture of the spleen was preserved along the experimental followup. White pulp was seen as large secondary follicles with an image of a “double halo” due to the prominence of the marginal zone. It comprised an assemblage of two vaguely delimited round or oval structures that were superimposed, with an axial profile of the penicillated artery in the center of the inner structure. Each layer contained a mixture of cells, primarily small lymphocytes without any evidence of nuclear atypia or activation (mitotic/apoptotic activity, clearing of chromatin-prominent nucleoli, and macrophages with cytoplasmic tangible bodies) (Figures
Photomicrographs of spleen from Wistar-Kyoto rats: (a and b) without treatment; (c) treated with restraint stress for 1 week; (d) after 7 weeks of immobilization; (e) after 1 week with hydrocortisone; (f) after 7 weeks of hydrocortisone; (g) after 1 and 7 weeks of mixed treatment (hematoxylin and eosin stain; magnification, 100x).
Notably, in the immobilization group, there was an expansion of the red pulp compartment and involution of the white pulp, with loss of the prominent marginal zone. In contrast, at seven weeks, the red pulp continued to expand, without significant changes in composition, at the expense of the surface that was occupied by the white pulp (Figure
At one week, the hydrocortisone group had essentially the same histological appearance as the control group. The cellular composition of the red and white pulp did not vary. There was no evidence of hematopoietic activity in this microscopic field (Figure
Versus the control group, the rats that received mixed treatment experienced a loss of white pulp and a decrease in the size of the follicles and the marginal zone. The greatest difference between groups occurred by seven weeks, wherein the pulp was distorted, favoring the red pulp in all groups. The surface ratio favored the red pulp nearly to 4 : 1 in the mixed group, maintaining the spacing of the tissue elements (Figure
Despite the subtle changes between immobilization- and hydrocortisone-treated groups, we noted a consistent trend in the disarray of the pulp. The mixed treatment group experienced more significant alterations, favoring an increase in red pulp and in the structure and size of the follicles. After four weeks of stress, there were no histological differences compared with that at one week.
Glucocorticoids are characteristic mediators of stress responses, and the detrimental effects of chronic stress on health have been proposed to be attributed to their immunosuppressive properties [
We observed that all experimental groups display a decrease in body and adrenal gland weight, associated with a decline in corticosterone levels; the least extensive changes were observed in the group of immobilization after one week. Notably, the magnitude of these decreases was dependent on the stressor (mixed > hydrocortisone > immobilization). These results are consistent with other animal studies that have demonstrated that repeat restraint and exogenous corticosterone lower body and adrenal gland weight [
The mixed treatment lowered all parameters significantly; we expected that the two component stressors would synergize. The significant reduction in corticosterone levels might be attributed to the length (days to weeks) and extent of the stressor, which should have desensitized the HPA axis-desensitization that might have been caused by impaired glucocorticoid receptor expression and function [
Few studies have investigated the effects of chronic stressors on lymphoid organs and the immune response [
We noted alterations in red and white pulps with the various treatments (IMM, HC, and IMM + HC). Restraint stress can cause the spleen to involute [
As discussed, chronic administration of corticosterone in rats effects behavioral and neurobiological alterations that mirror several of the core symptoms and neurobiological changes that are associated with psychiatric illnesses. Clinical studies have shown that major depressive patients express a pattern of anti-inflammatory cytokines, in association with high cortisol levels [
Based on the function of the spleen as a site of antigen presentation, B- and T-lymphocyte activation, and polarization of cytokine responses, chronic stress induces morphological changes that might alter cellular and humoral immune responses.
Funding for this study was provided by Proyecto Factor de Transferencia-IPN IC-10-002 and INPRF Project NC092318.0. M. E. Hernandez and N. G. Hernandez-Chan were supported by a scholarship from RedFarmed-CONACYT.