Exploration of inflammation-related markers in hens experiencing chronic stress

Abstract

Reduced adult hippocampal plasticity in hens has recently been demonstrated to be a consequence of chronic stress. This has been observed in multiple studies measured by reduced cellular density of doublecortin (DCX) neurons in the rostral and caudal hippocampal formation (HF). This includes hens with severe keel bone fractures (KBF), poor physical condition and unpredictable chronic mild stress (UCMS). Chronic stress is known to trigger activation of the hypothalamus pituitary adrenal (HPA) axis and ultimately lead to the rise in secretion of glucocorticoid hormones. However, there was no consistent increase in glucocorticoid hormones across these studies. This thesis investigates the involvement of inflammation in the periphery and the rostral and caudal HF in birds where chronic stress led to reduced DCX+ density. As a first step, this thesis measured gene expression of inflammatory cytokines in the spleen and HF where DCX+ neurons were reduced in birds with severe KBF and poor physical condition. There was no change in expression or any correlation with DCX+ density. Secondly this thesis measured density of microglia cells which have neuroprotective and neurotoxic characteristics by staining hippocampal sections with microglia marker IBA-1. This was to determine if the phenotype of microglia cells differs between birds with severe and minimal KBF who had reduced DCX+ density, towards an inflammatory ameboid microglia. The severity of the KBF does not lead to morphological changes between ameboid, rod and ramified microglia. Finally, this thesis utilized UCMS as a method of inducing chronic stress in a timecontrolled manner to determine if inflammatory cytokine expression changes throughout, which in this instance it does not. As there was no evidence of inflammatory cytokines and microglia involvement, astrocytes and their related markers were measured in the HF. This thesis found UCMS led to increased gene expression of astrocyte marker GFAP in the caudal HF and increased complement-3 protein across the rostral and caudal HF. These results suggest chronic stress could lead to increased presence of astrocytes and activation of the complement pathway in the HF. Astrocytes are known to secrete complement proteins which can lead to neuronal dysfunction. This points toward a potential role for astrocytes and the complement system in the HF due to chronic stress in hens.