Altered Thyroid Hormone Regulation and Behavioural Change in a Sub-population of Rats following Injury

Abstract

Overview Of This Thesis. This thesis documents an investigation of thyroid hormones and the regulation of thyroid hormones in an animal model of neuropathic pain. The technique used in this model, Chronic Constriction Injury (CCI), produces an injury that creates an inescapable mechanical and inflammatory stress for the rat. Various forms of stress, and in particular unavoidable stress, have been shown to alter thyroid hormones in both rats and humans. This rat model has been developed to more closely resemble the neuropathic pain state in humans where a proportion of patients develop a syndrome of social and behavioural changes, which has been termed ‘disability’. As well as demonstrable sensory dysfunction, as a result of the nerve injury, a sub-population of the rats exhibit altered patterns of complex social behaviours. The results of three experiments are presented. In each case a cohort of rats was subjected to the same procedure – Chronic Constriction Injury – and changes in social behaviour measured using a Resident-Intruder test. Rats were assigned to one of three groups based on the results of the behavioural test: No Disability (ND), Transient Disability (TD) or Persistent Disability (PD). In experiment 1, documented in Chapter 4, peripheral thyroid and corticosteroid hormones were measured pre and post-CCI together with Thyroid Stimulating Hormone (TSH) levels. Results for the three different behavioural groups were compared. In experiment 2, documented in Chapter 5, RNA was extracted from the hypothalamuses of rats sacrificed six days post CCI. Relative gene expression was determined for a number of factors involved in thyroid hormone and corticosteroid regulation. In experiment 3, documented in Chapter 6, at the end of the experimental period, seven days post-CCI, the rats were perfused to remove blood and the brains preserved in paraformaldehyde. The hypothalamuses were examined using immunohistochemical techniques for evidence of differing protein expression for molecules involved in thyroid hormone regulation. Summary of Findings. Behavioural Testing (Chapter 3): The results of behavioural testing for the experimental groups of animals used in this study conform to those previously found using this model (Monassi et al., 2003). Based on previously derived criteria 70% were classified as ‘No Disability’ (ND); 10% of the rats and were classified as ‘Transient Disability’ (TD) and 20% of rats were classified as ‘Permanent Disability’ (PD) animals. Rats classified as PD have significantly decreased levels of Dominance behaviour on days 1-3 and 4-6 post-CCI when compared to ND rats whereas TD rats have decreased Dominance behaviour on days 1-3 only. Pre-CCI behaviour is not predictive of post-CCI behavioural change. Peripheral Thyroid Hormones (Chapter 4): When compared to pre-injury levels, the mean total thyroxine (TT4), total triiodothyronine (TT3) and free thyroxine (fT4) levels were significantly decreased in the PD rats whereas the post-injury levels of these hormones did not change in the ND and TD rats. There was no difference between groups in mean free triiodothyronine (fT3) and thyroid stimulating hormone (TSH) levels pre and post-injury. The pattern of secretion of hormones differed in the ND and PD rats over the six days post-injury. In the ND rats, there was a surge in TSH post-injury and thyroid hormone levels were maintained at pre-injury levels. However, in the PD group, the TSH response was delayed, blunted and inadequate as thyroid hormone levels decreased. There was a significant correlation between decreased Dominance behaviour and both TT4 and fT4 but no correlation with TT3 or fT3. Peripheral Corticosteroids (Chapter 4): There was an increase in corticosterone in all animals post-CCI. The Transient Disability rats showed the greatest increase in corticosteroid and this was significantly different to the other two groups of rats. There was no difference in post-CCI corticosterone levels between the PD and ND rats. Hypothalamo-pituitary-thyroid (HPT Axis) regulation (Chapters 5 & 6): RT-qPCR measurement of relative mRNA expression in the hypothalamus showed that there was no difference between No Disability, Transient Disability and two groups of control rats (Behavioural Controls (BC) and naïve group caged controls) for Thyrotrophin Releasing Hormone (TRH), Thyroid Hormone Receptor β (TR β), Deiodinase 2 (Dio2) and Deiodinase 3 (Dio3). Compared to the BC rats, the Permanent Disability rats had significantly decreased relative expression of TRH and Deiodinase 2 and significantly increased expression of Deiodinase 3 mRNA. Immunohistochemical techniques used to label TRH, TR β, Deiodinase2 and Deiodinase 3 protein distribution in the Paraventricular Nucleus (PVN) of the hypothalamus showed significantly fewer immunoreactive profiles for Deiodinase 3 and TRβ in the group with changed behaviour (PD group) compared to the Behavioural Control rats. These differences were shown to be specific for the sections of the PVN where the hypophysiotrophic TRH secreting neurons are known to be primarily located. Hypothalamo-pituitary-adrenal (HPA axis) regulation (Chapter 5): RT-qPCR measurement of relative mRNA expression in the hypothalamus showed that there was no difference between ND, TD, PD and Behavioural Controls rats for Corticotrophin Releasing Hormone (CRH), Glucocorticoid Receptor (GR) and Vasopressin. Compared to the Behavioural Controls, group caged Control animals had significantly increased relative expression of CRH and Vasopressin mRNA.