EFFECTS OF A UNILATERAL INTRASTRIATAL 6-HYDROXYDOPAMINE MODEL OF EARLY PARKINSON'S DISEASE ON MIDBRAIN DOPAMINE NEURONS IN RATS: A STEREOLOGICAL STUDY

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by loss of dopaminergic function, leading to the classical clinical signs of tremor, bradykinesia and loss of postural balance. These motor symptoms occur late in the disease, and since the treatments for late-stage PD are largely ineffective, a better understanding of the early stages of PD is needed in order to prevent and treat the disease. The early disease process is still poorly understood, however, and current difficulties in diagnosing prodromal or very early-stage PD make clinical studies challenging. Therefore, animal models of early PD are an invaluable resource in discovering the neuropathological, behavioral and biochemical features of the early stages of neurodegeneration found in PD. Early PD is associated with non-motor clinical signs such as changes to sleep patterns, olfactory functions, cognition and mood; while gross motor function is largely compensated for until the later stages of dopaminergic neurodegeneration. The nature of these early clinical signs presents a challenge when assessing PD models, however, as subtle sensory and affective changes can be difficult to quantify in animals. The goal of this work, therefore, was to investigate morphological changes to the dopamine neurons most implicated in the development of PD. Morphological endpoints, which can be robustly quantified using unbiased stereological analysis, provide information about the changes occurring to the neuronal structure during the neurodegenerative process, and offer promise as an objective method to assess the conditions which render dopamine neurons vulnerable in PD, as well as to evaluate new neuroprotectants and therapeutic interventions in early PD animal models. A deficiency in n-3 polyunsaturated fatty acids (n-3 PUFAs), which have been shown to be neuroprotective, has been proposed as a potential factor in the vulnerability of dopamine neurons to PD. In order to determine the effects of n-3 PUFA deficiency on substantia nigra pars compacta (SNpc) dopamine neurons, morphological, behavioral and biochemical endpoints were investigated in the unilateral intrastriatal 6-hydroxydopamine (6-OHDA) model of early to moderate Parkinson's disease in Aim 1. In addition, a method was developed to use a novel staining method combining tyrosine hydroxylase (TH) staining with silver nucleolar (AgNOR) staining and stereological analysis techniques to quantify the morphological changes induced by 6-OHDA lesion in dopaminergic neurons in Aim 2. In Aim 3, this method was then expanded to investigate morphological changes to the dopaminergic nucleoli stained with AgNOR, which may lend valuable insights into the role of the nucleolus in early PD. In Aim 4 the cumulative techniques developed in the previous aims were employed to determine the differential morphological changes to the dopamine neurons and their nucleoli in the A8, A9 and A10 subpopulations of midbrain dopamine neurons, which correspond to the retrorubral field (RRF), SNpc, and ventral tegmental area (VTA), respectively. Although the functions of these different neuronal groups is complex and still not fully understood, these regions are differentially affected in PD; and in particular the A8 and A10 groups have been associated with the early non-motor effects of PD. Therefore the characterization of the differential morphological changes to these neurons and their nucleoli could provide valuable insights into the structural changes to the neurons induced by the neurodegenerative process, and may be useful in assessing early PD interventions in the future. Overall, this dissertation project contributed to a better understanding of the morphological changes to midbrain dopaminergic neurons as determined by stereological analysis in an early model of PD.