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Type: Theses
Title: The cerebrovascular response to metastatic melanoma and Clostridium perfringens type D epsilon toxin
Author: Mander, Kimberley Anne
Issue Date: 2017
School/Discipline: Adelaide Medical School
Abstract: The principal focus of this thesis is the cerebral vasculature and, more specifically, its fundamental and important role in two disease processes, namely cerebral metastatic melanoma and Clostridium perfringens type D epsilon neurotoxicity. Firstly, blood vessels are critical for both impeding and facilitating the penetration, colonisation, and spread of metastatic tumours such as melanomas in the brain and, secondly, the microvasculature is the major target of the potent bacterial neurotoxin, Clostridium perfringens type D epsilon toxin, which causes a severe, and frequently fatal, naturally-occurring, neurological disorder in domestic livestock and is a potential bioterrorism agent for human populations. There are important structural and functional differences between blood vessels in the brain and other tissues and the regional distribution is inhomogeneous. These features also have consequences for patterns of disease expression, for example lodgement of tumour emboli. Moreover, the dynamic microvascular interface between blood and brain parenchyma, termed the blood-brain barrier (BBB), differs in important structural detail from capillaries elsewhere and is critical in maintaining homeostasis in the central nervous system. In Part A of this thesis, the different patterns of neovascularisation in archival, human melanomas metastatic to the brain were characterised, given that acquisition of a new vascular supply is essential for these neoplasms to survive, proliferate, and disseminate. These new blood vessels are frequently structurally and functionally aberrant and those examined in the metastatic melanoma cohort herein were classified using histological and immunohistochemical techniques. It was also determined whether there was any correlation between vascular subtype and histological category of melanoma, mitotic index, extent of tumour necrosis, and intratumoural haemorrhage. Since the substance P (SP)/NK-1 receptor (NK-1R) system plays an important role in tumour survival, proliferation, and progression, its distribution was examined immunohistochemically in these metastatic melanomas, both in tumour-associated blood vessels and melanocytes. The NK-1 receptor was expressed by most melanocytes and endothelium in a small subset of tumour blood vessels, but there was no detectable immunoreactivity of the tachykinin peptide, SP, in tumour cells or blood vessels. The distribution of caveolin-1, the main structural component of caveolae, was also examined in these melanomas. Its immunoexpression was reduced in tumour-associated blood vessels, concordant with increased neoangiogenesis, and CAV-1 was commonly expressed in melanocytes, particularly in cell membranes, reflecting its important role in both tumour progression and suppression. Since melanomas generally metastasise via the haematogenous route and finally encounter the BBB when they reach the brain, it was decided to examine the transendothelial migration of melanocytes using in vitro and in vivo models. In a culture system, the migration of melanocytes from a melanoma cell line across a membrane representing a “blood-brain barrier” was quantified and the manner of their passage across this endothelial barrier examined by light and electron microscopy, the ultrastructural assessment being one of the very few studies of this type conducted to date. In order to examine how melanocytes in the systemic circulation enter the brain, a melanoma cell line was injected into rat carotid arteries and the distribution of melanocytes in the brain assessed at different time intervals post-injection. Unfortunately, very few tumour cells penetrated into the brain parenchyma and this technique proved to be unsatisfactory for examining transendothelial migration of metastatic melanocytes and evaluation of drugs that might impede this process. In Part B of this thesis, several vascular features of the neurological disorder produced by Clostridium perfringens type D epsilon toxin (ETX) were studied. In the principal, and novel, study, the aim was to determine whether ETX produced a direct and damaging effect on cerebral microvascular endothelial cells in vitro. While previous histological and ultrastructural studies suggested that the fundamental lesion in this neurotoxicity was ETX-induced microvascular injury, with subsequent BBB breakdown, increased vascular permeability and severe, generalised cerebral vasogenic oedema, the effect of ETX on brain-derived endothelial cells in culture had not been examined. The present study found, for the first time, that EXT produces a dose- dependent cytopathic effect on cultured human brain microvascular endothelial cells, confirming the importance of microvascular endothelial damage in the pathogenesis of this neurological disorder. In an animal model of ETX neurotoxicity using Sprague-Dawley rats, extravasation of endogenous albumin was used as a surrogate immunohistochemical marker of increased vascular permeability; loss of endothelial barrier antigen was evaluated after exposure to ETX as it is a marker of an intact BBB in this species; and the role of the major water channel protein in the brain, aquaporin-4, in the development/resolution of EXT-induced cerebral oedema was studied. Since the BBB is a prime target for ETX-induced brain damage and the blood-retinal barrier (BRB) resembles the BBB in many respects, the action of ETX on the BRB was also examined in rats using albumin immunohistochemistry to assess enhanced vascular permeability and electron microscopy to study retinal blood vessels. Retinal microvascular endothelial damage resembled that found in ETX-disrupted BBB and there was widespread retinal oedema as indicated by diffuse albumin extravasation. Studies carried out in this thesis aimed to better characterise the cerebral microvasculature alterations and the associated mechanisms, in response to two distinct insults; metastatic melanoma, and ETX. A range of investigative modalities facilitated the detailed exploration of vascular reactions in these 2 neuropathological states and findings from this thesis will direct further research in the field of cerebrovascular pathology.
Advisor: Vink, Robert
Thornton, Emma
Harford-Wright, Elizabeth
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2017.
Keywords: melanoma
metastasis
cerebrovascular
ETX
mechanisms
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals
DOI: 10.4225/55/5b10c2b692ea0
Appears in Collections:Research Theses

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