The plasma pentraxin proteins serum amyloid P component (SAP) and C-reactive protein (CRP) bind specifically to nuclear autoantigens. There is a blunted acute phase response of human CRP in systemic lupus erythematosus (SLE), and of mouse SAP in NZB/W murine SLE. SAP deficiency in (129/Sv X C57BL/6)F2 mice is associated with spontaneous antinuclear autoimmunity. The pentraxins may thus function in preventing autoimmunity. Pure line C57BL/6 SAP knockout mice, studied here for the first time, spontaneously developed broad spectrum anti-nuclear autoimmunity resembling human SLE, and, females in particular had proliferative immune complex glomerulonephritis but without proteinuria or renal failure. Mice hemizygous for the SAP gene deletion had an intermediate autoimmune phenotype. Injected apoptotic cells and isolated chromatin were more immunogenic in SAP-/- than in wild-type mice. Extrinsic chromatin was catabolised predominantly in hepatocytes, Kupffer's cells and renal parenchymal cells. Plasma clearance of long chromatin and core particles was marginally slower in SAP-/- mice, with significantly greater splenic uptake of nucleosome core particles, which may have immunological significance. SAP bound to apoptotic cells but had no effect on their phagocytosis in mice in vivo, or by human macrophages in vitro. CRP also bound to apoptotic cells but did not compete with SAP, indicating recognition of different ligands. In contrast, to the C57BL/6 strain, pure line 129/Sv SAP knockout mice did not produce autoantibodies. Formation of antinuclear autoantibodies is thus markedly strain dependent. Interestingly, transgenic expression of human SAP in the C57BL/6 SAP knockouts did not abrogate the autoimmune phenotype. Transgenic reconstitution of the knockouts with mouse SAP is currently in progress to confirm that autoimmunity is caused by SAP deficiency and not by 129/Sv genes transferred into the C57BL/6 background during homologous recombination, or by disruption of loci associated with murine SLE that are close to the mouse SAP gene.

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Vascular cognitive impairment describes a heterogeneous condition in which cognitive decline is precipitated by underlying cerebrovascular dysfunction. Ageing, as well as vascular diseases such as hypertension, stroke, cerebral small vessel disease and cerebral amyloid angiopathy, are risk factors for vascular cognitive impairment. The precise mechanisms by which these conditions impact the cerebral vasculature to drive cognitive decline, however, are unknown. Previous research has indicated that vascular risk factors can lead to microvascular oxidative stress, inflammation and endothelial dysfunction that can lead to tissue hypoperfusion, the development of white and grey matter vascular lesions (microinfarcts and microbleeds) and cognitive impairment. It was hypothesised that ageing, a prominent risk factor for cognitive decline, would induce impairments on neurovascular coupling resulting from neurovascular unit disruption. It was further hypothesised that induction of chronic cerebral hypoperfusion would mediate neurovascular dysfunction and vascular lesion development through increased oxidative stress, resulting in cognitive decline. Finally, it was also hypothesised that neurovascular impairments resulting from ageing and chronic cerebral hypoperfusion would be exacerbated in the presence of amyloid deposition. Four studies were performed in order to test these hypotheses. Vascular risk factors can be reproduced using experimental mouse models and provide a valuable basis in which to test hypotheses and therapeutic interventions. As such, a primary aim of this thesis was to develop and validate sensitive MRI approaches that would allow the detection of vascular alterations in vivo. In the first series of studies, MRI techniques to assess resting cerebral blood flow, vessel number, vascular lesions and inflammation in experimental mice were validated using established in vivo and ex vivo techniques, so that these techniques could be used in subsequent studies for vascular assessments in vivo. Arterial spin labelling was developed to assess resting cerebral blood flow, and was able to detect reductions in blood flow following cerebral hypoperfusion that correlated well with those obtained from laser speckle imaging. Q-map imaging was able to detect reductions in vessel number in acute lesions, and in non-lesioned mice measures of vessel number correlated well with histopathological measures. Structural T2 imaging was performed in order to detect ischaemic and haemorrhagic lesions in chronically hypoperfused mice, and was validated using H&E and Perls’ staining. Finally, contrast-enhanced T2* imaging was used to detect iron oxide uptake by macrophages in the brains of hypoperfused mice, which was further validated by the identification of iron-containing macrophages in immunostained brain sections. The second study was conducted to test the hypothesis that ageing would impair neurovascular unit function and structure, and that these impairments would be exacerbated in the presence of amyloid pathology. The aim of the study was to incorporate previously developed in vivo imaging approaches in the assessment of vascular function and alterations in neurovascular unit structure in both wild type and TgSwDI mice. As predicted, ageing caused a pronounced deficit on measures of neurovascular coupling, however this was not exacerbated by accumulation of amyloid in TgSwDI mice and was not associated with alterations in baseline blood flow measured by arterial spin labelling. Structural assessment of the neurovascular unit revealed a loss of contact between astrocytic endfeet and vasculature, which was significantly associated with the impairment on neurovascular coupling, in addition to other markers of breakdown of the neurovascular unit such as loss of pericyte coverage and microglial activation. Age and thalamic vascular amyloid accumulation were also associated with an increase in the NADPH oxidase (NOX) subunit p47, indicative of increased oxidative stress. Data from this experiment indicate that ageing can profoundly impair neurovascular coupling, mediated by gliosis and loss of astrocytic contacts with vasculature. The third study aimed to test the hypothesis that chronic cerebral hypoperfusion (a prominent early feature of vascular cognitive impairment) would impair vascular function and induce the development of vascular lesions and cognitive decline. The impact of hypoperfusion on neurovascular coupling, ischaemic and haemorrhagic lesion burden and cognition was investigated in wild type and TgSwDI mice. Hypoperfusion induced deficits on neurovascular coupling, increased lesion burden and inflammation assessed with T2 and contrast-enhanced T2* imaging, and caused impairment on measures of learning and memory. Hypoperfusion was also associated with an increase in the levels of NOX2, NOX4 and 3-NT at 3 months following surgery, indicating persistent reactive oxygen species production and oxidative damage in hypoperfused mice. The findings from this study indicate that vascular dysfunction and cognitive impairment following hypoperfusion may be mediated by increased NADPH oxidase activity and resulting oxidative stress. The previous studies indicated that markers of oxidative stress were induced in response to ageing, vascular amyloid accumulation and cerebral hypoperfusion. The final study sought to determine whether increased NOX activity mediates downstream pathological effects on vascular function, vascular lesion development and cognitive decline following hypoperfusion. NOX activity was inhibited pharmacologically by administration of apocynin to hypoperfused TgSwDI mice for 3 months following surgery. Treatment with apocynin significantly restored neurovascular coupling to a level similar to sham-operated mice, and there was a trend toward reduction of ischaemic vascular lesions. However, it was unable to rescue the prominent inflammatory response or decline in cognitive ability, as apocynin-treated mice were no different on these measures to non-treated hypoperfused mice. The data indicate that whilst inhibiting NOX may have potential therapeutic value in improving vascular function, additional interventions, for example to reduce inflammation, may also be required in order to prevent cognitive decline. Overall, the work outlined within the thesis indicate that vascular risk factors of ageing, cerebral amyloid angiopathy and cerebral hypoperfusion may converge on common pathways involving oxidative stress and increased inflammation in order to drive vascular dysfunction and lead to cognitive decline. Inhibition of NOX activity was able to rescue vascular function, however the results indicate that this was not sufficient to protect against cognitive impairment, suggesting additional therapeutic targets may need to be sought in order to fully preserve vascular health and prevent cognitive decline.