Imaging of blood-brain barrier integrity and inflammatory tracers during Alzheimer’s disease

Alenezi, Basmah M. 2026. Imaging of blood-brain barrier integrity and inflammatory tracers during Alzheimer’s disease. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Background: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterised by extracellular amyloid-β plaques, intracellular neurofibrillary tangles, and chronic neuroinflammation. The complement system, particularly component 5(C5), has been implicated in AD pathogenesis, contributing to synaptic loss and neurodegeneration through its role as the first protein of the Membrane Attack Complex. Despite the therapeutic success of anti-C5 antibodies in peripheral inflammatory conditions, their application as diagnostic imaging tools for neuroinflammation has not been explored. Furthermore, modifiable lifestyle factors, including diet and gut microbiota composition, may influence AD progression through the gut–brain axis. Aims: This thesis aimed to develop and validate zirconium-89 (⁸⁹Zr)-labelled BB5.1 monoclonal antibody as a novel positron emission tomography (PET) imaging biomarker for assessing blood–brain barrier (BBB) integrity and complement activation in a transgenic mouse model of AD. Secondary aims included investigating the effects of high-fat diet (HFD) and probiotic supplementation on the behavioural phenotype, cerebral glucose metabolism, and microglial activation in 3xTg-AD mice. Methods: BB5.1, an anti-mouse C5 monoclonal antibody, was produced using the Integra CELLine bioreactor system and purified by Protein G affinity chromatography. The antibody was conjugated with desferrioxamine (DFO) and radiolabelled with ⁸⁹Zr. Structural integrity was assessed by SDS-PAGE and western blotting, whilst biological activity was confirmed through complement haemolysis assays. Radiochemical purity was evaluated using radio-thin layer chromatography and radio-high performance liquid chromatography. Male 3xTg-AD mice were allocated to four dietary groups at six months of age: chow control, chow with Lab4P probiotic, HFD control, and HFD with Lab4P probiotic. Following a 12-week intervention, longitudinal PET/computed tomography (CT) imaging was performed using ⁸⁹Zr-DFO-BB5.1 and an isotype control (⁸⁹Zr-DFO-D1.3), with serial imaging at 24, 72, and 144 hours post-injection at baseline (6 months) and endpoint (9 months). Cerebral glucose metabolism was assessed using 2 ¹⁸F-FDG PET imaging. Behavioural assessments included open field testing and novel object recognition. Post-mortem immunohistochemical analysis quantified microglial markers (CD11b and P2Y12) in cortical, hippocampal, and cerebellar regions. Results: BB5.1 was produced and analysed by SDS-PAGE to confirm purity. Complement haemolysis assays demonstrated that the antibody was functionally active and that this was maintained after radiolabelling . PET/CT imaging revealed differential brain uptake patterns between ⁸⁹Zr-DFO-BB5.1 and the isotype control ⁸⁹Zr-DFO-D1.3. Following dietary intervention, a statistically significant increase in brain concentration of ⁸⁹Zr-DFO-D1.3 was observed in HFD-fed mice compared with chow controls (p ≤ 0.001), supporting the hypothesis that HFD induces increased BBB permeability. ¹⁸F-FDG PET imaging demonstrated that probioticsupplemented groups exhibited significant increases in cerebral glucose uptake over the study period, whereas non-probiotic groups did not show comparable increases, suggesting that probiotic supplementation may support neuronal metabolic function. Behavioural assessments revealed no statistically significant differences in discrimination index between dietary groups. Immunohistochemical analysis demonstrated region-specific patterns of microglial activation, with CD11b and P2Y12 expression showing differential responses to dietary interventions across brain regions. Conclusions: The monoclonal antibody ⁸⁹Zr-BB5.1 targeted against the C5 complement protein was assessed as a BBB patency and complement activation PET tracer in a mouse model of AD. In contrast to what was expected, the percentage of injected dose found in the brain inversely correlated with AD progression, as measured by microglial activation, suggesting a confounding influence of HFD on the BBB penetration potential of ⁸⁹Zr-BB5.1. The differential brain uptake patterns observed between ⁸⁹Zr-BB5.1 and the isotype control ⁸⁹Zr-D1.3 demonstrate that this approach can distinguish between antigen-specific retention and passive BBB permeation, although the relationship between complement-targeted antibody uptake and disease state is more complex than initially hypothesised. Furthermore, the findings give some support to the neuroprotective potential of probiotic supplementation, as evidenced by preserved cerebral glucose metabolism in probiotic-treated groups. These findings contribute to our understanding of complement-mediated neuroinflammation in AD and highlight the need for further mechanistic investigation into the interplay between dietary factors, BBB integrity, and complement activation in neurodegeneration.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Medicine
Date of First Compliant Deposit:	16 July 2026
Last Modified:	20 Jul 2026 09:03
URI:	https://orca.cardiff.ac.uk/id/eprint/188262

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