This thesis describes studies of cerebral oxygenation, autoregulation and metabolism carried out on human volunteers and patients. These studies are intended to aid both the development and the validation of a new physiology based mathematical model of the cerebral circulation and metabolism. The thesis contains comparisons between the experimentally derived data and predictions from this model. The experimental studies involve the measurement of systemic and cerebral haemodynamic parameters and their response to physiological challenges. In particular, near-infrared spectroscopy (NIRS) is used to monitor cerebral blood volume, oxygenation and flow. NIRS is a non-invasive technique, which uses the differing optical absorption of oxy-and deoxy-haemoglobin in the near infrared to monitor variations in oxygenation and blood volume deep within the tissue. A 2 channel NIRS instrument with spatially-resolved capabilities (NIRO 300, Hamamatsu Photonics KK), was used to monitor cerebral changes in response to physiological challenges such as hypercapnia, hypoxia and passive tilt in healthy volunteers. Furthermore we studied patients with primary autonomic failure with severe orthostatic hypotension undergoing a tilt test. The main reason for using data from these patients is that a comparatively minor physiological challenge (a 60 tilt) produces a major drop in arterial blood pressure and cerebral haemodynamics. The computational model, against which the experimental data is compared, is being developed at UCL. The model is capable of accepting experimentally determined data as an input and predicting changes in other measured and non-measured parameters. The thesis describes the model and illustrates its abilities using both theoretical and experimental data, in particular examining the measured and predicted changes in cerebral tissue oxygenation. Finally, practical aspects of clinical data monitoring are addressed and the capabilities and limitations of the cerebral modelling work are discussed. The findings and conclusions of these studies should be relevant to the wider mathematical, physiology and biomedical optics community.

Cerebral endothelial cells (CEC) constitute a key cellular element of the blood- brain barrier (BBB) due to their bidirectional selective permeability that contributes to central nervous system (CNS) homeostasis. However, their specialized barrier function is altered during neuroinflammatory disorders such as multiple sclerosis (MS). Yet, the molecular mechanisms implicated in the loss of brain endothelial barrier function remain to be fully elucidated. To address this question, we first established an in vitro BBB inflammation model using the human cerebral microvascular endothelial cell line, hCMEC/D3 cell, we then determined the mRNA signature of hCMEC/D3 cells in the absence and presence of pro-inflammatory cytokines and compared results to previously published data on mouse CEC transcriptome analysis. We classified genes expressed at the mRNA level into inter-brain endothelial junctions, transporter systems, cytoskeletal associated molecules, integrin-focal adhesions complexes, cell adhesion molecules and chemokines. We report that long term exposure of hCMEC/D3 cells to TNFa/IFNy results in a new CEC gene expression pattern associated with barrier dysfunction. We then investigated the signalling pathways implicated in cytokine-induced increase in paracellular permeability. We propose that during neuroinflammation the concentration of cytokines in the CNS micfoenviroment to which CECs are exposed determines the extent of caspase-mediated barrier permeability changes which may be generalized, as a result of brain endothelial apoptosis, or more subtle, as a result of alterations in the organization of junctional complex molecules. In addition, we found that the PKC-JNK axis might be an important "control node" to regulate the paracellular pathway of CECs during inflammation. In the third chapter, we have investigated whether the BBB-signature of CECs might be in part regulated by microRNAs (miRNAs). MiRNAs are endogenous non-coding small RNAs that suppress gene expression at a post-transcriptional level and have been shown to modulate several biological processes. As a first approach we determined the changes in miRNA levels induced by pro-inflammatory cytokines in hCMEC/D3 cells. We then confirmed the deregulation of brain endothelial miRNAs in MS brain tissue and in experimental auto immune encephalomyelitis, an animal model of MS. Interestingly, we observed a temporal-pattern of miRNA expression that might correspond to pro- and anti- inflammatory miRNAs that fine tune gene expression in cerebral endothelium during an inflammatory response. In the fourth chapter, we investigated the role of miR-l55 on brain endothelial barrier integrity. MiR-l55 was shown to be rapidly and highly increased in hCMEC/D3 cells after cytokine stimulation. Our findings showed that increased levels of miR-l55 in brain endothelium might participate in cell activation during inflammation resulting in a moderate increase in leukocyte adhesion and a robust effect on modulating brain endothelial paracellular permeability. We propose that the paracellular permeability effect of miR-l55 involves targeting a set of interrelated genes that regulate cell-cell junctions and focal adhesions. In addition, we characterized the cytokine-induced signalling pathways mediating upregulation of miR-155 in cultured CECs. Our results suggest that miRNAs modulate key features of CECs and may constitute major players in the pathogenesis of CNS inflammatory disorders that affect the BBB. In summary, we have determined the role of some key brain endothelial miRNAs and signalling pathways in a critical pathogenic feature of neuroinflammation, BBB dysfunction, which may have important consequences for the rational development of therapies for CNS inflammatory disorders.

No description available.

Collateral blood flow is the compensatory flow of blood to the tissue through secondary channels when the primary channel is compromised. It is of vital importance in cerebrovascular disease where collateral flow can maintain large regions of brain tissue which would otherwise have suffered ischaemic damage. Traditional x-ray based techniques for visualising collateral flow are invasive and carry risks to the patient. In this thesis novel magnetic resonance imaging techniques for performing vessel-selective labelling of brain feeding arteries are explored and developed to reveal the source and extent of collateral flow in the brain non-invasively and without the use of contrast agents. Vessel-encoded pseudo-continuous arterial spin labelling (VEPCASL) allows the selective labelling of blood water in different combinations of brain feeding arteries that can be combined in post-processing to yield vascular territory maps. The mechanism of VEPCASL was elucidated and optimised through simulations of the Bloch equations and phantom experiments, including its sensitivity to sequence parameters, blood velocity and off-resonance effects. An implementation of the VEPCASL pulse sequence using an echo-planar imaging (EPI) readout was applied in healthy volunteers to enable optimisation of the post-labelling delay and choice of labelling plane position. Improvements to the signal-to-noise ratio (SNR) and motion-sensitivity were made through the addition of background suppression pulses and a partial-Fourier scheme. Experiments using a three-dimensional gradient and spin echo (3D-GRASE) readout were somewhat compromised by significant blurring in the slice direction, but showed potential for future work with a high SNR and reduced dropout artefacts. The VEPCASL preparation was also applied to a dynamic 2D angiographic readout, allowing direct visualisation of collateral blood flow in the brain as well as a morphological and functional assessment of the major cerebral arteries. The application of a balanced steady-state free precession (bSSFP) readout significantly increased the acquisition efficiency, allowing the generation of dynamic 3D vessel-selective angiograms. A theoretical model of the dynamic angiographic signal was also derived, allowing quantification of blood flow through specified vessels, providing a significant advantage over qualitative x-ray based methods. Finally, these methods were applied to a number of patient groups, including those with vertebro-basilar disease, carotid stenosis and arteriovenous malformation. These preliminary studies demonstrate that useful clinical information regarding collateral blood flow can be obtained with these techniques.