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It is now widely recognised that α-Synuclein (αSyn) oligomers are the main pathogenic species in Parkinson's Disease (PD). In addition to being cytotoxic they are also thought to play a central role in the interneuronal spread of αSyn pathology. There is compelling evidence to suggest that these proteoforms are released into cerebrospinal fluid, where differences in their abundance and ability to seed the formation of larger species have been used to differentiate PD patients from aged matched controls. However, αSyn oligomers are a highly heterogeneous population and the characteristics of those present in biofluids have not been clearly established, nor which species are most neurotoxic and contribute to disease onset and progression. This is largely due to the fact that these small clusters of αSyn are notoriously difficult to study using classical biochemical ensemble methods, owing to their heterogeneity, transient nature and low abundance relative to the monomeric protein (<5% of a solution of protein). For this reason, single-molecule fluorescence microscopy has been used to study them; by probing molecules one-by-one, the rare oligomeric species can be identified amongst the monomeric protein, counted and characterised. Typically, to study species in single-molecule experiments, the biomolecule of interest is covalently tagged with a fluorophore. This may affect the behaviour of the molecule, and also prevents the techniques from being applied to human samples. Attempts to study native oligomers at the single-molecule level, have relied upon amyloid binding dyes, which are non-specific, labelling other βsheet rich structures present in cerebrospinal fluid as well as αSyn. The αSyn content of aggregates secreted by cells was investigated using an iPS cell line derived from a Parkinson's diseased patient harbouring a triplication of the SNCA gene, which encodes for αSyn, on one allele and a derivative of this cell line with the gene deleted, SNCA knock-out. The iPS cells were differentiated into midbrain dopaminergic neurons, the primary neuronal subtype associated with the pathogensis of PD and the conditioned media was collected and used as an analogue of cerebrospinal fluid. Initial, investigations focused on utilising amyloid binding dye, thioflavin-T, to probe media samples for the presence of secreted αSyn aggregates. This involved studying aggregates using single-molecule confocal microscopy coupled with microfluidics to count and characterise thioflavin-T active species, as well as total internal refection microscopy to directly visualise and measure their structure. In order to facilitate specific detection of αSyn species in complex biological fluids using single-molecule confocal microscopy, the amyloid binding dye was replaced with an αSyn antibody. The need for constitutivly active dyes to be used at low picomolar concentrations in order to discretise burst of fluorescence intensity has prevented this from being achieved before now. With access to an antibody with low picomolar affinity for fibrillar αSyn, I was able to study the oligomersation of unlabelled αSyn. Despite this technique being considerably more sensitive than previously reported methods, I was unable to detect αSyn oligomers in conditioned media, even in conditions where intracellular pathology was induced via treatment with pre-formed αSyn fibrils, suggesting aggregates are rapidly internalised by cells but are then trapped and unable to escape. Observing unlabelled αSyn oligomers directly at the single-molecule level is challenging, due to the fact that specific imaging probes such antibodies are adsorb onto glass surfaces, generating a large degree of non-specific signal. Single-molecule pull-down (SiMPull) is a recently developed technique that circumvents this issue, by immobilising the protein of interest on a passivated surface that is capable of blocking antibody binding. By combining SiMPull with the principles of two-colour coincidence detection, I was able to develop a highly sensitive method for directly visualising αSyn aggregates. Applying this new methodology to conditioned media samples, I was able infer the presence of αSyn oligomers. The predicted size of αSyn oligomers is in the range of 12-16 nm, which is substantially below the resolving power of any conventional fluorescence microscopy technique due to the diffraction limit of light. Therefore, in order to view αSyn oligomers at an appropriate scale, I combined SiMPull with a super-resolution technique, DNA-PAINT, which enabled αSyn structures to be viewed and characterised with nanometer resolution (∼ 20 nm). Overall, the work presented here describes methodologies developed to detect and characterise native αSyn oligomers with a high degree of specificity and improved sensitivity in comparison with established techniques. Although these developments were performed on α-synuclein, they have now been adopted by others to investigate other pathogenic proteins implicated in neurodegeneration. The advance ments made will enable further investigations into the molecular mechanisms involved in neurodegerative diseases, and also for potential biomarkers to be detected and studied. This should lead to the development of diagnostic tools for neurode-generative conditions, which will enable clinicians to follow the disease course in patients, and test new therapeutic interventions.

Within the human body, vitreous humour, cerebrospinal fluid and synovial fluid are all contained within anatomical compartments that, by virtue of their relative avascularity, provide considerable protection from contamination during the embalming process. Analysing samples taken from embalmed medical school cadavers, and using an entirely novel headspace GC/MS method, the concentration of formaldehyde in all three fluids is shown to be significantly less than has previously been assumed in the literature. Using a newly-developed LC/Q-ToF method, the stability of fifteen drugs of forensic interest in representative concentrations of formaldehyde is then reported. The study is prompted by the fact that United Kingdom nationals are dying abroad with increasing frequency. Following repatriation of their bodies, an autopsy may be undertaken before permission for a funeral is granted. Although toxicological screening may form an important part of the investigative processes, there exists a large degree of uncertainty surrounding the reactive nature of formaldehyde, the main constituent of embalming fluids, with substances commonly implicated in drug-related deaths. It is the case, therefore, that in repatriated bodies, all of which are routinely embalmed before leaving the country in which death occurred, the presence of formaldehyde can cause significant problems for the forensic toxicologist. Although a number of papers in the past twenty years have investigated the challenges of drug detection in embalmed tissues, they have largely examined the problem from the perspective of histological samples preserved in formalin. While some of this work has, by implication, considered the analysis of samples taken from embalmed bodies, no studies have addressed directly the specific problems arising in the toxicological analysis of embalmed and repatriated bodies. The studies reported within the thesis demonstrate that there is considerable merit in undertaking further work on the usefulness of these fluids in the toxicological analysis of embalmed and repatriated nationals.

This dissertation is devoted to dynamics of brain liquids in patients with altered CSF circulation and pressure-volume compensation. Since the introduction of intracranial pressure (ICP) monitoring, the studies of CSF dynamics have revealed unique information about the intracranial circulation and opened new opportunities for diagnosis and treatment of hydrocephalus and pseudotumour cerebri. The adaptation of infusion tests in clinical practice over 45 years ago has introduced a practical tool to benefit both patients and research into altered CSF dynamics. Objective testing of intracranial circulation in patients with clinical symptoms constitutes a unique situation, where the discovery of new patterns and reasons for disturbed intracranial circulation can be quantified. Such macroscopic yet practical quantifications can easily be translated to clinically useful information, and back, in real time or alternative past and future synchronicities. The aim of this dissertation is to demonstrate the value of testing CSF dynamics in vivo and how it could provide pathophysiological and clinical insights into hydrocephalus and pseudotumour cerebri syndrome (PTCS). My intention was to describe and reflect the main themes involved in the study of CSF dynamics: a) their role in diagnosis and treatment, b) their use in understanding shunts and shunt malfunction c) the need to optimise our understanding of the contents of ICP, meaning that long-term ICP monitoring or dynamic tests are required in CSF disorders, not snapshot ICP measurements and finally d) the mapping and quantification of the interaction between CSF circulation and cerebral blood flow (CBF). As the above foundations and results of my work lead to the formation of a required, albeit expected, long doctoral treatise, I have structured the later in 9 chapters containing a comprehensive literature review of the Resistance to CSF outflow as well as a systematic literature review of the CBF and autoregulation of the CBF in NPH. I have also dedicated a methods chapter, Chapter 3, into introducing and explaining the variable tested during a CSF infusion test, such as the fundamental amplitude of ICP and the compensatory reserve indices. Following this is the presentation of the data and clinical material used for my original projects. Specifically, my results contain the following: I) Autoregulation of cerebral blood flow in hydrocephalus CSF infusion tests provide a unique setting where both ICP and cerebral blood flow and autoregulation can be measured in ambulatory patients utilising many different methods. Autoregulation has been studied by quantifying the interaction between the CSF and cerebral blood circulation has revealed diagnostic and outcome implications that could perhaps describe the natural course of a CSF disorder, or differentiate between a CSF disorder and a vascular disorder, or the coexistence of the two, opening new chapters to the comprehension of shunt responsive NPH. I have explored the state of global autoregulation in patients undergoing infusion tests, in an attempt to set out a reference for investigations related to NPH, Resistance, autoregulation and their clinical implications. In the 5th chapter, I have: • Described the relationship between Rout, cerebral autoregulation and arterial blood pressure. Rout demonstrates a negative linear relationship with global autoregulation. When I combined these parameters and accounted for the patients’ age, I was able to show a good correlation with outcome, much improved compared to Rout alone. II) CSF dynamics in normal pressure hydrocephalus and pseudotumour cerebri. CSF dynamics in different conditions have shown that parameters such as the Resistance to CSF outflow in NPH and ICP at baseline combined with compensatory reserve indices in PTCS, could provide important diagnostic and management information. This could be a valuable addition of objective evidence to imaging and clinical examination. Using large cohorts of patients, I have explored the Resistance to CSF outflow (Rout) in NPH in the context of different aetiologies of NPH, its relationship with age as well as its overall correlation with outcome after shunting. I have also explored these relationships in relevance to clinical practice. In PTCS, I have described the findings from infusion tests in both adult and paediatric patients and have highlighted the differences with hydrocephalus. In chapter 6, , I have described the following: • Davson’s equation in NPH: The so-called Davson’s equation describes the relationship between ICP, Rout, CSF formation rate and sagittal sinus pressure under physiological circumstances. I have validated the existence of such a linear relationship in NPH. • CSF dynamics in post-traumatic hydrocephalus: Traumatic brain injury, as a cause of secondary NPH, shows some differences in Rout and ICP amplitude compared to idiopathic NPH. I have also described the effect of decompressive craniectomy and of cranioplasty on CSF dynamics. In chapter 7, I have explored the CSF dynamics of PTCS and in particular: • The coupling between CSF pressure and Sagittal sinus pressure (SSp) in PTCS patients at baseline and during infusion tests. I have also shown how this relates to Davson’s equation under an unstable SSp and the possible pathophysiological consequences of this finding. • The CSF dynamics of paediatric patients with PTCS. Those included all patients assessed in Cambridge and classified as definite, probable and not PTCS. III) Shunt testing in vivo. Shunts are currently the mainstay for the management of hydrocephalus, as well as an important part of the management of PTCS. They change CSF dynamic parameters in a way that is easily assessed with shunt infusion tests. The knowledge of the post-shunting CSF circulation contains crucial information on the state of the shunt function, as well as the adequate restoration of the patients’ intracranial circulation. I have described how objective knowledge from shunt testing in vivo impacts clinical practice and patients’ outcomes. In chapter 8, I have presented two studies about testing shunt function in-vivo: • Shunt testing in vivo using infusion tests is important in avoiding unnecessary revisions of patent shunts and allows patients to be managed conservatively, with good outcomes. This also translates to financial benefits for healthcare systems. • In paediatric hydrocephalus, shunt infusion studies are an accurate and useful tool for investigating insidious shunt obstruction. IV) Slow waves of Intracranial Pressure. Reliable, long-term overnight monitoring is the gold standard in monitoring and analysing ICP and its contents. Slow waves, compensatory reserve and relationship with the venous circulation contain reliable information that are again correlated to clinical practice and can be compared and incorporated into the shorter-term infusion test. I have explored the behaviour of slow waves in anaesthetized patients In chapter 9, I have investigated the influence of general anaesthesia on slow waves of ICP in NPH and traumatic brain injury (TBI) patients. Conclusion: Infusion tests are a practical tool for research and possibly diagnosis and treatment in patients with PTCS and NPH. CSF dynamics provide a quantitative description of cerebral pathophysiology in CSF disorders, both for CSF and potentially for cerebral blood flow. After shunting, infusion tests are a reliable and cost-effective tool for identifying or excluding shunt malfunction. Further studies are needed to verify the clinical implications of CSF infusion tests and cerebral blood flow and autoregulation in those patients.

The biological basis of the clinical heterogeneity in Parkinson's Disease (PD) is unclear. It is likely to involve complex interactions between genetic and environmental factors and between a range of pathological processes, including protein homeostasis and immune system function. Microglial activation in the brain and peripheral innate immune changes are known to occur in PD. Recently genetic, animal and cellular studies have linked several innate immune related genes and proteins (e.g. HLADR, TREM2, TLR2, TLR4, caspase-1) to PD and provided evidence that they may have a role in PD pathogenesis. Alpha-synuclein is central to PD, with evidence from neuropathology, genetics and animal/cell models indicating that it plays a significant pathogenic role. There is developing evidence directly linking innate immune activity and alpha-synuclein pathology. For example, inflammation, particularly in response to microbial infection, is associated with increased alpha-synuclein accumulation in the periphery and activation of the innate immune inflammasome related caspase-1 leads to increased cleavage and aggregation of alpha-synuclein. Overall Hypothesis- "Parkinson's disease (PD) and its clinical heterogeneity are associated with systemic changes in innate immune and associated microbial factors and in alpha-synuclein". This was investigated from the perspective of an epidemiological study, a study of peripheral blood monocyte, innate immune/microbial markers and a cerebrospinal fluid (CSF) study in PD patients. *The epidemiological study, involved the longitudinal PICNICS cohort of 290 Idiopathic PD patients, and showed that the use of medication known to influence alpha-synuclein and immune function is associated with motor heterogeneity in PD. *The peripheral immune study involved 41 early PD patients and 41 age, gender and MAPT genotype matched paired controls, with the PD patients categorised into 2 groups based on the presence of previously identified clinical and genetic risk factors for the development of an early dementia (impaired semantic fluency, pentagon copying and MAPT H1/H1 haplotype). This study demonstrated that the phenotypic profile of peripheral monocytes and the level of serum alpha-synuclein and relevant innate immune and microbial markers do differ in early PD compared to controls and that there are differential changes in those patients at higher versus lower risk for early dementia. The systemic alpha-synuclein related changes appear to be present overall in PD patients compared to controls, while the more microbial/innate immune related changes appear to be more prominent in the dementia higher risk group. *The CSF study involved samples from 35 PD patients and has demonstrated evidence of relationships between neurodegeneration-linked CSF tau species and inflammatory cytokines, and between CSF alpha-synuclein and cognitive function, suggesting that these factors may be involved in PD heterogeneity within the central nervous system as well. Overall, these studies provide evidence that variations in alpha synuclein/ tau homeostasis and innate immune and microbial factors are related to PD and its clinical heterogeneity.

Alzheimer's disease (AD) is the most common cause of dementia; a problem that is growing in size and cost as the population ages. Early soluble aggregates composed misfolded 'amyloid' peptide sequences have been implicated as key to the initiation and onset of AD pathology, although little is definitively known as to when and how these assemblies form or interact to instigate pathology. The primary focus of this study was to evaluate whether L-Tryptophanol (Trol) signal, which has been shown to be induced via soluble amyloid species, increases with AD severity in a range of ex vivo human samples. Testing of this hypothesis was carried out in several stages: Initially synthetic versions of the amyloid beta (Aβ) peptide were tested in vitro to corroborate Trols propensity to associate to amyloid assemblies and allow for method development. Next, a range of brain lysates from several transgenic mouse lines and aged human AD cases and controls were assessed using the reporter. These experiments demonstrated Trols sensitivity to Aβ and tau, and provided compelling evidence that Trol signal tracks disease progression in brain lysates. During the final stage of testing cerebrospinal fluid (CSF) from AD and Parkinson's disease (PD) patients, and blood plasma samples from PD patients was evaluated. Results from this phase of testing indicated that Trol was able to detect differences in sample composition between healthy and diseased individuals, however differences were not clear cut and could have been affected by confounding factors. Overall, the data presented here suggest that Trol may be able to track disease progression in amyloidopathies when implemented in brain lysates. However, further testing is required to completely validate this finding. These findings highlight the potential of simple techniques for amyloid detection to aid within the diagnosis, evaluation of disease progression and study of AD and other neurodegenerative diseases.

Delirium, or “acute confusion” is a common and serious acute neuropsychiatric syndrome mainly affecting older people. It is associated with multiple adverse outcomes, including an increased risk of developing dementia and increased mortality. The underlying mechanisms of delirium are poorly understood, and there are currently no specific treatments. This thesis investigated the roles of the hypothalamic-pituitary adrenal axis and inflammation in the pathophysiology of delirium, persistent delirium and cognitive decline following delirium. It investigated whether levels of cortisol in blood and cerebrospinal fluid (CSF) are elevated in delirium, with elevated pro-inflammatory and reduced anti-inflammatory cytokines. It also investigated whether there is loss of cortisol diurnal rhythm (in saliva) with elevated afternoon cortisol levels. The thesis investigated whether any hypercortisolaemia was sustained during the year after delirium, and whether this was associated with deterioration in cognition during the year after hip fracture. Finally, it also tested whether there are high levels of a marker of central nervous system damage (S100B) and of a dementia marker (tau) in CSF in delirium. A prospective observational cohort study was conducted in N=108 patients aged over 60 who had sustained a hip fracture, in whom 40% developed delirium. Participants gave informed consent or if they lacked capacity to give informed consent, this was given by their next of kin. Participants were assessed regularly for delirium, according to DSM IV criteria, during the two weeks after hip fracture. A sample of CSF was collected during the spinal anaesthetic performed for the operation to repair their fracture. Samples of blood and saliva were collected during the two weeks after the hip fracture operation. Participants were visited three, six and twelve months after their hip fracture for further delirium assessment, and a cognitive test battery was completed. Further samples of blood and saliva were collected at these visits. The study found evidence of high levels of cortisol and of S100B in CSF in those with active delirium, but there were no differences in levels of tau or cytokines in CSF. Those with delirium had elevated serum cortisol during the perioperative period, and elevated afternoon salivary cortisol, suggesting flattening of cortisol diurnal rhythm with failure to reach the normal diurnal nadir. After adjusting for confounders in a multivariate logistic regression analysis, serum cortisol was still predictive of delirium, but salivary cortisol AM:PM ratio had a trend towards significance. Those who had persistent delirium features in the months after hip fracture had significantly higher serum cortisol three months after hip fracture. There was a change in serum inflammatory profile in those with delirium, with a shift towards a pro-inflammatory state. Testing the study hypotheses surrounding cognition after delirium was very challenging, due to patient attrition and other factors. Some participants showed a trajectory of cognitive improvement, which was probably due to resolution of delirium during the year after hip fracture. Those with resolved delirium had deficits in verbal and visual memory. This study has improved understanding of the mechanisms of delirium, suggested further avenues for research and identified possible new therapeutic targets.

This critical synopsis of prior work by Clive Beggs is submitted in support of a PhD by published work. The work focuses on venous and cerebrospinal fluid (CSF) anomalies associated with multiple sclerosis (MS) and other neurological diseases. MS is characterized by focal inflammatory lesions, which are often venocentric. Recently a vascular syndrome, chronic cerebrospinal venous insufficiency (CCSVI) has been linked with MS. This syndrome, which is characterized by constricted cerebral venous outflow, has become mired in controversy, with various studies producing conflicting findings, with the result that the science associated with CCSVI has become obscured. Clive Beggs work seeks to bring clarity to the debate surrounding CCSVI by characterizing physiological changes associated with constricted cerebral venous outflow. The work submitted here involves collaborative studies with Robert Zivadinov (University of Buffalo), Paolo Zamboni (University of Ferrara), and Chih- Ping Chung (National Yang Ming University of Medicine). The key findings of these studies are: (i) MS patients, diagnosed with CCSVI, exhibit greatly increased hydraulic resistance of the cerebral venous drainage system; (ii) MS patients experience loss of the small cerebral veins; (iii) MS patients exhibit reduced CSF bulk flow, consistent with mild venous hypertension; (iv) MS patients exhibit increased CSF pulsatility in the Aqueduct of Sylvius, which appears to be linked with mild venous hypertension associated with CCSVI; and (v) jugular venous reflux is associated with white matter and parenchymal volumetric changes in Alzheimer’s patients. Collectively, these findings suggest that extracranial venous anomalies are associated with changes in the intracranial physiology.

Cerebrospinal fluid (CSF) fills a system of cavities at the centre of the brain, known as ventricles, and the subarachnoid space surrounding the brain and the spinal cord. In addition, CSF is in free communication with the interstitial fluid of the brain tissue. Disturbances in CSF dynamics can lead to diseases that cause severe brain damage or even death. So-called infusion tests are frequently performed in the diagnosis of such diseases. In this type of test, changes in average CSF pressure are related to changes in CSF volume through infusion of known volumes of additional fluid. Traditionally, infusion tests are analysed with single compartment models, which treat all CSF as part of one compartment and balance fluid inflow, outflow and storage through a single ordinary differential equation. Poroelastic models of the brain, on the other hand, have been used to simulate spatial changes with disease, particularly of the ventricle size, on larger time scales of days, weeks or months. Wirth and Sobey (2008) developed a two-fluid poroelastic model of the brain in which CSF pressure pulsations are linked to arterial blood pressure pulsations. In this thesis, this model is developed further and simulation results are compared to clinical data. At first, the functional form of the compliance, which governs the storage of CSF in single compartment models, is examined by comparison of two different compliance models with clinical data. The derivations of a single-fluid and a two-fluid poroelastic model of the brain in spherical symmetry are laid out in detail and some of the parameters are related to the compliance functions considered earlier. The finite element implementation of the two-fluid model is described and finally simulation results of the average CSF pressure response and the pressure pulsations are compared to clinical data.

The aim of this study is to procure a quantitative measure of the intracranial pressure by tympanic membrane displacement test by comparing volume displacement of the tympanic membrane on acoustic stapedial contraction with direct intracranial pressure measurement. The study also defines the sensitivity and specificity of the test as a diagnostic modality and assesses the test's reproducibility. The tympanic membrane displacement test was first introduced clinically as an audiometric technique in the assessment of intracranial pressure in hydrocephalics in 1987. A pilot study in 1989 qualitatively showed that consistent results could be obtained in young patients with benign intracranial hypertension with hydrocephalus. A prospective comparative evaluation of 68 clinical episodes of shunt malfunction was assessed by tympanic membrane displacement test and direct intracranial pressure measurement via the Rickham ventriculostromy reservoir in 40 patients (mean age 13.3 years) with shunted hydrocephalus between January 1995 and June 1996. In addition, 31 clinical episodes of shunt malfunction were evaluated by serial tympanic membrane displacement test and direct intracranial pressure measurement in 8 shunted hydrocephalic children between January 1995 and Feb 1996. The patients were classified into three groups by tympanic membrane displacement test and direct intracranial pressure measurement as raised intracranial pressure, low intracranial pressure and normal intracranial pressure. The inter-rater agreement between the groups was very good, κ=0.88, with a P value of < 0.001. The sensitivity of the test was 93%, specificity was 100%, positive predictive value was 100% and negative predictive value was 73%. The test was reproducible. The objective measure of the tympanic membrane displacement for raised intracranial pressure was -200nl or less and for low intracranial pressure was +200nl or greater. The tympanic membrane displacement test is a reliable, reproducible, non-invasive investigation for the assessment of intracranial pressure in shunted hydrocephalics. It can be used on regular bases as a routine test, thereby reducing the need for invasive intracranial pressure monitoring. The test may well provide a useful serial guide to intracranial pressure abnormalities in children with shunted hydrocephalus.

This thesis describes the use of modern engineering and computational techniques to investigate the complex biomechanical relationships between the main cerebral components in normal pressure hydrocephalus and other complex disorders of cerebrospinal fluid (CSF) circulation. CSF infusion tests and overnight intracranial pressure (ICP) monitoring were combined with simultaneous transcranial Doppler ultrasongoraphy (TCD) to investigate the relationship between CSF dynamics and cerebral haemodynamics. ICP increases induced during CSF infusion studies, provoked an increase in the pulsatility of the haemodynamic waveform. As the increase was significantly greater in patients with increased resistance to CSF outflow TCD may be used to enhance or replace ICP monitoring during CSF infusion studies. Overnight ICP and TCD monitoring demonstrated that haemodynamic fluctuations are the driver for hydrodynamic B waves seen in some hydrocephalic patients during sleep. This study also demonstrated that the transmission of haemodynamic fluctuation to the CSF is dependent on changes in compliance of the cerebral mantle. The relationship between increased CSF volume and the properties of the cerebral mantle was explored using image analysis and mathematical modeling. An innovative technique was developed for the quantitative assessment of hyperintensities on magnetic resonance images. The results from this study demonstrated that patients with minimal disruption to the brain tissue are more likely to improve with insertion of a CSF shunt. A finite element model was established to investigate the stress-strain relationship in the brain tissue as CSF volume increases. The tissue changes simulated in the model correlated well with the pathopyhsiological and anatomical changes seen in hydrocephalus, providing insight into the possible biomechanical process that may produce these changes.

With the mean age of the population increasing, the number of patients with dementia is likely to increase. Although Alzheimer's disease is the most common form of dementia, it is recognised that other forms of dementia are more prevalent than initially thought. As drug therapies become available, it will become important to obtain an accurate clinical diagnosis. Creutzfeldt-Jakob disease (CJD) is a rare type of dementia but with the emergence of the new variant form of this disease which affects young patients, and is thought to be related to the ingestion of meat or meat products from cattle affected with bovine spongiform encephalopathy (BSE), it is possible that it may become more common. These concerns have led to the search for diagnostic tests which may help in the early diagnosis of these forms of dementia. This study investigated whether measurement of cerebrospinal fluid (CSF) brain- specific proteins could be used in the assessment of patients with dementia. Four proteins were investigated: S-100b, an astrocytic protein; and three neuronal proteins, neurone-specific enolase, tau protein and 14-3-3. A two site sandwich ELISA was developed and evaluated for the measurement of S-100b. Levels of all four brain-specific proteins were raised in the majority of patients with sporadic CJD. The presence of CSF 14-3-3 had the best combination of sensitivity and specificity for detecting sporadic CJD. CSF 14-3-3 has recently been included in the diagnostic criteria for probable sporadic CJD. The value of measurement of these proteins in the new variant, iatrogenic and familial forms of CJD was also investigated. None of the CSF brain-specific proteins investigated was of value in the diagnosis of Alzheimer's disease.

In our quickly aging world today, the worldwide population is experiencing an increased risk of Alzheimer’s Disease, a neurodegenerative disease known to have a higher risk with age. With alleviation of symptoms being the best that can be done for patients suffering from Alzheimer’s Disease, there is an imperative need for early diagnosis of the disease for early intervention efforts to reduce mortality rates. As such, there is a great interest in better understanding the dynamics of cerebrospinal fluid (CSF) and its exchange with the vasculature. Currently, noninvasive in-vivo human methods are limited; therefore, most existing results originate from animal models. One promising approach is probing the kinetics of dynamic contrast using PET imaging and different modeling methods. In addition to the traditional compartmental modeling method, in this study, we develop a CFD model that resolves spatial transport of the radiopharmaceutical contrast agent used in PET imaging. This study provides a comparative analysis of these two modeling approaches. To establish the efficacy of each method, we solved an inverse problem by identifying the optimal turnover rates that minimized the difference between each model prediction and clinical measurements. The result of this process indicates that the compartmental model is more aligned with clinical measurements than the CFD model (average compartmental model error 6.9% versus 12% for CFD). This higher CFD error is likely reflective of the inaccurate boundary conditions adopted in this study that are difficult to extract from clinical measurements. There was no significant correlation found between turnover time and studied parameters: age, ventricle volume, body mass index (BMI), amyloid-β and tau accumulation, and Apolipoprotein E (APOE) genotype. Despite its higher overall error, the CFD modeling approach presents a unique advantage as it is the only method that is able to capture the dependence of turnover time on the simulated brain compartment volume.

The aging population is growing, both in the United States and globally. And with aging, the prevalence of chronic, non-communicable diseases is increasing, as well as the associated health care costs expended to provide treatment, aide services, and medications. One fascinating molecule of interest is the Klotho protein, which has been shown to have anti-aging, anti-inflammatory, anti-oxidative, and anti-tumor properties. Since its serendipitous discovery in 1997, Klotho, whose levels decline with age, has been implicated in many cellular pathways and just as many disease states. As an example of one of its many investigated functions, Klotho serves as a co-receptor for fibroblast growth factor-23. This thereby implicates Klotho in the regulation of mineral homeostasis. Furthermore, Klotho levels have been found to be downregulated in a variety of diseases, including chronic kidney disease, Alzheimer’s disease, and most types of cancer. Given the important roles that Klotho plays in normal homeostatic conditions and illness, it is imperative to study the diagnostic and therapeutic potential of this intriguing protein. The objective of the present study is to perform a meta-analysis of studies that have measured Klotho in blood, cerebrospinal fluid, urine, and whole biopsy/necropsy tissue. It is hoped that such an analysis will provide a comprehensive review of trends in Klotho levels across age groups in healthy and diseased individuals. The elucidated patterns in Klotho concentration will provide insight into how Klotho can potentially be used as a biomarker and diagnostic tool, and in which types of diseases. Moreover, the therapeutic capabilities of Klotho will be better defined, as profiling Klotho levels in different types of tissue will illuminate if and how Klotho concentrations vary in differing components of the body. This will help guide the development of future therapeutics, especially in terms of the pharmacokinetics and pharmacodynamics of potential drugs. In addition, the prognostic value of Klotho cannot be ignored. Knowing how Klotho levels correlate with the severity of a disease, from kidney disease to various cancers, will be immensely useful when utilizing Klotho as a biomarker. Among healthy individuals and in blood, this study found a statistically significant decrease in mean Klotho levels with age, when comparing infants aged 0-1 years (n=23), children aged 2-16 years (n=116), and adults aged 18 years and above (n=971). Differences in mean Klotho between these groups all had a p-value

Abstract: Lipids are a large class of hydrophobic molecules involved in energy storage, signaling, modulation of gene expression and membranes. Lipidomics focuses on the comprehensive analysis of lipids and their interactions. Although the potential of lipidomics to study physiological and pathological processes is undeniable, reliable methodologies for the comprehensive assessment of the lipidome in biological samples are still needed. Many biological samples are available in limited amounts and display very complex compositions. Nano-scale liquid chromatography allied to mass spectrometry (nanoLC-MS) offers extremely high sensitivity, although it is known to be technically more challenging than conventional liquid chromatography. The first part of this work describes the optimization of a nanoLC-MS method for routine analysis of the lipidome of small volumes of biological samples (1.0 to 2.5 µL) with high sensitivity. The method was employed to study the composition of blood serum, cerebrospinal fluid and intraparenchymal microdialysate samples. An application of the method to study extremely diluted exosome samples is also discussed. The second part of this work focuses on untargeted, comprehensive lipidomics of biological samples by ultra-high-performance liquid chromatography (UHPLC) coupled to mass spectrometry to study the physiological and pathological effects of different conditions. First, the mechanism of action of a novel vaccine against respiratory syncytial virus was investigated in a rat model by a combination of untargeted lipidomics and chemical isotope labeling metabolomics. Second, untargeted lipidomics of serum was further employed to identify novel biomarkers of Parkinson’s disease and predict the progression to dementia up to three years before noticeable symptoms, emphasizing the relationship of lipids with neurological diseases. Third, alterations in the lipid metabolism related to cystic fibrosis, a severe genetic condition that causes permanent lung damage, showcases the importance of odd-chain fatty acids, previously seen as irrelevant in humans due to their inherent low concentrations, for pathological processes. This work illustrates that lipidomic profiling can provide a comprehensive understanding of metabolic alterations caused by physiological and pathological processes in the organism. The potential of high-quality untargeted lipidomics studies for physiological and pathogenesis analyses, as well as for biomarker discovery, is further discussed.

Studying human brains and spinal cords, or many other complex body parts for that matter, come with great challenges and risks when hoping to get accurate data, hopefully in real-time, in vivo, as opposed to in vitro or from a corpse. Of even more interest, are diseased members, since they possess unique traits that shine significant light into the medical community to help understand and treat countless disorders and pathologies. Having this information greatly improves the quality of life for the afflicted, but not to mention, the whole human race. Of particular interest here, is the cerebrospinal fluid flow, a water-like substance, that encases the spinal cord and pulses in and out of the brain’s intracranial spaces. Upon entrance into the brain cavity from the spinal cord, it first encounters the cerebellum, the posterior lobes at the bottom of the brain. A lot of studies have been done, in vivo and in vitro, to understand its impact on the movement of the cerebellum, specifically in relation to a Chiari malformation which is characterized by herniation of the cerebellar tonsils. Pahlavian et al shows data from magnetic resonance imaging where the cerebellum of healthy patients moves approximately 100 microns. The work herein uses numerical software tools to hypothesize the cause of the cerebellum’s movement. Sources vary widely about the actual material properties of the spinal cord and brain. But with the later data found from Klatt et al, estimates show material elasticity on the order of 1 kPa. With said material elasticity, numerical studies here concluded the cerebellum moves approximately 200 microns. Considering the broad variance among scientists about human tissue elasticities, fluid substance properties, geometry, etc., this was very close to Pahlavian’s conclusion, thus fruitful. Two main contributors can lead to cerebellum movement, pressure and wall shear. It was determined from these numerical studies that pressure is the main contributor, while wall shear plays a very secondary role in the cerebellum movement.

The presence of thousands of autoantibodies (aABs) in the human sera is typical, and therefore it is possible to identify an aAB profile for each individual. In the first part of this thesis, we will show the cerebrospinal fluid also exhibits an extraordinarily complex immunoglobulin G aAB profile that is composed of thousands of aABs. We show that the pattern of expression of individual aABs in CSF closely mimics that in the blood, indicative of a blood-based origin for CSF aABs. In addition, using longitudinal serum samples obtained over a span of nine years, we show remarkable stability in aAB profiles over time, establishing that in the absence of stressors, individual aAB profiles show fidelity over time. We next explore the effect of one specific physiological stressor, pregnancy, on an aAB profile, using longitudinal samples taken before, during and after pregnancy. We are able to show a global reduction in aAB titers, followed by almost a full recovery, indicating that aAB profiles have a set-point that they try to re-establish. Lastly, though the presence of glycosylated antibodies in serum has already been established, there are no studies that have looked at the prevalence and distribution of glycosylated aABs. Glycosylation of antibodies infers different consequences based on the location of the glycosylation on the antibody molecule. During pregnancy, there is shift in the distribution of aAB glycosylation, from the majority of aAB initially glycosylated at 10-39% in early pregnancy to 30-69% in late pregnancy. This number then shifts dramatically post-pregnancy with the majority of aABs glycosylated at 10-19%. The increase in glycosylation during pregnancy is speculated to play an immunomodulatory role to foster immune tolerance of the fetus.

Stress granules (SGs) are membraneless organelles that form in response to cellular stress. Chronic cellular stress associated with neurodegenerative disease results in the persistence of SG structures. Chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of Huntington’s disease (HD) neuropathology. For my dissertation, I investigated localization and levels of the SG nucleating protein G3BP1 and found a significant increase in the density of G3BP1-positive SGs in the cortex and hippocampus of R6/2 transgenic mice, as well as in the superior frontal cortex of HD patient brains. I also examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of HD patients and show that a subset of their target mRNAs is differentially expressed in the prefrontal cortex of HD patients. Of these targets, there is an enrichment of SG components, including G3BP1. These findings suggest that SG dynamics might play a role in the pathophysiology of HD. Lastly, because the majority of the CSF is produced by choroid plexus epithelial cells, and a significant portion of CSF EVs likely originate from CPECs, I initiated the generation induced pluripotent stem cell (iPSC)-derived CPECs from HD and control lines that will allow for future mechanistic studies of EV secretion. Together, my work provides evidence of a potential role for CSF EV miRNAs in the regulation of SGs, identifies specific miRNAs that modulate the SG component G3BP1, and demonstrates feasibility for the generation of an iPSC-derived CPEC model.

Prion disease is a fatal, incurable neurodegenerative disease caused by a conformational change in the prion protein (PrP). Reducing brain PrP expression is a well-supported therapeutic strategy, and may be achievable in humans using intrathecally delivered antisense oligonucleotides (ASOs). Preclinical evidence indicates that some therapies may be more effective, or may only be effective, before symptom onset. Predictive genetic testing provides an opportunity to intervene early and preserve full quality of life in healthy individuals with prion protein gene (PRNP) mutations. Here, we report four advances that simultaneously strengthen the rationale for clinical trials in these pre-symptomatic individuals, and provide key data to enable such trials. First, by comparing allele frequencies in prion disease cases and population controls, we quantify PRNP variant penetrance and determine which individuals are at high lifetime risk — a potential patient population for trials. We also characterize healthy humans with heterozygous PRNP loss-of-function variants, supporting the safety of PrP-lowering strategies. Second, we characterize age of onset in genetic prion disease, and show that randomization to a disease endpoint is infeasible. This motivates development of biomarkers as surrogate endpoints, and our natural history dataset could aid in long-term confirmation of clinical benefit. Third, we develop a method for quantifying cerebrospinal fluid (CSF) PrP by mass spectrometry. CSF PrP decreases in active prion disease, potentially confounding any pharmacodynamic readout for a PrP-lowering drug in symptomatic patients, thus further motivating pre-symptomatic trials. Accordance between mass spectrometry and immunoassay results builds confidence in CSF PrP as an analyte, supporting its use as a biomarker. Fourth, we assess the efficacy of PrP-lowering ASOs against established brain prion infection in mice. We observe efficacy even at timepoints with detectable neuropathology and near the onset of obvious symptoms, encouraging further development of this modality. We also find that efficacy is maximized when treatment is initiated earlier, emphasizing the need to establish a clinical pathway for pre-symptomatic trials. Our findings help to establish both the need for, and feasibility of, early therapeutic intervention in genetic prion disease.

Chiari malformation type I is a disorder in which the cerebellar tonsils herniate through the foramen magnum. A majority of patients with this condition also develop a fluid filled cavity (syrinx) within the spinal cord. Syrinxes are associated with both sensory and motor disturbances, with extreme cases leading to autonomic dysfunction. The mechanisms that lead to the accumulation of fluid remain unknown. There is no universally accepted treatment and current surgical treatments have variable, but often unsatisfactory, outcomes and side effects are common. There is a need to understand cerebrospinal fluid (CSF) circulation in Chiari malformation to identify the mechanism(s) that cause syrinx formation. This would enable a mechanistically based treatment to be developed. This thesis presents six interrelated studies investigating CSF circulation and the influence of tonsillar herniation on CSF dynamics, to better understand potential mechanisms for syrinx formation. Methods used include novel real-time magnetic resonance imaging (MRI) of CSF and blood flow, morphological assessments of anatomical MRI, subject-specific computational models of the spinal subarachnoid space, and idealised models of the perivascular space. The subject-specific models showed that in Chiari patients the peak systolic CSF pressure was increased and occurred earlier in the cardiac cycle, compared with controls. The perivascular space model suggested that these subarachnoid pressure characteristics may cause increased flow into the cord that is favourable for syrinx formation in Chiari patients. Increased overcrowding below the foramen magnum in syrinx free subjects led to an earlier systolic pulse, whereas in syrinx subjects it caused a delay. This difference in behaviour may be related to syrinx patients having a smaller midsagittal cross-sectional area and could explain why not all patients develop a syrinx. Real-time imaging of CSF flow in controls were inconsistent with the currently accepted mechanism for respiratory CSF circulation, suggesting instead that respiratory CSF flow is primarily dependent on the balance of the thoracic and lumbar spinal pressures. Real-time imaging in Chiari patients found coughing and straining produced high velocity cranial flow at the foramen magnum, which may be a marker for Chiari associated headache, but whether this influences syrinx formation requires further investigation.

Epilepsy affects about 3.4 million people in the United States, with 14% under 18 years of age. The disease causes sudden recurrent unprovoked seizures and accounts for almost 2% of all emergency hospital visits, with a yearly total estimate in direct/indirect costs of about $14 billion. Over 60% of the 200,000 new cases diagnosed yearly are children under 5 years of age. Infantile spasms represent one of the most devastating seizure disorders of pediatric epilepsy and are more frequently associated with poor intellectual outcomes. The onset of infantile spasms is known to usually happen between 4-8 months within the first year of life. However, very little is known about the biochemical changes surrounding this phenomenon. Low levels of brain ?-aminobutyric acid (GABA) have been reported to be associated with poor seizure control, but exploitation of this knowledge in seizure management had been limited by several challenges including the absence of a reliable method for measuring brain GABA. The main purpose of the current project is to investigate the utility of brain GABA levels as a harbinger in the timeline to the onset/development, and remission of infantile spasms, with the help of a new, robust sample preparation and analysis method for quantifying low levels of GABA in small amounts of CSF. This method, which was recently described by Nkinin et al. in 2013 [62] will be employed to investigate if a sensitive marker, GABA may be used for screening and identifying potentially susceptible infants at risk of developing spasms prior to disease onset.

Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF) and regulate its electrolyte composition. CPECs express both the Na+/ K+ ATPase and the Na+/ K+/ 2Cl- cotransporter 1 (NKCC1) on their apical membrane (CSF facing), deviating from the typical basolateral membrane location in chloride secretory epithelia. Given this unusual location of NKCC1 and the unknown intracellular Na+ and Cl- concentrations of CPECs, the cotransporter function in these cells is not understood. Further, the direction of net ion and associated water fluxes mediated by NKCC1 under basal physiological conditions in CPECs is controversial. Determining the direction of NKCC1- mediated fluxes is critical to understanding the function of this cotransporter in CPECs. If NKCC1 works in the net efflux mode, it may be involved in CSF secretion as suggested by some investigators. Conversely, if NKCC1 works in the net influx mode, as it does in the basolateral membrane of other chloride secreting epithelia, then it would have an absorptive function in CPECs and could be involved in CSF K+ regulation, as we and others propose. NKCC1-mediated ion and water fluxes are tightly linked; thus, their direction is inferred by measuring cell water volume (CWV) changes in calcein-loaded CPECs following NKCC1 inactivation. Both genetic and pharmacological inactivation of NKCC1 produces CPEC shrinkage of ~16 %, due to unbalanced solute and water effluxes. [Na+]i and [Cl-]i measurements using the fluorescent indicator dyes ANG-2 and MQAE show that [Na+]i = 8.4 ± 1.0 mM and [Cl-]i = 62.1 ± 3.5 mM. NKCC1 -/- CPECs have ~ 50% less [Cl-]i than WT. WT CPECs are exquisitely sensitive to isosmotic changes in [K+]o (± 2 mM), as shown by CWV responses. CPECs of NKCC1 -/- mouse lose the response to [K+]o. These results suggest that under basal conditions, NKCC1 is constitutively active and works in the inward mode near its thermodynamic equilibrium in CPECS. We propose NKCC1 has absorptive functions and contributes to the maintenance of normal and constant CWV. The Km for [K+]o and the thermodynamic forces driving NKCC1 transport in CPECs suggest that it functions as a sensor and regulator of CSF [K+].

C-type natriuretic peptide (CNP) has high abundance in cerebrospinal fluid (CSF) and central tissues, and has been implicated in the regulation of important processes in the central nervous system (CNS) including the stimulation of neuronal branching, regulation of blood-brain barrier permeability and neuroprotection. Despite the many important putative roles that have been attributed to CNP during development and in adult life through in vitro studies, little is known about the regulation of CNP in vivo — and its usefulness in clinical settings of pathophysiological CNS disorders remains untapped. The aim of this PhD project was to explore the regulation of CSF concentrations of CNP and the amino-terminal fragment of proCNP (NTproCNP) in sheep, and relate this to central sources. To determine whether CSF concentrations of CNP peptides are affected by altered levels in plasma, concurrent CSF and plasma samples were collected from 15 pregnant sheep throughout the gestation period (days 4, 87 and 116) — a time when plasma concentrations become markedly increased. Compared with non pregnant sheep (n = 15), plasma concentrations of CNP peptides were elevated by 30-fold at days 87 and 116 in pregnant sheep, yet CSF concentrations of CNP and NTproCNP did not differ between the groups — which indicated that central levels of CNP peptides were independently regulated from those in plasma in sheep. As there were no known compounds or physiological setting capable of acutely affecting CSF concentrations of CNP peptides, a series of pilot studies was carried out to screen a number of candidate drugs in sheep, and to examine the effect of changes in live weight and appetite in red deer stags. Despite the dramatic changes in appetite and live weight that occurred throughout the breeding season in red deer stags (> 30 kg live weight gain), CSF concentrations of CNP peptides remained stable. Similarly, CNP and NTproCNP concentration in CSF of sheep remained unchanged following administration with all compounds (including anaesthetics, morphine, a pyrogen and l-deprenyl) except for one; a single iii i.v. dose of dexamethasone (0.25 mg/live weight) was shown to reliably induce a marked increase in CNP and NTproCNP concentration in CSF within 8 h. Following the successful identification of a secretagogue for CNP, subsequent studies were carried out to examine the increases in CNP peptide concentration in CSF and plasma following different doses of dexamethasone (0, 0.025, 0.063, 0.125, 0.25 mg/kg live weight), and to associate dexamethasone-stimulated increases in peptide levels in CSF with changes in peptide concentration and gene expression of NPPC, NPRB and NPRC in tissues sampled from the brain and anterior and posterior pituitary glands. Whereas plasma concentrations of CNP peptides were increased following all doses of dexamethasone, concentrations in CSF were elevated only at the two highest doses. Compared with saline-treated sheep, CNP and NTproCNP content in nervous tissue of dexamethasone-treated sheep (0.25 mg/kg, i.v.) was significantly higher throughout the brain in 6 and 11 of the 14 tissues considered, respectively. Gene expression of NPPC was upregulated in several tissues (anterior pituitary gland, posterior pituitary gland, hypothalamus, hippocampus and pons), which is supportive of increased synthesis of CNP following dexamethasone. The pituitary gland (anterior and posterior), which is known for its enriched levels of CNP content — had a similar concentration of NTproCNP when compared with most tissues sampled from the brain. Furthermore, the NTproCNP:CNP concentration ratio in pituitary gland (1:1) differed from brain (5:1 to 10:1), suggesting that little CNP degradation occurs in the pituitary gland — however the expression of NPRC did not differ between pituitary and brain tissue. Gene expression of NPPC was upregulated in both the anterior and posterior pituitary gland following dexamethasone, despite no significant increase in CNP or NTproCNP concentration in these tissues. Analysis of the molecular forms present in various fluids and tissues using size-exclusion high performance liquid chromatography revealed similar profiles for anterior pituitary gland and plasma extracts, whereby proCNP was present, CNP-53 was the prevalent form and CNP-22 was virtually absent. Together, these findings suggest that CNP processing differs between brain, anterior and posterior pituitary glands and supports the possibility that CNP is secreted directly from the anterior pituitary gland into the circulation. The widespread increase of CNP secretion in multiple tissues across the brain in response to dexamethasone implicates CNP in glucocorticoid actions — possibly related to inflammation or local fluid dynamics mediated by glial cells. These findings pave the way for future studies designed to establish the role of CNP in the CNS in normal iv health in vivo, and to establish a clinical application for this peptide in central settings of pathophysiological disorders in the CNS.

Fundamental to mammalian brain development is the integration of cell intrinsic and extrinsic signals that direct the proliferation and differentiation of neural stem cells. Precise expression of transcription factors together with other intracellular components instruct progenitor cell fate, whereas interaction with extracellular signaling factors refines this process. We have elucidated the composition of the cerebrospinal fluid that is the source of multiple extrinsic cues during brain development. The choroid plexus, a highly vascularized tissue located in each ventricle of the brain, actively secretes cerebrospinal fluid. By RNA sequencing, we obtained transcriptome data on the choroid plexi from lateral and fourth ventricles of the mouse brain and discovered that they include transcripts unique to each tissue. Transcription factor expression in the macaque and human choroid plexi suggests that positional identities of these tissues are conserved in the primate brain. Based on transcriptional results, we defined the choroid plexus secretome, a prediction of secreted factors from the choroid plexus. By quantitative mass spectrometry, we detected proteins secreted by each choroid plexus, and comparison of these proteomic results with transcriptional profiling suggests that choroid plexus transcriptomes contribute to availability of regionalized cerebrospinal fluid factors during development. In the second part of my dissertation research, I studied the role of DNA repair mechanisms in regulating neural stem cells. These studies focused on DNA LigaseIV, an essential component of DNA double-stranded break repair, during cerebral cortical development. Deficiency of LigaseIV activity caused by a missense mutation leads to LigaseIV syndrome, in which a key clinical feature is microcephaly. Using the Lig4 R278H mouse mutant, we found increased cell death in the developing cortex, contributing to reduced cortical thickness and cellularity in the anterior cerebral cortex. These results indicate that DNA LigaseIV is essential for proper cortical development. Together, these findings illustrate the complexity of regulatory mechanisms that guide brain development, requiring the integration of mechanisms from within and outside the cell. We have investigated two such mechanisms, extrinsic cues from regionalized cerebrospinal fluid and DNA LigaseIV. These results should provide greater insight into mechanisms of normal brain development and neuropathological states.

The dynamic environment of the cranial vault relies on normal brain pulsatility to maintain homeostasis. Small deviations in pressure or flow of either blood or cerebrospinal fluid (CSF) can drastically alter normal brain function. This thesis will investigate alterations in brain pulsatility in normal pressure hydrocephalus (NPH) patients; however, many of the conclusions/analysis methods have a broader impact and can be applied to other neurologic conditions, including traumatic brain injury and subarachnoid hemorrhage. Invasive intracranial pressure (ICP) monitoring and noninvasive Phase-contrast MRI (PC-MRI) will be used to measure brain pulsatility for this thesis, which is divided into three sections with three specific aims.Study 1: It is commonly accepted that there is some degree of CSF dysfunction in NPH. The first study investigates the stroke volume, latency, and variability changes that occur in the cerebral aqueduct and prepontine cistern of NPH patients and age-matched controls. Results show significant alterations in all aspects of CSF dynamics, which suggests decrease cisternal buffering, decreased compliance, and increased variability in the NPH cohort.Study 2: Differentiating shunt responders and non-responders has been a cornerstone of NPH research for over 50 years. Several aspects of ICP monitoring have been investigated as possible prognostic tests for NPH; however, results remain inconsistent and inaccurate. The advanced morphological analysis techniques used in this study show higher accuracy than traditional ICP analysis. These features, once further validated, could replace existing methods for more expensive, less accurate shunt selections. Study 3: Changes in pressure and flow dominate regulation of the internal dynamic environment of the brain; despite the physical relationship between the two phenomena, little is known about their impact on one another. Using CSF flow measured by PC-MRI, regional and individual ICP metrics that correlate with CSF flow were easily identified. This is a first step toward a better understanding of the characteristic peaks of the ICP pulse pressure waveform.Conclusion: The aforementioned studies reveal that modest changes in brain pulsatility can be detected in NPH patients. Although it is unlikely that these changes are the sole reason for the symptoms of NPH, these studies make important advances in understanding the pathoetiology of NPH. These discoveries need further investigation, but have the potential to change the way NPH is diagnosed and treated in the future.

The advantages of high sensitivity, low detection, large dynamic range and multi-element/isotope capability for most elements in the periodic table have enabled inductively coupled plasma mass spectrometry (ICPMS) to become a popular technique for elemental analysis. However, there are challenges from sample loss, matrix effects, interferences, or contamination when applying this technique in various analysis. To explore methods for reducing these difficulties in ICPMS-based analysis, the first part of this thesis is focused on removing inorganic phosphate interferences for the determination of organophosphate nerve agent degradation products based on the detection of 31P+. Calcium chloride and ammonium hydroxide were chosen as the coagulant and for pH adjustment to remove inorganic phosphate interferences. Applications to apple juice and cola drink indicate that this method is suitable for enhancing the determination of organophosphate species in high inorganic phosphate matrices. The growing interest in metals and their biological functions have promoted the utilization of ICPMS in the biological area for trace element determination and metal-containing speciation studies. The remaining focus of this dissertation is to investigate and characterize the metal-protein associations in various biological systems, which is referred as metallomics. To achieve overall screening and sufficient confidence in protein identifications, state-of-the-art metallomics techniques via two-dimensional chromatographic separations in combination with elemental and molecular mass spectrometries were utilized. Two biological systems were studied with different purposes and possible applications. Human cerebrospinal fluid (CSF) from healthy control patients and patients who suffered subarachnoid hemorrhage and its complication, cerebral vasospasm was screened at different metal detection points (Fe, Ni, Cu, Zn and Pb) for molecular distribution patterns and was investigated for possible markers. The identification and comparisons across the three types of CSF resulted in six protein families of possible biomarkers, which are now possible focus areas to study further for predicting, diagnosing and treating the hemorrhage to preclude the debilitating or deadly vasospasm. Bacteriophage lambda was investigated for possible phage-metal association at proteome level. Of a large number of metals, Mn, Fe, Co, Ni, Cu and Zn were observed to best associate with lambda phage sample from SEC-ICPMS screening. Further identification indicates that gpE, gpD and gpV have the highest likelihood of association with these transition metals to provide valuable information for lambda phage engineered technologies.

A variety of neurologic illnesses including peripheral and cranial neuropathies, central vestibular disease, seizures and coma have been associated with hypothyroidism in dogs. Repeated studies have shown that there is loss of blood brain barrier (BBB) integrity in these animals. Current research has also shown the development cerebrospinal fluid abnormalities in neurologically normal hypothyroid dogs; a finding that is related to BBB degradation. This derangement may be secondary to atherosclerosis and vascular accidents. One possible mediator of vasospasm and ischemic brain injury is endothelin-1 (ET-1). Another group of mediators of vascular dysfunction that has been found in CSF of dogs with various other CNS diseases is matrix metalloproteinases (MMP). The purpose of this study was to assay molecular markers that may contribute to disruption in the blood brain barrier in chronically hypothyroid canines. We hypothesized that BBB disruption in hypothyroidism is mediated by ET-1 and MMPs, as evidenced by increased concentrations of these proteins in CSF compared to controls. Cerebrospinal fluid (CSF) previously collected from 9 control and 9 experimentally induced hypothyroid dogs was used. Administration of I-131 was used to create the experimental model. CSF from time points 0, 6, 12, and 18 months post-induction were evaluated using an ELISA kit for endothelin-1. CSF from each time point was also evaluated using gelatinase zymography to detect MMP-2,9, and 14. The endothelin assay was able to detect ET-1 in CSF as determined by a spike and recovery method. However, ET-1 was undetectable in CSF of control and hypothyroid dogs at all time points. Constitutively expressed MMP-2 was detectable in all dogs at all time points. No other MMPs were detectable in CSF. ET-1 and gelatinase MMP,-9, and -14 are not primary mediators of BBB damage in chronically hypothyroid dogs. They could be involved secondarily and may be better evaluated with different assays or in temporal association with the development of clinical signs of neurologic dysfunction. Additional research is needed to confirm this finding and to evaluate biomarkers of non-vascular components of the BBB.

Chronic hydrocephalus (CH) is characterized by the presence of ventricular enlargement, decreased cerebral blood flow (CBF), and brain tissue oxygen delivery. Although the underlying pathophysiological role of vascular endothelial growth factor (VEGF) is not clear, ischemic-hypoxic events in CH are known to trigger its release. We investigated changes in neuronal and glial VEGFR-2 density and blood vessel density (BVd) in two clinically relevant areas of the brain, the hippocampus and caudate nucleus in an experimental model of CH. We also investigated changes in neuronal and glial VEGF receptor -2 density and BVd in the caudate nucleus after cerebrospinal fluid (CSF) shunting. Animals with CH were divided into short term (ST, 2-4 weeks) and long term (LT, 12-16 weeks) and were compared with surgical controls (SCs, 12-16 weeks) and CH-shunted animals (CH-S, 12-16 weeks). The cellular and BVds were estimated using immunohistochemical and stereological counting methods. Overall, percentage (%) VEGFR-2 neurons were approximately two–six times greater in CH (ST, LT) than in SC. There were differences in glial VEGFR-2 and BVd expression in the hippocampus and caudate nucleus. There was a six fold increase in VEGFR-2 glia in all regions of the hippocampus. By comparison, glial cell %VEGFR-2 was greater by 4-17% in CH compared with that in SC. Blood vessel density was six times greater in the hippocampus after CH but significantly lower in CH caudate compared to SC. There was a decrease in VEGFR-2 neuronal expression after shunting in the caudate regions compared to CH. After CSF shunting, the BVd initially decreased followed by an increase but only in the superficial caudate. Changes in CSF ventricular volume correlated with VEGFR-2 or BVd in the hippocampus but only partially in the superficial caudate region.VEGFR-2 may play an adaptive role in angiogenesis after CH-induced hypoxia. The dissociation of BVd and VEGF expression observed in the caudate differs from the hippocampus where uniform increases in BVd may have been facilitated by greater VEGFR-2 activation and better collateral blood supply. Modulation of the VEGF ligand and/or VEGFR-2 receptor may be important in developing treatments for hypoxic conditions including hydrocephalus and other forms of ischemia.

Previous studies have failed to reach consensus on insulin levels in cerebrospinal fluid of Alzheimer's disease (AD) patients and on its relation to pathological features. We performed a new analysis in patients at different stages of AD, and investigated the relationship of insulin levels with biochemical disease markers and with cognitive score. We included 99 patients from our Memory Clinic (Karolinska University Hospital, Sweden), including: 27 patients with mild AD, 13 that progressed from mild cognitive impairment (MCI) to AD in two years time, 26 with MCI stable after two years, and 33 with subjective cognitive impairment. Insulin was significantly decreased in the cerebrospinal fluid of both women and men with mild AD. Insulin deficits were seen in women belonging to both MCI groups, suggesting that this occurs earlier than in men. Insulin was positively associated with amyloid-β 1-42 (Aβ1-42) levels and cognitive score. Furthermore, total-tau/(Aβ1-42*insulin) ratio showed strikingly better sensitivity and specificity than the total-tau/Aβ1-42 ratio for early AD diagnosis in women.

Cerebrospinal fluid (CSF) surrounds and serves as a protective media for the brain and central nervous system (CNS). This fluid remains isolated from other biological matrices in normal bodily conditions, therefore, an in depth analysis of CSF has the potential to reveal important details and malfunctions of many diseases that plague the nervous system. Because phosphorylation of a wide variety of proteins governs the activity of biological enzymes and systems, a method for the detection of 31P in proteins found in human cerebrospinal fluid by high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS) is described. Specifically, it is of interest to compare phosphorylated proteins/peptides from patients suffering from post subarachnoid hemorrhage (SAH) arterial vasospasms against CSF from non-diseased patients. HPLC provides a way to separate many of the components of the CSF matrix, while ICPMS allows for the simultaneous detection of various elements, specifically phosphorus, at high sensitivity. Because structural elucidation is lost in the ICPMS experiment, softer ionization techniques such as Electrospray Ionization (ESI) are thus utilized for potential protein/peptide identification. Database searching software (Spectrum Mill) is then used in this study as a means of identification and validation. SEC-ICPMS confirms the presence of phosphorylated peptides within normal CSF as well as vasospastic and non-vasospastic SAH CSF samples. Non-enzymatically digested samples with ESI-MS analysis, in conjunction with Spectrum Mill database searching, indicates a variance among phosphorylated protein species across the sample batches, however, no correlations are drawn between disease types and phosphoprotein presence or absence.

The intracranial system consists of three main basic components - the brain, the blood and the cerebrospinal fluid. The physiological processes of each of these individual components are complex and they are closely related to each other. Understanding them is important to explain the mechanisms behind neurostructural disorders such as hydrocephalus. This research project consists of three interrelated studies, which examine the mechanical properties of the brain at the macroscopic level, the mechanics of the brain during hydrocephalus and the study of fluid hydrodynamics in both the normal and hydrocephalic ventricles. The first of these characterizes the porous properties of the brain tissues. Results from this study show that the elastic modulus of the white matter is approximately 350Pa. The permeability of the tissue is similar to what has been previously reported in the literature and is of the order of 10-12m4/Ns. Information presented here is useful for the computational modeling of hydrocephalus using finite element analysis. The second study consists of a three dimensional finite element brain model. The mechanical properties of the brain found from the previous studies were used in the construction of this model. Results from this study have implications for mechanics behind the neurological dysfunction as observed in the hydrocephalic patient. Stress fields in the tissues predicted by the model presented in this study closely match the distribution of histological damage, focused in the white matter. The last study models the cerebrospinal fluid hydrodynamics in both the normal and abnormal ventricular system. The models created in this study were used to understand the pressure in the ventricular compartments. In this study, the hydrodynamic changes that occur in the cerebral ventricular system due to restrictions of the fluid flow at different locations of the cerebral aqueduct were determined. Information presented in this study may be important in the design of more effective shunts. The pressure that is associated with the fluid flow in the ventricles is only of the order of a few Pascals. This suggests that large transmantle pressure gradient may not be present in hydrocephalus.

Hydrocephalus (HC) is a condition involving an increased volume of cerebral spinal fluid (CSF) in the ventricular system, caused by a variety of prenatal and postnatal aetiologies. In obstructive H C there is an obstruction to the flow of CSF and in communicating HC there is an interference with the reabsorption of the CSF while the ventricular pathways remain functional. The compressive effects of HC may lead to thinning and stretching of the corpus callosum, and also damage projection fibres, optic tracts and olfactory pathways. Consistent with this neuropathology, progressive HC in early childhood may result in neuropsychological impairment such as gross and fine motor deficits, spatial perceptual problems, and slowed processing speed. To date there is a paucity of research that compares the neuropsychological functioning of children with obstructive and communicating H C . This study aimed to compare attention, processing speed and executive functions in children with obstructive and communicating HC.

Cerebrospinal fluid analysis (CSF) is commonly performed in clinical neurology, and is a sensitive, but non-specific indicator of central nervous system (CNS) pathology. Blood contaminated CSF samples have the potential to adversely affect results of cytologic, serologic, microbiologic, and molecular biologic diagnostics. A clear consensus of the effects of blood contamination on CSF analysis could not be drawn following a review of the existing veterinary literature. Based on data from earlier reports, it was hypothesized that iatrogenic blood contamination of CSF would result in significant increases in both the CSF total protein (TP) concentration and nucleated cell count (WBC). As hypothesized, in vitro CSF blood contamination resulted in statistically significant (p < 0.01) linear increases in both the CSF TP and WBC with increasing RBC concentration in CSF from sixteen normal dogs. Although increases in TP and WBC are statistically significant, their clinical impact is negligible. Results of this study demonstrate that in normal dogs, the mean CSF TP concentration collected from the cerebellomedullary cistern, is lower than previously reported. D-dimers are plasminolytic cleavage products formed by the cross-linkage of fibrin by Factor XIIIa. In humans, D-dimer analysis can be used to differentiate iatrogenic from pathologic CNS hemorrhage. An additional objective of this study was to determine if canine D-dimers could be assayed using commercially available latex agglutination (LA) and enzymatic immunoassay (EIA) kits in normal and diseased subjects. It was hypothesized that qualitative and quantitative determinations of blood and CSF D-dimer activities could be aid in the diagnosis of dogs with altered CNS and/or systemic coagulation. D-dimers were able to be assayed in all subjects studied. D-dimer concentrations in CSF samples, when analyzed using a qualitative LA assay system, from healthy dogs with iatrogenically blood contaminated CSF were consistently negative. Quantitation of CSF D-dimer concentrations in normal dogs using an EIA assay resulted in lower values (mean 16.2 + 4.3 ng/ml; range, 0 to 54 ng/ml) than detected in the peripheral blood of dogs and humans (normal cutoff value < 250 ng/ml). These findings suggest that D-dimer formation does not occur in canine CSF freshly contaminated with blood. Significantly (p < 0.001) higher mean blood D-dimer concentrations were present in dogs with systemic coagulation disorders (1,093.4 + 172.3 ng/ml; range, 0 to > 2,000 ng/ml) when compared to normal dogs (54.6 + 19.8 ng/ml; range, 0 to 190 ng/ml), when assayed with the EIA. When used as an adjunct in the diagnosis of systemic coagulation abnormalities, the EIA assay had an overall sensitivity of 92%, specificity of 100%, positive predictive value (PPV) of 100%, and negative predictive value (NPV) of 94%. When applied to the same dogs, the LA D-dimer was less sensitive and specific (sensitivity of 73%, specificity of 100%, PPV of 100%, and NPV of 80%) than the EIA. Evidence of intrathecal fibrinolysis in the absence of systemic abnormalities was also demonstrated using CSF LA and EIA D-dimer assays in some dogs with a variety of infectious (Rocky mountain spotted fever), non-infectious inflammatory (granulomatous meningoencephalitis, steroid-responsive meningitis), traumatic (intervertebral disc disease, spinal fracture), and neoplastic (meningioma) diseases. When all dogs with CNS diseases were examined together, the mean EIA D-dimer concentration was significantly (p = 0.03) higher (511.6 + 279.8 ng/ml) than normal dogs (mean 16.2 + 4.3 ng/ml). Future studies will be required before the definitive role of D-dimer analysis can be defined in veterinary medicine.

Cerebrospinal fluid (CSF) analysis is a standard part of a diagnostic evaluation. Commonly evaluated components include the cell count, protein concentration, glucose, and cytology. CSF analysis can be diagnostic in some diseases such as fungal infections and CNS lymphoma. Often, CSF analysis is not specific, but more information can be obtained. Matrix Metalloproteinases (MMPs) are enzymes that have been found in human CSF. They are calcium and zinc dependent endoproteinases with overlapping substrates. They hydrolyze at least one component of tissue extracellular matrix (ECM), such as collagen or elastin. They are important in normal physiologic processes such as angiogenesis, reproduction and wound healing. One class of MMPs, the gelatinases, degrade gelatins and type IV collagen include MMP 2 and MMP 9. MMPs are important in many pathological processes that involve unregulated matrix destruction such as arthritis, neoplasia and CNS diseases. MMP2 is known to be constituitively produced in CSF while MMP 9 is present only in certain pathologic conditions such as multiple sclerosis, neoplasia and inflammatory diseases. We hypothesize that MMP2 is present in normal canine CSF while MMP 9 is absent. Cerebrospinal fluid samples were taken from 23 normal dogs that were being used for other research purposes. Each CSF sample was evaluated immediately for red blood cells (RBCs), white blood cells (WBCs), protein, and glucose, and then stored at -70°C. Cytological examination was also performed. CSF samples were considered normal if the protein was less than 25 mg/dl, WBCs were less than 6 µl, and RBCs were less than 25 µl. Each dog was euthanized and the brains processed for routine histopathology. MMP analysis was done using gelatin zymography and an enzyme linked immunosorbent assay (ELISA). Bands of enzyme activity were visible following staining due to enzyme degradation of the gelatin. A commercially available polyclonal sandwich ELISA was used to identify the pro form of MMP2. The mean WBC count for the CSF samples was 0.96 WBC/ml with a range of 0-3 WBC/ml. The mean protein was 12 mg/dl, with a range of 8-17 mg/dl. The mean RBC count was 3.65 RBC/ml with a range of 0-21 RBC/ml. All normal samples of CSF contained a band of clearing that corresponded to the human commercial standard of proMMP2. No other major bands of clearing were noted on normal samples. The commercial human standards also contained ProMMP2. Other bands were present, but were faint and variable. Using a polyclonal antibody based sandwich ELISA, with samples run in triplicate, the mean pro MMP 2 levels were determined to be 5.61 ng/ml with a range of 3.36 - 10.83 ng/ml. We conclude that normal CSF values are narrower than what has been previously reported for protein concentration and WBC count. Also, the pro form of MMP 2 is present in normal canine CSF based on results of gelatin zymography and ELISA.

Horses that are exposed to Sarcocystis neurona, a causative agent of equine protozoal myeloencephalitis, produce antibodies that are detectable in serum by western blot (WB). A positive test is indicative of exposure to the organism. Positive tests in young horses can be complicated by the presence of maternal antibodies. Passive transfer of maternal antibodies to S. neurona from seropositive mares to their foals was evaluated. Foals were sampled at birth (presuckle), at 24 hours of age (postsuckle), and at monthly intervals. All foals sampled before suckling were seronegative. Thirty-three foals from 33 seropositive mares became seropositive with colostrum ingestion at 24 hours of age, confirming that passive transfer of S. neurona maternal antibodies occurs. Thirty-one of the 33 foals became seronegative by 9 months of age, with a mean seronegative conversion time of 4.2 months. These results indicate that evaluation of exposure to S. neurona by WB analysis of serum may be misleading in young horses. Cerebrospinal fluid (CSF) samples from 15 neonatal (2-8 day) foals were examined for the presence of antibodies to S. neurona by WB analysis. Twelve of 13 foals that were seropositive were also CSF positive, suggesting that maternal antibodies to S. neurona cross the blood-CSF barrier in neonatal foals resulting in a positive CSF WB. Repeat taps were performed on 5 of the foals which showed that the immunoreactivity of the western blot decreases over time. Two of the 5 foals were CSF negative at 83 and 84 days of age, with 1 foal still positive at 90 days, and 2 foals positive at 62 days. These results indicate that maternal antibodies to S. neurona in the CSF can confound WB results in neonatal foals up to several months of age.