Monitoring Alzheimer's disease in transgenic mice with ultra high field magnetic resonance imaging

While aging remains one of the most significant risk factors for development of Alzheimer disease (AD), increasing evidence strongly points to the potential roles of cerebrovascular and white matter abnormalities in the disease development. A better understanding of the manner in which these abnormalities contribute to disease progression can be achieved by in vivo characterization of AD related pathologies. To this end, MR based techniques serve as effective non-invasive tools to longitudinally monitor changes in AD brain. In this thesis, a variety of MR based techniques were optimized and employed to longitudinally monitor the AD progression in transgenic mouse models of the disease at 9.4T and 17.6T. In Chapter 2, age-dependent blood flow alterations were examined in a Tg2576 mouse model of Alzheimer's disease using MR angiography at 17.6T. AD is linked to abnormalities in the vascular system. In Chapter 3, in vivo T2 changes were longitudinally monitored in the corpus callosum, of the Tg2576 mice. In Chapter 4, age-dependent regional brain T1 and T2 changes in healty mice were established at 17.6T. In vivo imaging of these mouse models at ultra-high magnetic field strengths can permit a better understanding of the underlying cellular mechanism of AD.The Centre for Medical Systems Biology (CMSB), Internationale Stichting Alzheimer Onderzoek and Alzheimer NederlandSolid state NMR/Biophysical Organic Chemistr

Imaging mouse models of neurodegeneration using multi-parametric MRI

Alzheimer’s disease (AD) is a devastating condition characterised by significant cognitive impairment and memory loss. Transgenic mouse models are increasingly being used to further our knowledge of the cause and progression of AD, and identify new targets for therapeutic intervention. These mice permit the study of specific pathological hallmarks of the disease, including intracellular deposits of hyperphosphorylated tau protein and extracellular amyloid plaques. In order to characterise these transgenic mice, robust biomarkers are required to evaluate neurodegenerative changes and facilitate preclinical evaluation of emerging therapeutics. In this work, a platform for in vivo structural imaging of the rTg4510 mouse model of tauopathy was developed and optimised. This was combined with a range of other clinically relevant magnetic resonance imaging (MRI) biomarkers including: arterial spin labelling, diffusion tensor imaging and chemical exchange saturation transfer. These techniques were applied in a single time-point study of aged rTg4510 mice, as well as a longitudinal study to serially assess neurodegeneration in the same cohort of animals. Doxycycline was administered to a subset of rTg4510 mice to suppress the tau transgene; this novel intervention strategy permitted the evaluation of the sensitivity of MRI biomarkers to the accumulation and suppression of tau. Follow-up ex vivo scans were acquired in order to assess the sensitivity of in vivo structural MRI to the current preclinical gold standard. High resolution structural MRI, when used in conjunction with advanced computational analysis, yielded high sensitivity to pathological changes occurring in the rTg4510 mouse. Atrophy was reduced in animals treated with doxycycline. All other MRI biomarkers were able to discriminate between doxycycline-treated and untreated rTg4510 mice as well as wildtype controls, and provided insight into complimentary pathological mechanisms occurring within the disease process. In addition, this imaging protocol was applied to the J20 mouse model of familial AD. This mouse exhibits widespread plaque formation, enabling the study of amyloid-specific pathological changes. Atrophy and deficits in cerebral blood flow were observed; however, the changes occurring in this model were markedly less than those observed in the rTg4510 mouse. This study was expanded to investigate the early-onset AD observed in individuals with Down’s syndrome (DS) by breeding the J20 mouse with the Tc1 mouse model of DS, permitting the relationship between genetics and neurodegeneration to be dissected. This thesis demonstrates the application of in vivo multi-parametric MRI to mouse models of neurodegeneration. All techniques were sensitive to pathological changes occurring in the models, and may serve as important biomarkers in clinical studies of AD. In addition, in vivo multi-parametric MRI permits longitudinal studies of the same animal cohort. This experimental design produces more powerful results, whilst contributing to worldwide efforts to reduce animal usage with respect to the 3Rs principles

Brain Injury

The present two volume book "Brain Injury" is distinctive in its presentation and includes a wealth of updated information on many aspects in the field of brain injury. The Book is devoted to the pathogenesis of brain injury, concepts in cerebral blood flow and metabolism, investigative approaches and monitoring of brain injured, different protective mechanisms and recovery and management approach to these individuals, functional and endocrine aspects of brain injuries, approaches to rehabilitation of brain injured and preventive aspects of traumatic brain injuries. The collective contribution from experts in brain injury research area would be successfully conveyed to the readers and readers will find this book to be a valuable guide to further develop their understanding about brain injury

Western diet and its effect on motivation, learning and memory

In Western society, the prevalence of a hyper-caloric diet consisting of a high consumption of fat and simple sugars has coincided with an exponential rise in diabetes as well as cardiovascular diseases and several types of cancer. While some of these medical comorbidities are themselves associated with adverse cognitive effects, recent studies have also linked the western diet to an increased incidence of Alzheimer’s disease and mild cognitive impairment. Moreover, these disorders are considered to be major risk factors for dementia indicating that these metabolic effects have both peripheral and central effects. Rats that have been a fed high fat diet (HFD) have shown indications to be cognitively impaired compared to those fed a normal chow diet. Research suggests that HFD consumption has a deleterious effect on spatial learning and memory, and this effect consequently may be mediated by damage to the hippocampus. To date however, there is conflicting results regarding the motivational and other types of learning implications after HFD consumption. The primary animal model of obesity used in this thesis is the western diet (WD) model of obesity in rats. This model mimics the ‘western’ diet typically consumed in developed ‘western’ countries by feeding rats a WD chow (containing 22% w/w fat) or a control chow diet (containing 6% w/w fat). Using this model, we explored the ability of 8 weeks of WD consumption to influence changes to Pavlovian & instrumental conditioning as well as motivation. This study used well characterised tests to assess whether learnt feeding behaviour can be affected by WD consumption. The ability for WD consumption to alter motivational drive in varying states of food deprivation was also investigated. Results from this study found that rats fed a WD for 8 weeks did not affect Pavlovian conditioning or motivational state. The effect of WD consumption on instrumental conditioning is still indeterminate with conflicting results. There was no change in instrumental conditioning in rats fed a WD. However, WD fed rats were impaired in progressive ratio instrumental conditioning acquisition. Additionally, WD exposed rats were no different to changes in states of food deprivation compared to control diet counterparts. A further study investigated whether a period of 12 weeks WD consumption can affect spatial working and reference memory. No changes in spatial working or reference memory were observed in WD rats. Due to the assumed role of c-Fos, an immediate-early gene and corresponding protein, in learning and use as a surrogate marker of neuronal activation, neuronal activation in selected brain regions was evaluated. We demonstrated that WD consumption increased neuronal activation after environmental novelty in the striatum. Other brain regions involved in memory and learning were also investigated with no differences in neuronal activation before and after environmental novelty between control and WD animals. In a series of experiments, we explored the ability of WD consumption to influence change in neurotransmitters involved in memory and learning. The expression of serotonin (5-HT) receptors 5-HT2A, 5-HT2C and the 5-HT transporter within the striatum was also investigated, as previous studies have shown that serotonin is implicated in feeding behaviour following WD consumption. Both 5-HT2C receptor and 5-HT transporter expression were found to be increased in WD rats. In contrast, 5-HT2A receptor expression was unchanged in the striatum. This suggests that WD consumption has a selective capacity to alter the serotoninergic system. Furthermore due to the well-recognised role of dopamine in cognition, including motivation, reward, punishment and working memory, the modification of dopamine metabolism was evaluated. High performance liquid chromatography analysis found reduced levels of striatal dopamine, with alterations in dopamine metabolism and turnover also evident in the hippocampus after WD consumption. These neurotransmitter changes were also observed to be independent of any change in cognitive ability. This suggests that WD consumption may instigate dopaminergic and serotoninergic adaptations before cognitive impairment transpire. The early life nutritional environment was also investigated to ascertain if early life obesity may contribute to cognitive impairment using a neonatal overfeeding rat model of obesity. As early life is a critical window of vulnerability to long-term programming of health, cognitive assessment was performed by utilizing spatial memory function in the Y-maze test and also spatial reference and working memory using the delayed win-shift task (DWSh) in the radial arm maze. Neonatally overfed rats took longer to learn the DWSh task indicating a poorer memory acquisition compared to control. No change of spatial memory in the less cognitively demanding Y-maze test was observed in neonatally overfed rats. The potential of a synergistic effect of WD consumption in the APPswe/PS1dE9 double transgenic Alzheimer’s mice model (APDE9) animal model memory and anxiety-like behaviour was assessed. Metabolically, this study identified that APDE9 mice fed a WD showed impaired glucose tolerance but not in wild-type WD mice or ADPE9 mice fed the control diet indicating impaired insulin receptor signalling. Both APDE9 mice fed control or WD showed a spatial memory deficit in the Y-maze when compared to their wild-type counterparts. There was no observed synergistic effect of WD consumption and APDE9 phenotype in the Y-maze. Additionally no change in anxiety-like behaviour was discerned using the open field test and the light/dark preference test. Findings from this thesis indicate that WD consumption alone does not affect cognition using a variety of behavioural tasks. Whilst central changes in the dopaminergic and serotoninergic system ensue following WD consumption however, whether these changes occur before cognitive impairment is still unclear. The time period in which the obese phenotype transpires appears to play a factor in cognitive impairment as shown by the results in the neonatal overfeeding study. Additionally we demonstrated that WD consumption does not affect spatial memory but a possible synergistic interplay between the APDE9 mice phenotype and WD consumption may have a deleterious effect of spatial memory. Further work is necessary to elucidate the factors that contribute to the onset of cognitive impairment observed in rat models of obesity

Does lobar intracerebral haemorrhage differ from non-lobar intracerebral haemorrhage?

Spontaneous (non-traumatic) intracerebral haemorrhage accounts for ~10% of all strokes in Western populations. Investigations may identify intracerebral haemorrhage (ICH) as ‘secondary’ to underlying causes such as tumours or aneurysms, but ~80% of ICHs which have no apparent underlying cause (so-called ‘primary’ ICH) tend to be attributed to small vessel vasculopathies such as arteriolosclerosis or cerebral amyloid angiopathy (CAA), on the basis of an adult’s risk factors and clinical and radiographic features of the ICH. The commonly accepted hypothesis is that CAA contributes to lobar ICH and arteriolosclerosis causes non-lobar ICH. In the following thesis, I set out to explore whether (a) the baseline demographic, clinical features and apolipoprotein E genotype of adults with lobar and non-lobar ICH differ, (b) the prognosis of adults with lobar and non-lobar ICH differ and (c) the neuroimaging correlates of small vessel disease in adults with lobar and non-lobar ICH differ since this might provide clues to the vasculopathies underlying lobar and non-lobar ICH. I explored (d) the strength of the association between CAA and ICH by systematically reviewing neuropathological case control studies and (e) the radiological and pathological features of lobar ICH to examine the nature of CAA in persons with lobar ICH and whether any computed tomography (CT) features of ICH are associated with CAA-related lobar ICH. I set up a prospective, community-based inception cohort study of adults with ICH in South East Scotland. Adults with spontaneous primary definite ICH had the opportunity to consent to participate in the Lothian Study of IntraCerebral Haemorrhage, Pathology, Imaging and Neurological Outcome (LINCHPIN), an ethically-approved, prospective community-based research study examining the causes of ICH using apolipoprotein E genotyping, brain MRI and research autopsy in case of death. Of 128 adults with first-ever spontaneous primary definite ICH diagnosed during 2010- 2011, age and pre-morbid hypertension did not differ by ICH location but a history of dementia was more common in adults with lobar ICH. The proportion of adults with one or more non-lobar brain microbleed (BMB) was significantly higher in adults with non-lobar ICH but I did not find any other differences in the severity or distribution of other neuroimaging correlates of small vessel disease between lobar and non-lobar ICH. The apolipoprotein e4 allele was more common in participants with lobar ICH in comparison to those with non-lobar ICH but the frequency of the e2 allele did not differ by ICH location. Adults with lobar ICH were significantly more likely to survive one year after their ICH in comparison to those with non-lobar ICH after adjustment for other known predictors of outcome. From a systematic review of neuropathological case control studies of CAA and ICH, stratified by ICH location, I found a significant association between CAA and lobar ICH but not with ICH in other locations. I examined the radiological and pathological features of 33 adults with first-ever lobar ICH. The presence of CAA or vasculopathy and the severity of CAA in a lobe affected by ICH was concordant with that of the corresponding contralateral unaffected lobe. Capillary CAA was associated with severe CAA. Subarachnoid extension of the ICH tended to be more frequent in those with CAA-related strictly lobar ICH. Having explored the incidence, risk factors and prognosis of lobar and non-lobar ICH, in future work I would aim to establish the strength of the association between CAA and ICH in different brain locations in a neuropathological case control study. Future work should examine the radiopathological features of lobar ICH in a larger cohort and the coexistence of other small vessel diseases, in particular arteriolosclerosis in persons with ICH

Well-defined polyglutamates as carriers for the treatment of neurodegenerative diseases

Alzheimer’s disease (AD) is a neurodegenerative multiple process of the central nervous system, which currently represents the most common cost of Dementia. The already high incidence of AD is predicted to dramatically increase over the years. In fact, the experts claim that it will become a global epidemy by 2050. Consequently, direct and indirect costs related to AD are doomed to dramatically increase. For instance, only in America, AD related burden will overcome the trillion of dollars by 2050. Moreover, available medication (Exelon®, Namenda®, Aricept®, and Razadyne®) produce moderate symptomatic benefits, but do not stop disease progression. Hence, AD, among other neurodegenerative disorders, can be considered an unmet medical need. Neuroprotective drugs, such as, curcuminoids are been taking in high consideration in order to approach these fatal disorders from a protective and preventive point of view. In this context, nanomedicine and, in particular, Polymer Therapeutics (PT) emerge as a powerful alternative to overcome the limitations of low MW drugs including their poor pharmacokinetic and pharmacodynamic profiles and low solubility in aqueous solvents, required for i.v. administration. Nonetheless, in the PT field, there is a need to develop new and innovative polymer carriers to be used as drug delivery systems and/or imaging agents owing to the fact that there is no universal polymeric system that can be used in the treatment of all diseases. Apart from biodegradability, the development of novel well-defined architectures with higher MW (in order to increase passive targeting provided by the EPR effect), predictable structure and conformation (defined three-dimensional architecture in solution), higher homogeneity, greater drug loading capacity and increased multivalency is considered crucial. To this respect, polypeptides are envisaged to achieve a major impact on a number of different relevant areas including nanomedicine. Thus, new PT based on amino acids are excellent candidates for drug delivery, as they do not suffer from the previously mentioned limitations. Concretely, polyglutamates constitute a versatile platform, which has been effectively used as building blocks in polymer drug conjugates and polymeric micelles for various medical applications ranging from cancer to regenerative medicine. Moreover, it is expected its FDA approval after approval of PGA-paclitaxel conjugate, OpaxioTM for the treatment of various cancers alone or in combination (OpaxioTM has been recently designated as orphan drug in combination with radiotherapy and temozolomide for the treatment of glioblastoma multiforme). Nevertheless, control on polymer chain length, polydispersities and stereochemistry has been the major challenge in the development of synthetic polypeptides over the past years. Besides, the use of branched polymers is emerging in order to accomplish the previously described requisites. They exhibit special properties when compared to their linear counterparts. As a result of their different architectures, solution conformation, size and shape as well as greater multivalency, different therapeutic outputs could be gained. Due to their compact and globular shapes they are postulated to perform better regarding to overcome biological barriers, a pre-requisite in neurodegenerative disorders treatment as well as diagnostics due to the presence of the blood-brain barrier (BBB), one of the most challenging to surpass. Therefore, the main aim of this thesis was the design of new versatile polyglutamate-based nanotherapeutics to be used in the treatment and/or diagnosis of devastating neurodegenerative pathologies such as AD. In order to accomplish our final goal, firstly, we report the development of synthetic pathways to a plethora of functional polyglutamates with well-defined structure, adjustable MW and low polydispersities (Đ <1.2) applying the ring opening polymerization (ROP) of N-Carboxyanhydrides (NCA) with novel initiators. Furthermore, this methodology has been extended to reach a number of architectures based on PGA, including stars, grafts, and hybrid di-block copolymers. In addition, a versatile post-polymerization modification method to introduce a variety of functionalities such as alkyne, azides, reactive disulfides, maleimide groups or protected amines has been developed, yielding a set of orthogonal reactive attachment sites suitable for further bioconjugations. The physico-chemical properties of the obtained polyglutamates have been exhaustively investigated, in terms of size and solution conformation by the use of a battery of complex techniques including DLS, DOSY-NMR, CD, TEM and SANS. Furthermore, we have developed a novel PGA-based family of systems that, according to their physico-chemical characterization, underwent a self-assembly process where it did exist a structure/conformation-concentration dependency encountering at low concentrations “unimers” of 5-10 nm size, whereas bigger structures of around 100-180 nm were formed at high concentrations. After covalent entrapment of these bigger structures by means of click chemistry, the concentration dependence conformation was clearly eliminated. We have taken profit from that special behavior to develop a strategy in order to reach complex polypeptide based architectures through bottom-up approaches Preliminary in vitro evaluation in selected cell models in terms of biodegradability, biocompatibility and cellular uptake is presented. Furthermore, after an adequate labeling with fluorescence/NIR probes or/and cation complexing moieties towards the use of MRI and/or PET techniques, the in vivo fate (pharmacokinetics and biodistribution) of our polyglutamates is also described. Preliminary results suggest that they were non-toxic entities, validating them as possible carriers for drug delivery. The covalently entrapped unique architectures have been ultimately used to reach carriers for BBB crossing by means of surface modifications with targeting units and imaging agents. Their BBB crossing properties have being explored in vivo, reaching at least 1.2 % of injected dose in the brain. Thus, those results make them optimal candidates to be used in AD treatment. Among all the biological hallmarks of AD, we are centering our efforts in the amyloid pathway, by the use of curcuminoids and with a neuroprotective approach by combining them with the presence of propagyl moieties within the construct. Their biological output regarding cellular uptake, cell viability, drug release profile and biodistribution has been investigated. Moreover, proof of concept of their activity was achieved in vitro, in organotypic hippocampal cultures and is currently being validated in vivo. Finally, the potential of PGA-based conjugates as tissue-specific smart imaging probes is also explored within the frame of the European consortium LIVIMODE. The combination of NIRF enzyme specific smart probes together with the tissue specificity provided by PGA as carrier is explored to be applied in the early detection of disease-related events in vitro as well as in vivo. This strategy could be used for the development of theranostics towards the early detection and treatment of neurodegenerative disorder

Monitoring blood flow alterations in the Tg2576 mouse model of Alzheimer's disease by in vivo magnetic resonance angiography at 17.6 T

Neuro Imaging Researc

The Deleterious Effects of Glucocorticoids: From epidemiology to molecular perspective

Background: Cushing’s syndrome (CS) is caused by prolonged and inappropriately excessive tissue exposure to glucocorticoids (GC) [1]. CS results in significant morbidity and excess mortality. Increased 11β-hydroxysteroid dehydrogenase type1 (11β-HSD1) activity at local tissue has been documented for adverse cortisol effects. Aim: To explore the deleterious effects of systemic and local GC excess in man at molecular and epidemiological levels, which focuses on the outcomes that enable the quantification of disease burden and further avoidable premature death or morbidity. Methods: The epidemiological studies focused on a meta-analysis of mortality and causes of death in endogenous and exogenous CS. Mortality is a crucial health problem, and meta-analyses systematically explore the issue. The molecular study investigates 11β-HSD1 expression in hypoxia in human dermal fibroblasts. This is the preliminary research of 11β-HSD1 role in ischaemic/diabetic wounds, the worldwide health burden. The understanding of 11β-HSD1 in hypoxic skin may yield a new treatment for diabetic/ischaemic wounds. Results: The pooled proportion of death for endogenous CS was 5%, 4% in Cushing's disease (CD), 2% in adrenal adenoma, but 8% in bilateral adrenal hyperplasia. The standardised mortality ratio (SMR) was 3.0 for all CS. ACS was associated with a worse SMR than CD (p=0.003). Mortality was higher in publications published before 2000, active disease, and macroadenomas. Cumulative, average, and initial GC doses are associated with increased mortality in exogenous CS. Cardiovascular diseases, infection and malignancy, are the major contributors to deaths for all CS. Hypoxia increases 11β-HSD1 expression and activity in HDF, particularly in inflammatory conditions for the molecular study. Conclusion: CS confirmed the association with an increase in mortality. The causes of death highlight the need for aggressive management. The 11β-HSD1 role in hypoxia requires further research in ischaemic or diabetic human skin with is the new hope for curing the wound