Optics Based Label-Free Techniques and Applications in Brain Monitoring

AbstractFunctional near-infrared spectroscopy (fNIRS) has been utilized already around three decades for monitoring the brain, in particular, oxygenation changes in the cerebral cortex. In addition, other optical techniques are currently developed for in vivo imaging and in the near future can be potentially used more in human brain research. This paper reviews the most common label-free optical technologies exploited in brain monitoring and their current and potential clinical applications. Label-free tissue monitoring techniques do not require the addition of dyes or molecular contrast agents. The following optical techniques are considered: fNIRS, diffuse correlations spectroscopy (DCS), photoacoustic imaging (PAI) and optical coherence tomography (OCT). Furthermore, wearable optical brain monitoring with the most common applications is discussed. Abstract Functional near-infrared spectroscopy (fNIRS) has been utilized already around three decades for monitoring the brain, in particular, oxygenation changes in the cerebral cortex. In addition, other optical techniques are currently developed for in vivo imaging and in the near future can be potentially used more in human brain research. This paper reviews the most common label-free optical technologies exploited in brain monitoring and their current and potential clinical applications. Label-free tissue monitoring techniques do not require the addition of dyes or molecular contrast agents. The following optical techniques are considered: fNIRS, diffuse correlations spectroscopy (DCS), photoacoustic imaging (PAI) and optical coherence tomography (OCT). Furthermore, wearable optical brain monitoring with the most common applications is discussed

Early Improvement in Interstitial Fluid Flow in Patients With Severe Carotid Stenosis After Angioplasty and Stenting

Background and Purpose This study aimed to investigate early changes in interstitial fluid (ISF) flow in patients with severe carotid stenosis after carotid angioplasty and stenting (CAS). Methods We prospectively recruited participants with carotid stenosis ≥80% undergoing CAS at our institute between October 2019 and March 2023. Magnetic resonance imaging (MRI), including diffusion tensor imaging (DTI), and the Mini-Mental State Examination (MMSE) were performed 3 days before CAS. MRI with DTI and MMSE were conducted within 24 hours and 2 months after CAS, respectively. The diffusion tensor image analysis along the perivascular space (DTI-ALPS) index was calculated from the DTI data to determine the ISF status. Increments were defined as the ratio of the difference between post- and preprocedural values to preprocedural values. Results In total, 102 participants (age: 67.1±8.9 years; stenosis: 89.5%±5.7%) with longitudinal data were evaluated. The DTI-ALPS index increased after CAS (0.85±0.15; 0.85 [0.22] vs. 0.86±0.14; 0.86 [0.21]; P=0.022), as did the MMSE score (25.9±3.7; 24.0 [4.0] vs. 26.9±3.4; 26.0 [3.0]; P<0.001). Positive correlations between increments in the DTI-ALPS index and MMSE score were found in all patients (rs=0.468; P<0.001). Conclusion An increased 24-hour post-CAS DTI-ALPS index suggests early improvement in ISF flow efficiency. The positive correlation between the 24-hour DTI-ALPS index and 2-month MMSE score increments suggests that early ISF flow improvement may contribute to long-term cognitive improvement after CAS

Investigating Glymphatic Function In Alzheimer’s Disease Pathology

Alzheimer’s disease is fast becoming the greatest healthcare challenge of our time, with no known cure to-date. Brought about by the toxic formation of plaques of amyloid-β and tangles of tau in the brain, much is still unknown about the precise mechanisms that initiate these protein accumulations, thought to occur decades before clinical manifestation of symptoms. One theory is that an imbalance between the production of these proteins and their removal from the brain promotes retention that eventually aggregates into entities that devastate molecular and cellular machinery. Thus, targeting waste clearance mechanisms responsible for removing cerebral metabolites, including amyloid-β and tau, present novel, enthralling research targets. The glymphatic system is one such pathway that has been recently characterised. Considered a surrogate for lymphatics which are largely lacking in the brain, this fluid network relies on the water channel aquaporin-4, expressed highly on glia, thus being named “glymphatics”. In this work, first, a surgical protocol was established in the mouse brain to facilitate the delivery of tracer molecules into the cerebrospinal fluid. Direct, single time-point, histological assessment of fluorescent tracer distribution was performed to check consistency with previous characterisation of glymphatics in the mouse brain. Glymphatics were then visualised dynamically across the whole brain using magnetic resonance imaging. Glymphatic patterns were investigated in real-time by imaging fluid dynamics in the brain alongside a potent blocker of aquaporin-4. Next, imaging was used to characterise glymphatic changes and aquaporin-4 profiles in mouse models of Alzheimer’s pathology. This revealed a time-dependant relationship between glymphatics and tau accumulation. Finally, the findings were extrapolated onto humans by studying aquaporin-4 modifications in subjects with and without cognitive deficits. Here, the crucial relationship between aquaporin-4 and pathological aggregates of tau and amyloid-β was determined. Furthermore, dystrobrevin, a membrane protein linked to aquaporin-4, was also profiled in the setting of aging and amyloid-β pathology. The work presented herein elucidates the role of glymphatic perturbances in the context of Alzheimer’s disease and clarifies the implications of aquaporin-4 mediated clearance in neurodegeneration

Systemic delivery of a specific antibody targeting the pathological N-terminal truncated tau peptide reduces retinal degeneration in a mouse model of Alzheimer’s Disease

Retina and optic nerve are sites of extra-cerebral manifestations of Alzheimer’s Disease (AD). Amyloid-β (Aβ) plaques and neurofibrillary tangles of hyperphosphorylated tau protein are detected in eyes from AD patients and transgenic animals in correlation with inflammation, reduction of synapses, visual deficits, loss of retinal cells and nerve fiber. However, neither the pathological relevance of other post-translational tau modifications—such as truncation with generation of toxic fragments—nor the potential neuroprotective action induced by their in vivo clearance have been investigated in the context of AD retinal degeneration. We have recently developed a monoclonal tau antibody (12A12mAb) which selectively targets the neurotoxic 20–22&nbsp;kDa NH2-derived peptide generated from pathological truncation at the N-terminal domain of tau without cross-reacting with its full-length normal protein. Previous studies have shown that 12A12mAb, when intravenously (i.v.)-injected into 6-month-old Tg2576 animals, markedly improves their AD-like, behavioural and neuropathological syndrome. By taking advantage of this well-established tau-directed immunization regimen, we found that 12A12mAb administration also exerts a beneficial action on biochemical, morphological and metabolic parameters (i.e. APP/Aβ processing, tau hyperphosphorylation, neuroinflammation, synaptic proteins, microtubule stability, mitochondria-based energy production, neuronal death) associated with ocular injury in the AD phenotype. These findings prospect translational implications in the AD field by: (1) showing for the first time that cleavage of tau takes part in several pathological changes occurring in vivo in affected retinas and vitreous bodies and that its deleterious effects are successfully antagonized by administration of the specific 12A12mAb; (2) shedding further insights on the tight connections between neurosensory retina and brain, in particular following tau-based immunotherapy. In our view, the parallel response we detected in this preclinical animal model, both in the eye and in the hippocampus, following i.v. 12A12mAb injection opens novel diagnostic and therapeutic avenues for the clinical management of cerebral and extracerebral AD signs in human beings

Development and Application of MRI Techniques for Non-Invasive Assessment of Blood-Cerebrospinal Fluid Barrier Function

The choroid plexus (CP) tissue forms the blood-cerebrospinal fluid barrier (BCSFB) - a unique interface which plays a critical role in effective homeostasis of the central nervous system. To date, exploration of the BCSFB’s role in health and disease has been hindered by a lack of non-invasive, translatable methodologies. The recent development of BCSFB-ASL MRI by Evans et al. has permitted the non-invasive, surrogate measurement of BCSFB function. The work presented herein develops and applies the BCSFB-ASL method to investigate BCSFB function in rodent models of ageing and disease. Chapter 2 describes a novel platform for simultaneous recording of BCSFB function and brain tissue perfusion using interleaved echo-time ASL, which provided insight into alterations of vessel tone at the BBB and BCSFB under the influence of pharmacological agents, as well as how reactivity towards a vasopressin challenge is impaired in the aged mouse brain. In Chapter 3, I reproduce, optimise, and characterise the BCSFB-ASL MRI approach on a Bruker 9.4T system, that was heretofore applied only on an Agilent 9.4T MRI system. This work seeks to utilise the improved hardware and software on the Bruker system to increase measurement precision with minimised scan times. Chapter 4 describes efforts to further characterise the contributing sources and kinetics of ultra-long echo-time ASL signals arising from brain-wide CSF regions. These experiments seek to determine the reliability of the estimated labelled blood water delivery rates, alongside potential factors which may contribute to the appearance of these signals, in regions distal to the caudal lateral ventricles. In Chapter 5, BCSFB function was then investigated in the context of systemic hypertension. Spontaneously hypertensive rats displayed a reduction in BCSFB function, which highlights the potential for such measures to serve as a sensitive early biomarker for hypertension-driven neurodegeneration. Overall, we demonstrate the scope of BCSFB-ASL to capture changes to BCSFB function, which not only has value in providing a useful biomarker for downstream neurodegeneration, but also provides an insight into mechanisms which may increase the brain’s susceptibility towards neurodegenerative outcomes

Intracranial Pressure Monitoring in Cerebrospinal Fluid Dynamics Disturbances

There are numerous gaps in the knowledge of Intracranial Pressure (ICP) physiology and Cerebrospinal Fluid (CSF) dynamics. This PhD answers some of the research questions posed by these gaps, through the use of invasive ICP monitoring in patients with suspected CSF dynamics disturbances. Research on CSF dynamics disturbances has mainly focused on conditions that cause high ICP, whilst only sparse attention has been centred on low CSF pressure/volume states. Chapter 3 and chapter 4 of this thesis are focused on Spontaneous Intracranial Hypotension (SIH). Chapter 3 is a comprehensive systematic review and meta-analysis of the clinical presentation, investigation findings and treatment outcomes of this disease. Chapter 4 is an observational study that investigated the utility of invasive ICP monitoring when there is diagnostic uncertainty for SIH. This study demonstrates that, in selected cases, ICP monitoring can be useful and confirm a low-pressure state in 8% of the patients and identify a paradoxical clinical presentation with an underlying high-pressure state in 16% of the patients. Chapters 5 and 6 provide evidence on the way that ICP and brain compliance respond to external variables, such as changes in posture and shunt setting adjustments. Chapter 5 is a retrospective observational study that describes the changes of ICP and pulse amplitude with different postures. Chapter 6 is a retrospective observational study investigating the effect of valve setting adjustments on ICP. This study demonstrates that paradoxical changes in ICP following differential pressure valves setting changes can occur. Chapters 7 and 8 investigate the possibility of replacing invasive ICP monitoring with non-invasive biomarkers of raised ICP. Chapter 7 demonstrated the association between higher ICP measurements and the absence of spontaneous retinal venous pulsations detected with infrared video recordings. Chapter 8 demonstrates the utility of integrating ophthalmic and imaging biomarkers to predict raised ICP

Investigating glymphatic system and AQP4 water channels with novel drugs and MRI techniques

The glymphatic system serves as a vital low resistance pathway for the efficient removal of toxic waste products from the brain and its malfunction is implicated in numerous neuropathological conditions. Aquaporin-4 (AQP4) water channels are membrane-tied and highly expressed at the end-feet of astrocytic cells in the brain. They are thought to be crucial to the glymphatic clearance system, water circulation, and homeostasis of the brain. Pharmacologically targeting AQP4 presents a promising therapeutic strategy for various neurological diseases. In 2009, Huber et al. developed TGN-020, a potent AQP4 inhibitor that significantly reduced cerebral oedema in stroke models. In 2018, they introduced TGN-073, a novel AQP4 facilitator that enhanced fluid turnover and interstitial fluid clearance. This thesis investigates the effects of these AQP4 modulators, with a particular focus on TGN-073, using advanced MRI techniques and immunofluorescence staining in rat models to elucidate their potential therapeutic benefits. The initial objective was to employ an H2 17O tracer to evaluate the effect of the novel AQP4 facilitator TGN-073 on glymphatic transport. Despite extensive optimization efforts, the tracer signal remained low and unreliable, precluding its use in conducting our studies. Consequently, we assessed the impact of TGN-073 on glymphatic transport using dynamic contrast-enhanced MRI. This involved catheterizing the cisterna magna to infuse the MRI contrast agent Gd-DTPA into the cerebrospinal fluid. Our findings indicated that rats treated with TGN-073 exhibited a more extensive distribution and higher parenchymal uptake of GdDTPA compared to the vehicle group, suggesting TGN-073's potential in enhancing glymphatic function. Following this, I developed and established an immunohistochemistry protocol for AQP4 staining using immunofluorescence, a first in our department. The aim was to optimize this technique to its fullest potential, ensuring precision and reliability for the following experiments. Given the invasive nature of the method used to investigate the impact of TGN073 on glymphatic transport, which requires cisterna magna cannulation, noninvasive alternatives were explored. Therefore, the impact of both AQP4 modulators, TGN-020 and TGN-073, was assessed without the necessity of exogenous contrast agents. These evaluations utilized T2 mapping and stimulated echo diffusion-weighted echo planar imaging (STE-DW-EPI), followed by immunofluorescence labelling of AQP4. No significant changes in the diffusion coefficient were observed across all observation times in any animal group, indicating no substantial alterations in brain microstructure. However, T2 values significantly decreased following the administration of TGN-073, suggesting enhanced water exchange. In contrast, T2 values significantly increased following the administration of TGN-020, while remaining unchanged in the vehicle group. These findings underscore the role of AQP4 in modulating water exchange between tissue compartments. Immunofluorescence staining revealed significantly higher AQP4 expression in the brains treated with TGN-073, contrasting with a significant decrease in AQP4 expression in the brains treated with TGN-020, compared to the vehicle-treated group. To advance our understanding of the positive effects of AQP4 facilitators on glymphatic function, we investigated the impact of TGN-073 in a rat model of vascular cognitive impairment, specifically the bilateral common carotid artery stenosis (BCAS) model. This model, known for inducing chronic cerebral hypoperfusion and vascular dementia, represents a novel application within our institution. To our knowledge, this is the first study to evaluate glymphatic transport in BCAS rat models and to assess the impact of an AQP4 facilitator in this context. We successfully established cerebral hypoperfusion in the BCAS model, as evidenced by a significant reduction in cerebral blood flow (CBF). Our findings demonstrated glymphatic dysfunction and altered AQP4 expression associated with BCAS. Importantly, TGN-073, effectively mitigated these effects by restoring AQP4 expression, enhancing glymphatic function, and alleviating CBF reduction. This study highlights the potential of AQP4 facilitators in ameliorating the adverse effects of cerebral hypoperfusion and associated glymphatic dysfunction. TGN-073 shows promise for preventing the progression of neurodegenerative diseases and improving the quality of life for affected individuals

Inflammatory cells and blood-brain barrier leakage in cerebral small vessel diseases

This thesis focussed on cerebral small vessel (cSVD) disease, which is an umbrella term that covers a heterogeneous group of disorders that affect small arteries in the brain. CSVD is the main cause of vascular cognitive impairment and lacunar stroke. This thesis aimed to gain more insights in the pathophysiology of cSVD by investigating the role of local brain immune cells and leakage of brain blood vessels. In animal models of cSVD it was demonstrated that leaky blood vessels in the brain and activation of brain immune cells play an important role in cSVD pathophysiology. Studies in cSVD patients using advanced MRI techniques showed that increased leakage of brain small blood vessels is associated with over time brain tissue damage and cognitive decline. The results of this thesis suggest that there is a key role for local immune cell activation and blood vessel leakage in cSVD, which will give new opportunities for future therapeutic studies