Review of laser speckle contrast techniques for visualizing tissue perfusion

When a diffuse object is illuminated with coherent laser light, the backscattered light will form an interference pattern on the detector. This pattern of bright and dark areas is called a speckle pattern. When there is movement in the object, the speckle pattern will change over time. Laser speckle contrast techniques use this change in speckle pattern to visualize tissue perfusion. We present and review the contribution of laser speckle contrast techniques to the field of perfusion visualization and discuss the development of the techniques

Laser speckle analysis synchronised with cardiac cycle

We present an improved Laser speckle imaging approach to investigate the cerebral blood flow response following function stimulation of a single vibrissa. By synchronising speckle analysis with the cardiac cycle we are able to obtain robust averaging of the correlation signals while at the same time removing the contributions due to the pulsation of blood flow and associated tissue adaptation. With our inter-pulse correlation analysis we can follow second-scale dynamics of the cortical vascular system in response to functional brain activation. We find evidence for two temporally separated processes in the blood flow pattern following stimulation we tentatively attribute to vasodilation and vasoconstriction phases, respectivel

Functional Connectivity of the Rodent Brain Using Optical Imaging

RÉSUMÉ L'objectif de cette thèse de doctorat est d’appliquer la connectivité fonctionnelle dans une variété de modèles animaux, à l’aide de plusieurs techniques d’imagerie optique. Le cerveau, même au repos, montre une activité métabolique élevée : la corrélation des fluctuations spontanées lentes permet d’identifier des régions cérébrales distantes mais connectées; d’où le terme connectivité fonctionnelle. Les changements dans l’activité spontanée peuvent donner un aperçu des processus neuronaux qui comprennent la majorité de l’activité métabolique du cerveau, et constituent en conséquent une vaste source de changements reliés aux maladies. L’hémodynamique du cerveau peut être modifiée grâce à des affections neurovasculaires et avoir un effet sur l’activité au repos. Cette thèse vise la compréhension des changements de connectivité fonctionnelle induits par des maladies, à l’aide de l’imagerie optique fonctionnelle. Les techniques d’imagerie explorées dans les deux premières contributions de cette thèse sont l’Imagerie Optique Intrinsèque et l’Imagerie par Granularité Laser. Ensemble, elles peuvent estimer les changements de consommation d'oxygène, étroitement liés à l’activité neuronale. Ces techniques possèdent des résolutions temporelles et spatiales adéquates et bien adaptées pour imager la convexité du cortex cérébral. Dans le dernier article, une modalité d’imagerie en profondeur, la Tomographie Photoacoustique a été utilisée chez le rat nouveau-né. La Tomographie par Cohérence Optique et la Tomographie Laminaire Optique font également partie de la gamme des techniques d’imagerie développées et appliquées dans d’autres collaborations. La première partie des résultats mesure les changements de connectivité fonctionnelle dans un modèle d’activité épileptiforme aiguë chez le rongeur. Il y a des augmentations ainsi que des diminutions entre les corrélations homologues, avec une faible dépendance aux crises épileptiques. Ces changements suggèrent un découplage potentiel entre les paramètres hémodynamiques dans les réseaux au repos, en soulignant l’importance d’investiguer les réseaux épileptiques à l’aide de plusieurs mesures hémodynamiques indépendantes. La deuxième partie des travaux étudie un nouveau modèle de rigidité artérielle chez la souris : la calcification unilatérale de la carotide droite. L’analyse de connectivité basé sur les régions d’intérêt montre une tendance décroissante de corrélation homologue dans les cortex moteur et cingulum. L’analyse de graphes montre une randomisation des réseaux corticaux, ce qui suggère une perte de connectivité; plus spécifiquement, dans le cortex moteur ipsilateral à la carotide----------ABSTRACT The aim of this thesis is to apply functional connectivity in a variety of animal models, using several optical imaging modalities. Even at rest, the brain shows high metabolic activity: the correlation in slow spontaneous fluctuations identifies remotely connected areas of the brain; hence the term “functional connectivity”. Ongoing changes in spontaneous activity may provide insight into the neural processing that takes most of the brain metabolic activity, and so may provide a vast source of disease related changes. Brain hemodynamics may be modified during disease and affect resting-state activity. The thesis aims to better understand these changes in functional connectivity due to disease, using functional optical imaging. The optical imaging techniques explored in the first two contributions of this thesis are Optical Imaging of Intrinsic Signals and Laser Speckle Contrast Imaging, together they can estimate the metabolic rate of oxygen consumption, that closely parallels neural activity. They both have adequate spatial and temporal resolution and are well adapted to image the convexity of the mouse cortex. In the last article, a depth-sensitive modality called photoacoustic tomography was used in the newborn rat. Optical coherence tomography and laminar optical tomography were also part of the array of imaging techniques developed and applied in other collaborations. The first article of this work shows the changes in functional connectivity in an acute murine model of epileptiform activity. Homologous correlations are both increased and decreased with a small dependence on seizure duration. These changes suggest a potential decoupling between the hemodynamic parameters in resting-state networks, underlining the importance to investigate epileptic networks with several independent hemodynamic measures. The second study examines a novel murine model of arterial stiffness: the unilateral calcification of the right carotid. Seed-based connectivity analysis showed a decreasing trend of homologous correlation in the motor and cingulate cortices. Graph analyses showed a randomization of the cortex functional networks, suggesting a loss of connectivity, more specifically in the motor cortex ipsilateral to the treated carotid; however these changes are not reflected in differentiated metabolic estimates. Confounds remain due to the fact that carotid rigidification gives rise to neural decline in the hippocampus as well as unilateral alteration of vascular pulsatility; howeve

Optical imaging of acute epileptic networks in mice

The potential of intrinsic optical imaging and resting-state analysis under anesthetized conditions as a tool to study brain networks associated with epileptic seizures is investigated. Using an acute model of epileptiform activity, the 4-aminopyridine model in live mice, we observe the changes in resting-state networks with the onset of seizure activity and in conditions of spiking activity. Resting-state networks identified before and after the onset of epileptiform activity show both decreased and increased homologous correlations, with a small dependence on seizure intensity. The observed changes are not uniform across the different hemodynamic measures, suggesting a potential decoupling between blood flow and metabolism in the low-frequency networks. This study supports the need for a more extensive investigation of epileptic networks including more than one independent hemodynamic measurement

Multi-Contrast Photoacoustic Computed Tomography

Imaging of small animals has played an indispensable role in preclinical research by providing high dimensional physiological, pathological, and phenotypic insights with clinical relevance. Yet pure optical imaging suffers from either shallow penetration (up to ~1–2 mm) or a poor depth-to-resolution ratio (~3), and non-optical techniques for whole-body imaging of small animals lack either spatiotemporal resolution or functional contrast. A stand-alone single-impulse photoacoustic computed tomography (PACT) system has been built, which successfully mitigates these limitations by integrating high spatiotemporal resolution, deep penetration, and full-view fidelity, as well as anatomical, dynamical, and functional contrasts. Based on hemoglobin absorption contrast, the whole-body dynamics and large scale brain functions of rodents have been imaged in real time. The absorption contrast between cytochrome and lipid has enabled PACT to resolve MRI-like whole brain structures. Taking advantage of the distinct absorption signature of melanin, unlabeled circulating melanoma cells have been tracked in real time in vivo. Assisted by near-infrared dyes, the perfusion processes have been visualized in rodents. By localizing single-dyed droplets, the spatial resolution of PACT has been improved by six-fold in vivo. The migration of metallic-based microrobots toward the targeted regions in the intestines has been monitored in real time. Genetically encoded photochromic proteins benefit PACT in detection sensitivity and specificity. The unique photoswitching characteristics of different photochromic proteins allow quantitative multi-contrast imaging at depths. A split version of the photochromic protein has permitted PA detection of protein-protein interactions in deep-seated tumors. The photochromic behaviors have also been utilized to guide photons to form an optical focus inside live tissue. As a rapidly evolving imaging technique, PACT promises pre-clinical applications and clinical translation.</p

Role of Tissue Plasminogen Activator in Central Nervous System Physiology and Pathology

Tissue plasminogen activator (tPA) is a serine protease classically known for its endogenous activity promoting fibrinolysis and for its clinical role as a thrombolytic agent for treating ischemic stroke. This singular function for tPA in the vasculature contrasts with the numerous reported actions of tPA in the central nervous system (CNS); including, synaptic plasticity, neurodegeneration, and blood-brain barrier (BBB) permeability. Within each of these processes a variety of substrates and receptors have been implicated in mediating tPA’s effects, suggesting that tPA is a pleiotropic mediator whose actions are restricted in space and time. The specific localization of tPA, therefore, can provide useful information about its function. Accordingly, we utilized two new transgenic reporter mice – PlatBetaGAL and tPABAC-Cer – to provide a detailed characterization of tPA expression in the adult murine brain. The PlatBetaGAL reporter mouse houses the beta-galactosidase gene in the tPA locus and the tPABAC-Cer mouse has a cerulean-fluorescent protein fused in-frame to the tPA C-terminus. A comparison of these reporter mice demonstrates that neuronal tPA is primarily trafficked away from its somatic site of synthesis to nerve fibers in limbic brain structures, such as the hippocampus, amygdala, and basal ganglia. This differential expression pattern is most apparent in the hippocampus where tPA-BetaGAL expression is present in the dentate gyrus, while tPA-Cer is localized to giant mossy fiber boutons (MFBs) in the mossy fiber pathway. To understand the functional implications of tPA in the MFBs we assessed synchronous activity in the CA3 hippocampal subfield using a “no magnesium/high potassium” model of “seizure-like” activity. As previous work from our lab implicated tPA in mediating seizure progression in vivo via its role regulating BBB permeability, we dissected the BBB component to seizure progression and specifically tested tPA’s effect on neuronal communication. We found brain slices from tPA deficient mice to have an enhanced synchronous activity onset time, suggesting that the “seizure-resistance” observed in tPA deficient mice in vivo is likely a result of improved barrier function, not tPA’s role in modulating synaptic transmission. Lastly, in this dissertation, using sophisticated imaging and analytical tools we provide a rigorous assessment of vascular morphometry in wild-type mice, the original Carmeliet-tPA null mice, and in newly-generated tPA deficient mice on a pure C57BL/6J background (Szabo-tPA null mice). Through this examination we report that the lognormal distribution is a good model for cerebral vessel diameter and length and that there is a weak negative correlation between vessel diameter and length. We also find that the increased vascular density in Carmeliet-tPA null mice is possibly a compound result of constitutive loss of tPA and/or some strain-dependent modifier genes. Cumulatively, our data supports a model whereby tPA acts a pleiotropic mediator in the CNS whose actions are highly spatially and temporally compartmentalized. This compartmentalized localization is appreciable in the differential expression pattern seen for tPA between the PlatBetaGAL and tPABAC-Cer transgenic mice; and functionally, we show that in ex vivo hippocampal slices tPA modulates synchronous activity, but in an in vivo model of seizure, the dominant effect of tPA is on regulating BBB permeability. Our vascular morphometry data also suggests a possible developmental effect of tPA on cerebrovascular patterning. Future work using the models developed here should help to clarify the relative contribution of the various substrates and pathways associated with tPA in CNS physiology and pathology.PHDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/146064/1/tamaraks_1.pd

Advances in Hyperspectral and Multispectral Optical Spectroscopy and Imaging of Tissue

The purpose of this SI is to provide an overview of recent advances made in the methods used for tissue imaging and characterization, which benefit from using a large range of optical wavelengths. Guerouah et al. has contributed a profound study of the responses of the adult human brain to breath-holding challenges based on hyperspectral near-infrared spectroscopy (hNIRS). Lange et al. contributed a timely and comprehensive review of the features and biomedical and clinical applications of supercontinuum laser sources. Blaney et al. reported the development of a calibration-free hNIRS system that can measure the absolute and broadband absorption and scattering spectra of turbid media. Slooter et al. studied the utility of measuring multiple tissue parameters simultaneously using four optical techniques operating at different wavelengths of light—optical coherence tomography (1300 nm), sidestream darkfield microscopy (530 nm), laser speckle contrast imaging (785 nm), and fluorescence angiography (~800 nm)—in the gastric conduit during esophagectomy. Caredda et al. showed the feasibility of accurately quantifying the oxy- and deoxy-hemoglobin and cytochrome-c-oxidase responses to neuronal activation and obtaining spatial maps of these responses using a setup consisting of a white light source and a hyperspectral or standard RGB camera. It is interest for the developers and potential users of clinical brain and tissue optical monitors, and for researchers studying brain physiology and functional brain activity

Structural and functional brain imaging using extended-focus optical coherence tomography and microscopy

Neuroimaging techniques aim at revealing the anatomy and functional organisation of cerebral structures. Over the past decades, functional magnetic resonance imaging (fMRI) has revolutionized our understanding of human cerebral physiology through its ability to probe neural activity throughout the entire brain in a non-invasive fashion. Nevertheless, despite recent technological improvements, the spatial resolution of fMRI remains limited to a few hundreds of microns, restricting its use to macroscopic studies. Microscopic imaging solutions have been proposed to circumvent this limitation and have enabled revealing the existence of various cerebral structures, such as neuronal and vascular networks and their contribution to information processing and blood flow regulation within the brain. Optical imaging has proven, through its increased resolution and available contrast mechanisms, to be a valuable complement to fMRI for cellular-scale imaging. In this context, we demonstrate here the capabilities of an extension of optical coherence tomography, termed extended-focus optical coherence tomography (xf-OCT), in imaging cerebral structure and function at high resolution and very high acquisitions rates. Optical coherence tomography is an interferometric imaging technique using a low-coherence illumination source to provide fast, three-dimensional imaging of the back-scattering of tissue and cells. By multiplexing the interferometric ranging over several spectral channels, Fourier-domain OCT performs depth-resolved imaging at very high acquisition rates and high sensitivity. Increasing the lateral resolution of optical systems typically reduces the available depth-of-field and thus hampers this depth multiplexing advantage of OCT. Extended-focus systems aim at alleviating this trade-off between imaging depth and lateral resolution through the use of diffraction-less beams such as Bessel beams, providing high resolution imaging over large depths. The xf-OCT system therefore combines fast acquisition rates and high resolution, both characteristics required to image and study the structure and function of microscopic constituents of cerebral tissue. In this work, we performed functional brain imaging using the ability of xf-OCT to obtain quantita- tive measurements of blood flow in the brain. Changes in blood velocity evoked by neuronal activation were monitored and maps of hemodynamic activity were generated by adapting tools routinely used in fMRI to xf-OCT imaging. Additionally, three novel xf-OCT instruments are presented, wherein the advantages of different spectral ranges are exploited to reach specific imaging parameters. The increased contrast and resolution afforded by an illumination in the visible spectral range was used in two extended-focus optical coherence microscopy (xf-OCM) implementations for subcellular imaging of ex-vivo brain slices and cellular imaging of neurons, capillaries and myelinated axons in the superficial cortex in-vivo. Subsequently, an xf-OCT system is presented, operating in the infrared spectral range, wherein the reduced scattering enabled imaging the smallest capillaries deep in the murine cortex in-vivo

Probing mechanisms by which cerebral vascular disease may influence cognitive impairment and dementia

Introduction Vascular cognitive impairment (VCI) describes a full spectrum of cognitive deficits caused by underlying cerebral vascular alterations, regardless of the specific mechanisms involved. Several factors such as ageing, stroke, hypertension and cerebral hypoperfusion are associated with an increased risk of developing VCI. Vascular dementia (VaD) is the second most common cause of dementia after Alzheimer’s disease (AD). It is now recognised that considerable overlaps exist between the features of VaD and AD. Key pathological and neuroimaging features including cerebral amyloid angiopathy (CAA), white matter lesions (WML), microinfarcts and microbleeds are evident in both VaD and AD. Furthermore, brain infarction has been reported to influence the presence and severity of clinical expressions such as cognitive performance of AD, suggesting a common pathophysiological mechanism that contributes to the development of cognitive deficits. However, gaps remain in understanding the exact mechanisms by which vascular risk factors contribute to cognitive decline and neurodegenerative processes leading to dementia. Given the importance of blood supply to the brain for maintaining its structural and functional integrity, it has been proposed that vascular risk factors may affect the cerebral haemodynamic and alter the vascular function resulting in damages to the brain. These changes may involve altered neurovascular coupling that is a critical mechanism for regulating the dynamic changes of local cerebral circulation. Further, impaired vascular function, amyloid-β (Aβ) accumulation in the cerebral vasculature and disrupted neurovascular unit are found in VCI. The glymphatic pathway, a clearance route for removing soluble waste from the brain to periphery, has been proposed to play a role in the pathogenesis of VCI. Mounting evidence has suggested that cerebral hypoperfusion, by large vessel occlusion and stenosis, is emerging as a major contributor to cognitive impairment. This has led to the development of mouse models of bilateral common carotid stenosis (BCAS), a model narrowing both common carotid arteries by placing microcoils. The BCAS model has been reported to produce many features of VCI, including white matter damage, microglial activation, gliovascular disruption, increased oxidative stress (by increased NADPH oxidase 2 (NOX2) levels) as well as memory impairment. To investigate whether there is an interaction between cerebral hypoperfusion and microvascular Aβ accumulation, a mixed model that demonstrates both microvascular amyloid (Tg-SwDI mouse model) and BCAS has been developed. In this thesis, it is hypothesized that the complex interaction of Aβ and BCAS leads to cognitive impairment via an impaired glymphatic function in addition to perfusion deficits that promote vascular related lesions and neurodegenerative changes. Second to this, given clear links between NOX2, hypoperfusion and amyloid accumulation, it was further hypothesised that NOX2 is a central mechanism leading to VCI. Methods Mice were given BCAS surgery to mimic cerebral hypoperfusion for a period of 3 months. In vivo laser speckle imaging was performed to evaluate the changes in cortical blood flow. This was followed by additional CBF measurements using arterial spin labelling (ASL)-based magnetic resonance imaging (MRI), which gave a non-invasive access to CBF information in the cerebral cortex, hippocampus and thalamus. Neurovascular coupling was assessed by performing whisker stimulation. Barnes maze was used to assess the spatial learning and memory function at 3 months following BCAS or sham surgery. For the examination of glymphatic function, in vivo intracisternal injection and ex vivo imaging of CSF fluorescent tracers were performed. Histological assessment and immunohistochemistry were used to examine vascular related pathology, Aβ burden, astrogliosis and basement membrane changes following BCAS. Results Part 1: To examine the effect of BCAS on cerebral perfusion deficits, glymphatic function and cognition in Tg-SwDI mice compared to wild-type mice. The first studies in the thesis sought to examine the effect of BCAS and microvascular amyloid on the extent of cerebral perfusion deficits and cognitive impairment. The first step was to validate whether the BCAS model has an effect on cerebral perfusion. Cortical cerebral blood flow (CBF) was examined by laser speckle imaging. This revealed sustained reductions of CBF at 24 hours, 1 and 3 months following the establishment of BCAS (p<0.001) but no effect of the microvascular Aβ was found to affect cortical perfusion (p>0.05). To further explore the CBF changes in other brain regions following BCAS, Arterial spin labelling (ASL), a technique widely used in clinical imaging, was performed. A significant effect of BCAS was confirmed in the dorsolateral cortex and hippocampus (p<0.001, respectively) but no genotype effect of the microvascular Aβ or any interaction was found (p>0.05, respectively). In order to investigate whether long-term carotid stenosis has a further effect on cognitive function in the experimental animals, assessment of Barnes maze demonstrated that BCAS mice spent longer escape latency than the sham mice in both wild-type and Tg-SwDI animals (p<0.05, respectively) indicating visuo-spatial learning was significantly impaired at 3 months following BCAS. To determine the effect of BCAS and Aβ on long-term memory, a probe test was taken to examine whether mice remember the previous training target after a period of time. This test revealed that all groups spent a significantly higher percentage of time than chance (25%). Exclusively in wild-type BCAS mice, the percentage of time spent in the target quadrant was significantly lower than by chance (p<0.05). In addition, there was no significant effect of BCAS or Aβ on the percentage of time spent in the correct quadrant (p>0.05, respectively). These results suggested long-term memory was not impaired in BCAS and the presence of amyloid. Further to enhance the detection of spatial learning and memory impairment, reversal trials were taken to evaluate the ability of experimental animals to learn a new location. Compared to wild-type mice that still learned the new tests showing significantly improved performance over time (p<0.05), both the Tg-SwDI sham and BCAS mice no longer learned the task (p>0.05). The long-term memory tested in reversal tests showed impairment in both wildtype and Tg-SwDI BCAS as well as in the presence of amyloid after increasing the difficulties in reversal probe tests. The results indicated the only mice from wild-type sham (37.40 ± 12.63) (p<0.05) spent a significantly higher percentage of time by 12.40 (95%CI, 1.84 to 22.96) than by chance, t(7)=2.8, p=0.027 and a significantly higher percentage of time than Tg-SwDI BCAS mice (p<0.05) with all the other groups spending a lower percentage of time than chance (wild-type BCAS: 27.57 ± 11.12%, Tg-SwDI sham: 20.36 ± 15.50%, Tg-SwDI BCAS: 26.79 ± 16.79%). To further explore the potential mechanisms by which BCAS causes cognitive impairment, the glymphatic entry was further assessed. This revealed that the global influx of CSF tracers was different across the anatomical levels (p<0.001) but unaltered post-BCAS in wild-type and Tg-SwDI mice (p>0.05, respectively). To explore whether BCAS influences CSF glymphatic influx, ex vivo images of the CSF tracer influx in the dorsolateral cortex (DL CTX) and hippocampus (CA1-DG molecular layer) on the D-3 tracer were measured. The results showed in both regions, altered CSF influx was found in the BCAS and Tg-SwDI mice due to the main effect of BCAS (p=0.037 and p=0.011, DL CTX and CA-DG regions respectively) but not Aβ (p>0.05, respectively). Taken together, these first studies support the original hypothesis that BCAS causes cognitive impairment via reduced cerebral perfusion and impaired glymphatic function. However, there was no exacerbation of these effects in Tg-SwDI mice. Part 2: To examine the effect of BCAS on neurovascular function, degenerative changes and amyloid accumulation in Tg-SwDI mice compared to wild-type mice. To begin with, responses of cortical blood vessels to whisker stimulation were recorded and quantified as the mean CBF percentage increase from the baseline. There was a significant effect of BCAS (p<0.001), whereby impaired neurovascular coupling was observed in the BCAS mice from both wild-type and Tg-SwDI mice. However, there was no significant effect of Aβ in these mice (p>0.05). Vascular related lesions including microinfarcts and microbleeds were compared by measuring the frequency in experimental animals. No vascular lesions were detectable in wild-type and Tg-SwDI sham mice. 4/10 mice were found to have vascular lesions in the wild-type BCAS mice following 3 months of surgery. 6/10 mice were identified with vascular lesions in the Tg-SwDI mice. No significant difference in proportions (p>0.05) was found between Tg-SwDI BCAS and wild-type BCAS mice. To discern the mechanisms by which BCAS and microvascular amyloid may impact on the glymphatic function, the extent of astrogliosis was further studied. GFAP immunostaining was undertaken to investigate the extent of reactive gliosis post-BCAS. Increased astrogliosis following BCAS was found (p<0.05), but no effect of Aβ or interaction was found in the dorsolateral cortex. The hippocampal CA1-DG molecular layer was further analysed, and this showed a significant effect of Aβ (p=0.002) but no effect of BCAS (p>0.05) and interactions (p>0.05) on astrogliosis. Further, Aβ load was evaluated in the cortex and colabelled with collagen 4 (COL4) (a marker of the basement membrane of blood vessels) to enable the assessment of microvascular amyloid in the Tg-SwDI mouse model. A significant increase in the total amount of amyloid as well as the percentage of vascular amyloid was detected post-stenosis (p<0.05, respectively). No changes of COL4 levels were found in the mice post-BCAS (p>0.05). In summary, these results demonstrated that BCAS impaired neurovascular coupling and promoted amyloid accumulation in the cerebral microvasculature. Part 3: To determine whether targeting NOX2 has an effect on cerebral perfusion, degenerative changes and cognitive impairment in Tg-SwDI mice compared to wildtype mice. The third aim of the thesis was to determine the effect of NOX inhibitor (apocynin) on the previously reported cerebral hypoperfusion, impaired neurovascular coupling, development of neurodegenerative pathologies and cognitive deficits caused by BCAS in the Tg-SwDI mice. Following BCAS surgery, mice were immediately fed with either apocynin or vehicle in their drinking water for 3 months. Cortical CBF changes after the treatment of apocynin were assessed using laser speckle imaging, in apocynin treated mice, a recovery of CBF from the BCAS apocynin group after 3 months of treatment was found. The further investigation of neurovascular coupling revealed that apocynin restored vascular function following carotid stenosis. A significant interaction between BCAS surgery and apocynin treatment (p<0.05) was found after 3 months of treatment. The mice that received 3 months of apocynin treatment showed a robust response during the stimulation. The frequency of vascular lesions was counted to compare whether inhibiting NOX activity could provide any beneficial effect on the development of vascular pathology. However, there was no significant difference in proportions between the mice treated with vehicle and apocynin (p>0.05). The cortical amyloid load was assessed by double labelling of COL4 and 6E10. The results revealed no effect of treatment on the Aβ burden and vessel densities compared to vehicle treated group (p>0.05). Finally, the cognitive function was assessed using Barnes maze. It demonstrated that apocynin did not improve spatial learning and memory in the behavioural tests (p>0.05, respectively). Conclusions The findings in this thesis demonstrate novel evidence of how carotid stenosis damages the cerebral microcirculation and structure, contributing to the pathogenesis of cognitive impairment. Specifically, long-term BCAS caused chronic cerebral hypoperfusion and impaired glymphatic function, which is likely to contribute to the accumulation of Aβ in the microvasculature. Additionally, carotid stenosis caused sustained cerebral hypoperfusion and led to impaired neurovascular coupling, neurodegenerative changes and cognitive deficits. However, despite evidence supporting a basis for targeting NADPH oxidase, there was only a modest beneficial effect of the NOX inhibitor on neurovascular function. Collectively, this thesis provides evidence that following the carotid stenosis, while reducing cerebral perfusion, the glymphatic drainage pathway may be affected, leading to cognitive impairment. This new data adds credence to a growing body of human studies that alternate mechanisms may exist in addition to cerebral hypoperfusion leading to VCI. The treatment with non-selective NOX inhibitor successfully restored blood perfusion and vascular function with no ultimate improvement in cognitive function, suggesting a limited role by targeting NOX to restore the full pathological processes in VCI. Thus, further studies using more specific method targeting post-carotid stenosis events will help to understand the proposed mechanisms and provide a therapeutic strategy

Novel contrasts in photoacoustic tomography

Photoacoustic tomography (PAT) combines rich optical contrast and high ultrasonic resolution in optically scattering tissue at depths. Taking advantage of its 100% sensitivity to optical absorption, PAT has been widely applied to structural, functional and molecular imaging, with both endogenous and exogenous contrasts, at superior depths than pure optical methods. This dissertation explores novel absorption contrast mechanisms of PAT based on optical/thermal patterns, endogenous cellular chromophores, nanoparticles, small-molecule dyes and genetically-encoded proteins. With these novel contrasts, the proof-of-concept applications of PAT have been extended to include homogenous flow measurements, targeted angiogenesis imaging and therapy, label-free white blood cell imaging, 3D-whole-organ cell nuclei imaging with a subcellular resolution, and in vivo neural activity imaging with voltage/calcium-sensitive indicators. Specifically, Chapter 1 introduces photoacoustic microscopy (PAM) and photoacoustic computed tomography (PACT) systems and discuss the motivation of the dissertation. Chapter 2 describes two photoacoustic (PA) flow measurement methods with optical and thermal patterns, which are applicable to homogenous flowing medium. In the first method, a Doppler frequency shift in PA signals of the flow was detected and used to calculate flow speeds. In the second method, unique features in an externally imposed thermal pattern of the flow, captured by repeated B-scans along the flow direction with a PAM system, revealed different flow speeds. Chapter 3 explores the unique PA contrast of macrophages, an important type of white blood cells. Macrophages were imaged by PAM without any label, and their measured PA spectrum was distinctive from the hemoglobin spectrum, so they can be potentially differentiated from red blood cells in the blood stream. Next, with a microtomy-assisted PAM system, cell nuclei distribution in whole organs, including mouse brain and mouse lung, were imaged with subcellular resolution. Chapter 4 introduces a type of target copper nanoparticles, which are less expensive and more biocompatible than its counterpart gold nanoparticles. The PA signals of neovasculature in the mouse flank were enhanced by the ___3-targeted copper nanoparticles. Moreover, the work shows the first example of a systemically targeted antiangiogenic drug delivery with a photoacoustic contrast nanoparticle in vivo. Chapter 5 demonstrates the voltage imaging capability of PA. A voltage sensitive dye with sufficient signal change was discovered and used as a PA voltage indicator for the first time. The mechanism was characterized through both PA imaging and spectroscopic methods. Its use was explored in a mouse epilepsy model and cortical electrical stimulation model in vivo. Finally, the deep imaging potential of PA was realized by imaging the voltage response of cells under 4.5 mm thick slice of rat brain tissue using a PACT system. Chapter 6 proves the neural calcium imaging capability of PA with a genetically encoded calcium indicator. In a fly model, I ambiguously demonstrated for the first time that PA can be used to imaging neural activities in the fly brain without the interference signals from hemoglobin. In the a live-mouse-brain-slice model, I successfully demonstrated the deep imaging capability of PA for calcium imaging by imaging through a 2-mm-thick scattering medium with a PACT system