Laser speckle imaging of the hippocampus.

Research on hippocampal blood flow is essential for gaining insight into its involvement in learning and memory and its role in age-related cognitive impairment and dementia. In this study, we applied laser speckle contrast imaging (LSCI) and dynamic light scattering imaging (DLSI) to monitor perfusion in mouse hippocampus via a chronic, optically transparent window. LSCI scans showed hippocampal blood vessels appear more out of focus than similar caliber vessels in the mouse cortex. We hypothesize that it is caused by the inverse vascular topology and increased contribution of multiply-scattered photons detected from the upper layers of the hippocampus. We support the hypothesis with DLSI, showing a 1300% increased contribution of multiple-scattering unordered dynamics regime in large hippocampal vessels.

Choosing a polarisation configuration for dynamic light scattering and laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) is applied in various biomedical applications for full-field characterization of blood flow and tissue perfusion. The accuracy of the contrast interpretation and its conversion to the blood flow index depends on specific parameters of the optical system and scattering media. One such parameter is the polarisation of detected light, which is often adjusted to minimize specular reflections and image artefacts. The polarisation's effect on the detected light scattering dynamics and, therefore, the accuracy of LSCI data interpretation requires more detailed investigation. In this study, we used LSCI and Dynamic Light Scattering Imaging to evaluate the effects of the detected light polarisation when imaging perfusion in the mouse cortex. We found that cross-polarisation results in a shorter decorrelation time constant, a higher coherence degree and stronger dynamic scattering compared to the parallel-polarisation or no-polariser configurations. These results support the cross-polarisation configuration as the most optimal for brain cortex imaging and suggest against direct or calibrated comparisons between the contrast recordings made with different polarisation configurations.

In vivo mapping of hemodynamic responses mediated by tubuloglomerular feedback in hypertensive kidneys.

The kidney has a sophisticated vascular structure that performs the unique function of filtering blood and managing blood pressure. Tubuloglomerular feedback is an intra-nephron negative feedback mechanism stabilizing single-nephron blood flow, glomerular filtration rate, and tubular flow rate, which is exhibited as self-sustained oscillations in single-nephron blood flow. We report the application of multi-scale laser speckle imaging to monitor global blood flow changes across the kidney surface (low zoom) and local changes in individual microvessels (high zoom) in normotensive and spontaneously hypertensive rats in vivo. We reveal significant differences in the parameters of TGF-mediated hemodynamics and patterns of synchronization. Furthermore, systemic infusion of a glucagon-like-peptide-1 receptor agonist, a potential renoprotective agent, induces vasodilation in both groups but only alters the magnitude of the TGF in Sprague Dawleys, although the underlying mechanisms remain unclear.

Optimizing the precision of laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) is a rapidly developing technology broadly applied for the full-field characterization of tissue perfusion. Over the recent years, significant advancements have been made in interpreting LSCI measurements and improving the technique's accuracy. On the other hand, the method's precision has yet to be studied in detail, despite being as important as accuracy for many biomedical applications. Here we combine simulation, theory and animal experiments to systematically evaluate and re-analyze the role of key factors defining LSCI precision-speckle-to-pixel size ratio, polarisation, exposure time and camera-related noise. We show that contrary to the established assumptions, smaller speckle size and shorter exposure time can improve the precision, while the camera choice is less critical and does not affect the signal-to-noise ratio significantly.

Lossless temporal contrast analysis of laser speckle images from periodic signals.

Laser speckle contrast imaging is a technique that provides valuable physiological information about vascular topology and blood flow dynamics. When using contrast analysis, it is possible to obtain detailed spatial information at the cost of sacrificing temporal resolution and vice versa. Such a trade-off becomes problematic when assessing blood dynamics in narrow vessels. This study presents a new contrast calculation method that preserves fine temporal dynamics and structural features when applied to periodic blood flow changes, such as cardiac pulsatility. We use simulations and in vivo experiments to compare our method with the standard spatial and temporal contrast calculations and demonstrate that the proposed method retains the spatial and temporal resolutions, resulting in the improved estimation of the blood flow dynamics.

The assessment of cortical hemodynamic responses induced by tubuloglomerular feedback using in vivo imaging.

The tubuloglomerular feedback (TGF) mechanism modulates renal hemodynamics and glomerular filtration rate in individual nephrons. Our study aimed to evaluate the TGF-induced vascular responses by inhibiting Na-K-2Cl co-transporters and sodium-glucose co-transporters in rats. We assessed cortical hemodynamics with high-resolution laser speckle contrast imaging, which enabled the evaluation of blood flow in individual microvessels and analysis of their dynamical patterns in the time-frequency domain. We demonstrated that a systemic administration of furosemide abolishes TGF-mediated hemodynamic responses. Furthermore, we showed that the local microcirculatory blood flow decreased, and the TGF-induced hemodynamic oscillations were sustained but weakened after inhibiting sodium-glucose co-transporters in Sprague-Dawley rats.

Multi-scale laser speckle contrast imaging of microcirculatory vasoreactivity.

Laser speckle contrast imaging is a robust and versatile blood flow imaging tool in basic and clinical research for its relatively simple construction and ease of customization. One of its key features is the scalability of the imaged field of view. With minimal changes to the system or analysis, laser speckle contrast imaging allows for high-resolution blood flow imaging through cranial windows or low-resolution perfusion visualization of perfusion over large areas, e.g. in human skin. We further utilize this feature and introduce a multi-scale laser speckle contrast imaging system, which we apply to study vasoreactivity in renal microcirculation. We combine high resolution (small field of view) to segment blood flow in individual vessels with low resolution (large field of view) to monitor global blood flow changes across the renal surface. Furthermore, we compare their performance when analyzing blood flow dynamics potentially associated with a single nephron and show that the previously published approaches, based on low-zoom imaging alone, provide inaccurate results in such applications.

Choosing a model for laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) is a real-time full-field non-invasive technique, which is broadly applied to visualize blood flow in biomedical applications. In its foundation is the link between the speckle contrast and dynamics of light scattering particles-erythrocytes. The mathematical form describing this relationship, which is critical for accurate blood flow estimation, depends on the sample's light-scattering properties. However, in biological applications, these properties are often unknown, thus requiring assumptions to be made to perform LSCI analysis. Here, we review the most critical assumptions in the LSCI theory and simulate how they affect blood flow estimation accuracy. We show that the most commonly applied model can severely underestimate the flow change, particularly when imaging brain parenchyma or other capillary perfused tissue (e.g. skin) under ischemic conditions. Based on these observations and guided by the recent experimental results, we propose an alternative model that allows measuring blood flow changes with higher accuracy.

Does Src Kinase Mediated Vasoconstriction Impair Penumbral Reperfusion?

Despite successful recanalization, a significant number of patients with ischemic stroke experience impaired local brain tissue reperfusion with adverse clinical outcome. The cause and mechanism of this multifactorial complication are yet to be understood. At the current moment, major attention is given to dysfunction in blood-brain barrier and capillary blood flow but contribution of exaggerated constriction of cerebral arterioles has also been suggested. In the brain, arterioles significantly contribute to vascular resistance and thus control of perfusion. Accordingly, pathological changes in arteriolar wall function can, therefore, limit sufficient reperfusion in ischemic stroke, but this has not yet received sufficient attention. Although an increased vascular tone after reperfusion has been demonstrated in several studies, the mechanism behind it remains to be characterized. Importantly, the majority of conventional mechanisms controlling vascular contraction failed to explain elevated cerebrovascular tone after reperfusion. We propose here that the Na,K-ATPase-dependent Src kinase activation are the key mechanisms responsible for elevation of cerebrovascular tone after reperfusion. The Na,K-ATPase, which is essential to control intracellular ion homeostasis, also executes numerous signaling functions. Under hypoxic conditions, the Na,K-ATPase is endocytosed from the membrane of vascular smooth muscle cells. This initiates the Src kinase signaling pathway that sensitizes the contractile machinery to intracellular Ca2+ resulting in hypercontractility of vascular smooth muscle cells and, thus, elevated cerebrovascular tone that can contribute to impaired reperfusion after stroke. This mechanism integrates with cerebral edema that was suggested to underlie impaired reperfusion and is further supported by several studies, which are discussed in this article. However, final demonstration of the molecular mechanism behind Src kinase-associated arteriolar hypercontractility in stroke remains to be done.

Dynamic light scattering imaging.

We introduce dynamic light scattering imaging (DLSI) to enable the wide-field measurement of the speckle temporal intensity autocorrelation function. DLSI uses the full temporal sampling of speckle fluctuations and a comprehensive model to identify the dynamic scattering regime and obtain a quantitative image of the scatterer dynamics. It reveals errors in the traditional theory of laser Doppler flowmetry and laser speckle contrast imaging and provides guidance on the best model to use in cerebral blood flow imaging.

Functional Ultrasound Speckle Decorrelation-Based Velocimetry of the Brain.

A high-speed, contrast-free, quantitative ultrasound velocimetry (vUS) for blood flow velocity imaging throughout the rodent brain is developed based on the normalized first-order temporal autocorrelation function of the ultrasound field signal. vUS is able to quantify blood flow velocity in both transverse and axial directions, and is validated with numerical simulation, phantom experiments, and in vivo measurements. The functional imaging ability of vUS is demonstrated by monitoring the blood flow velocity changes during whisker stimulation in awake mice. Compared to existing Power-Doppler- and Color-Doppler-based functional ultrasound imaging techniques, vUS shows quantitative accuracy in estimating both axial and transverse flow speeds and resistance to acoustic attenuation and high-frequency noise.

Chronic Imaging of Mouse Brain: From Optical Systems to Functional Ultrasound.

Utilization of functional ultrasound (fUS) in cerebral vascular imaging is gaining popularity among neuroscientists. In this article, we describe a chronic surgical preparation method that allows longitudinal studies and therefore is applicable to a wide range of studies, especially on aging, stroke, and neurodegenerative diseases. This method can also be used with awake mice; hence, the deleterious effects of anesthesia on neurovascular responses can be avoided. In addition to fUS imaging, this surgical preparation allows researchers to take advantage of common optical imaging methods to acquire complementary datasets to help increase the technical rigor of studies. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Surgical preparation of mouse chronic cranial windows using polymethylpentene Basic Protocol 2: Imaging of mice with chronic cranial windows.

Chronic Cranial Windows for Long Term Multimodal Neurovascular Imaging in Mice.

Chronic cranial windows allow for longitudinal brain imaging experiments in awake, behaving mice. Different imaging technologies have their unique advantages and combining multiple imaging modalities offers measurements of a wide spectrum of neuronal, glial, vascular, and metabolic parameters needed for comprehensive investigation of physiological and pathophysiological mechanisms. Here, we detail a suite of surgical techniques for installation of different cranial windows targeted for specific imaging technologies and their combination. Following these techniques and practices will yield higher experimental success and reproducibility of results.

Modification of oxygen consumption and blood flow in mouse somatosensory cortex by cell-type-specific neuronal activity.

Gamma activity arising from the interplay between pyramidal neurons and fast-spiking parvalbumin (PV) interneurons is an integral part of higher cognitive functions and is assumed to contribute significantly to brain metabolic responses. Cerebral metabolic rate of oxygen (CMRO2) responses were evoked by optogenetic stimulation of cortical PV interneurons and pyramidal neurons. We found that CMRO2 responses depended on neuronal activation, but not on the power of gamma activity induced by optogenetic stimulation. This implies that evoked gamma activity per se is not energy demanding. Optogenetic stimulation of PV interneurons during somatosensory stimulation reduced excitatory neuronal activity but did not potentiate O2 consumption as previously hypothesized. In conclusion, our data suggest that activity-driven CMRO2 responses depend on neuronal excitation rather than the cerebral rhythmic activity they induce. Excitation of both excitatory and inhibitory neurons requires energy, but inhibition of cortical excitatory neurons by interneurons does not potentiate activity-driven energy consumption.

Abnormal neurovascular coupling as a cause of excess cerebral vasodilation in familial migraine.

AIMS: Acute migraine attack in familial hemiplegic migraine type 2 (FHM2) patients is characterized by sequential hypo- and hyperperfusion. FHM2 is associated with mutations in the Na, K-ATPase α2 isoform. Heterozygous mice bearing one of these mutations (α2+/G301R mice) were shown to have elevated cerebrovascular tone and, thus, hypoperfusion that might lead to elevated concentrations of local metabolites. We hypothesize that these α2+/G301R mice also have increased cerebrovascular hyperaemic responses to these local metabolites leading to hyperperfusion in the affected part of the brain. METHODS AND RESULTS: Neurovascular coupling was compared in α2+/G301R and matching wild-type (WT) mice using Laser Speckle Contrast Imaging. In brain slices, parenchymal arteriole diameter and intracellular calcium changes in neuronal tissue, astrocytic endfeet, and smooth muscle cells in response to neuronal excitation were assessed. Wall tension and smooth muscle membrane potential were measured in isolated middle cerebral arteries. Quantitative polymerase chain reaction, western blot, and immunohistochemistry were used to assess the molecular background underlying the functional changes. Whisker stimulation induced larger increase in blood perfusion, i.e. hyperaemic response, of the somatosensory cortex of α2+/G301R than WT mice. Neuronal excitation was associated with larger parenchymal arteriole dilation in brain slices from α2+/G301R than WT mice. These hyperaemic responses in vivo and ex vivo were inhibited by BaCl2, suggesting involvement of inward-rectifying K+ channels (Kir). Relaxation to elevated bath K+ was larger in arteries from α2+/G301R compared to WT mice. This difference was endothelium-dependent. Endothelial Kir2.1 channel expression was higher in arteries from α2+/G301R mice. No sex difference in functional responses and Kir2.1 expression was found. CONCLUSION: This study suggests that an abnormally high cerebrovascular hyperaemic response in α2+/G301R mice is a result of increased endothelial Kir2.1 channel expression. This may be initiated by vasospasm-induced accumulation of local metabolites and underlie the hyperperfusion seen in FHM2 patients during migraine attack.

The nephron-arterial network and its interactions.

Tubuloglomerular feedback and the myogenic mechanism form an ensemble in renal afferent arterioles that regulate single-nephron blood flow and glomerular filtration. Each mechanism generates a self-sustained oscillation, the mechanisms interact, and the oscillations synchronize. The synchronization generates a bimodal electrical signal in the arteriolar wall that propagates retrograde to a vascular node, where it meets similar electrical signals from other nephrons. Each signal carries information about the time-dependent behavior of the regulatory ensemble. The converging signals support synchronization of the nephrons participating in the information exchange, and the synchronization can lead to formation of nephron clusters. We review the experimental evidence and the theoretical implications of these interactions and consider additional interactions that can limit the size of nephron clusters. The architecture of the arterial tree figures prominently in these interactions.

Choosing a laser for laser speckle contrast imaging.

The use of laser speckle contrast imaging (LSCI) has expanded rapidly for characterizing the motion of scattering particles. Speckle contrast is related to the dynamics of the scattering particles via a temporal autocorrelation function, but the quality of various elements of the imaging system can adversely affect the quality of the signal recorded by LSCI. While it is known that the laser coherence affects the speckle contrast, it is generally neglected in in vivo LSCI studies and was not thoroughly addressed in a practical matter. In this work, we address the question of how the spectral width of the light source affects the speckle contrast both experimentally and through numerical simulations. We show that commonly used semiconductor laser diodes have a larger than desired spectral width that results in a significantly reduced speckle contrast compared with ideal narrow band lasers. This results in a reduced signal-to-noise ratio for estimating changes in the motion of scattering particles. We suggest using a volume holographic grating stabilized laser diode or other diodes that have a spectrum of emitted light narrower than ≈1 nm to improve the speckle contrast.

Rat mesenteric small artery neurogenic dilatation is predominantly mediated by ß1 -adrenoceptors in vivo.

KEY POINTS: The prevailing dogma about neurogenic regulation of vascular tone consists of major vasodilatation caused by CGRP (and possibly substance P) released from sensory-motor nerves and vasoconstriction caused by noradrenaline, ATP and neuropeptode Y release from sympathetic nerves. Most studies on perivascular nerve-mediated vasodilatation are made in vitro. In the present study, we provide evidence indicating that in vivo electrical perivascular nerve stimulation in rat mesenteric small arteries causes a large ß1-adrenoceptor-mediated vasodilatation, which contrasts with a smaller vasodilatation caused by endogenous CGRP that is only visible after inhibition of Y1 NPY receptors. ABSTRACT: Mesenteric arteries are densely innervated and the nerves are important regulators of vascular tone and hence blood pressure and blood flow. Perivascular sensory-motor nerves have been shown to cause vasodilatation in vitro. However, less is known about their function in vivo. Male Wistar rats (10-12 weeks old; n = 72) were anaesthetized with ketamine (3 mg kg-1 ) and xylazine (0.75 mg kg-1 ) or pentobarbital (60 mg kg-1 ). After a laparotomy, a section of second-order mesenteric artery was visualized in an organ bath after minimal removal of perivascular adipose tissue. The effects of electrical field stimulation (EFS) and drugs on artery diameter and blood flow were recorded with intravital microscopy and laser speckle imaging. EFS caused vasodilatation in arteries constricted with 1 µm U46619 in the presence of 140 µm suramin and 1 µm prazosin. The vasodilatation was inhibited by 1 µm tetrodotoxin and 5 µm guanethidine, although not by the 1 µm of the CGRP receptor antagonist BIBN4096bs. In the presence of 0.3 µm Y1 receptor antagonist BIBP3226, BIBN4096bs partly inhibited the vasodilatation. Atenolol at a concentration 1 µm inhibited the vasodilatation, whereas 0.1 µm of the ß2 -adrenoceptor selective antagonist ICI-118,551 had no effect. Increasing the extracellular [K+ ] to 20 mm caused vasodilatation but was converted to vasoconstriction in the presence of 1 µm BIBN4096bs, and constriction to 30 mm potassium was potentiated by BIBN4096bs. Atenolol but not BIBN4096bs increased contraction to EFS in the absence of suramin and prazosin. In mesenteric small arteries of anaesthetized rats, EFS failed to stimulate major dilatation via sensory-motor nerves but induced sympathetic ß1 -adrenoceptor-mediated dilatation.

Dairy products viscosity estimated by laser speckle correlation.

Dairy products exhibit several physical properties that are crucial to define whether we like the food or not: firmness, creaminess, thickness, or lightness. Viscosity changes the flow properties of food and influences the appearance and the consistency of a product; this control variable is important in most production stages-manufacture, processing, and storage. Viscosity of heterogeneous products at a given temperature depends on its composition and physical state of its substances. Although rheology provides a method to access the product viscosity, it lacks non-contact full-field monitoring. We apply a simple correlation analysis of laser speckle images to evaluate viscosity properties of dairy products. Our approach ensures robust measurements with high degree of detectability.

Cardiac pulsatility mapping and vessel type identification using laser speckle contrast imaging.

Systemic flow variations caused by the cardiac cycle can play a role or be an important marker in both normal and pathological conditions. The shape, magnitude and propagation speed of the flow pulse reflect mechanical properties of the vasculature and are known to vary significantly with vascular diseases. Most conventional techniques are not capable of imaging cardiac activity in the microcirculation due to spatial and/or temporal resolution limitations and instead make inferences about propagation speed by making measurements at two points along an artery. Here, we apply laser speckle contrast imaging to images with high spatial resolution in the high frequency harmonics of cardiac activity in the cerebral cortex of a mouse. We reveal vessel dependent variation in the cardiac pulse activity and use this information to automatically identify arteries and veins.