Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes.

The role of high mobility group box 1 (HMGB1) in inflammation is well characterized in the immune system and in response to tissue injury. More recently, HMGB1 was also shown to initiate an "inflammatory signaling cascade" in the brain parenchyma after a mild and brief disturbance, such as cortical spreading depolarization (CSD), leading to headache. Despite substantial evidence implying a role for inflammatory signaling in prevalent neuropsychiatric disorders such as migraine and depression, how HMGB1 is released from healthy neurons and how inflammatory signaling is initiated in the absence of apparent cell injury are not well characterized. We triggered a single cortical spreading depolarization by optogenetic stimulation or pinprick in naïve Swiss albino or transgenic Thy1-ChR2-YFP and hGFAP-GFP adult mice. We evaluated HMGB1 release in brain tissue sections prepared from these mice by immunofluorescent labeling and immunoelectron microscopy. EzColocalization and Costes thresholding algorithms were used to assess the colocalization of small extracellular vesicles (sEVs) carrying HMGB1 with astrocyte or microglia processes. sEVs were also isolated from the brain after CSD, and neuron-derived sEVs were captured by CD171 (L1CAM). sEVs were characterized with flow cytometry, scanning electron microscopy, nanoparticle tracking analysis, and Western blotting. We found that HMGB1 is released mainly within sEVs from the soma of stressed neurons, which are taken up by surrounding astrocyte processes. This creates conditions for selective communication between neurons and astrocytes bypassing microglia, as evidenced by activation of the proinflammatory transcription factor NF-ĸB p65 in astrocytes but not in microglia. Transmission immunoelectron microscopy data illustrated that HMGB1 was incorporated into sEVs through endosomal mechanisms. In conclusion, proinflammatory mediators released within sEVs can induce cell-specific inflammatory signaling in the brain without activating transmembrane receptors on other cells and causing overt inflammation.

Neurovascular coupling is preserved in chronic stroke recovery after targeted photothrombosis.

Functional neuroimaging, which measures hemodynamic responses to brain activity, has great potential for monitoring recovery in stroke patients and guiding rehabilitation during recovery. However, hemodynamic responses after stroke are almost always altered relative to responses in healthy subjects and it is still unclear if these alterations reflect the underlying brain physiology or if the alterations are purely due to vascular injury. In other words, we do not know the effect of stroke on neurovascular coupling and are therefore limited in our ability to use functional neuroimaging to accurately interpret stroke pathophysiology. To address this challenge, we simultaneously captured neural activity, through fluorescence calcium imaging, and hemodynamics, through intrinsic optical signal imaging, during longitudinal stroke recovery. Our data suggest that neurovascular coupling was preserved in the chronic phase of recovery (2 weeks and 4 weeks post-stoke) and resembled pre-stroke neurovascular coupling. This indicates that functional neuroimaging faithfully represents the underlying neural activity in chronic stroke. Further, neurovascular coupling in the sub-acute phase of stroke recovery was predictive of long-term behavioral outcomes. Stroke also resulted in increases in global brain oscillations, which showed distinct patterns between neural activity and hemodynamics. Increased neural excitability in the contralesional hemisphere was associated with increased contralesional intrahemispheric connectivity. Additionally, sub-acute increases in hemodynamic oscillations were associated with improved sensorimotor outcomes. Collectively, these results support the use of hemodynamic measures of brain activity post-stroke for predicting functional and behavioral outcomes.

Measurements of cerebral microvascular blood flow, oxygenation, and morphology in a mouse model of whole-brain irradiation-induced cognitive impairment by two-photon microscopy and optical coherence tomography: evidence for microvascular injury in the cerebral white matter.

Whole-brain irradiation (WBI, also known as whole-brain radiation therapy) is a mainstay treatment modality for patients with multiple brain metastases. It is also used as a prophylactic treatment for microscopic tumors that cannot be detected by magnetic resonance imaging. WBI induces a progressive cognitive decline in ~ 50% of the patients surviving over 6 months, significantly compromising the quality of life. There is increasing preclinical evidence that radiation-induced injury to the cerebral microvasculature and accelerated neurovascular senescence plays a central role in this side effect of WBI. To better understand this side effect, male C57BL/6 mice were first subjected to a clinically relevant protocol of fractionated WBI (5 Gy, two doses per week, for 4 weeks). Nine months post the WBI treatment, we applied two-photon microscopy and Doppler optical coherence tomography to measure capillary red-blood-cell (RBC) flux, capillary morphology, and microvascular oxygen partial pressure (PO2) in the cerebral somatosensory cortex in the awake, head-restrained, WPI-treated mice and their age-matched controls, through a cover-glass-sealed chronic cranial window. Thanks to the extended penetration depth with the fluorophore - Alexa680, measurements of capillary blood flow properties (e.g., RBC flux, speed, and linear density) in the cerebral subcortical white matter were enabled. We found that the WBI-treated mice exhibited a significantly decreased capillary RBC flux in the white matter. WBI also caused a significant reduction in capillary diameter, as well as a large (although insignificant) reduction in segment density at the deeper cortical layers (e.g., 600-700 µm), while the other morphological properties (e.g., segment length and tortuosity) were not obviously affected. In addition, we found that PO2 measured in the arterioles and venules, as well as the calculated oxygen saturation and oxygen extraction fraction, were not obviously affected by WBI. Lastly, WBI was associated with a significant increase in the erythrocyte-associated transients of PO2, while the changes of other cerebral capillary PO2 properties (e.g., capillary mean-PO2, RBC-PO2, and InterRBC-PO2) were not significant. Collectively, our findings support the notion that WBI results in persistent cerebral white matter microvascular impairment, which likely contributes to the WBI-induced brain injury and cognitive decline. Further studies are warranted to assess the WBI-induced changes in brain tissue oxygenation and malfunction of the white matter microvasculature as well.

Overview of extracellular vesicle characterization techniques and introduction to combined reflectance and fluorescence confocal microscopy to distinguish extracellular vesicle subpopulations.

Extracellular vesicles (EVs) are nanoparticles (30 to 1000 nm in diameter) surrounded by a lipid-bilayer which carry bioactive molecules between local and distal cells and participate in intercellular communication. Because of their small size and heterogenous nature they are challenging to characterize. Here, we discuss commonly used techniques that have been employed to yield information about EV size, concentration, mechanical properties, and protein content. These include dynamic light scattering, nanoparticle tracking analysis, flow cytometry, transmission electron microscopy, atomic force microscopy, western blotting, and optical methods including super-resolution microscopy. We also introduce an innovative technique for EV characterization which involves immobilizing EVs on a microscope slide before staining them with antibodies targeting EV proteins, then using the reflectance mode on a confocal microscope to locate the EV plane. By then switching to the microscope's fluorescence mode, immunostained EVs bearing specific proteins can be identified and the heterogeneity of an EV preparation can be determined. This approach does not require specialist equipment beyond the confocal microscopes that are available in many cell biology laboratories, and because of this, it could become a complementary approach alongside the aforementioned techniques to identify molecular heterogeneity in an EV preparation before subsequent analysis requiring specialist apparatus.

Confocal reflectance microscopy for metal and lipid nanoparticle visualization in the brain.

Aim: A requirement for nanoparticle (NP) research is visualization of particles within cells and tissues. Limitations of electron microscopy and low yields of NP fluorescent tagging warrant the identification of alternative imaging techniques. Method: Confocal reflectance microscopy (CRM) in combination with fluorescence imaging was assessed for visualizing rhodamine B-conjugated silver and fluorescein isothiocyanate-conjugated lipid core-stearylamine NP uptake in vitro and in vivo. Results: CRM successfully identified cellular uptake and blood-brain barrier penetration of NPs owing to their distinguishing refractive indices. NP-dependent reflectance signals in vitro were dose and incubation time dependent. Finally, CRM facilitated the distinction between nonspecific fluorescence signals and NPs. Conclusion: These findings demonstrate the value of CRM for NP visualization in tissues, which can be performed with a standard confocal microscope.

Nanoparticles (NPs) are extremely small materials utilized in the healthcare sector mainly for the delivery of drugs into tissues that are not easily accessible with regular pharmaceuticals. One such tissue is the brain, which has a barrier between it and the bloodstream that prevents the passage of most drugs. For NP research, the successful entry of NPs into target tissues must be demonstrated, but this is complicated by the small size and weak labeling of NPs. In this article, the authors demonstrate a low-cost, complementary microscopy technique that is readily available in most biological research laboratories and that can be used to detect and analyze the entry of different NP types into brain tissue and their uptake by brain tumor cells. These data create new opportunities for research on NP-assisted drug delivery to the central nervous system.

Parenchymal neuroinflammatory signaling and dural neurogenic inflammation in migraine.

BACKGROUND: Pain is generally concomitant with an inflammatory reaction at the site where the nociceptive fibers are activated. Rodent studies suggest that a sterile meningeal inflammatory signaling cascade may play a role in migraine headache as well. Experimental studies also suggest that a parenchymal inflammatory signaling cascade may report the non-homeostatic conditions in brain to the meninges to induce headache. However, how these signaling mechanisms function in patients is unclear and debated. Our aim is to discuss the role of inflammatory signaling in migraine pathophysiology in light of recent developments. BODY: Rodent studies suggest that a sterile meningeal inflammatory reaction can be initiated by release of peptides from active trigeminocervical C-fibers and stimulation of resident macrophages and dendritic/mast cells. This inflammatory reaction might be needed for sustained stimulation and sensitization of meningeal nociceptors after initial activation along with ganglionic and central mechanisms. Most migraines likely have cerebral origin as suggested by prodromal neurologic symptoms. Based on rodent studies, a parenchymal inflammatory signaling cascade has been proposed as a potential mechanism linking cortical spreading depolarization (CSD) to meningeal nociception. A recent PET/MRI study using a sensitive inflammation marker showed the presence of meningeal inflammatory activity in migraine with aura patients over the occipital cortex generating the visual aura. These studies also suggest the presence of a parenchymal inflammatory activity, supporting the experimental findings. In rodents, parenchymal inflammatory signaling has also been shown to be activated by migraine triggers such as sleep deprivation without requiring a CSD because of the resultant transcriptional changes, predisposing to inadequate synaptic energy supply during intense excitatory transmission. Thus, it may be hypothesized that neuronal stress created by either CSD or synaptic activity-energy mismatch could both initiate a parenchymal inflammatory signaling cascade, propagating to the meninges, where it is converted to a lasting headache with or without aura. CONCLUSION: Experimental studies in animals and emerging imaging findings from patients warrant further research to gain deeper insight to the complex role of inflammatory signaling in headache generation in migraine.

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.

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.

Seeking predictors for paroxysmal atrial fibrillation in stroke with an online clinical database.

OBJECTIVE: A considerable fraction of ischemic stroke cases remain cryptogenic and there is increasing data suggesting the role of missed paroxysmal atrial fibrillations (pAF) in at least a number of these cases. Since electrophysiological identification of pAFs can be challenging, there has been an accumulation of proposed predictors and biomarkers for pAFs. The predictive values of these is varying and sometimes conflicting among studies. Therefore, we aimed to verify a fraction of previously reported parameters for pAF detection by investigating an independent clinical sample. METHODS: Using a publicly available data downloaded from the MIMIC-3 intensive care unit database, we tested the predictive role of particular risk factors and biomarkers for pAF detection after ischemic stroke in 124 patients with ischemic stroke admitted within 24 hours of stroke onset. RESULTS: Our evaluation revealed a strong association of older age in women, as well as admission National Institutes of Health Stroke Scale (NIHSS) and discharge modified Rankin Scores (mRS) in both sexes for pAFs, in patients that were in sinus rhythm on admission. We also detected a trend for lower gender-adjusted hemoglobin in patients with pAF, although the difference was insignificant. On the other hand, we did not find any significant association of pAF detection with some other previously reported biomarkers: serum magnesium level, leukocyte count, neutrophil/lymphocyte ratio or left atrial dilatation. CONCLUSION: Even though our analysis did not reveal a strong and specific biomarker to predict pAFs after stroke, it identified key risk factors. It may be necessary to consider the possibility of pAFs and perform rigorous evaluation to prevent further events of embolic stroke in female patients older than 75 years, with more severe neurological deficits on admission, higher disability on discharge and also with relatively lower hemoglobin level. This first study from Turkey using clinical data from the MIMIC-3 database also demonstrates the value of publicized clinical data for confirmatory studies on various medical fields across the World.

KCl-induced cortical spreading depression waves more heterogeneously propagate than optogenetically-induced waves in lissencephalic brain: an analysis with optical flow tools.

Although cortical spreading depolarizations (CSD) were originally assumed to be homogeneously and concentrically propagating waves, evidence obtained first in gyrencephalic brains and later in lissencephalic brains suggested a rather non-uniform propagation, shaped heterogeneously by factors like cortical region differences, vascular anatomy, wave recurrences and refractory periods. Understanding this heterogeneity is important to better evaluate the experimental models on the mechanistics of CSD and to make appropriate clinical estimations on neurological disorders like migraine, stroke, and traumatic brain injury. This study demonstrates the application of optical flow analysis tools for systematic and objective evaluation of spatiotemporal CSD propagation patterns in anesthetized mice and compares the propagation profile in different CSD induction models. Our findings confirm the asymmetric angular CSD propagation in lissencephalic brains and suggest a strong dependency on induction-method, such that continuous potassium chloride application leads to significantly higher angular propagation variability compared to optogenetically-induced CSDs.

F-actin polymerization contributes to pericyte contractility in retinal capillaries.

Although it has been documented that central nervous system pericytes are able to contract in response to physiological, pharmacological or pathological stimuli, the underlying mechanism of pericyte contractility is incompletely understood especially in downstream pericytes that express low amounts of alpha-smooth muscle actin (α-SMA). To study whether pericyte contraction involves F-actin polymerization as in vascular smooth muscle cells, we increased retinal microvascular pericyte tonus by intravitreal injection of a vasoconstrictive agent, noradrenaline (NA). The contralateral eye of each mouse was used for vehicle injection. The retinas were rapidly extracted and fixed within 2 min after injections. Polymeric/filamentous (F-actin) and monomeric/globular (G-actin) forms of actin were labeled by fluorescently-conjugated phalloidin and deoxyribonuclease-I, respectively. We studied 108 and 83 pericytes from 6 NA- and 6 vehicle-treated retinas and, found that F/G-actin ratio, a microscopy-based index of F-actin polymerization, significantly increased in NA-treated retinas [median (IQR): 4.2 (3.1) vs. 3.5 (2.1), p = .006], suggesting a role for F-actin polymerization in pericyte contractility. Shift from G-actin monomers to polymerized F-actin was more pronounced in 5th and 6th order contracted pericytes compared to non-contracted ones [7.6 (4.7) vs. 3.2 (1.2), p < .001], possibly due to their dependence on de novo F-actin polymerization for contractile force generation because they express α-SMA in low quantities. Capillaries showing F-actin polymerization had significantly reduced diameters compared to the ones that did not exhibit increased F/G-actin ratio in pericytes [near soma / branch origin diameter; 0.67 (0.14) vs. 0.81 (0.34), p = .005]. NA-responsive capillaries generally did not show nodal constrictions but a tide-like diameter decrease, reaching a maximum near pericyte soma. These findings suggest that pericytes on high order downstream capillaries have F-actin-mediated contractile capability, which may contribute to the vascular resistance and blood flow regulation in capillary bed.

Awake chronic mouse model of targeted pial vessel occlusion via photothrombosis.

Animal models of stroke are used extensively to study the mechanisms involved in the acute and chronic phases of recovery following stroke. A translatable animal model that closely mimics the mechanisms of a human stroke is essential in understanding recovery processes as well as developing therapies that improve functional outcomes. We describe a photothrombosis stroke model that is capable of targeting a single distal pial branch of the middle cerebral artery with minimal damage to the surrounding parenchyma in awake head-fixed mice. Mice are implanted with chronic cranial windows above one hemisphere of the brain that allow optical access to study recovery mechanisms for over a month following occlusion. Additionally, we study the effect of laser spot size used for occlusion and demonstrate that a spot size with small axial and lateral resolution has the advantage of minimizing unwanted photodamage while still monitoring macroscopic changes to cerebral blood flow during photothrombosis. We show that temporally guiding illumination using real-time feedback of blood flow dynamics also minimized unwanted photodamage to the vascular network. Finally, through quantifiable behavior deficits and chronic imaging we show that this model can be used to study recovery mechanisms or the effects of therapeutics longitudinally.

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.

Small Vessels Are a Big Problem in Neurodegeneration and Neuroprotection.

The cerebral microcirculation holds a critical position to match the high metabolic demand by neuronal activity. Functionally, microcirculation is virtually inseparable from other nervous system cells under both physiological and pathological conditions. For successful bench-to-bedside translation of neuroprotection research, the role of microcirculation in acute and chronic neurodegenerative disorders appears to be under-recognized, which may have contributed to clinical trial failures with some neuroprotectants. Increasing data over the last decade suggest that microcirculatory impairments such as endothelial or pericyte dysfunction, morphological irregularities in capillaries or frequent dynamic stalls in blood cell flux resulting in excessive heterogeneity in capillary transit may significantly compromise tissue oxygen availability. We now know that ischemia-induced persistent abnormalities in capillary flow negatively impact restoration of reperfusion after recanalization of occluded cerebral arteries. Similarly, microcirculatory impairments can accompany or even precede neural loss in animal models of several neurodegenerative disorders including Alzheimer's disease. Macrovessels are relatively easy to evaluate with radiological or experimental imaging methods but they cannot faithfully reflect the downstream microcirculatory disturbances, which may be quite heterogeneous across the tissue at microscopic scale and/or happen fast and transiently. The complexity and size of the elements of microcirculation, therefore, require utilization of cutting-edge imaging techniques with high spatiotemporal resolution as well as multidisciplinary team effort to disclose microvascular-neurodegenerative connection and to test treatment approaches to advance the field. Developments in two photon microscopy, ultrafast ultrasound, and optical coherence tomography provide valuable experimental tools to reveal those microscopic events with high resolution. Here, we review the up-to-date advances in understanding of the primary microcirculatory abnormalities that can result in neurodegenerative processes and the combined neurovascular protection approaches that can prevent acute as well as chronic neurodegeneration.

Normalized field autocorrelation function-based optical coherence tomography three-dimensional angiography.

Optical coherence tomography angiography (OCTA) has been widely used for en face visualization of the microvasculature, but is challenged for real three-dimensional (3-D) topologic imaging due to the "tail" artifacts that appear below large vessels. Further, OCTA is generally incapable of differentiating descending arterioles from ascending venules. We introduce a normalized field autocorrelation function-based OCTA (g1-OCTA), which minimizes the tail artifacts and is capable of distinguishing penetrating arterioles from venules in the 3-D image. g1 ( τ ) is calculated from repeated optical coherence tomography (OCT) acquisitions for each spatial location. The decay amplitude of g1 ( τ ) is retrieved to represent the dynamics for each voxel. To account for the small g1 ( τ ) decay in capillaries where red blood cells are flowing slowly and discontinuously, Intralipid is injected to enhance the OCT signal. We demonstrate that the proposed technique realizes 3-D OCTA with negligible tail projections and the penetrating arteries are readily identified. In addition, compared to regular OCTA, the proposed g1-OCTA largely increased the depth-of-field. This technique provides a more accurate rendering of the vascular 3-D anatomy and has the potential for more quantitative characterization of vascular networks.

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.

Spatio-temporal dynamics of cerebral capillary segments with stalling red blood cells.

Optical coherence tomography (OCT) allows label-free imaging of red blood cell (RBC) flux within capillaries with high spatio-temporal resolution. In this study, we utilized time-series OCT-angiography to demonstrate interruptions in capillary RBC flux in mouse brain in vivo. We noticed ∼7.5% of ∼200 capillaries had at least one stall in awake mice with chronic windows during a 9-min recording. At any instant, ∼0.45% of capillaries were stalled. Average stall duration was ∼15 s but could last over 1 min. Stalls were more frequent and longer lasting in acute window preparations. Further, isoflurane anesthesia in chronic preparations caused an increase in the number of stalls. In repeated imaging, the same segments had a tendency to stall again over a period of one month. In awake animals, functional stimulation decreased the observance of stalling events. Stalling segments were located distally, away from the first couple of arteriolar-side capillary branches and their average RBC and plasma velocities were lower than nonstalling capillaries within the same region. This first systematic analysis of capillary RBC stalls in the brain, enabled by rapid and continuous volumetric imaging of capillaries with OCT-angiography, will lead to future investigations of the potential role of stalling events in cerebral pathologies.

Improving Microcirculatory Reperfusion Reduces Parenchymal Oxygen Radical Formation and Provides Neuroprotection.

BACKGROUND AND PURPOSE: Reperfusion is the most significant determinant of good outcome after ischemic stroke. However, complete reperfusion often cannot be achieved, despite satisfactory recanalization. We hypothesized that microvascular protection was essential for achieving effective reperfusion and, hence, neuroprotection. To test this hypothesis, we have developed an in vivo model to differentially monitor parenchymal and vascular reactive oxygen species (ROS) formation. By comparing the ROS-suppressing effect of N-tert-butyl-α-phenylnitrone (PBN) with its blood-brain barrier impermeable analog 2-sulfo-phenyl-N-tert-butylnitrone (S-PBN), we assessed the impact of vascular ROS suppression alone on reperfusion and stroke outcome after recanalization. METHODS: The distal middle cerebral artery was occluded for 1 hour by compressing with a micropipette and then recanalized (n=60 Swiss mice). ROS formation was monitored for 1 hour after recanalization by intravital fluorescence microscopy in pial vasculature and cortical parenchyma with topically applied hydroethidine through a cranial window. PBN (100 mg/kg) or S-PBN (156 mg/kg) was administered shortly before recanalization, and suppression of the vascular and parenchymal hydroethidine fluorescence was examined (n=22). Microcirculatory patency, reperfusion, ischemic tissue size, and neurological outcome were also assessed in a separate group of mice 1 to 72 hours after recanalization (n=30). RESULTS: PBN and S-PBN completely suppressed the reperfusion-induced increase in ROS signal within vasculature. PBN readily suppressed ROS produced in parenchyma by 88%. S-PBN also suppressed the parenchymal ROS by 64% but starting 40 minutes later. Intriguingly, PBN and S-PBN comparably reduced the size of ischemic area by 65% and 48% (P>0.05), respectively. S-PBN restored the microvascular patency and perfusion after recanalization, suggesting that its delayed parenchymal antioxidant effect could be secondary to improved microcirculatory reperfusion. CONCLUSIONS: Promoting microvascular reperfusion by protecting vasculature can secondarily reduce parenchymal ROS formation and provide neuroprotection. The model presented can be used to directly assess pharmacological end points postulated in brain parenchyma and vasculature in vivo.

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.