Cortical Microvasculature Research Articles

Dynamic changes in the structure and function of brain mural cells around chronically implanted microelectrodes

Integration of neural interfaces with minimal tissue disruption in the brain is ideal to develop robust tools that can address essential neuroscience questions and combat neurological disorders. However, implantation of intracortical devices provokes severe tissue inflammation within the brain, which requires a high metabolic demand to support a complex series of cellular events mediating tissue degeneration and wound healing. Pericytes, peri-vascular cells involved in blood-brain barrier maintenance, vascular permeability, waste clearance, and angiogenesis, have recently been implicated as potential perpetuators of neurodegeneration in brain injury and disease. While the intimate relationship between pericytes and the cortical microvasculature have been explored in other disease states, their behavior following microelectrode implantation, which is responsible for direct blood vessel disruption and dysfunction, is currently unknown. Using two-photon microscopy we observed dynamic changes in the structure and function of pericytes during implantation of a microelectrode array over a 4-week implantation period. Pericytes respond to electrode insertion through transient increases in intracellular calcium and underlying constriction of capillary vessels. Within days following the initial insertion, we observed an influx of new, proliferating pericytes which contribute to new blood vessel formation. Additionally, we discovered a potentially novel population of reactive immune cells in close proximity to the electrode-tissue interface actively engaging in encapsulation of the microelectrode array. Finally, we determined that intracellular pericyte calcium can be modulated by intracortical microstimulation in an amplitude- and frequency-dependent manner. This study provides a new perspective on the complex biological sequelae occurring at the electrode-tissue interface and will foster new avenues of potential research consideration and lead to development of more advanced therapeutic interventions towards improving the biocompatibility of neural electrode technology.

Comparison of compartmental analytical Blood-Oxygen-Level-Dependent functional Magnetic Resonance Imaging models against Monte Carlo simulations performed over cortical micro-angiograms.

Blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) arises from a physiological and physical cascade of events taking place at the level of the cortical microvasculature which constitutes a medium with complex geometry. Several analytical models of the BOLD contrast have been developed, but these have not been compared directly against detailed bottom-up modeling methods. Using a 3D modeling method based on experimentally measured images of mice microvasculature and Monte Carlo simulations, we quantified the accuracy of two analytical models to predict the amplitude of the BOLD response from 1.5 to 7T, for different echo time (TE) and for both gradient echo and spin echo acquisition protocols. We also showed that accounting for the tridimensional structure of the microvasculature results in more accurate prediction of the BOLD amplitude, even if the values for SO2 were averaged across individual vascular compartments. A secondary finding is that modeling the venous compartment as two individual compartments results in more accurate prediction of the BOLD amplitude compared with standard homogenous venous modeling, arising from the bimodal distribution of venous SO2 across the microvasculature in our data.

Label free, capillary-scale blood flow mapping in vivo reveals that low intensity focused ultrasound evokes persistent dilation in cortical microvasculature.

Non-invasive, low intensity focused ultrasound (FUS) is an emerging neuromodulation technique that offers the potential for precision, personalized therapy. An increasing body of research has identified mechanosensitive ion channels that can be modulated by FUS and support acute electrical activity in neurons. However, neuromodulatory effects that persist from hours to days have also been reported. The brain's ability to provide targeted blood flow to electrically active regions involve a multitude of non-neuronal cell types and signaling pathways in the cerebral vasculature; an open question is whether persistent effects can be attributed, at least partly, to vascular mechanisms. Using a novel in vivo optical approach, we found that microvascular responses, unlike larger vessels which prior investigations have explored, exhibit persistent dilation following sonication without the use of microbubbles. This finding and approach offers a heretofore unseen aspect of the effects of FUS in vivo and indicate that concurrent changes in neurovascular function may partially underly persistent neuromodulatory effects.

Dynamic changes in structure and function of brain mural cells around chronically implanted microelectrodes.

Integration of neural interfaces with minimal tissue disruption in the brain is ideal to develop robust tools that can address essential neuroscience questions and combat neurological disorders. However, implantation of intracortical devices provokes severe tissue inflammation within the brain, which requires a high metabolic demand to support a complex series of cellular events mediating tissue degeneration and wound healing. Pericytes, peri-vascular cells involved in blood-brain barrier maintenance, vascular permeability, waste clearance, and angiogenesis, have recently been implicated as potential perpetuators of neurodegeneration in brain injury and disease. While the intimate relationship between pericytes and the cortical microvasculature have been explored in other disease states, their behavior following microelectrode implantation, which is responsible for direct blood vessel disruption and dysfunction, is currently unknown. Using two-photon microscopy we observed dynamic changes in the structure and function of pericytes during implantation of a microelectrode array over a 4-week implantation period. Pericytes respond to electrode insertion through transient increases in intracellular calcium and underlying constriction of capillary vessels. Within days following the initial insertion, we observed an influx of new, proliferating pericytes which contribute to new blood vessel formation. Additionally, we discovered a potentially novel population of reactive immune cells in close proximity to the electrode-tissue interface actively engaging in encapsulation of the microelectrode array. Finally, we determined that intracellular pericyte calcium can be modulated by intracortical microstimulation in an amplitude- and frequency-dependent manner. This study provides a new perspective on the complex biological sequelae occurring the electrode-tissue interface and will foster new avenues of potential research consideration and lead to development of more advanced therapeutic interventions towards improving the biocompatibility of neural electrode technology.

Elevation of miR-150-5p in Brain Microvascular Endothelial Cells as a Result of Prenatal Alcohol Exposure Is Mediated by Transcriptional Mechanisms

Fetal Alcohol Spectrum Disorders (FASD) encompass a variety of conditions impacting development in children exposed to alcohol in utero. Vascular alterations such as defective angiogenesis, blood brain barrier permeability, vessel reactivity, and blood flow have been found in various animal models of prenatal alcohol exposure (PAE). The underlying mechanisms of these vascular defects are not fully understood. Our lab previously showed that miR-150-5p was upregulated in brain microvascular endothelial cells (BMVECs) isolated from embryonic mouse cortices following moderate PAE. We identified downstream vascular targets of miR-150-5p in BMVECs and demonstrated that miR-150-5p inhibition could counter alcohol-mediated effects on BMVECS and the cortical microvasculature in vitro and in vivo, respectively. Here, we investigate upstream mechanisms that contribute to miR-150-5p elevation in BMVECs following PAE. We hypothesized that multiple mechanisms, including pathways at the transcriptional level, contribute to increased miR-150-5p abundance in BMVECs following ethanol (EtOH) exposure. Overall transcriptional activity at the miR-150 promoter was increased in BMVECs with EtOH treatment, and pri-miR-150, the primary transcript for miR-150-5p, was elevated in EtOH-treated BMVECs. Additionally, miR-150 promoter methylation was decreased in PAE cortices compared to saccharine-exposed controls (SAC). Current work aims to confirm the role of DNA methylation in transcriptional regulation of the miR-150 promoter in BMVECs and characterize DNA methyltransferase activity in BMVECs during PAE. We also assessed transcription factor binding at the miR-150 promoter and found that miR-150 promoter activation by several transcription factors was blunted with EtOH treatment. Current work involves the investigation of global transcription factor activation in BMVECs in PAE versus SAC. In summary, our research sheds light on multiple mechanisms of miR-150-5p regulation that may contribute to brain microvascular defects as a result of PAE. American Physiological Society Porter Physiology Development Fellowship University of New Mexico Health School of Medicine Department of Cell Biology and Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A dynamic multiscale model of cerebral blood flow and autoregulation in the microvasculature

Models of the micro-circulatory blood flow in the brain can play a key role in understanding the variety of cerebrovascular diseases that occur in the microvasculature. These conditions are often linked to structural modifications in the vessel network, alterations in the blood flow patterns, as well as impairment in the autoregulatory response, all of which are pathological changes that the model should be able to address if it were to have any clinical value. Furthermore, if the model results were to be validated against clinical MRI data, the model simulations need to be computationally feasible when used on networks on the scale of an MRI voxel. This requires some form of an upscaling approach that bypasses the need for an explicit architectural representation of the whole network while maintaining the relevant anatomical connections. To this end, we developed a hybrid multiscale model of blood flow and autoregulation that traces the dynamic changes in blood flow, volume, and pressure in the cortical microvasculature, where the discrete topology of the penetrating vessels is preserved, and these are then appropriately coupled to the homogenised capillary bed by a spatially distributing support function in the terminal endings. In contrast to the other multiscale models, the model developed here accounts for the dynamic physiological phenomena of the blood flow and the autoregulation processes in the microvessels. We show how the adaptive meshing scheme for the capillary bed developed in this study can be employed to ensure a scale-invariant coupling formulation and numerically accurate simulations, all without compromising the computational feasibility of the model. A statistically accurate cortical network on the scale of an MRI voxel is generated, and the model parameter values are calibrated using a Monte Carlo Filtering analysis to ensure that the model results are physiologically informed. The model is found to be able to capture the steep pressure gradients that have been reported to occur at coupling interfaces. Furthermore, in response to an upstream pressure drop, the network is found to be able to recover cerebral blood flow while exhibiting the characteristic autoregulatory behaviour in terms of changes in vessel calibre and the biphasic flow response. Overall, the model developed here offers a high-quality characterisation of dynamic flow and autoregulation in the microvasculature at improved computational efficiency and lays the ground for whole-brain dynamic simulations.

A comparison between the superb microvascular imaging technique and conventional Doppler ultrasound in evaluating chronic allograft damage in renal transplant recipients

The present study comparatively evaluates the performance of conventional Doppler ultrasound and superb microvascular imaging (SMI) in delineating the cortical microvessels of the transplanted kidney and compares the chronic allograft damage index (CADI) based on the examination of biopsy specimens with Doppler ultrasound and SMI findings. Sixty-eight renal transplant recipients underwent kidney biopsy with the pre-diagnosis of rejection before undergoing renal Doppler ultrasound examination between January 2020 and October 2020. The distance between the kidney capsule and the vascular structure closest to the kidney capsule was measured at the level of the lower pole in the transplanted kidney using color Doppler ultrasound (CDUS), power Doppler ultrasound (PDUS), and the SMI technique. The kidney size, resistive index at the level of the arcuate artery in the lower pole of the kidney, and renal artery flow rates were also measured. The mean distance between the kidney capsule and the vessel was 2.44 ± 2.0 mm on CDUS, 1.34 ± 1.2 mm on PDUS, 0.99 ± 1.8 mm using the color SMI (cSMI) technique, and 0.86 ± 1.8 mm using the monochrome SMI (mSMI) technique. The study found that the SMI technique was superior to CDUS and PDUS in delineating the cortical microvasculature of the kidney. Both Doppler ultrasound examinations and the SMI technique proved effective in predicting the CADI (P = 0.006 for CDUS, P = 0.002 for PDUS, P = 0.018 for cSMI, and P = 0.027 for mSMI). Among conventional Doppler ultrasound examinations and the SMI technique, PDUS had the highest sensitivity, and cSMI had the highest specificity in differentiating high and low CADI values. Both the cSMI and mSMI techniques had similar sensitivity values, whereas only cSMI exhibited high specificity. CDUS had the lowest specificity value (P = 0.003 for CDUS, P = 0.002 for PDUS, P = 0.005 for cSMI, and P = 0.004 for mSMI). The present study is the first in the literature to demonstrate the utility of the distance between the kidney capsule and the vessels in predicting the CADI score and to compare the Doppler ultrasound examinations and SMI technique in doing so.

Adhesion molecules and cerebral microvascular hemodynamic abnormalities in sickle cell disease.

Cerebrovascular abnormalities are a common feature of sickle cell disease that may be associated with risk of vaso-occlusive pain crises, microinfarcts, and cognitive impairment. An activated endothelium and adhesion factors, VCAM-1 and P-selectin, are implicated in sickle cell vasculopathy, including abnormal hemodynamics and leukocyte adherence. This study examined the association between cerebral expression of these adhesion factors and cortical microvascular blood flow dynamics by using in-vivo two-photon microscopy. We also examined the impact of blood transfusion treatment on these markers of vasculopathy. Results showed that sickle cell mice had significantly higher maximum red blood cell (RBC) velocity (6.80 ± 0.25 mm/sec, p ≤ 0.01 vs. 5.35 ± 0.35 mm/sec) and more frequent blood flow reversals (18.04 ± 0.95 /min, p ≤ 0.01 vs. 13.59 ± 1.40 /min) in the cortical microvasculature compared to controls. In addition, sickle cell mice had a 2.6-fold (RFU/mm2) increase in expression of VCAM-1 and 17-fold (RFU/mm2) increase in expression of P-selectin compared to controls. This was accompanied by an increased frequency in leukocyte adherence (4.83 ± 0.57 /100 μm/min vs. 2.26 ± 0.37 /100 μm/min, p ≤ 0.001). We also found that microinfarcts identified in sickle cell mice were 50% larger than in controls. After blood transfusion, many of these parameters improved, as results demonstrated that sickle cell mice had a lower post-transfusion maximum RBC velocity (8.30 ± 0.98 mm/sec vs. 11.29 ± 0.95 mm/sec), lower frequency of blood flow reversals (12.80 ± 2.76 /min vs. 27.75 ± 2.09 /min), and fewer instances of leukocyte adherence compared to their pre-transfusion imaging time point (1.35 ± 0.32 /100 μm/min vs. 3.46 ± 0.58 /100 μm/min). Additionally, we found that blood transfusion was associated with lower expression of adhesion factors. Our results suggest that blood transfusion and adhesion factors, VCAM-1 and P-selectin, are potential therapeutic targets for addressing cerebrovascular pathology, such as vaso-occlusion, in sickle cell disease.

Direct current stimulation increases blood flow and permeability of cortical microvasculature in vivo.

Transcranial direct current stimulation (DCS) structurally and functionally modulates neuronal networks and microglia dynamics. Neurovascular coupling adapts regional cerebral blood flow to neuronal activity and metabolic demands. In this study, we examined effects of anodal DCS on vessel morphology, blood flow parameters, permeability of cortical microvasculature, and perivascular microglia motility by time-lapse two-photon microscopy in anaesthetized mice. Low-intensity DCS significantly increased vessel diameter and blood flow parameters. These effects were transient and dependent on the spontaneous vasomotion characteristics of the individual vessel. Vessel leakage increased significantly after DCS at 1.1 and was more pronounced at 2.2A/m2 , indicating a dose-dependent increase in vascular permeability. Perivascular microglia exhibited increased soma motility post-DCS at both intensities, potentially triggered by the extravasation of intravascular substrates. Our findings demonstrate that DCS affected only vessels with spontaneous vasomotion. This rapid vascular response may occur as an adaptation of regional blood supply to neuronal excitability altered by DCS or as a direct effect on the vessel wall. In contrast to these immediate effects during stimulation, increases in cortical vessel permeability and perivascular microglia motility appeared after the stimulation had ended.

Abstract 15180: Cerebral Blood Flow Measured by Mri is a Key Translational Hallmark of Brain Injury in Experimental Models of Hypertension-Induced Cognitive Impairment

While several works pointed out the epidemiological associations between hypertension and dementia, the underlying mechanisms are still relatively unknown. In this work we focused on the application of translational tools like MRI to investigate the brain injury induced by hypertension and characterized by cognitive impairment and neuroinflammation. Cerebral hypertension was induced in mice by transverse aortic constriction (TAC) or sham procedure, as previously demonstrated by our group. We characterized cerebral blood flow (CBF) by 7T MRI, measuring the cerebral tissue blood prefusion. Morris Water Maze (MWM) was used to evaluate cognitive function in hypertensive and normotensive mice. Cortical microvasculature was analyzed in histological brain sections. A group of mice was treated with an antibody neutralizing IFNy, a key proinflammatory cytokine induced by hypertension. TAC mice showed a marked cerebral hypoperfusion, with CBF reduction in cortex (Sham: 96.58 vs TAC: 74.25 mL/100g/min p=0.024) and hypothalamus (Sham: 140 vs TAC: 121.4 mL/100g/min p=0.027). Tissue analysis revealed a reduction in capillary density defined as portion of field of view (FOV) occupied by capillaries (Sham: 11.3% vs TAC: 7.7%, p=0.004) and loss of their integrity in the cortex of hypertensive mice, defined as pericyte coverage percentage in the FOV (Sham: 79% vs TAC: 36%, p=0.01). Finally, the impairment in cognitive function measured by the MWM correlated with the CBF measures (r 2 :0.407, p=0.01). In the end, we subjected TAC mice to anti-IFNy treatment and analyzed the effects on brain vasculature assessed in vivo by MRI and ex vivo by histology. We observed that, while leaving unchanged the hemodynamic challenge on the brain, the treatment induced a global rescue in CBF (TAC: 99.85 vs TAC anti-IFNy: 116 mL/100g/min p=0. 008) and a strong correlation between MRI-measured CBF and microvascular density (r 2 :0.471, p=0.002). In conclusion, CBF evaluated by MRI is a key translational hallmark of hypertensive brain injury, and its evaluation in the clinical setting can provide insights on cerebral microvasculature health.

MicroRNA-150-5p is upregulated in the brain microvasculature during prenatal alcohol exposure and inhibits the angiogenic factor Vezf1.

Fetal alcohol spectrum disorders (FASD) occur in children who were exposed to alcohol in utero and are manifested in a wide range of neurocognitive deficits. These deficits could be caused by alterations to the cortical microvasculature that are controlled by post-transcriptional regulators such as microRNAs. Using an established mouse model of moderate prenatal alcohol exposure (PAE), we isolated cortices (CTX) and brain microvascular endothelial cells (BMVECs) at embryonic day 18 (E18) and examined the expression of miR-150-5p and potential downstream targets. Cellular transfections and intrauterine injections with LNA™ mimics or inhibitors were used to test miR-150-5p regulation of novel target vascular endothelial zinc finger 1 (Vezf1). Dual-luciferase assays were used to assess the direct binding of miR-150-5p to the Vezf1 3'UTR. The effects of miR-150-5p and Vezf1 on endothelial cell function were determined by in vitro migration and tube formation assays. We found that miR-150-5p was upregulated and Vezf1 was downregulated during PAE in the E18 CTX and BMVECs. Transfection with miR-150-5p mimics resulted in decreased Vezf1 expression in BMVECs, while miR-150-5p inhibition did the opposite. Dual-luciferase assays revealed direct binding of miR-150-5p with the Vezf1 3'UTR. Intrauterine injections showed that miR-150-5p regulates the expression of Vezf1 in vivo during PAE. miR-150-5p overexpression decreased BMVEC migration and tube formation, while miR-150-5p inhibition enhanced migration and tube formation. Vezf1 overexpression rescued the effects of the miR-150-5p mimic. Alcohol treatment of BMVECs increased miR-150-5p expression and inhibited migration and tube formation. Finally, miR-150-5p inhibition and Vezf1 overexpression rescued the negative effects of alcohol on migration and tube formation. miR-150-5p regulation of Vezf1 results in altered endothelial cell function during alcohol exposure. Further, miR-150-5p inhibition of Vezf1 may adversely alter the development of the cortical microvasculature during PAE and contribute to deficits seen in patients with FASD.

Large-depth three-photon fluorescence microscopy imaging of cortical microvasculature on nonhuman primates with bright AIE probe In vivo

Multiphoton microscopy has been a powerful tool in brain research, three-photon fluorescence microscopy is increasingly becoming an emerging technique for neurological research of the cortex in depth. Nonhuman primates play important roles in the study of brain science because of their neural and vascular similarity to humans. However, there are few research results of three-photon fluorescence microscopy on the brain of nonhuman primates due to the lack of optimized imaging systems and excellent fluorescent probes. Here we introduced a bright aggregation-induced emission (AIE) probe with excellent three-photon fluorescence efficiency as well as facile synthesis process and we validated its biocompatibility in the macaque monkey. We achieved a large-depth vascular imaging of approximately 1 mm in the cerebral cortex of macaque monkey with our lab-modified three-photon fluorescence microscopy system and the AIE probe. Functional measurement of blood velocity in deep cortex capillaries was also performed. Furthermore, the comparison of cortical deep vascular structure parameters across species was presented on the monkey and mouse cortex. This work is the first in vivo three-photon fluorescence microscopic imaging research on the macaque monkey cortex reaching the imaging depth of ∼1 mm with the bright AIE probe. The results demonstrate the potential of three-photon microscopy as primate-compatible method for imaging fine vascular networks and will advance our understanding of vascular function in normal and disease in humans.

In utero alcohol exposure impairs vessel-associated positioning and differentiation of oligodendrocytes in the developing neocortex

Prenatal alcohol exposure (PAE) is a major cause of nongenetic mental retardation and can lead to fetal alcohol syndrome (FAS), the most severe manifestation of fetal alcohol spectrum disorder (FASD). FASD infants present behavioral disabilities resulting from neurodevelopmental defects. Both grey and white matter lesions have been characterized and are associated with apoptotic death and/or ectopic migration profiles. In the last decade, it was shown that PAE impairs brain angiogenesis, and the radial organization of cortical microvessels is lost. Concurrently, several studies have reported that tangential migration of oligodendrocyte precursors (OPCs) originating from ganglionic eminences is vascular associated. Because numerous migrating oligodendrocytes enter the developing neocortex, the present study aimed to determine whether migrating OPCs interacted with radial cortical microvessels and whether alcohol-induced vascular impairments were associated with altered positioning and differentiation of cortical oligodendrocytes. Using a 3D morphometric analysis, the results revealed that in both human and mouse cortices, 15 to 40% of Olig2-positive cells were in close association with radial cortical microvessels, respectively. Despite perinatal vascular disorganization, PAE did not modify the vessel association of Olig2-positive cells but impaired their positioning between deep and superficial cortical layers. At the molecular level, PAE markedly but transiently reduced the expression of CNPase and MBP, two differentiation markers of immature and mature oligodendrocytes. In particular, PAE inverted their distribution profiles in cortical layers V and VI and reduced the thickness of the myelin sheath of efferent axons. These perinatal oligo-vascular defects were associated with motor disabilities that persisted in adults. Altogether, the present study provides the first evidence that Olig2-positive cells entering the neocortex are associated with radial microvessels. PAE disorganized the cortical microvasculature and delayed the positioning and differentiation of oligodendrocytes. Although most of these oligovascular defects occurred in perinatal life, the offspring developed long-term motor troubles. Altogether, these data suggest that alcohol-induced oligo-vascular impairments contribute to the neurodevelopmental issues described in FASD.

Antibody-based in vivo leukocyte label for two-photon brain imaging in mice.

.SignificanceTo study leukocyte-endothelial interactions in a living system, robust and specific leukocyte labeling techniques are needed for in vivo two-photon microscopy of the cerebral microvasculature.AimWe tested fluorophore-conjugated anti-CD45.2 monoclonal antibodies (mAb) to optimize dosing and two-photon imaging parameters for leukocyte labeling in healthy mice and a venous microstroke model.ApproachWe retro-orbitally injected anti-CD45.2 mAb at 0.04, 0.4, and into BALB/c mice and used flow cytometry to analyze antibody saturation. Leukocyte labeling in the cortical microvasculature was examined by two-photon imaging. We also tested the application of CD45.2 mAb in a pathological leukocyte-endothelial adhesion model by photothrombotically occluding cortical penetrating venules.ResultsWe found that of anti-CD45.2 antibody intravenously was sufficient to label 95% of circulating leukocytes. There was no depletion of circulating leukocytes after 24 h at the dosages tested. Labeled leukocytes could be observed as deep as from the cortical surface. The antibody reliably labeled rolling, crawling, and adherent leukocytes in venules around the stroke-affected tissues.ConclusionWe show that the anti-CD45.2 mAb is a robust reagent for acute labeling of leukocytes during in vivo two-photon microscopy of the cortical microvasculature.

395 Vascular Cognitive Impairment: Novel Endothelial Mechanisms and the Impact of Dietary PUFAs

OBJECTIVES/GOALS: Vascular cognitive impairment (VCI) is the leading cause of dementia behind Alzheimers Disease (AD) and is often the result of brain hypoxia. Diets rich in polyunsaturated fatty acids (PUFAs) can lower cognitive decline and AD incidence in human patients. Therefore, our goal is to determine the mechanisms that PUFAs influence in a mouse model of VCI. METHODS/STUDY POPULATION: We hypothesize that hypoxia promotes endothelial P-tau accumulation and vasotrophic uncoupling, impairing endothelial integrity. Additionally, we believe that a preventative PUFA-enriched diet blocks this uncoupling and subsequently prevents/delays neurovascular dysfunction and cognitive decline. Male and female mice will be administered a control or novel PUFA-enriched dietary intervention 1 month prior to hypoxic injury using the bilateral carotid artery stenosis model. Mice will continue their diet and be assessed for cerebral blood flow, cognitive function, and motor function at 1- & 3-month time points. Following euthanasia, tissue samples from deep cortical regions and microvasculature will be examined for endothelial- & neuronal-specific P-tau accumulation, inflammation, and cell death. RESULTS/ANTICIPATED RESULTS: Preliminary data in our lab indicates that hypoxia leads to a two-fold increase in endothelial P-tau accumulation and lowered mature BDNF (mBDNF) in brain microvascular endothelial cells (BMECs) compared to controls. Further, BMECs cultured in media with the PUFA docasahexaenoic acid (DHA) had lowered P-tau and increased mBDNF after hypoxia compared to controls. Based on this data and past research, we anticipate that mice on the PUFA-enriched diet will have enhanced cognitive and motor function alongside improved cerebral blood flow compared to controls. Also, we expect that mice on our PUFA-enriched diet will have lowered tau pathology, cell death, and neuroinflammation alongside increased blood brain barrier integrity and altered fatty acid composition in brain and vascular tissue samples. DISCUSSION/SIGNIFICANCE: An AHA Presidential Advisory identified cognitive function as modifiable through the management of cardiovascular risk factors, like diet. However, the mechanisms underlying the benefits of PUFA-enriched diets are unknown. Successful completion of these studies will provide insight into the vaso-neuronal protective effects of PUFAs in VCI.

A versatile toolbox for studying cortical physiology in primates

SUMMARYLesioning and neurophysiological studies have facilitated the elucidation of cortical functions and mechanisms of functional recovery following injury. Clinical translation of such studies is contingent on their employment in non-human primates (NHPs), yet tools for monitoring and modulating cortical physiology are incompatible with conventional lesioning techniques. To address these challenges, we developed a toolbox validated in seven macaques. We introduce the photothrombotic method for inducing focal cortical lesions, a quantitative model for designing experiment-specific lesion profiles and optical coherence tomography angiography (OCTA) for large-scale (~5 cm2) monitoring of vascular dynamics. We integrate these tools with our electrocorticographic array for large-scale monitoring of neural dynamics and testing stimulation-based interventions. Advantageously, this versatile toolbox can be incorporated into established chronic cranial windows. By combining optical and electrophysiological techniques in the NHP cortex, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, integrate physiological and behavioral findings, and develop neurorehabilitative treatments.MOTIVATION The primate neocortex encodes for complex functions and behaviors, the physiologies of which are yet to be fully understood. Such an understanding in both healthy and diseased states can be crucial for the development of effective neurorehabilitative strategies. However, there is a lack of a comprehensive and adaptable set of tools that enables the study of multiple physiological phenomena in healthy and injured brains. Therefore, we developed a toolbox with the capability to induce targeted cortical lesions, monitor dynamics of underlying cortical microvasculature, and record and stimulate neural activity. With this toolbox, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, test stimulation-based interventions, and integrate physiological and behavioral findings.

Prenatal intake of omega-3 promotes Wnt/β-catenin signaling pathway, and preserves integrity of the blood-brain barrier in preeclamptic rats.

BackgroundPreeclampsia is a systemic, multi‐organ endotheliopathy, associated with oxidative injury to the blood–brain barrier (BBB). Preeclampsia initiates a cascade of events that include neuroinflammation. Recently, it was documented that Wnt/β‐catenin signaling pathway exerts neuroprotective effects and maintain BBB integrity. We investigate the protective effect of omega‐3 against neurovascular complication of preeclampsia and its relation to Wnt/β‐catenin signaling pathway.MethodologyAfter confirmation of day 0 pregnancy (G0), 24 adult pregnant female Wistar rats were divided into four groups control pregnant, pregnant supplemented with omega‐3, preeclampsia (PE); female rats received N (ω)‐nitro‐L‐arginine methyl ester (L‐NAME) (50 mg/kg/day SC from day 7 to day 16 of pregnancy for induction of preeclampsia) and PE rats supplemented with omega‐3. The intake of omega‐3 started on day zero (0) of pregnancy until the end of the study (144 mg/kg\\day orally).ResultsWe found that omega‐3 supplementation significantly improved cognitive functions and EEG amplitude, decreased blood pressure, water contents of brain tissues, sFlt‐1, oxidative stress, proteinuria, and enhanced Wnt\\β‐catenin proteins. Histological examination showed improved cerebral microangiopathy, increased expression of claudin‐1 and ‐3, CD31, and VEGF in the cerebral cortical microvasculature and choroid plexus in PE rats treated with omega‐3. A positive correlation between protein expression level of Wnt \\β‐catenin and cognitive functions, and a negative correlation between claudin‐5 relative expression, claudin‐1 and ‐3 area % from one side and water content of the brain tissues from the other side were observed.ConclusionWnt/β‐catenin signaling pathway suspected to have an important role to improve BBB integrity. Neuroprotective, antioxidant, and anti‐inflammatory effects of omega‐3 were observed and can be suggested as protective supplementation for preeclampsia.

Prenatal alcohol exposure disrupts miR‐150‐5p regulation of angiogenesis and blood‐brain barrier integrity genes

Fetal alcohol spectrum disorders (FASD) occur after in utero ethanol (EtOH) exposure and manifest in a wide range of neurocognitive deficits. These deficits can be caused by EtOH-induced dysregulation of genes required for angiogenesis and vascular integrity which are being regulated by microRNAs (miRNAs). miRNAs silence gene expression by binding to the 3’UTRs of target mRNAs, inducing mRNA decay. In an established mouse model of moderate prenatal alcohol exposure (PAE), we found that cortices (CTX) and primary brain microvascular endothelial cells (pBMVECs) isolated from PAE pups at embryonic day 18 (E18) had significantly higher levels of miR-150-5p compared to saccharin-exposed controls. A novel angiogenic target of miR-150-5p, vascular endothelial zinc finger 1 (Vezf1), is an endothelial-specific transcription factor required for vascular development. Vezf1 levels were significantly reduced in E18 PAE CTX and pBMVECs compared to controls. pBMVECs also showed a decrease in expression of other genes important for vascular function and integrity, including Claudin-12, Claudin-5, Occludin, VE-cadherin and MMP14. To identify the effects of EtOH on endothelial cell gene expression, we treated a mouse BMVE cell line (BMVECs) with 25mM of EtOH for 24 hrs. miR-150-5p expression increased after EtOH treatment compared to no treatment controls. Transfection of miR-150-5p LNATM mimics in BMVECs decreased Vezf1, Claudin-12 and MMP14 expression, while miR-150-5p LNATM inhibitors increased their expression. To demonstrate direct interaction between miR-150-5p and Vezf1, we cloned the Vezf1 3’UTR into a firefly luciferase reporter. Dual luciferase assays showed reduced luciferase activity for the Vezf1 3’UTR reporter with miR-150-5p LNATM mimics. To investigate the effect of EtOH and miR-150-5p on endothelial cell function, we performed in vitro migration, tube formation and permeability assays. BMVECs treated with EtOH significantly attenuated migration and tube formation vs no EtOH treatment controls. In addition, miR-150-5p overexpression in BMVECs attenuated tube formation and cell migration, and significantly increased cell permeability. Correspondingly, miR-150-5p inhibition significantly enhanced tube formation and migration, and decreased cell permeability. Our research suggests that miR-150-5p may adversely alter the cortical microvasculature during PAE by inhibiting the expression of targets important for angiogenesis and blood-brain barrier integrity.

Visualization of brain microvasculature and blood flow in vivo: Feasibility study using confocal laser endomicroscopy.

Qualitative and quantitative analyses of blood flow in normal and pathologic brain and spinal cord microvasculature were performed using confocal laser endomicroscopy (CLE). Blood flow in cortical, dural, and spinal cord microvasculature was assessed in vivo in swine. We assessed microvasculature under normal conditions and after vessel occlusion, brain injury due to cold or surgical trauma, and cardiac arrest. Tumor-associated microvasculature was assessed in vivo and ex vivo in 20 patients with gliomas. We observed erythrocyte flow in vessels 5-500µm in diameter. Thrombosis, flow arrest and redistribution, flow velocity changes, agglutination, and cells rolling were assessed in normal and injured brain tissue. Microvasculature in in vivo CLE images of gliomas was classified as normal in 68% and abnormal in 32% of vessels on the basis of morphological appearance. Dural lymphatic channels were discriminated from blood vessels. Microvasculature CLE imaging was possible for up to 30minutes after a 1mg/kg intravenous dose of fluorescein. CLE imaging allows assessment of cerebral and tumor microvasculature and blood flow alterations with subcellular resolution intraoperative imaging demonstrating precise details of real-time cell movements. Research and clinical scenarios may benefit from this novel intraoperative in vivo microscopic fluorescence imaging modality.

Design and modeling of a two-magnet actuator for robotic micromanipulation

In this paper we optimized the design of a magnetic actuator prototype based on permanent magnets for navigation of magnetic microparticles in cortical microvasculature networks. The proposed design can push-and-pull microparticles in an open-loop strategy in a wide range of several cm with force in the range of few hundred of μN. To do so, a successive quadratic programming (SQP) method is used to solve the optimization problem in terms of stability, displacement and force. To demonstrate the optimized actuator performances, we built a prototype and validated it experimentally. Finally, robotic experiments were conducted in order to investigate the micromanipulation capabilities of the designed actuator in realistic and vascular-shaped microfluidic phantoms mimicking the delivery of drugs to brain tumor sites.