Cortical Capillaries Research Articles

Sulforaphane Ameliorate Cadmium-Induced Blood-Thymus Barrier Disruption by Targeting the PI3K/AKT/FOXO1 Axis.

Cadmium (Cd) is a transition metal ion that is extremely harmful to human and animal biological systems. Cd is a toxic substance that can accumulate in the food chain and cause various health issues. Sulforaphane (SFN) is a natural bioactive compound with potent antioxidant properties. In our study, 80 1 day-old chicks were fed with Cd (140 mg/kg BW/day) and/or SFN (50 mg/kg BW/day) for 90 days. The blood-thymus barrier (BTB) is a selective barrier separating T-lymphocytes from blood and cortical capillaries in the thymus cortex. Our research revealed that Cd could destroy the BTB by downregulating Wnt/β-catenin signaling and induce immunodeficiency, leading to irreversible injury to the immune system. The study emphasizes the health benefits of SFN in the thymus. SFN could ameliorate Cd-triggered BTB dysfunction and pyroptosis in the thymus tissues. SFN modulated the PI3K/AKT/FOXO1 axis, improving the level of claudin-5 (CLDN5) in the thymus to alleviate BTB breakdown. Our findings indicated the toxic impact of Cd on thymus, and BTB could be the specific target of Cd toxicity. The finding also provides evidence for the role of SFN in maintaining thymic homeostasis for Cd-related health issues.

An-Gong-Niu-Huang-Wan (AGNHW) regulates cerebral blood flow by improving hypoperfusion, cerebrovascular reactivity and microcirculation disturbances after stroke

BackgroundThe restoration of cerebrovascular regulation and improvement of cerebral blood flow in ischaemic regions are crucial for improving the clinical prognosis after stroke. An-Gong-Niu-Huang-Wan (AGNHW) is a famous traditional compound Chinese medicine that has been used for over 220 years to treat acute ischaemic stroke; however, its role in the regulation of cerebral blood flow is still unclear. The aim of the present study was to investigate the regulatory effect of AGNHW on cerebral blood flow and microcirculation after ischaemic stroke and to elucidate the underlying mechanisms involved.MethodsMale C57BL/6 mice were subjected to distal middle cerebral artery occlusion (dMCAO) and randomly assigned to the sham, MCAO, or AGNHW groups. AGNHW was administered intragastrically 1 h after dMCAO. The rotarod test was utilized to evaluate behavioural function; TTC was used to determine the infarct volume; and ischaemic injury was assessed by detecting brain levels of SOD, MDA and NO. Then, cortical perfusion and acetazolamide-induced cerebrovascular reactivity were assessed using laser speckle contrast imaging, and the velocity and flux of red blood cells in cortical capillaries were detected using two-photon laser scanning microscopy. In addition, we employed RNA-Seq to identify variations in gene expression profiles and assessed endothelium-dependent changes in microcirculatory dysfunction by measuring vasoactive mediator levels.ResultsAGNHW significantly increased cerebral blood flow, reduced the infarct volume, and promoted functional recovery after cerebral ischaemia. AGNHW increased the velocity and flux of red blood cells in capillaries and improved cerebrovascular reactivity in the ischaemic cortex. Furthermore, AGNHW regulated endothelium-dependent microcirculation, as evidenced by decreases in the expression of endothelins (Edn1, Edn3 and Ednrb) and the ratios of brain and serum TXB2/6-keto-PGF1α and ET-1/CGRP.ConclusionsAGNHW improved cerebral hypoperfusion, regulated cerebrovascular reactivity and attenuated microcirculatory dysfunction within the ischaemic cortex after stroke. This outstanding effect was achieved by modulating the expression of genes related to vascular endothelial cell function and regulating endothelium-dependent vasoactive mediators.

Amyloid-β aggregates activate peripheral monocytes in mild cognitive impairment

The peripheral immune system is important in neurodegenerative diseases, both in protecting and inflaming the brain, but the underlying mechanisms remain elusive. Alzheimer’s Disease is commonly preceded by a prodromal period. Here, we report the presence of large Aβ aggregates in plasma from patients with mild cognitive impairment (n = 38). The aggregates are associated with low level Alzheimer’s Disease-like brain pathology as observed by 11C-PiB PET and 18F-FTP PET and lowered CD18-rich monocytes. We characterize complement receptor 4 as a strong binder of amyloids and show Aβ aggregates are preferentially phagocytosed and stimulate lysosomal activity through this receptor in stem cell-derived microglia. KIM127 integrin activation in monocytes promotes size selective phagocytosis of Aβ. Hydrodynamic calculations suggest Aβ aggregates associate with vessel walls of the cortical capillaries. In turn, we hypothesize aggregates may provide an adhesion substrate for recruiting CD18-rich monocytes into the cortex. Our results support a role for complement receptor 4 in regulating amyloid homeostasis.

Combination of intraoperative indocyanine green video-angiography FLOW 800 and computed tomography perfusion to assess the risk of cerebral hyperperfusion syndrome in chronic internal carotid artery occlusion patients after revascularization surgery.

To study the changes of corticocerebral hemodynamics in surgical area and postoperative hyperperfusion syndrome in patients with chronic internal carotid artery occlusion (CICAO) by intraoperative indocyanine green videoangiography (ICGA)-FLOW 800 and CT perfusion after superficial temporal artery (STA)-middle cerebral artery (MCA) bypass surgery. From October 2019 to January 2021, 77 patients diagnosed with CICAO underwent direct bypass surgery at Huadong hospital (affiliated with Fudan University) were enrolled. Regions of interest (ROIs) at STA, proximal MCA (PMCA), distal MCA (DMCA), cortical blood capillary (CBC), and cortical vein (CV) were identified after anastomosis by ICGV-FLOW 800 including peak fluorescence intensity (PFI), time to peak (TTP), and area under the time curve (AUC) of fluorescence intensity. All patients underwent perfusion-weighted CT before bypass surgery and those patients with HPS were verified by CTP after bypass. 14 patients with HPS were verified by perfusion-weighted CT after bypass. In HPS group, the AUCTTP of DMCA was significantly larger (T = -3.301, p = 0.004) and TTP of CBC was shorter (T = -2.929, p = 0.005) than patients in non-HPS group. The larger AUCTTP of DMCA (OR = 3.024, 95%CI 1.390-6.578, p = 0.0050) was an independent risk factor by further multivariate logistic regression analysis. The hemodynamic changes of cortical vessels during STA-MCA bypass surgery could be recorded accurately by ICGV-FLOW 800. Furthermore, the increased AUCTTP of DMCA and shorter TTP of CBC may be potential risk factors of HPS.

Whisker-evoked neurovascular coupling is preserved during hypoglycemia in mouse cortical arterioles and capillaries

Hypoglycemia is a serious complication of insulin treatment of diabetes that can lead to coma and death. Neurovascular coupling, which mediates increased local blood flow in response to neuronal activity, increases glucose availability to active neurons. This mechanism could be essential for neuronal health during hypoglycemia, when total glucose supplies are low. Previous studies suggest, however, that neurovascular coupling (a transient blood flow increase in response to an increase in neuronal activity) may be reduced during hypoglycemia. Such a reduction in blood flow increase would exacerbate the effects of hypoglycemia, depriving active neurons of glucose. We have reexamined the effects of hypoglycemia on neurovascular coupling by simultaneously monitoring neuronal and vascular responses to whisker stimulation in the awake mouse somatosensory cortex. We find that neurovascular coupling at both penetrating arterioles and at 2nd order capillaries did not change significantly during insulin-induced hypoglycemia compared to euglycemia. In addition, we show that the basal diameter of both arterioles and capillaries increases during hypoglycemia (10.3 and 9.7% increases, respectively). Our results demonstrate that both neurovascular coupling and basal increases in vessel diameter are active mechanisms which help to maintain an adequate supply of glucose to the brain during hypoglycemia.

Microvascular blood-brain barrier alterations in isolated brain capillaries of mice over-expressing alpha-synuclein (Thy1-aSyn line 61)

Dysfunction of the blood-brain barrier (BBB) is suggested to play a critical role in the pathological mechanisms of Parkinson's disease (PD). PD-related pathology such as alpha-synuclein accumulation and inflammatory processes potentially affect the integrity of the BBB early in disease progression, which in turn may alter the crosstalk of the central and peripheral immune response. Importantly, BBB dysfunction could also affect drug response in PD. Here we analyzed microvascular changes in isolated brain capillaries and brain sections on a cellular and molecular level during disease progression in an established PD mouse model that overexpresses human wild-type alpha-synuclein (Thy1-aSyn, line 61). BBB alterations observed in Thy1-aSyn mice included reduced vessel density, reduced aquaporin-4 coverage, reduced P-glycoprotein expression, increased low-density lipoprotein receptor-related protein 1 expression, increased pS129-alpha-synuclein deposition, and increased adhesion protein and matrix metalloprotease expression together with alterations in tight junction proteins. Striatal capillaries presented with more dysregulated BBB integrity markers compared to cortical capillaries. These alterations of BBB integrity lead, however, not to an overt IgG leakage in brain parenchyma. Our data reveals intricate alterations in key proteins of BBB function together with histological evidence for altered structure of the brain vasculature. Thy1-aSyn mice represent a useful model to investigate therapeutic targeting of BBB alterations in synucleinopathies.

Imaging the time course, morphology, neuronal tissue compression, and resolution of cerebral microhemorrhages in mice using intravital two-photon microscopy: insights into arteriolar, capillary, and venular origin.

Cerebral microhemorrhages (CMHs, microbleeds), a manifestation of age-related cerebral small vessel disease, contribute to the pathogenesis of cognitive decline and dementia in older adults. Histological studies have revealed that CMHs exhibit distinct morphologies, which may be attributed to differences in intravascular pressure and the size of the vessels of origin. Our study aimed to establish a direct relationship between the size/morphology of CMHs and the size/anatomy of the microvessel of origin. To achieve this goal, we adapted and optimized intravital two-photon microscopy-based imaging methods to monitor the development of CMHs in mice equipped with a chronic cranial window upon high-energy laser light-induced photodisruption of a targeted cortical arteriole, capillary, or venule. We assessed the time course of extravasation of fluorescently labeled blood and determined the morphology and size/volume of the induced CMHs. Our findings reveal striking similarities between the bleed morphologies observed in hypertension-induced CMHs in models of aging and those originating from different targeted vessels via multiphoton laser ablation. Arteriolar bleeds, which are larger (> 100μm) and more widely dispersed, are distinguished from venular bleeds, which are smaller and exhibit a distinct diffuse morphology. Capillary bleeds are circular and smaller (< 10μm) in size. Our study supports the concept that CMHs can occur at any location in the vascular tree, and that each type of vessel produces microbleeds with a distinct morphology. Development of CMHs resulted in immediate constriction of capillaries, likely due to pericyte activation and constriction of precapillary arterioles. Additionally, tissue displacement observed in association with arteriolar CMHs suggests that they can affect an area with a radius of ~ 50μm to ~ 100μm, creating an area at risk for ischemia. Longitudinal imaging of CMHs allowed us to visualize reactive astrocytosis and bleed resolution during a 30-day period. Our study provides new insights into the development and morphology of CMHs, highlighting the potential clinical implications of differentiating between the types of vessels involved in the pathogenesis of CMHs. This information may help in the development of targeted interventions aimed at reducing the risk of cerebral small vessel disease-related cognitive decline and dementia in older adults.

Optical opening of the blood-brain barrier for targeted and ultra-sparse viral infection of cells in mouse cortex

Adeno-associated viruses (AAVs) are used in a wide array of experimental situations for driving expression of biosensors, recombinases, and opto-/chemo-genetic actuators in the brain. However, conventional approaches for minimally invasive, spatially precise, and ultra-sparse AAV-mediated transduction of cells during imaging experiments have remained a significant challenge. Here, we show that intravenous injection of commercially available AAVs at different doses, combined with laser-based perforation of cortical capillaries through a cranial widow, allows for ultra-sparse, titratable, and micron-level precision for delivery of viral vectors with relatively little inflammation or tissue damage. Further, we show the utility of this approach for eliciting sparse expression of GCaMP6, channelrhodopsin, or fluorescent reporters in neurons and astrocytes within specific functional domains in normal and stroke-damaged cortex. This technique represents a facile approach for targeted delivery of viral vectors that should assist in the study of cell types and circuits in the cortex.

High affinity of β-amyloid proteins to cerebral capillaries: implications in chronic lead exposure-induced neurotoxicity in rats

Lead (Pb) is a known environmental risk factor in the etiology of Alzheimer’s disease (AD). The existing reports suggest that Pb exposure increases beta-amyloid (Aβ) levels in brain tissues and cerebrospinal fluid (CSF) and facilitates the formation of amyloid plaques, which is a pathological hallmark for AD. Pb exposure has long been associated with cerebral vasculature injury. Yet it remained unclear if Pb exposure caused excessive Ab buildup in cerebral vasculature, which may damage the blood–brain barrier and cause abnormal Ab accumulation. This study was designed to investigate the impact of chronic Pb exposure on Aβ accumulation in cerebral capillary and the expression of low-density lipoprotein receptor protein-1 (LRP1), a critical Aβ transporter, in brain capillary and parenchyma. Sprague–Dawley rats received daily oral gavage at doses of 0, 14 (low-dose), and 27 (high-dose) mg Pb/kg as Pb acetate, 5 d/wk, for 4 or 8 wks. At the end of Pb exposure, a solution containing Aβ40 was infused into the brain via the cannulated internal carotid artery. Data by ELISA showed a strikingly high affinity of Ab to cerebral vasculature, which was approximately 7–14 times higher than that to the parenchymal fractions collected from control brains. Pb exposure further aggravated the Aβ accumulation in cerebral vasculature in a dose-dependent manner. Western blot analyses revealed that Pb exposure decreased LRP1 expression in cortical capillaries and hippocampal parenchyma. Immunohistochemistry (IHC) studies further revealed a disrupted distribution of LRP1 alongside hippocampal vasculature accompanied with a decreased expression in hippocampal neurons by Pb exposure. Taken together, the current study demonstrated that the cerebral vasculature naturally possessed a high affinity to Aβ present in circulating blood. Pb exposure significantly increased Aβ accumulation in cerebral vasculature; such an increased Aβ accumulation was due partly to the diminished expression of LRP1 in response to Pb in tested brain regions. Perceivably, Pb-facilitated Ab aggravation in cerebral vasculature may contribute to Pb-associated amyloid alterations.

Dynamic 3D imaging of cerebral blood flow in awake mice using self-supervised-learning-enhanced optical coherence Doppler tomography

Cerebral blood flow (CBF) is widely used to assess brain function. However, most preclinical CBF studies have been performed under anesthesia, which confounds findings. High spatiotemporal-resolution CBF imaging of awake animals is challenging due to motion artifacts and background noise, particularly for Doppler-based flow imaging. Here, we report ultrahigh-resolution optical coherence Doppler tomography (µODT) for 3D imaging of CBF velocity (CBFv) dynamics in awake mice by developing self-supervised deep-learning for effective image denoising and motion-artifact removal. We compare cortical CBFv in awake vs. anesthetized mice and their dynamic responses in arteriolar, venular and capillary networks to acute cocaine (1 mg/kg, i.v.), a highly addictive drug associated with neurovascular toxicity. Compared with awake, isoflurane (2-2.5%) induces vasodilation and increases CBFv within 2-4 min, whereas dexmedetomidine (0.025 mg/kg, i.p.) does not change vessel diameters nor flow. Acute cocaine decreases CBFv to the same extent in dexmedetomidine and awake states, whereas decreases are larger under isoflurane, suggesting that isoflurane-induced vasodilation might have facilitated detection of cocaine-induced vasoconstriction. Awake mice after chronic cocaine show severe vasoconstriction, CBFv decreases and vascular adaptations with extended diving arteriolar/venular vessels that prioritize blood supply to deeper cortical capillaries. The 3D imaging platform we present provides a powerful tool to study dynamic changes in vessel diameters and morphology alongside CBFv networks in the brain of awake animals that can advance our understanding of the effects of drugs and disease conditions (ischemia, tumors, wound healing).

Running exercise improves spatial learning and memory ability and enhances angiogenesis in the cerebral cortex via endogenous nitric oxide

BackgroundThe molecular mechanisms by which exercise improves brain function and capillaries in the cerebral cortex are unclear. Exercise can increase the expression of nitric oxide (NO) in the brain, and endogenous NO is thought to exert beneficial effects on proangiogenic factors, antiangiogenic factors and brain function. Therefore, we hypothesized that running exercise might improve brain function and enhance angiogenesis through endogenous NO. Methods and resultsThe following three groups of rats were administered intracerebroventricular (i.c.v.) injections before running exercise each day for 4 weeks: exercise+L-NAME group (i.c.v. L-NAME, an NO synthase blocker, dose: 1 μmol/μl and 5 μl/day; treadmill exercise, 20 min/day), exercise group (i.c.v. normal saline, 5 μl/day; treadmill exercise, 20 min/day), and sham group (i.c.v. normal saline, 5 μl/day; no treadmill exercise). Subsequently, the spatial learning and memory abilities were tested using a Morris water maze, and the nitric oxide synthase (NOS) activity in the cerebral cortex in each group of rats was measured using a method involving nitric acid reductase and metabolic chemistry. The parameters of the cortical capillaries were quantitatively investigated using an immunohistochemistry technique and stereological methods. The expression levels of proangiogenic factors (VEGF and FGF-2) and an antiangiogenic inhibitor (endostatin) in the cerebral cortex were tested using a Western blot analysis. Running exercise significantly improved the rats' spatial learning and memory abilities and increased NOS activity in the cortex. Running exercise also subsequently improved the expression of proangiogenic factors (VEGF and FGF-2) and the length, volume and surface area of capillaries and reduced the expression of antiangiogenic factors (endostatin) in the cortex. In contrast, the L-NAME treatment attenuated the effects of running exercise. ConclusionsRunning exercise regulates proangiogenic factors, antiangiogenic factors and angiogenesis in the cerebral cortex via a partially NO-dependent mechanism, and influencing endogenous NO might potentially affect the exercise-related beneficial effects on cognitive ability and cortical capillaries.

Piezo1 Is a Mechanosensor Channel in Central Nervous System Capillaries.

Capillaries are equipped to sense neurovascular coupling agents released onto the outer wall of a capillary, translating these external signals into electrical/Ca2+ changes that play a crucial role in blood flow regulation and ensuring that neuronal demands are met. However, control mechanisms attributable to forces imposed onto the lumen are less clear. Here, we show that Piezo1 channels act as mechanosensors in central nervous system capillaries. Electrophysiological analyses confirmed expression and function of Piezo1 channels in brain cortical and retinal capillaries. Activation of Piezo1 channels evoked currents that were sensitive to endothelial cell-specific Piezo1 deletion. Using genetically encoded Ca2+ indicator mice and an ex vivo pressurized retina preparation, we found that activation of Piezo1 channels by mechanical forces triggered Ca2+ signals in capillary endothelial cells. Collectively, these findings indicate that Piezo1 channels are capillary mechanosensors that initiate crucial Ca2+ signals and could, therefore, have a profound impact on central nervous system blood flow control.

Pericyte-mediated constriction of renal capillaries evokes no-reflow and kidney injury following ischaemia.

Acute kidney injury is common, with ~13 million cases and 1.7 million deaths/year worldwide. A major cause is renal ischaemia, typically following cardiac surgery, renal transplant or severe haemorrhage. We examined the cause of the sustained reduction in renal blood flow ('no-reflow'), which exacerbates kidney injury even after an initial cause of compromised blood supply is removed. Adult male Sprague-Dawley rats, or NG2-dsRed male mice were used in this study. After 60 min kidney ischaemia and 30-60 min reperfusion, renal blood flow remained reduced, especially in the medulla, and kidney tubule damage was detected as Kim-1 expression. Constriction of the medullary descending vasa recta and cortical peritubular capillaries occurred near pericyte somata, and led to capillary blockages, yet glomerular arterioles and perfusion were unaffected, implying that the long-lasting decrease of renal blood flow contributing to kidney damage was generated by pericytes. Blocking Rho kinase to decrease pericyte contractility from the start of reperfusion increased the post-ischaemic diameter of the descending vasa recta capillaries at pericytes, reduced the percentage of capillaries that remained blocked, increased medullary blood flow and reduced kidney injury. Thus, post-ischaemic renal no-reflow, contributing to acute kidney injury, reflects pericytes constricting the descending vasa recta and peritubular capillaries. Pericytes are therefore an important therapeutic target for treating acute kidney injury.

Brain capillary obstruction during neurotoxicity in a mouse model of anti-CD19 chimeric antigen receptor T-cell therapy.

Immunotherapy for haematologic malignancies with CD19-directed chimeric antigen receptor T cells has been highly successful at eradicating cancer but is associated with acute neurotoxicity in ∼40% of patients. This neurotoxicity correlates with systemic cytokine release syndrome, endothelial activation and disruption of endothelial integrity, but it remains unclear how these mechanisms interact and how they lead to neurologic dysfunction. We hypothesized that dysfunction of the neurovascular unit is a key step in the development of neurotoxicity. To recapitulate the interaction of the intact immune system with the blood–brain barrier, we first developed an immunocompetent mouse model of chimeric antigen receptor T-cell treatment-associated neurotoxicity. We treated wild-type mice with cyclophosphamide lymphodepletion followed by escalating doses of murine CD19-directed chimeric antigen receptor T cells. Within 3–5 days after chimeric antigen receptor T-cell infusion, these mice developed systemic cytokine release and abnormal behaviour as measured by daily neurologic screening exams and open-field testing. Histologic examination revealed widespread brain haemorrhages, diffuse extravascular immunoglobulin deposition, loss of capillary pericyte coverage and increased prevalence of string capillaries. To measure any associated changes in cerebral microvascular blood flow, we performed in vivo two-photon imaging through thinned-skull cranial windows. Unexpectedly, we found that 11.9% of cortical capillaries were plugged by Day 6 after chimeric antigen receptor T-cell treatment, compared to 1.1% in controls treated with mock transduced T cells. The capillary plugs comprised CD45+ leucocytes, a subset of which were CD3+ T cells. Plugging of this severity is expected to compromise cerebral perfusion. Indeed, we found widely distributed patchy hypoxia by hypoxyprobe immunolabelling. Increased serum levels of soluble ICAM-1 and VCAM-1 support a putative mechanism of increased leucocyte–endothelial adhesion. These data reveal that brain capillary obstruction may cause sufficient microvascular compromise to explain the clinical phenotype of chimeric antigen receptor T-cell neurotoxicity. The translational impact of this finding is strengthened by the fact that our mouse model closely approximates the kinetics and histologic findings of the chimeric antigen receptor T-cell neurotoxicity syndrome seen in human patients. This new link between systemic immune activation and neurovascular unit injury may be amenable to therapeutic intervention.

Blood-brain barrier-restricted translocation of Toxoplasma gondii from cortical capillaries.

The cellular barriers of the central nervous system proficiently protect the brain parenchyma from infectious insults. Yet, the single-celled parasite Toxoplasma gondii commonly causes latent cerebral infection in humans and other vertebrates. Here, we addressed the role of the cerebral vasculature in the passage of T. gondii to the brain parenchyma. Shortly after inoculation in mice, parasites mainly localized to cortical capillaries, in preference over post-capillary venules, cortical arterioles or meningeal and choroidal vessels. Early invasion to the parenchyma (days 1-5) occurred in absence of a measurable increase in blood-brain barrier (BBB) permeability, perivascular leukocyte cuffs or hemorrhage. However, sparse focalized permeability elevations were detected adjacently to replicative parasite foci. Further, T. gondii triggered inflammatory responses in cortical microvessels and endothelium. Pro- and anti-inflammatory treatments of mice with LPS and hydrocortisone, respectively, impacted BBB permeability and parasite loads in the brain parenchyma. Finally, pharmacological inhibition or Cre/loxP conditional knockout of endothelial focal adhesion kinase (FAK), a BBB intercellular junction regulator, facilitated parasite translocation to the brain parenchyma. The data reveal that the initial passage of T. gondii to the central nervous system occurs principally across cortical capillaries. The integrity of the microvascular BBB restricts parasite transit, which conversely is exacerbated by the inflammatory response.

Dilation of cortical capillaries is not related to astrocyte calcium signaling.

The brain requires an adequate supply of oxygen and nutrients to maintain proper function as neuronal activity varies. This is achieved, in part, through neurovascular coupling mechanisms that mediate local increases in blood flow through the dilation of arterioles and capillaries. The role of astrocytes in mediating this functional hyperemia response is controversial. Specifically, the function of astrocyte Ca2+ signaling is unclear. Cortical arterioles dilate in the absence of astrocyte Ca2+ signaling, but previous work suggests that Ca2+ increases are necessary for capillary dilation. This question has not been fully addressed in vivo, however, and we have reexamined the role of astrocyte Ca2+ signaling in vessel dilation in the barrel cortex of awake, behaving mice. We recorded evoked vessel dilations and astrocyte Ca2+ signaling in response to whisker stimulation. Experiments were carried out on WT and IP3R2 KO mice, a transgenic model where astrocyte Ca2+ signaling is substantially reduced. Compared to WT mice at rest, Ca2+ signaling in astrocyte endfeet contacting capillaries increased by 240% when whisker stimulation evoked running. In contrast, Ca2+ signaling was reduced to 9% of WT values in IP3R2 KO mice. In all three conditions, however, the amplitude of capillary dilation was largely unchanged. In addition, the latency to the onset of astrocyte Ca2+ signaling lagged behind dilation onset in most trials, although a subset of rapid onset Ca2+ events with latencies as short as 0.15 s occurred. In summary, we found that whisker stimulation-evoked capillary dilations occurred independent of astrocyte Ca2+ increases in the cerebral cortex.

Contribution of Vascular Cell Adhesion Molecule to Hemodynamics in Sickle Cell Disease

Vaso-occlusive events (VOEs) and pain crises are common clinical features of sickle cell disease (SCD) that result from sickle-shaped erythrocytes and leukocytes blocking blood flow, particularly in small vessels (Kato 2018). Activated endothelium also plays a role in the pathogenesis of VOEs in SCD. For example, VCAM-1 is expressed on blood vessels after activation by chemical and/or mechanical stimulation, which results in cytokine release. Studies have shown that patients with SCD have higher steady-state serum levels of soluble VCAM-1 compared to controls and that these levels are elevated during VOEs (White 2020). Furthermore, overexpression of these adhesion molecules on the endothelium results in prolonged adherence of white blood cells (WBCs), which has been shown to contribute to the development of VOEs and possibly cerebral vasculopathy. These findings raise the need to explore further the role of aberrant WBC- and/or RBC-endothelial interaction, mediated via VCAM-1, in the pathophysiology of cerebral microvascular hemodynamics and vasculopathy leading to cerebral microinfarcts in SCD. Therefore, we hypothesized that sickle cell mice will show greater cerebral cortical expression of VCAM-1 compared with age-matched controls and that this deposition will be associated with significant evidence of abnormal cerebral microvascular hemodynamic abnormalities.To examine the relationship between abnormalities in cerebral microvascular hemodynamics and VCAM-1 deposition in the cerebral microvasculature, we utilized a humanized sickle cell (with HbSS) and corresponding control (with HbAA). After cranial-window procedures, cortical capillaries, precapillary arterioles, and post-capillary venules were imaged using two-photon microscopy at two time points. In addition, this experiment included pre-and post-transfusion groups as we intend to study the impact of blood transfusion on hemodynamics. Using custom-written but well-validated MATLAB scripts, we analyzed line scans to identify the number and duration of rolling or adherent WBCs and RBCs, the RBC velocity in cerebral microvasculature, and the frequency and magnitude (mL/sec) of cerebral microvascular blood flow reversals. Rolling WBCs were defined as lasting two seconds or more, and adherent RBCs were defined as lasting 0.5 seconds or more. To quantify the expression of VCAM-1, we used immunohistochemistry to stain 50-micron sections of brain tissue for VCAM-1, Lectin to localize the vasculature, and Neun to localize neuronal nuclei. Images were analyzed using Phenochart and ImageJ software to examine the deposition of VCAM-1 throughout the brain tissue.As shown in Figure 1, at the first time point (baseline), we observed a significantly higher maximum RBC velocity (p<0.001) in the sickle cell mice compared to controls (figure 1a). We also found that there was significantly higher expression of VCAM-1 (p<0.001) (figure 1b) as well as significantly more leukocyte rolling (p<0.001) (figure 1c) in the sickle cell mice compared to controls. Additionally, we noted that the sickle cell mice have a significantly higher frequency of blood flow reversals (p<0.01) (figure 1d) as well as higher magnitude of microvascular blood flow reversals (p<0.001) (figure 1e) compared to controls. Interestingly, the sickle cell mice have a slightly lower average or mean capillary blood flow velocity compared to control (figure 1f), but this was not statistically significant (p=0.079). Since the mean capillary velocity is obtained as a smoothened difference between the forward flow and reversals, this decrease was surprising given the significant differences in frequency and magnitude of microvascular blood flow reversal in the sickle cell mice compared to controls (figs 1d and 1e).In conclusion, we see that the high velocity of blood flow might be a mechanical force, among other factors contributing to cerebral microvascular VCAM-1 expression in sickle cell mice. This might be responsible for the increased leukocyte-endothelial interactions and adhesion, ultimately leading to higher frequency and magnitude of cerebral microvascular blood flow reversal. Taken together, this may contribute to the observed slightly lower mean or effective microvascular forward blood flow. These pathophysiological changes might contribute to the reported higher rate of cerebral microinfarct and silent infarct in sickle cell disease. [Display omitted] DisclosuresArcher: Global Blood Therapeutics: Consultancy, Research Funding; Forma Therapeutics: Research Funding. Hyacinth: Novartis: Consultancy; Acuta Capital: Consultancy.

Pathomorphological changes of capillaries in the cerebral cortex in type 2 diabetes mellitus

The aim of the study was to determine microscopic, immunohistochemical, electron microscopic, morphometric parameters of capillaries in the cerebral cortex in deceased patients with type 2 diabetes mellitus (DM) in comparison with the conditional control group and the group of deceased patients with dyscirculatory ischemic encephalopathy. Materials and methods. Microscopic, immunohistochemical, electron microscopic and morphometric studies of the cerebral cortex capillaries were performed in 3 groups: group I – 20 deceased patients with type 2 DM, group II conditional control (CC) – 20 deceased patients without clinical morphological signs of DM and cerebrovascular pathology, group III – 20 patients with dyscirculatory ischemic encephalopathy (DIEP). Results. It was found that in type 2 DM, the capillaries in the cerebral cortex lose pericytes due to their apoptosis: the number of pericytes in the cortical capillaries in type 2 DM was statistically significantly lower by 57.14 % compared to CC group and 50.00 % lower compared to DIEP group, the level of caspase-3 expression in the cortical microvessels in type 2 DM was significantly higher by 230.85 % compared with CC group and 81.67 % higher than in DIEP group. According to electron microscopy in type 2 DM, apoptosis of pericytes and single endothelial cells in the cerebral cortex capillaries was determined as well as significant expansion of basement membranes with the accumulation of electron-dense amorphous material and collagen fibrils. According to the results of morphometry, the outer diameter of the cortical capillaries in type 2 DM group was 4.90 % significantly larger, the inner diameter was 9.78 % smaller and the walls were 66.62 % thicker (compared with CC group) due to the accumulation of PAS-positive substances of blood serum and fibrosis, confirmed by 22.96 % greater area of type IV collagen expression in the microvessel walls. Conclusions. The pathomorphological changes of microvessels identified in deceased patients with type 2 diabetes mellitus are signs of diabetic cerebral microangiopathy.

Loss with ageing but preservation of frontal\xa0cortical capillary pericytes in post-stroke dementia, vascular dementia and Alzheimer\u2019s disease

Cerebral pericytes are an integral component of the neurovascular unit, which governs the blood–brain barrier. There is paucity of knowledge on cortical pericytes across different dementias. We quantified cortical pericytes in capillaries in 124 post-mortem brains from subjects with post-stroke dementia (PSD), vascular dementia (VaD), Alzheimer’s disease (AD) and AD-VaD (Mixed) and, post-stroke non-demented (PSND) stroke survivors as well as normal ageing controls. Collagen 4 (COL4)-positive nucleated pericyte soma were identified as protrusions on capillaries of the frontal cortex. The COL4-positive somata or nodule-like cell bodies were also verified by platelet derived growth factor receptor-β (PDGFR-β) immunohistochemistry. The mean (± SEM) pericyte somata in frontal cortical capillaries in normal young controls (46–65 years of age) was estimated as 5.2 ± 0.2 per mm capillary length. This number was reduced by 45% in older controls (> 78 years) to 2.9 ± 0.1 per mm capillary length (P < 0.001). We further found that the numbers of pericyte cell bodies per COL4 mm2 area or per mm capillary length were not decreased but rather preserved or increased in PSD, AD and Mixed dementia groups compared to similar age older controls (P < 0.01). Consistent with this, we noted that capillary length densities identified by the endothelial marker glucose transporter 1 or COL4 were not different across the dementias compared to older controls. There was a negative correlation with age (P < 0.001) suggesting fewer pericyte somata in older age, although the % COL4 immunoreactive capillary area was increased in older controls compared to young controls. Using a proven reliable method to quantify COL4-positive nucleated pericytes, our observations demonstrate ageing related loss but mostly preserved pericytes in the frontal cortex of vascular and AD dementias. We suggest there is differential regulation of capillary pericytes in the frontal lobe between the cortex and white matter in ageing-related dementias.

Matcha and Its Components Control Angiogenic Potential.

The brain needs the appropriate capillary networks to maintain normal brain function. Since previous studies showed age-related decrease in the cortical capillaries, it is suggested that protection against capillary aging is critical for maintaining brain function. Epidemiological studies have indicated that brain functions were protected from age-related decline by the long-term consumption of matcha. However, whether matcha has protective effects on capillary aging has not been studied yet. In this study, we utilized Flt1-DsR mice that expressed a red fluorescent protein in vascular endothelial cells to visualize cortical capillaries clearly. We found that cortical capillary density decreased in aging Flt1-DsR mice. Our results of the aortic ring assay and tube formation assay revealed that matcha and its components vitamin K1 and lutein, which are abundant in matcha powder, enhanced the angiogenic potential. Moreover, we evaluated the effect of long-term ingestion of matcha on mouse cortical capillary aging by using imaging experiments. The capillary density of the Flt1-DsR mice, which were fed matcha-containing food, indicated the protective effects of matcha ingestion on capillary aging in a limited cortical layer. These results suggest that biological regulation of matcha and its components affect the angiogenic potential, which is related to the prevention of capillary aging.