IgG Extravasation Research Articles

Histo-ELISA technique for quantification and localization of tissue components

A novel Histo-ELISA technique is intended to facilitate quantification of target tissue proteins in a tissue section and involves the selection of target regions in the tissue section, application of streptavidin-conjugated HRP (horseradish peroxidase), coupled with peroxidase substrate—TMB (3,3′,5,5′-tetramethylbenzidine), and staining dye evaluation with ELISA reader. The target protein content (weight per volume unit) was translated from optical densities by a reference standard curve, obtained via parallel staining of the targeted protein-coated slides. To validate the technique, we carried out quantifications of IgG extravasation in ischemic and nonischemic brain sections in a mouse stroke model. With those obtained data and the reference of immunohistochemistry scores assessed on the adjacent sections, accuracy, sensitivity, and precision for the technique were evaluated. For all evaluated parameters, Histo-ELISA performance was either comparable to or better than the standard immunohistochemistry. A comparison with the data from the repeated measurements yielded a rather low coefficient of variation. The results confirmed that the technique is a fairly reliable quantitative test with rather high sensitivity, accuracy, precision, and reproducibility for detecting target protein content in tissue sections and that its tissue distribution and related subsequent morphological changes can be observed at the same time.

Desert Hedgehog-Driven Endothelium Integrity Is Enhanced by Gas1 (Growth Arrest-Specific 1) but Negatively Regulated by Cdon (Cell Adhesion Molecule-Related/Downregulated by Oncogenes).

Evidences accumulated within the past decades identified hedgehog signaling as a new regulator of endothelium integrity. More specifically, we recently identified Dhh (desert hedgehog) as a downstream effector of Klf2 (Kruppel-like factor 2) in endothelial cells (ECs). The purpose of this study is to investigate whether hedgehog coreceptors Gas1 (growth arrest-specific 1) and Cdon (cell adhesion molecule-related/downregulated by oncogenes) may be used as therapeutic targets to modulate Dhh signaling in ECs. Approach and Results: We demonstrated that both Gas1 and Cdon are expressed in adult ECs and relied on either siRNAs- or EC-specific conditional knockout mice to investigate their role. We found that Gas1 deficiency mainly phenocopies Dhh deficiency especially by inducing VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) overexpression while Cdon deficiency has opposite effects by promoting endothelial junction integrity. At a molecular level, Cdon prevents Dhh binding to Ptch1 (patched-1) and thus acts as a decoy receptor for Dhh, while Gas1 promotes Dhh binding to Smo (smoothened) and as a result potentiates Dhh effects. Since Cdon is upregulated in ECs treated by inflammatory cytokines, including TNF (tumor necrosis factor)-α and Il (interleukin)-1β, we then tested whether Cdon inhibition would promote endothelium integrity in acute inflammatory conditions and found that both fibrinogen and IgG extravasation were decreased in association with an increased Cdh5 (cadherin-5) expression in the brain cortex of EC-specific Cdon knockout mice administered locally with Il-1β. Altogether, these results demonstrate that Gas1 is a positive regulator of Dhh in ECs while Cdon is a negative regulator. Interestingly, Cdon blocking molecules may then be used to promote endothelium integrity, at least in inflammatory conditions.

Abstract P088: The Gut Microbiome Contributes To The Cerebral Small Vessel Disease Phenotype In Spontaneously Hypertensive Stroke Prone Rats

In recent years, it has become apparent that the gut microbiome can influence the functioning and pathological states of organs and systems throughout the body. In this study we tested the hypothesis that the gut microbiome has a major role in the cascade of pathological events, including inflammation and hypertension, leading to the onset of cerebral small vessel disease (CSVD). To test this hypothesis, we used an animal model for hypertensive CSVD, the spontaneously hypertensive stroke prone rat (SHRSP). At birth, SHRSP pups were placed with foster dams of the SHRSP strain or dams of the WKY strain, the control strain that does not develop hypertensive CSVD. Similarly, WKY pups were placed with dams of the same or opposite strain. The rationale for cross fostering is that gut microbiomes are shaped by environmental bacteria of the foster dam and the nesting surroundings. Analysis of the bacterial genera in feces using 16S rRNA analysis demonstrated that the gut microbiome in the rat pups was strongly influenced by the foster dam. SHRSP offspring fostered on WKY dams had systolic blood pressures (SBPs) that were significantly decreased by 26 mmHg (P<0.001, N=5-7/group) at 20 weeks, compared to SHRSP offspring fostered on SHRSP dams. Similarly, WKY offspring fostered on SHRSP dams had significantly increased SBP compared to WKY offspring fostered on WKY dams, although the magnitude of SBP change was not as robust (9 mmHg, P<0.05, N=6-8/group). At ~20 weeks of age, rats fostered on SHRSP dams showed increased expression of IL-1a, TLR-2, E-CAD, Muc-2, and Il-17a in ileum regardless of the strain of the offspring (N=6/group, P<0.05). Loss of blood-brain barrier (BBB) integrity, an early marker of CSVD onset, was assessed by extravasation of IgG, which is confined to the plasma space under normal conditions. Extravasation of IgG was increased four-fold in SHRSP fostered on SHRSP dams compared to WKY rats fostered on WKY dams; however, extravasation of IgG was decreased in SHRSP fostered on WKY dams by ~ 50% (P=0.005, N=5/group). These findings demonstrate that although SHRSP is a genetic model for CSVD, the makeup of the gut microbiota has a major influence on gut and brain physiology, and ultimately in shaping the phenotype and onset of CSVD.

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Induce Ischemic Neuroprotection by Modulating Leukocytes and Specifically Neutrophils.

Background and Purpose- Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods- Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV-treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results- sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O2) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O2) but also reduced serum IgG extravasation-a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions- Leukocytes and specifically PMNs contribute to MSC-sEV-induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview- An online visual overview is available for this article.

Cerebral venous congestion promotes blood‐brain barrier disruption and neuroinflammation, impairing cognitive function in mice.

Cognitive impairment is one of the most common co‐occurring chronic conditions among elderly heart failure patients (incidence: up to ~ 80%); however, the underlying mechanisms are not completely understood. It is hypothesized that in addition to decreased cardiac output, increases in central‐and consequentially, cerebral‐venous pressure (backward failure) also contribute significantly to the genesis of cognitive impairment. To test this hypothesis and elucidate the specific pathogenic role of venous congestion in the brain, we have established a novel model of increased cerebral venous pressure: mice with jugular vein ligation (JVL). To test the hypothesis that increased venous pressure in the brain contributes to the development of cognitive deficits by causing blood‐brain barrier disruption, dysregulation of blood flow, and/or promoting neuroinflammation, in C57BL/6 mice, the internal and external jugular veins were ligated. Cognitive function (radial arm water maze), gait function (CatWalk), and motor coordination (rotarod) were tested post‐JVL. Neurovascular coupling responses were assessed by measuring changes in cerebral blood flow in the whisker barrel cortex in response to contralateral whisker stimulation by laser speckle contrast imaging through a closed cranial window. Blood‐brain barrier integrity (IgG extravasation) and microglia activation (Iba1 staining) were assessed in brain slices by immunohistochemistry. Neuroinflammation‐related gene expression profile was assessed by a targeted qPCR array. After jugular vein ligation, mice exhibited impaired spatial learning and memory, altered motor coordination, and impaired gait function, mimicking important aspects of altered brain function observed in human heart failure patients. JVL did not alter neurovascular coupling responses. In the brains of mice with JVL, significant extravasation of IgG was detected, indicating blood‐brain barrier disruption, which was associated with histological markers of neuroinflammation (increased presence of activated microglia) and a proinflammatory shift in gene expression profile. Thus, cerebral venous congestion per se can cause blood‐brain barrier disruption and neuroinflammation, which likely contribute to the genesis of cognitive impairment. These findings have relevance to the pathogenesis of cognitive decline associated with heart failure as well as increased cerebal venous pressure due to increased jugular venous reflux in elderly human patients.Support or Funding InformationThis work was supported by grants the Oklahoma Center for the Advancement of Science and Technology, the Oklahoma Shared Clinical and Translational Resources (OSCTR) program funded by the National Institute of General Medical Sciences (GM104938), the NIA‐supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), and the NIGMS supported Center of Biomedical Research Excellence (CoBRE)..

β-hydroxybutyrate Impedes the Progression of Alzheimer's Disease and Atherosclerosis in ApoE-Deficient Mice.

β-hydroxybutyrate (β-OHB) has been shown to exert an anti-inflammatory activity. Apolipoprotein-E (ApoE) is strongly associated with atherosclerosis and Alzheimer’s disease (AD). This study aimed to explore the therapeutic effect of β-OHB in the brain and the aorta of high-fat diet (HFD)-fed ApoE-deficient mice. We found in Apo-E deficient mice that β-OHB attenuated lipid deposition in the choroid plexus (ChP) and decreased amyloid plaque in the substantia nigra pars compacta. We also found decreased CD68-positive macroglia infiltration of the ChP in β-OHB-treated ApoE-deficient mice. β-OHB treatment ameliorated IgG extravasation into the hippocampal region of the brain. In vitro study using ChP mice cell line revealed that β-OHB attenuated oxidized low-density lipoprotein-induced ApoE-specific differentially expressed inflammatory ChP genes. Treatment with β-OHB reduced aortic plaque formation without affecting blood lipid profiles and decreased serum production of resistin, a well-established risk factor for both AD and atherosclerosis. Thus, the current study suggests and describes the therapeutic potential of β-OHB for the treatment of AD and atherosclerosis.

EXTH-35. LASER INTERSTITIAL THERMAL THERAPY INCREASES BLOOD-BRAIN BARRIER AND BLOOD-TUMOR BARRIER PERMEABILITY IN A MOUSE MODEL OF GLIOBLASTOMA

Abstract INTRODUCTION A central challenge in glioblastoma treatment is the presence of the blood-brain barrier (BBB) and blood-tumor barrier (BTB), which prevent access of drugs to the brain and tumor respectively. Recent evidence in patients suggests laser interstitial thermal therapy (LITT), used clinically for tumor ablation, locally disrupts BBB integrity, potentially creating a therapeutic window to deliver otherwise brain-impermeant agents. METHODS A mouse model for LITT, established using a Nd-YAG laser coupled to a 600 mm fiber optic and thermocouple probe, was inserted via burrhole to target the somatosensory cortex. Syngeneic GL261 tumor cells were stereotactically implanted prior to LITT. BBB and BTB permeability were assessed through measurement of fluorescein and doxorubicin after IV injection. Permeability of IV dextran (10 and 70 kDa) and human IgG was monitored by immunohistochemistry (IHC) analysis. Mechanisms of BBB breakdown in vivo were explored utilizing electron microscopy and IHC. RESULTS By fluorescein assay, LITT-induced BBB and BTB permeability began one day post-treatment and was sustained for at least 2 weeks. Additionally, both normal brain and brain tumors demonstrated an increase in Dextran 10 kDa, Dextran 70 kDa, and human IgG extravasation after IV injection in vivo. Mechanistically, we provide evidence that LITT triggers both a decrease in tight junction integrity and an increase in brain endothelial cell transcytosis. As proof-of-concept that LITT can enhance tumor delivery of systemic drugs, LITT increased IV doxorubicin permeability in brain in vivo. Moreover, LITT plus doxorubicin significantly increased survival in brain tumor-bearing mice compared to doxorubicin or LITT alone. CONCLUSIONS Our data suggest that LITT increases BBB and BTB permeability over a defined time window to large molecular weight agents, including antibodies, through multiple cellular mechanisms. Our preclinical results with LITT plus doxorubicin, which mirror a current clinical trial, indicate LITT can enhance the efficacy of systemically delivered drugs.

Neonatal cerebral hypoxia-ischemia in mice triggers age-dependent vascular effects and disabilities in adults; implication of tissue plasminogen activator (tPA)

Neonatal encephalopathy frequently results from hypoxia-ischemia (HI) or inflammation in preterm or term neonates. Neuropathology depends on cerebral development at insult time, but the poor correlation of neuromotor, cognitive, and behavioral disabilities in infancy with initial imaging and clinical records precludes early prognosis. The Rice–Vannucci HI procedure was applied to wild type and tissue plasminogen activator knockout (tPA-KO) mice as surrogates for human preterm (with five-day-old postnatal (P5) mice) or human term (with ten-day-old postnatal (P10) mice). Acute and delayed T2-magnetic resonance imaging (T2-MRI) signals and cognitive deficits in adulthood (spatial memory and social interaction) were investigated in the same animals. Early vascular tPA and matrix metalloproteinase-9 (MMP-9) activities, blood–brain barrier permeability to water or IgG, and microglial activation were assessed separately.HI in P5 or P10 mice induced early hemisphere swelling in T2-MRI scans, and a delayed atrophy of the cortex and hippocampus, but affected white matter in the P5 group only, irrespective of the wild type or tPA-KO genotype. Adults had no motor disabilities, but we did find HI-induced age-dependent deficits, preferentially social interaction and activity in P5 mice, and spatial learning in P10 mice. In P5 mice, tPA-KO prevented MMP-9 activation, IgG extravasation, microglial activation, and behavior impairments. In P10 mice, MMP-9 activation and inflammatory processes remained in the hippocampus of the tPA-KO group, and also contributed to persistent spatial learning deficits.Perinatal HI in mice mimicked the unpredictability of outcomes from imaging in human clinics. Delayed deficits appeared associated to vascular dysfunction-induced inflammation, which recalls our previous work showing major vascular maturation between P5 and P10 stages. Using omics to explore neural, glial, or brain vessel markers in neonate blood may be a promising perspective to identify pertinent prognostic tools.

Glimepiride and glibenclamide have comparable efficacy in treating acute ischemic stroke in mice

Glibenclamide protects against ischemic injury in both preclinical and clinical studies, presumably by blocking the de novo assembled sulfonylurea receptor 1-transient receptor potential M4 (Sur1-Trpm4) channel induced by ischemia. However, glibenclamide may cause unexpected serious hypoglycemia. Here, we tested whether glimepiride, another sulfonylurea with better safety, has comparable efficacy with glibenclamide and whether gene deletion of Trpm4 (Trpm4−/−) exerts similar effect. Wild-type (WT) mice subjected to temporary middle cerebral artery occlusion (tMCAO) were randomized to receive glibenclamide (an initial dose of 10 μg/kg and additional doses of 1.2 μg every 8 h), three different doses of glimepiride (10 μg/kg, 100 μg/kg and 1 mg/kg) or vehicle after ischemia, while tMCAO-treated Trpm4−/− mice were randomized to receive vehicle or glimepiride. Neurological function, infarct volume, edema formation, the integrity of blood-brain barrier and inflammatory reaction were evaluated at 24 h after ischemia. In tMCAO-treated WT mice, 10 μg/kg and 100 μg/kg glimepiride had comparable efficacy with glibenclamide in improving longa score and grip test score, reducing infarct volume, mitigating brain edema, lessening extravasation of Evans blue dye and IgG, restoring tight junction protein expression as well as suppressing inflammatory cytokines. Compared with WT mice, Trpm4−/− mice showed less neurological deficit, smaller cerebral infarction, lighter brain edema and more integrity of blood-brain barrier. As expected, glimepiride did not provide additional neuroprotection compared with vehicle in the tMCAO-treated Trpm4−/− mice. Glimepiride shows comparable efficacy with glibenclamide in alleviating brain injury after ischemic stroke in mice, possibly via targeting the Sur1-Trpm4 channel.

2287Endothelial SIRT6 exerts a beneficial role in cerebral ischemia/reperfusion injury by preserving blood-brain barrier integrity

Abstract Introduction Stroke is a major cause of mortality and morbidity worldwide. Yet, therapeutic strategies are limited to the early reperfusion which can, on the other hand, worsen the brain damage trough ischemia/reperfusion (I/R) injury. Post-stroke blood-brain barrier (BBB) impairment is associated with worsened outcome. Aging is a major risk factor for stroke and genes regulating lifespan also contribute to the determination of cerebral damage during I/R injury. Purpose Given the pivotal role of endothelial cells in BBB, we hypothesized that the endothelial-specific expression of the longevity gene SIRT6 may protect the BBB from ischemia/reperfusion damage thus having a beneficial role on stroke outcome. Methods Endothelial-specific SIRT6 knockout (eSIRT6−/−) mice and control littermates (CTRL) underwent transient middle cerebral artery occlusion (tMCAO) for 45 min followed by 48 hours of reperfusion. Immunohistochemistry (IHC) was used to investigate BBB permeability by IgG extravasation and molecular mechanisms. Primary human brain microvascular endothelial cells (HBMVECs) transfected with either SIRT6 (siSIRT6) or scrambled (siSCR) small interfering RNA were subjected to hypoxia/reoxygenation (H/R). An in vitro BBB model consisting of a monolayer of siRNA-treated HBMVECs was established and barrier function was assessed by 48 h-lasting transendothelial electrical resistance measurement. SIRT6 expression in monocytes from stroke patients was correlated with the short-term neurological outcome [ΔNIHSS% = (NIHSS discharge-NIHSS admission)/ NIHSS admission*100]. Results eSIRT6−/− displayed higher infarct volumes and lower survival rate compared to WT mice 48 h after tMCAO. The increased infarct volume was functionally relevant as eSIRT6−/− also showed worse post-stroke neurological impairment. Analysis of brain sections revealed increased BBB damage and increased endothelial expression of cleaved caspase-3 in eSIRT6−/− as compared to control littermates. In vitro, H/R reduced SIRT6 expression in HBMVECs. Mirroring the animal results, SIRT6 silencing impaired the barrier function of HBMVECs 48 h after exposure to H/R. In line with this, SIRT6-silenced HBMVECs showed reduced viability, increased cleaved caspase-3 expression and reduced activation of the anti-apoptotic survival pathway Akt as compared to control cells after H/R. The direct interaction between SIRT6 and Akt was confirmed by co-immunoprecipitation. In ischemic stroke patients, SIRT6 expression was higher in those with short-term neurological improvement (ΔNIHSS% >0) and negatively correlated with ΔNIHSS%. Conclusion Endothelial SIRT6 exerts a beneficial role in ischemic stroke by blunting I/R-mediated BBB damage. Specifically, SIRT6 reduces endothelial I/R-induced apoptotic death through activation of the protective Akt pathway. The longevity gene SIRT6 may represent a novel therapeutic target for the treatment of ischemic stroke. Acknowledgement/Funding Swiss National Science Foundation

Cerebral venous congestion promotes blood-brain barrier disruption and neuroinflammation, impairing cognitive function in mice.

Cognitive impairment is one of the most common co-occurring chronic conditions among elderly heart failure patients (incidence: up to ~ 80%); however, the underlying mechanisms are not completely understood. It is hypothesized that in addition to decreased cardiac output, increases in central-and consequentially, cerebral-venous pressure (backward failure) also contribute significantly to the genesis of cognitive impairment. To test this hypothesis and elucidate the specific pathogenic role of venous congestion in the brain, we have established a novel model of increased cerebral venous pressure: mice with jugular vein ligation (JVL). To test the hypothesis that increased venous pressure in the brain contributes to the development of cognitive deficits by causing blood-brain barrier disruption, dysregulation of blood flow, and/or promoting neuroinflammation, in C57BL/6 mice, the internal and external jugular veins were ligated. Cognitive function (radial arm water maze), gait function (CatWalk), and motor coordination (rotarod) were tested post-JVL. Neurovascular coupling responses were assessed by measuring changes in cerebral blood flow in the whisker barrel cortex in response to contralateral whisker stimulation by laser speckle contrast imaging through a closed cranial window. Blood-brain barrier integrity (IgG extravasation) and microglia activation (Iba1 staining) were assessed in brain slices by immunohistochemistry. Neuroinflammation-related gene expression profile was assessed by a targeted qPCR array. After jugular vein ligation, mice exhibited impaired spatial learning and memory, altered motor coordination, and impaired gait function, mimicking important aspects of altered brain function observed in human heart failure patients. JVL did not alter neurovascular coupling responses. In the brains of mice with JVL, significant extravasation of IgG was detected, indicating blood-brain barrier disruption, which was associated with histological markers of neuroinflammation (increased presence of activated microglia) and a pro-inflammatory shift in gene expression profile. Thus, cerebral venous congestion per se can cause blood-brain barrier disruption and neuroinflammation, which likely contribute to the genesis of cognitive impairment. These findings have relevance to the pathogenesis of cognitive decline associated with heart failure as well as increased cerebal venous pressure due to increased jugular venous reflux in elderly human patients.

Integrin α5β1 inhibition by ATN-161 reduces neuroinflammation and is neuroprotective in ischemic stroke

Stroke remains a leading cause of death and disability with limited therapeutic options. Endothelial cell β1 integrin receptors play a direct role in blood-brain barrier (BBB) dysfunction through regulation of tight junction proteins and infiltrating leukocytes, potentially mediated by β1 integrins. Following tandem transient common carotid artery/middle cerebral artery occlusion on wild-type mice, we administered the integrin a5b1 inhibitor, ATN-161, intraperitoneal (IP) injection at 1 mg/kg acutely after reperfusion, on post-stroke day (PSD)1 and PSD2. Systemic changes (heart rate, pulse distension, and body temperature) were determined. Additionally, infarct volume and edema were determined by 2,3-triphenyltetrazolium chloride and magnetic resonance imaging, while neurological changes were evaluated using an 11-point Neuroscore. Brain immunohistochemistry was performed for claudin-5, α5β1, IgG, and CD45 + cells, and quantitative polymerase chain reaction (qPCR) was performed for matrix metalloproteinase-9 (MMP-9), interleukin (IL)-1β, collagen IV, and CXCL12. ATN-161 significantly reduced integrin α5β1 expression in the surrounding peri-infarct region with no systemic changes. Infarct volume, edema, and functional deficit were significantly reduced in ATN-161-treated mice. Furthermore, ATN-161 treatment reduced IgG extravasation into the parenchyma through conserved claudin-5, collagen IV, CXCL12 while reducing MMP-9 transcription. Additionally, IL-1β and CD45 + cells were reduced in the ipsilateral cortex following ATN-161 administration. Collectively, ATN-161 may be a promising novel stroke therapy by reducing post-stroke inflammation and BBB permeability.

Abl family tyrosine kinases govern IgG extravasation in the skin in a murine pemphigus model

The pathway of homeostatic IgG extravasation is not fully understood, in spite of its importance for the maintenance of host immunity, the management of autoantibody-mediated disorders, and the use of antibody-based biologics. Here we show in a murine model of pemphigus, a prototypic cutaneous autoantibody-mediated disorder, that blood-circulating IgG extravasates into the skin in a time- and dose-dependent manner under homeostatic conditions. This IgG extravasation is unaffected by depletion of Fcγ receptors, but is largely attenuated by specific ablation of dynamin-dependent endocytic vesicle formation in blood endothelial cells (BECs). Among dynamin-dependent endocytic vesicles, IgG co-localizes well with caveolae in cultured BECs. An Abl family tyrosine kinase inhibitor imatinib, which reduces caveolae-mediated endocytosis, impairs IgG extravasation in the skin and attenuates the murine pemphigus manifestations. Our study highlights the kinetics of IgG extravasation in vivo, which might be a clue to understand the pathological mechanism of autoantibody-mediated autoimmune disorders.

Laser Therapy Enhances Blood-Brain Barrier and Blood-Tumor Barrier Permeability in a Mouse Model of Glioblastoma

Abstract INTRODUCTION A main challenge in the treatment of glioblastoma is the blood-brain barrier (BBB). In this study, we established a laser interstitial thermotherapy (LITT) platform in mice to test the hypothesis that LITT can increase BBB and blood-tumor barrier (BTB) permeability, which could potentially be exploited to enhance delivery of antitumor agents. METHODS We established a preclinical mouse model for LITT using an Nd-YAG laser coupled to a 600 μm fiber optic and thermocouple probe in C57BL/6 mice. Both the fiber optic and temperature probes were inserted through a small burrhole to target somatosensory cortex. In other experiments, mice were stereotactically implanted with syngeneic GL261 tumor cells prior to LITT. Post-LITT, brain tissue was assessed for BBB and BTB permeability through quantitative measurement of brain fluorescein (FL) after intraperitoneal injection and immunohistochemistry (IHC) after intravenously injected dextran (MWs 10 and 70 kDa) and human IgG (MW 150 kDa) to define spatial characteristics. Electron microscopy and IHC was used to delineate the mechanisms of BBB break down. RESULTS Using BBB permeability tracers, noninjected mice and mice orthotopically implanted with GL261 tumor cells were used to characterize the effect of laser therapy on the BBB and BTB, respectively. We found LITT substantially disrupts the BBB in the first day post-treatment and is sustained for 4 wk. Additionally, tumor-implanted mice demonstrated an increase in Dextran 10 and 70 kDa and human IgG extravasation after IV injection of these tracers. Mechanistically, we provide evidence, by IHC and electron microscopy, that LITT triggers both a decrease in tight junction integrity and an increase in brain endothelial cell transcytosis to increase overall BBB permeability. CONCLUSION Our data suggest that LITT increases BBB and BTB permeability over a defined time window in a mouse model and that large molecular size agents, including human IgG, can infiltrate brain parenchyma and brain tumors after systemic administration. Future experiments will focus on testing delivery of antitumor agents following LITT to enhance survival in brain tumor-bearing mice.

Seizures Are Associated with Blood-Brain Barrier Disruption in a Piglet Model of Neonatal Hypoxic-Ischaemic Encephalopathy

Seizures in the neonatal period are most often symptomatic of central nervous system (CNS) dysfunction and the most common cause is hypoxic-ischaemic encephalopathy (HIE). Seizures are associated with poor long-term outcomes and increased neuropathology. Blood-brain barrier (BBB) disruption and inflammation may contribute to seizures and increased neuropathology but are incompletely understood in neonatal HIE. The aim of this study was to investigate the impact of seizures on BBB integrity in a preclinical model of neonatal hypoxic-ischaemic (HI) injury. Piglets (age: <24 h) were subjected to a 30-min HI insult followed by recovery to 72 h post-insult. Amplitude-integrated electroencephalography (aEEG) was performed and seizure burden and background aEEG pattern were analysed. BBB disruption was evaluated in the parietal cortex and hippocampus by means of immunohistochemistry and Western blot. mRNA and protein expression of tight-junction proteins (zonula-occludens 1 [ZO1], occludin [OCLN], and claudin-5 [CLDN5]) was assessed using quantitative polymerase chain reaction (qPCR) and Western blot. In addition, mRNA from genes associated with BBB disruption vascular endothelial growth factor (VEGF) and matrix metalloproteinase 2 (MMP2) as well as inflammatory cytokines and chemokines was assessed with qPCR. Piglets that developed seizures following HI (HI-Sz) had significantly greater injury, as demonstrated by poorer aEEG background pattern scores, lower neurobehavioural scores, and greater histopathology. HI-Sz animals had severe IgG extravasation into brain tissue and uptake into neurons as well as significantly greater levels of IgG in both brain regions as assessed by Western blot. IgG protein in both brain regions was significantly associated with seizure burden, aEEG pattern scores, and neurobehavioural scores. There was no difference in mRNA expression of the tight junctions, however a significant loss of ZO1 and OCLN protein was observed in the parietal cortex. The inflammatory genes TGFβ, IL1β, IL8, IL6, and TNFα were significantly upregulated in HI-Sz animals. MMP2 was significantly increased in animals with seizures compared with animals without seizures. Increasing our understanding of neuropathology associated with seizure is vital because of the association between seizure and poor outcomes. Investigating the BBB is a major untapped area of research and a potential avenue for novel treatments.

Multimodal Imaging Analyses of Brain Hippocampal Formation Reveal Reduced Cu and Lipid Content and Increased Lactate Content in Non-Insulin-Dependent Diabetic Mice.

Non-insulin-dependent diabetes mellitus (NIDDM) is reported to increase the risk of cognitive impairment and dementia. However, the underlying mechanisms are not fully understood. While the brain homeostasis of metals and lipids is pivotal to maintaining energy metabolism and redox homeostasis for healthy brain function, no studies have reported hippocampal metal and biochemical changes in NIDDM. Therefore, we here utilized direct spectroscopic imaging to reveal the elemental distribution within the hippocampal subregions of an established murine model of NIDDM, db/db mice. In 26-week-old insulin resistant db/db mice, X-ray fluorescence microscopy revealed that the Cu content within the dentate gyrus and CA3 was significantly greater than that of the age-matched nondiabetic control mice. In addition, Fourier transform infrared (FTIR) spectroscopy analysis indicated a significant increase in the abundance of lactate within the corpus callosum (CC), dentate gyrus, CA1, and CA3 regions of diabetic db/db mice compared to that of the control, indicating altered energy metabolism. FTIR analysis also showed a significant decrease in the level of lipid methylene and ester within the CC of db/db mice. Furthermore, immunomicroscopy analyses demonstrated the increase in the level of glial fibrillary acidic protein expression and peri-vascular extravasation of IgG, indicating astrogliosis and blood-brain barrier dysfunction, respectively. These data suggest that astrogliosis-induced alterations in the supply of Cu, lipids, and energy substrates may be involved in the mechanisms of NIDDM-associated cognitive decline.

Abstract TMP104: Upregulation of Tissue-Nonspecific Alkaline Phosphatase in Endothelial Cells Leads to Intracranial Vascular Calcification: A Useful Model of Primary Familial Brain Calcification

Introduction: Vascular calcification is an important pathophysiological factor contributing to neurodegenerative diseases such as primary familial brain calcification (PFBC) and is a potential therapeutic target. Hypothesis: Given the essential role of tissue-nonspecific alkaline phosphatase (TNAP) in biomineralization, we tested the hypothesis that upregulation of TNAP activity can lead to intracranial calcification. Methods: We previously reported that overexpression of TNAP in endothelial cells (eTNAP) leads to arterial calcification. Here we analyzed intracranial calcification in eTNAP on the original B6;129 and on the C57BL/6 (B6) genetic backgrounds. Histology was performed on formalin-fixed cryo-preserved tissues. Locomotion testing was performed at 23 weeks and analyzed in Matlab. Gene expression was analyzed by qPCR. Results: On the B6;129 background, eTNAP mice developed progressive intracranial calcification (0% were affected at 8 weeks, 71% at 13 weeks, and 100% at 23 weeks, n=7 per group). At 23 weeks, calcification was undetectable in the middle cerebral arteries but was associated with microvasculature in the basal ganglia, thalamus, hindbrain, and cerebellum. Calcified lesions were accompanied by astro- and micro-gliosis. Extravasation of IgG into the brain parenchyma was evident in eTNAP; blood-brain barrier was intact in controls (n=3 per group; 23 weeks). There were no significant differences in the locomotion or behavior (open field exploration) between the eTNAP and controls on the B6;129 background (n=6 per group). On B6 background, eTNAP mice displayed significant motor deficits - reduced ambulation (p&lt;0.01), rearing (p&lt; 0.01), speed (p&lt;0.05), and acceleration (p&lt;0.05; n=7-9 per group). There were no changes in osteogenic gene expression or phosphate transporters in eTNAP brains compared with controls. However, we detected significant regional differences between the thalamus and cortex in the levels of RumX2 , Spp1 , SLC17a7 , SLC17a6 of both control and eTNAP mice. Conclusions: Upregulation of TNAP activity can lead to intracranial vascular calcification. Given the similarities in presentation between eTNAP mice and PFBC patients, this model can advance the understanding of PFBC disease progression.

Blood-Brain Barrier Leakage and Microvascular Lesions in Cerebral Amyloid Angiopathy.

Background and Purpose- Cerebral amyloid angiopathy (CAA) is a common small vessel disease that independently effects cognition in older individuals. The pathophysiology of CAA and CAA-related bleeding remains poorly understood. In this postmortem study, we explored whether blood-brain barrier leakage is associated with CAA and microvascular lesions. Methods- Eleven CAA cases (median [IQR] age=69 years [65-79 years], 8 males) and 7 cases without neurological disease or brain lesions (median [IQR] age=77 years [68-92 years], 4 males) were analyzed. Cortical sections were sampled from each lobe, and IgG and fibrin extravasation (markers of blood-brain barrier leakage) were assessed with immunohistochemistry. We hypothesized that IgG and fibrin extravasation would be increased in CAA cases compared with controls, that this would be more pronounced in parietooccipital brain regions compared with frontotemporal brain regions in parallel with the posterior predilection of CAA, and would be associated with CAA severity and number of cerebral microbleeds and cerebral microinfarcts counted on ex vivo magnetic resonance imaging of the intact brain hemisphere. Results- Our results demonstrated increased IgG positivity in the frontotemporal ( P=0.044) and parietooccipital ( P=0.001) cortex in CAA cases compared with controls. Within CAA cases, both fibrin and IgG positivity were increased in parietooccipital brain regions compared with frontotemporal brain regions ( P=0.005 and P=0.006, respectively). The percentage of positive vessels for fibrin and IgG was associated with the percentage of amyloid-β-positive vessels (Spearman ρ=0.71, P=0.015 and Spearman ρ=0.73, P=0.011, respectively). Moreover, the percentage of fibrin and IgG-positive vessels, but not amyloid-β-positive vessels, was associated with the number of cerebral microbleeds on magnetic resonance imaging (Spearman ρ=0.77, P=0.005 and Spearman ρ=0.70, P=0.017, respectively). Finally, we observed fibrin deposition in walls of vessels involved in cerebral microbleeds. Conclusions- Our results raise the possibility that blood-brain barrier leakage may be a contributory mechanism for CAA-related brain injury.

Abstract TMP118: The Gut Microbiota Can Protect Against the Onset of Symptoms in an Animal Model of Cerebral Small Vessel Disease

It has become increasingly evident that disruption of the gut microbiota plays an important role in multiple host disease states. We hypothesize that maintaining a healthy gut microbiome protects against the development of cerebral small vessel disease (CSVD), an inflammatory disease accompanied by blood-brain barrier (BBB) breakdown and neuroinflammation. To test our hypothesis, we altered the gut microbiota of spontaneously hypertensive stroke-prone rats (SHRSPs), a model of CSVD associated with hypertension, to resemble that of healthy/asymptomatic WKY control rats. At birth SHRSP pups were removed from their birth moms and nursed by either WKY (SW rats) or SHRSP (SS) foster dams until 21 days of age. At 20 weeks, markers associated with neuroinflammation were significantly decreased in SW rats compared to SS rats (NF-κB1 by 68%, TLR-2 by 58%, and TLR-4 by 54%, P≤ 0.01, n=5-6/ group). When compared to SS rats, SW rats showed a 33% (P=0.045) decrease in IgG extravasation into brain indicating greater BBB integrity. Systolic blood pressure at 20 weeks in SW was 173±6 mm Hg compared to 201±6 mm Hg in SS rats (P=0.003) and these pressures remained stable for until at least 30 weeks. Analysis of microbiota in feces revealed that gut microbiota of SW rats were significantly different from SS rats (P&lt;0.05, principal components analysis of weighted Unifrac distances, n=5-6), resembled the microbiome in WKY rats, and showed &gt; 2-fold increase in the abundance of the genus, Akkermansia, in SW compared to SS rats (6.1% vs 3.0%, P=0.01). Akkermansia can enhance gut integrity by stimulating epithelial cell mucus production and enhancing tight junctions. We conclude that the gut microbiota can influence the onset of CSVD-like symptoms even in the genetic SHRSP model. Maintaining a microbiota similar to WKY control rats, which includes an increased abundance of Akkermansia, appears to protect against the onset of CSVD. Altering the gut microbiota in humans could be an effective prophylactic measure to protect against CSVD.

Thrombin promotes pericyte coverage by Tie2 activation in a rat model of intracerebral hemorrhage

Pericyte coverage on the endothelial tubes leads to the formation of a mature and stable microvessel system, which is critical for brain repair after intracerebral hemorrhage (ICH). We report herein that thrombin promotes pericyte coverage by activating Tie2 and the downstream signaling pathway PI3K/Akt in a rat model of ICH. ICH was induced by injection of autologous blood with or without thrombin inhibitor hirudin. Rats were treated with thrombin alone or in combination with a Tie2 inhibitor. The expression of total- and phospho-Tie2, PI3K and phospho-Akt, blood perfusion, pericyte coverage, IgG extravasation, neuron survival and neurological deficits were evaluated by western blot, fluorescein-5-isothiocyanate-dextran staining, immunohistochemistry, Nissl staining and modified neurological severity scores respectively. Induction of ICH resulted in increased phosphorylation of Tie2 on endothelial cells and pericyte coverage, better formation of integral and functional microvessels, more surviving neurons and accelerated motor function recovery, all of which were significantly attenuated by hirudin at 7 and 14 days after ICH induction. Furthermore, thrombin increased phosphorylation of Tie2 and Akt, expression of PI3K, and pericyte coverage, which were however reversed by pharmacological inhibition of Tie2. Our results demonstrated that thrombin promotes pericyte coverage on microvessels following ICH by enhancing activation of Tie2, in which the downstream PI3K/Akt signaling pathway might be involved.