Brain endothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

[Soybean isoflavones alleviate cerebral ischemia/reperfusion injury in rats by inhibiting ferroptosis and inflammatory cascade reaction].

OBJECTIVE: To explore the mechanism that mediates the effect of soybean isoflavones (SI) against cerebral ischemia/reperfusion (I/R) injury in light of the regulation of regional cerebral blood flow (rCBF), ferroptosis, inflammatory response and blood-brain barrier (BBB) permeability. METHODS: A total of 120 male SD rats were equally randomized into sham-operated group (Sham group), cerebral I/R injury group and SI pretreatment group (SI group). Focal cerebral I/R injury was induced in the latter two groups using a modified monofilament occlusion technique, and the intraoperative changes of real-time cerebral cortex blood flow were monitored using a laser Doppler flowmeter (LDF). The postoperative changes of cerebral pathological morphology and the ultrastructure of the neurons and the BBB were observed with optical and transmission electron microscopy. The neurological deficits of the rats was assessed, and the severities of cerebral infarction, brain edema and BBB disruption were quantified. The contents of Fe2+, GSH, MDA and MPO in the ischemic penumbra were determined with spectrophotometric tests. Serum levels of TNF-α and IL-1ßwere analyzed using ELISA, and the expressions of GPX4, MMP-9 and occludin around the lesion were detected with Western blotting and immunohistochemistry. RESULTS: The rCBF was sharply reduced in the rats in I/R group and SI group after successful insertion of the monofilament. Compared with those in Sham group, the rats in I/R group showed significantly increased neurological deficit scores, cerebral infarction volume, brain water content and Evans blue permeability (P < 0.01), decreased Fe2+ level, increased MDA level, decreased GSH content and GPX4 expression (P < 0.01), increased MPO content and serum levels of TNF-α and IL-1ß (P < 0.01), increased MMP-9 expression and lowered occludin expression (P < 0.01). All these changes were significantly ameliorated in rats pretreated with IS prior to I/R injury (P < 0.05 or 0.01). CONCLUSION: SI preconditioning reduces cerebral I/R injury in rats possibly by improving rCBF, inhibiting ferroptosis and inflammatory response and protecting the BBB.

Methamphetamine enhances caveolar transport of therapeutic agents across the rodent blood-brain barrier.

The blood-brain barrier (BBB) restricts clinically relevant accumulation of many therapeutics in the CNS. Low-dose methamphetamine (METH) induces fluid-phase transcytosis across BBB endothelial cells in vitro and could be used to enhance CNS drug delivery. Here, we show that low-dose METH induces significant BBB leakage in rodents ex vivo and in vivo. Notably, METH leaves tight junctions intact and induces transient leakage via caveolar transport, which is suppressed at 4°C and in caveolin-1 (CAV1) knockout mice. METH enhances brain penetration of both small therapeutic molecules, such as doxorubicin (DOX), and large proteins. Lastly, METH improves the therapeutic efficacy of DOX in a mouse model of glioblastoma, as measured by a 25% increase in median survival time and a significant reduction in satellite lesions. Collectively, our data indicate that caveolar transport at the adult BBB is agonist inducible and that METH can enhance drug delivery to the CNS.

Brain endothelial PTEN/AKT/NEDD4-2/MFSD2A axis regulates blood-brain barrier permeability.

The low level of transcytosis is a unique feature of cerebrovascular endothelial cells (ECs), ensuring restrictive blood-brain barrier (BBB) permeability. Major facilitator superfamily domain-containing 2a (MFSD2A) is a key regulator of the BBB function by suppressing caveolae-mediated transcytosis. However, the mechanisms regulating MFSD2A at the BBB have been barely explored. Here, we show that cerebrovascular EC-specific deletion of Pten (phosphatase and tensin homolog) results in a dramatic increase in vesicular transcytosis by the reduction of MFSD2A, leading to increased transcellular permeability of the BBB. Mechanistically, AKT signaling inhibits E3 ubiquitin ligase NEDD4-2-mediated MFSD2A degradation. Consistently, cerebrovascular Nedd4-2 overexpression decreases MFSD2A levels, increases transcytosis, and impairs BBB permeability, recapitulating the phenotypes of Pten-deficient mice. Furthermore, Akt deletion decreases phosphorylated NEDD4-2 levels, restores MFSD2A levels, and normalizes BBB permeability in Pten-mutant mice. Altogether, our work reveals the essential physiological function of the PTEN/AKT/NEDD4-2/MFSD2A axis in the regulation of BBB permeability.

A human cell type similar to murine central nervous system perivascular fibroblasts.

The brain vasculature has several specific features, one of them being the blood-brain barrier (BBB), which supports and protects the brain by allowing for the passage of oxygen and nutrients, while at the same time preventing passage of pathogens and toxins. The BBB also prevents efficient delivery of drugs to the brain, e.g. for treatment of brain tumors. In the murine brain, perivascular fibroblasts were recently identified as a novel potential constituent of the BBB. Here we present the existence of human cells that could be the equivalent to the murine brain perivascular fibroblasts. Using RNA sequencing, we show a similar transcriptomic profile of cultured human brain cells and murine perivascular fibroblasts. These data open up a window for new hypotheses on cell types involved in human CNS diseases.

Burns Impair Blood-Brain Barrier and Mesenchymal Stem Cells Can Reverse the Process in Mice.

Neurological syndromes are observed in numerous patients who suffer burns, which add to the economic burden of societies and families. Recent studies have implied that blood-brain barrier (BBB) dysfunction is the key factor that induces these central nervous system (CNS) syndromes in peripheral traumatic disease, e.g., surgery and burns. However, the effect of burns on BBB and the underlying mechanism remains, largely, to be determined. The present study aimed to investigate the effect of burns on BBB and the potential of umbilical cord-derived mesenchymal stem cells (UC-MSCs), which have strong anti-inflammatory and repairing ability, to protect the integrity of BBB. BBB permeability was evaluated using dextran tracer (immunohistochemistry imaging and spectrophotometric quantification) and western blot, interleukin (IL)-6, and IL-1ß levels in blood and brain were measured by enzyme-linked immunosorbent assay. Furthermore, transmission electron microscopy (TEM) was used to detect transcellular vesicular transport (transcytosis) in BBB. We found that burns increased mouse BBB permeability to both 10-kDa and 70-kDa dextran. IL-6 and IL-1ß levels increased in peripheral blood and CNS after burns. In addition, burns decreased the level of tight junction proteins (TJs), including claudin-5, occludin, and ZO-1, which indicated increased BBB permeability due to paracellular pathway. Moreover, increased vesicular density after burns suggested increased transcytosis in brain microvascular endothelial cells. Finally, administering UC-MSCs at 1 h after burns effectively reversed these adverse effects and protected the integrity of BBB. These results suggest that burns increase BBB permeability through both paracellular pathway and transcytosis, the potential mechanism of which might be through increasing IL-6 and IL-1ß levels and decreasing Mfsd2a level, and appropriate treatment with UC-MSCs can reverse these effects and protect the integrity of BBB after burns.

Protective effect of (Xenical+GSF) against I/R-induced blood brain barrier disruption, ionic edema, lipid deregulation and neuroinflammation.

Ischemic stroke is a leading cause of mortality worldwide that occurs following the reduction or interruption of blood brain supply, characterized by a cascade of early events as oxidative stress and ensuing neuro-inflammation, energy failure and the burst of intracellular Ca++ resulting in activation of phospholipases and large increase in FFA including arachidonic acid, ultimately leading to nervous cell death. Grape Seed Flour (GSF) is a complex polyphenolic mixture harboring antioxidant, anti-inflammatory and neuroprotective properties. Orlistat (Xenical ™,Xe) is a gastro-intestinal lipase inhibitor and an anti-obesity agent. In an earlier study we reported the higher efficiency in neuroprotection against HFD-induced brain lipotoxicity when combining the two drugs (GSF + Xe). As a result repurposing Xe as an adjunct to GSF therapy against stroke appeared relevant and worthy of investigation. I/R insult disrupted the blood brain barrier (BBB) as assessed by EB dye extravasation, increased water and Na+ within the brain. Ultrastructurally I/R altered the brain blood capillaries at the vicinity of hippocampus dentate gyrus area as assessed by transmission and scanning electron microscopy. I/R altered lipid metabolism as revealed by LDL/HDL ratio, lipase activity, and FFA profiles. Moreover, I/R induced neuro-inflammation as assessed by down-regulation of anti-inflammatory CD 56 and up-regulation of pro-inflammatory CD 68 antigen. Importantly almost all I/R-induced disturbances were retrieved partially upon Xe or GSF on their own, and optimally when combining the two drugs. Xe per se is protective against I/R injury and the best neuroprotection was obtained when associating low dosage Xe with high dosage GSF, enabling neuroprevention and cell survival within hippocampus dentate gyrus area as revealed by increased staining of Ki 67 proliferation biomarker.

HOXB9 enhances the ability of lung cancer cells to penetrate the blood-brain barrier.

Even after multimodal therapy, the prognosis is dismal for patients with brain metastases from non-small cell lung cancer (NSCLC). Although the blood-brain barrier (BBB) limits tumor cell penetration into the brain parenchyma, some nevertheless colonize brain tissue through mechanisms that are not fully clear. Here we show that homeobox B9 (HOXB9), which is commonly overexpressed in NSCLC, promotes epithelial-to-mesenchymal transition (EMT) and tumor migration and invasion. Animal experiments showed that HOXB9 expression correlates positively with the brain metastatic potential of human NSCLC cells, while brain metastatic cells derived through in vivo selection showed greater HOXB9 expression than their cells of origin. Comparable results were obtained after immunohistochemical analysis of clinical primary NSCLC and matched brain metastasis samples obtained after surgery. Using an in vitro BBB model, knockdown and overexpression experiments showed that HOXB9-dependent expression of MMP9 in NSCLC cells leads to reduced expression of junctional proteins in cultured human vascular endothelial cells and enhanced transmigration of tumor cells. These data indicate that HOXB9 enables NSCLC cells to break away from the primary tumor by inducing EMT, and promotes brain metastasis by driving MMP9 production and degradation of intercellular adhesion proteins in endothelial cells comprising the BBB.

Biomanufacturing of a novel in vitro biomimetic blood-brain barrier model.

A glioma is a malignant tumor that severely threatens human health. However, it is difficult for most therapeutic agents to penetrate through the blood-brain barrier (BBB) and exhibit their antineoplastic activity in the brain. In this article, a biomimetic in vitro BBB model was created by a composite process, this model can provide a significant foundation for the research of drug transport, tumor treatment, tumor microenvironment and other fields. A series of tests and comparative experiments were performed to evaluate this model. The tests showed that the model enabled preliminary simulation of the structure and function of the BBB. Experimental results demonstrated: (1) the new technology enabled controlled release of growth factors and successfully induced endothelial progenitor cells into endothelial cells. Compared with the traditional gold standard, the Transwell model, the expression of four specific proteins that are related to the BBB characteristics was significantly increased (alkaline phosphatase(ALP) by 89.82%, γ-GT by 88.86%, zonula occludens-1 (ZO-1) by 57.40%, and Claudin-5 by 102.32%) in this model; (2) astrocytes had a promoting effect on the microvascular endothelial cells to form tight junction (ZO-1 increased by 249.35%, Claudin-5 increased by 184.99%), and there was a great difference between whether these two types of cells were contact cultured or not; (3) the gelatinous cell U118 had a destructive effect on the tight junction of BBB (ZO-1 decreased by 55.86%, Claudin-5 decreased by 37.84%).

Efficient Delivery of Nerve Growth Factors to the Central Nervous System for Neural Regeneration.

The central nervous system (CNS) plays a central role in the control of sensory and motor functions, and the disruption of its barriers can result in severe and debilitating neurological disorders. Neurotrophins are promising therapeutic agents for neural regeneration in the damaged CNS. However, their penetration across the blood-brain barrier remains a formidable challenge, representing a bottleneck for brain and spinal cord therapy. Herein, a nanocapsule-based delivery system is reported that enables intravenously injected nerve growth factor (NGF) to enter the CNS in healthy mice and nonhuman primates. Under pathological conditions, the delivery of NGF enables neural regeneration, tissue remodeling, and functional recovery in mice with spinal cord injury. This technology can be utilized to deliver other neurotrophins and growth factors to the CNS, opening a new avenue for tissue engineering and the treatment of CNS disorders and neurodegenerative diseases.

Opening the Blood-Brain Barrier and Improving the Efficacy of Temozolomide Treatments of Glioblastoma Using Pulsed, Focused Ultrasound with a Microbubble Contrast Agent.

OBJECTIVE: To explore the effects of pulsed, focused, and microbubble contrast agent-enhanced ultrasonography (mCEUS) on blood-brain barrier (BBB) permeability and the efficacy temozolomide for glioblastoma. METHODS: Wistar rats (n = 30) were divided into three groups (n = 10 per group) to determine optimal CUES conditions for achieving BBB permeability, as assessed by ultrastructure transmission electron microscopy (TEM) and western blot assays for the tight junction protein claudin-5. Optimized mCEUS effects on BBB permeability were subsequently confirmed with Evans blue staining (2 groups of 10 rats). The glioma cell line 9L was injected into the brain striatum of Wistar rats. After temozolomide chemotherapy, we detected glial fibrillary acidic protein (GFAP) levels in serum by enzyme-linked immunosorbent assay (ELISA) and in brain tissue by western blot, immunocytochemistry, and real-time quantitative polymerase chain reaction (qPCR). RESULTS: BBB permeability was maximized with 1 ml/kg contrast agent mCEUS delivered via 10-min intermittent launches with a 400-ms interval. Evans blue staining confirmed BBB permeability following ultrasonic cavitation in the control group (P < 0.05). Following temozolomide chemotherapy, levels of the tumor marker GFAP were increased in the group with ultrasonic cavitation compared with the control group (P < 0.05). CONCLUSIONS: When rats were treated by mCEUS with intermittent launches (interval, 400 ms) and injected with 1 mg/kg contrast agent, BBB permeability was increased and temozolomide BBB penetration was enhanced, therapeutic enhancement for glioblastoma.

Transplantation of human bone marrow stem cells into symptomatic ALS mice enhances structural and functional blood-spinal cord barrier repair.

Accumulating evidence shows alterations in the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) in ALS patients and in animal models of disease, mainly by endothelial cell (EC) damage. Repair of the altered barrier in the CNS by replacement of ECs via cell transplantation may be a new therapeutic approach for ALS. Recently, we demonstrated positive effects towards BSCB repair by intravenous administration of unmodified human bone marrow CD34+ (hBM34+) cells at different doses into symptomatic ALS mice. However, particular benefits of these transplanted cells on microvascular integrity in symptomatic ALS mice are still unclear. The aim of the present study was to determine the structural and functional spinal cord capillary integrity in symptomatic ALS mice after intravenous administration of hBM34+ cells. The G93A mice at 13 weeks of age intravenously received one of three different cell doses (5 × 104, 5 × 105, or 1 × 106) and were euthanized at 17 weeks of age (4 weeks post-transplant). Control groups were media-treated and non-carrier mutant SOD1 gene mice. Capillary ultrastructural (electron microscopy), immunohistochemical (laminin and HuNu), and histological (myelin and capillary density) analyses were performed in the cervical and lumbar spinal cords. Capillary permeability in the spinal cords was determined by Evans Blue (EB) injection. Results showed significant restoration of ultrastructural capillary morphology, improvement of basement membrane integrity, enhancement of axonal myelin coherence, and stabilization of capillary density in the spinal cords primarily of ALS mice receiving the high dose of 1 × 106 cells. Moreover, substantial reduction of parenchymal EB levels was determined in these mice, confirming our previous results on capillary permeability. Additionally, transplanted cells were detected in blood smears of sacrificed late symptomatic mice by HuNu marker. Altogether, these results provide novel evidence that unmodified bone marrow hematopoietic stem cell treatment at optimal dose might be beneficial for structural and functional repair of the damaged BSCB in advanced stage of ALS, potentially resulting in delayed disease progression by increased motor neuron survival.

The protective roles of L-borneolum, D-borneolum and synthetic borneol in cerebral ischaemia via modulation of the neurovascular unit.

OBJECTIVE: Borneol has been used to treat stroke in China since ancient times. In our previous research, we demonstrated the effect of borneol on cerebral ischaemia injury via meta-analysis. The neurovascular unit (NVU) is the structural basis of the preservation of the brain microenvironment and is believed to be a promising target in treating stroke. In this research, we explored the roles of three kinds of borneol, namely, L-borneolum (B1), D-borneolum (B2) and synthetic borneol (B3), in the NVU with permanent middle cerebral artery occluded (pMCAO) rats. METHODS: The Longa scoring method was used to evaluate nerve function deficits in the pMCAO rats. Awakening time, brain water content, brain index and brain edema rate were also measured. TTC staining was used to calculate the cerebral infarction rate. The morphology of the ischaemia penumbra brain tissue was observed via HE staining, and the neuronal denatured cell index (DCI) was calculated. An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of vascular endothelial growth factor VEGF and TNF-α in the serum. Moreover, the ultrastructures of the neurons and of the blood-brain barrier (BBB) were observed using transmission electron microscopy. The expression levels of Claudin-5, Bcl-2 and Bax in the ischaemia penumbra of pMCAO rats were detected using real-time PCR and immunohistochemistry. RESULTS: Pretreatment with B1, B2 and B3 delayed the recovery time (P < 0.01). B1 remarkably ameliorated neurological deficits 24 h after cerebral ischaemia (P < 0.05). Moreover, B1 and B3 were both able to ameliorate brain edema and the area of cerebral infarction. In addition, B1, B2 and B3 all increased serum VEGF levels and decreased serum TNF-α levels (P < 0.01). For the ultrastructure determination, the BBB and the nerve centre were significantly improved by B1, B2 and B3. The mechanistic exploration revealed that B2 and B3 protected the brain by reducing the Bax/Bcl-2 ratio (P < 0.05, P < 0.01, respectively). Immunohistochemistry suggested that B1, B2 and B3 could also enhance the expression of Claudin-5 (P < 0.01). CONCLUSION: The three kinds of borneol demonstrated different protective effects on cerebral ischaemia injury. L-Borneolum displayed the most prominent anti-cerebral ischaemia effect among them. The mechanism was most likely executed via anti-apoptosis and anti-inflammation effects and maintenance of the stability of the BBB and TJs to comprehensively improve NVU function.

Strain-dependent effects of clinical echovirus 30 outbreak isolates at the blood-CSF barrier.

BACKGROUND: Echovirus (E) 30 (E-30) meningitis is characterized by neuroinflammation involving immune cell pleocytosis at the protective barriers of the central nervous system (CNS). In this context, infection of the blood-cerebrospinal fluid barrier (BCSFB), which has been demonstrated to be involved in enteroviral CNS pathogenesis, may affect the tight junction (TJ) and adherens junction (AJ) function and morphology. METHODS: We used an in vitro human choroid plexus epithelial (HIBCPP) cell model to investigate the effect of three clinical outbreak strains (13-311, 13-759, and 14-397) isolated in Germany in 2013, and compared them to E-30 Bastianni. Conducting transepithelial electrical resistance (TEER), paracellular dextran flux measurement, quantitative real-time polymerase chain reaction (qPCR), western blot, and immunofluorescence analysis, we investigated TJ and AJ function and morphology as well as strain-specific E-30 infection patterns. Additionally, transmission electron and focused ion beam microscopy electron microscopy (FIB-SEM) was used to evaluate the mode of leukocyte transmigration. Genome sequencing and phylogenetic analyses were performed to discriminate potential genetic differences among the outbreak strains. RESULTS: We observed a significant strain-dependent decrease in TEER with strains E-30 Bastianni and 13-311, whereas paracellular dextran flux was only affected by E-30 Bastianni. Despite strong similarities among the outbreak strains in replication characteristics and particle distribution, strain 13-311 was the only outbreak isolate revealing comparable disruptive effects on TJ (Zonula Occludens (ZO) 1 and occludin) and AJ (E-cadherin) morphology to E-30 Bastianni. Notwithstanding significant junctional alterations upon E-30 infection, we observed both para- and transcellular leukocyte migration across HIBCPP cells. Complete genome sequencing revealed differences between the strains analyzed, but no explicit correlation with the observed strain-dependent effects on HIBCPP cells was possible. CONCLUSION: The findings revealed distinct E-30 strain-specific effects on barrier integrity and junctional morphology. Despite E-30-induced barrier alterations leukocyte trafficking did not exclusively occur via the paracellular route.

Blood-Brain Barrier in a Haemophilus influenzae Type a In Vitro Infection: Role of Adenosine Receptors A2A and A2B.

The blood-brain barrier (BBB) is mainly made up of tightly connected microvascular endothelial cells (BMECs), surrounded by pericytes (BMPCs) which regulate BBB tightness by providing soluble factors that control endothelial proliferation. Haemophilus influenzae type a (Hia) is able to reach the BBB, crossing it, thus causing meningitis. In this study, by using an in vitro model of BBB, performed with human BMECs and human BMPCs in co-culture, we demonstrated that, after Hia infection, the number of hBMPCs decreased whereas the number of hBMECs increased in comparison with non-infected cells. SEM and TEM images showed that Hia was able to enter hBMECs and reduce TEER and VE-cadherin expression. When the cells were infected in presence of SCH58261 and PSB603 but not DPCPX, an increase in TEER values was observed thus demonstrating that A2A and A2B adenosine receptors play a key role in BBB dysfunction. These results were confirmed by the use of adenosine receptor agonists CGS21680, CCPA, and NECA. In infected co-cultures cAMP and VEGF increased and TEER reduction was counter-balanced by VEGF-R1 or VEGF-R2 antibodies. Moreover, the phosphorylated CREB and Rho-A significantly increased in infected hBMECs and hBMPCs and the presence of SCH58261 and PSB603 significantly abrogated the phosphorylation. In conclusion, this study demonstrated that the infection stimulated A2A and A2B adenosine receptors in hBMECs and hBMPCs thus inducing the pericytes to release large amounts of VEGF. The latter could be responsible for both, pericyte detachment and endothelial cell proliferation, thus provoking BBB impairment.

Preparation and Characterization of Biocompatible Chitosan Nanoparticles for Targeted Brain Delivery of Peptides.

Here, we describe a nanocarrier system that can transfer chitosan nanoparticles loaded with either small peptides such as the caspase inhibitor Z-DEVD-FMK or a large peptide like basic fibroblast growth factor across the blood-brain barrier. The nanoparticles are selectively directed to the brain and are not measurably taken up by the liver and spleen. Intravital fluorescent microscopy provides an opportunity to study the penetration kinetics of nanoparticles loaded with fluorescent agents such as Nile red. Nanoparticles functionalized with anti-transferrin antibody and loaded with peptides efficiently provided neuroprotection when systemically administered either as a formulation bearing a single peptide or a mixture of them. Failure of brain permeation of the nanoparticles after inhibition of vesicular transcytosis by imatinib as well as when nanoparticles were not functionalized with anti-transferrin antibody indicates that this nanomedicine formulation is rapidly transported across the blood-brain barrier by receptor-mediated transcytosis.

Targeted inhibition of RAGE reduces amyloid-ß influx across the blood-brain barrier and improves cognitive deficits in db/db mice.

AIMS: To investigate restorative effects of the receptor for advanced glycation end products (RAGE)-specific inhibitor FPS-ZM1 on abnormal amyloid ß (Aß) influx across the blood brain-barrier (BBB) and cognitive deficits in db/db mice. METHODS: Aß influx across the BBB was determined by intra-arterial infusion of 125I-Aß1-40. Receptor for advanced glycation end products (RAGE), Aß, NF-κB p65, caspase-3, Bax, Bcl-2, PSD-95 and synaptophysin were assayed by Western blot, immunohistochemistry or RT-PCR. Apoptosis was quantified by TUNEL assay. In vivo hippocampal long term potentiation (LTP) recording, Golgi Staining, Morris water maze (MWM) task and Y-maze test were performed. RESULTS: FPS-ZM1 (1.0 mg/kg i.p.) inhibited Aß influx across the BBB and expression of RAGE participating in Aß influx, consequently decreased hippocampal Aß1-40 and Aß1-42 in db/db mice. After FPS-ZM1 treatment, NF-κB signaling was inhibited, and neuronal apoptosis was reduced, which revealed by less TUNEL + cells, reduced caspase-3 activity and higher ratio of Bcl-2/Bax. In addition, FPS-ZM1 improved hippocampal plasticity evidenced by enhanced in vivo LTP and the restoration of spine deficit and increased PSD-95 expression in hippocampal neuron. Further studies found that FPS-ZM1 treatment alleviated cognitive deficits shown by better performance in behavioral tests, without significant metabolic effects on blood glucose, insulin and cerebral AGEs. CONCLUSION: Downregulation of abnormal Aß influx across the BBB by FPS-ZM1 at higher dosage contributes to reduced neuronal apoptosis, improved hippocampal plasticity and cognitive impairment in db/db mice.

Bioluminescent Study of the Distribution of High-Molecular-Weight Protein Fraction of Cellex Daily Preparation in the Brain after Intranasal Administation.

Permeability of the blood-brain barrier for protein fractions 50-100 kDa (PF50-100) of Cellex Daily preparation labeled with fluorescent tracer FITC and non-conjugated FITC were compared after intranasal administration of the preparations to healthy rats. Fluorimetrical analysis of the serum and cerebrospinal fluid samples showed that Cellex Daily PF50-100-FITC administered intranasally penetrated into the blood and cerebrospinal fluid with maximum accumulation in 2 h after administration and persists in the circulation for 24 h probably due to binding with plasma proteins. The differences in the kinetic profile of PF50-100-FITC and free FITC indirectly suggest that the major part of the preparation is not degraded within 24 h and FITC is probably not cleaved from the protein components of the preparation. In vivo fluorescence analysis showed significant fluorescent signal in the olfactory bulbs in 6 h after intranasal administration; hence, the preparation administered via this route can bypass the blood-brain barrier. Scanning laser confocal microscopy of rat brain sections confirmed penetration of the high-molecular weight protein fraction PF50-100-FITC into CNS structures. The most pronounced accumulation of the labeled drug was observed in the olfactory bulb in 6 and 12 h after administration. In contrast to free FITC administered in the control group, significant accumulation of PF50-100-FITC in the olfactory cortex and frontal cortex neurons with functionally active nuclei was observed in 6, 12 and 24 h after intranasal administration.

Blood-brain Barrier Disruption Leads to Postoperative Cognitive Dysfunction.

BACKGROUND: Postoperative Cognitive Dysfunction (POCD) has received considerable attention as one of the main postoperative complications. The underlying mechanism of POCD in elderly subjects has not been fully elucidated to date. The Central Nervous System (CNS) is isolated from the bloodstream by the Blood Brain Barrier (BBB) that consists of endothelial cells, capillary blood vessels and tight junctions. The tight junctions carry out significant biological functions that are associated with the CNS and blood circulation. METHODS: In this review, I present a hypothesis that blood-brain barrier disruption leads to postoperative cognitive dysfunction. A total of 81 healthy male Wistar rats were used for the present study. All the experimental animals were randomly divided into 3 groups: normal control group, isoflurane group and splenectomy group. The control group was not subjected to any form of treatment. The rats in isoflurane group were given 1.5-2% isoflurane under intubation and mechanical ventilation. The rats in splenectomy group underwent splenectomy under the same anesthesia as the isoflurane group. The Morris water maze was used to examine the learning and memory ability of the animals. The expression of the Tight Junctions Proteins (TJPs) in the hippocampus was analyzed using Western blotting. The concentration of Evans Blue (EB) in the supernatant was analyzed using UV spectroscopy. Ultrastructure changes in the basal laminas, the Tight Junctions (TJs), mitochondria and the endoplasmic reticulum surrounding the capillaries were assessed by Transmission Electron Microscopy (TEM). RESULTS: Following splenectomy, the rats displayed concomitant significant cognitive deficits in the Morris water maze test. Taken together, the results indicate that the expression levels of occludin (65KD) following splenectomy were reduced on days one and three in aged rats. No significant difference was noted in the expression levels of claudin-5, except for a reduction after surgery on day one. The leakage of EB was higher following splenectomy than control group and isoflurane group. The ultrastructure of the neurovascular unit was monitored on the day prior to surgery and on the 1st, 3rd and 7th day following surgery using a transmission electronmicroscope. CONCLUSION: The alterations in the levels of tight junction proteins following splenectomy may contribute to the BBB permeability increase, which in turn will induce postoperative cognitive dysfunction.

Morphologic mechanisms of increased vascular permeability of triolein emulsion to the blood-brain barrier.

Triolein emulsion has been known to increase vascular permeability in the brain when it is infused into the carotid artery. The purpose of this study was to identify the morphologic mechanism of increased vascular permeability in brain induced by infusion of emulsified triolein into the carotid artery by transmission electron microscopy (TEM). Triolein emulsion was infused into the carotid artery of rats. TEM using lanthanum tracer was used to evaluate morphologic changes in endothelium with a focus on transcytotic vesicles and tight junction opening. The treat group showed multiple transcytotic vesicles containing lanthanum tracer within endothelium on TEM. TEM also revealed that lanthanum tracer entered neural interstitium through tight junctions between capillary endothelial cells infrequently in the treat group. No evidence of transcytotic vesicles containing lanthanum tracer or lanthanum leakage through tight junctions was observed in the control group. Transcytosis and the opening of tight junctions appears the pathway for vascular permeability enhancement by triolein. This result could be utilized in studies on the blood-brain barrier and by those searching for chemotherapeutic methods that deliver anti-tumor agents to normally drug inaccessible organs.