By The Blood-brain Barrier Permeability Research Articles

The NF-κB pathway negatively regulates the replication of rabies virus by triggering inflammatory responses

Rabies, which is caused by the rabies virus (RABV), is a neurological disease of mammals, including humans. The disease is untreatable, as drugs and antibodies are prevented from entering brain tissue by the blood-brain barrier (BBB). However, it has been reported that BBB permeability can be enhanced in mice infected with a laboratory-attenuated strain of RABV, which is not the case in those infected with wild-type RABV. Moreover, it is not the RABV infection directly that enhances BBB, but rather the inflammatory molecules that are induced by the virus. Understanding the mechanism underlying the RABV-induced production of chemokines and cytokines is crucial for the prevention and management of rabies. In this study, we found that mice infected with the RABV challenge strain CVS11 showed significant inflammatory infiltration in the later stages of infection. At the cellular level, we confirmed that the NF-κB signaling pathway was activated following RABV infection, leading to significant upregulation of the expression of downstream chemokines and inflammatory factors (CCL2, CCL5, CXCL10, TNF-α, and IL-6). Surprisingly, the replication of RABV was upregulated when the NF-κB pathway was inhibited, and the levels of its downstream chemokines and inflammatory factors were reduced following treatment with the inhibitor JSH-23. This suggests that RABV infection activates the NF-κB pathway, which is then able to negatively regulate the replication of RABV. This pathway is therefore a potential target for drugs and other therapies in the treatment of rabies.

Brain‐Penetrable Antibody Probes for in vivo Tau Imaging

AbstractBackgroundAntibody‐based PET ligands are desirable due to their high specificity; however, their brain uptakes are limited by the blood‐brain barrier (BBB). We previously demonstrated that transport of antibody across BBB can be facilitated through interaction with the transferrin receptor (TfR)1. We report here the first in vivo PET imaging studies using BBB permeable F‐18 radiolabeled bispecific antibodies specific to tau protein in mice.MethodWe performed multiple PET scans using each of the radio‐probes synthesized via conjugation of [18F]SFB with one of the four bispecific antibody constructs: 1) 6B2G12‐ScFv8D3 (full‐size Tau antibody conjugated with TfR fragment (ScFv8D3); abbreviated as TAUb), 2) ScFv235‐ScFv8D3 (small Tau‐TfR; TAUs), 3) 3D6‐ScFv8D3 (full‐size Aβ‐TfR; Aβb) or 4) ScFv3D6‐ScFv3D6 (small Aβ‐TfR; Aβs). We used a matched control design to compare a WT with one of the two types of transgenic (tg) tauopathy mice [PS19 (P301S) or JNPL3 (P301)] in each scan. Subjects were scanned on a MOLECUBES PET/CT scanner. An initial low‐dose (244 ± 54 μCi) dynamic scan (30 min) was performed, followed by two scans at 8hr and 12hr (20 min each static scans) after injection of a high dose (1832 ± 447 μCi) of the same probe. PMOD and FireVoxel were used for imaging processing. After co‐registration of PET/CT/atlas, data were quantified as SUV and SUVR [CB as a reference], and clearance was estimated as %change/hr.ResultTAUs probe displayed highest brain uptake with more specific binding retained in the brain, as compared to TAUb and Aβs. TAUs probe showed faster brain penetration in tau‐tg mice than in WT, with differences in amygdala, basal forebrain, and hypothalamus of 66%, 62% and 85%, respectively, at 8hr post‐injection (Fig. 1). Further, higher brain uptakes (TAUs > Aβs) in the same tau‐tg mouse (Fig. 2) suggest the specific binding of TAUs probe, while Aβs probe can serve as a control due to its lack of specific binding in tau‐tg mice.ConclusionWe have successfully synthesized and evaluated four novel F‐18 bispecific antibodies via in vivo PET. TAUs probe showed most promising BBB permeability and binding specificity to tau antigen with a reasonable clearance.

Blood-brain barrier permeability is associated with different neuroinflammatory profiles in Alzheimer's disease.

Inflammation is an important player in Alzheimer's disease (AD), whose effects can be influenced by the blood-brain barrier (BBB). Here, we investigated the relationship between BBB permeability, indicated by cerebrospinal fluid (CSF)/plasma albumin quotient (Qalb), and CSF indexes of neuroinflammation in a cohort of biologically defined AD patients. Fifty-nine consecutive patients with mild cognitive impairment (MCI) or early AD (Mini-Mental State Examination [MMSE] >22) underwent CSF analysis for inflammatory cytokines (interleukin [IL]-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, Il-10, IL-12, IL-13, IL-17, tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], granulocyte-monocyte colony-stimulating factor [GM-CSF], granulocyte colony-stimulating factor [G-CSF]). Using backward stepwise linear regression analysis, we explored the potential influence of each cytokine CSF level on Qalb considering age, sex, and apolipoprotein E (APOE) as covariates. Higher levels of IL-4 (β = 0.356, 0.005) and IL-8 (β = 0.249, 0.05) were associated with higher Qalb values, while macrophage inflammatory protein-1α (MIP-1β) (β = -0.274; p = 0.032) and TNF-α (β = -0.248; p = 0.031) showed a significant negative association with BBB permeability. Age was also positively associated with Qalb (β = 0.283; p = 0.016). Despite the overall integrity of the BBB, its permeability could either influence or be influenced by central neuroinflammation, reflected by CSF cytokine levels. This is in line with previous studies that showed that patients with a more intact barrier are those with more prominent neurodegeneration. Our findings suggest that different neuroinflammatory profiles can be associated with different levels of BBB permeability in AD.

Low-Level Laser Treatment Induces the Blood-Brain Barrier Opening and the Brain Drainage System Activation: Delivery of Liposomes into Mouse Glioblastoma.

The progress in brain diseases treatment is limited by the blood-brain barrier (BBB), which prevents delivery of the vast majority of drugs from the blood into the brain. In this study, we discover unknown phenomenon of opening of the BBBB (BBBO) by low-level laser treatment (LLLT, 1268 nm) in the mouse cortex. LLLT-BBBO is accompanied by activation of the brain drainage system contributing effective delivery of liposomes into glioblastoma (GBM). The LLLT induces the generation of singlet oxygen without photosensitizers (PSs) in the blood endothelial cells and astrocytes, which can be a trigger mechanism of BBBO. LLLT-BBBO causes activation of the ABC-transport system with a temporal decrease in the expression of tight junction proteins. The BBB recovery is accompanied by activation of neuronal metabolic activity and stabilization of the BBB permeability. LLLT-BBBO can be used as a new opportunity of interstitial PS-free photodynamic therapy (PDT) for modulation of brain tumor immunity and improvement of immuno-therapy for GBM in infants in whom PDT with PSs, radio- and chemotherapy are strongly limited, as well as in adults with a high allergic reaction to PSs.

Abstract 3461: Focused ultrasound induced blood-tumor barrier permeability of combinatorial chemotherapy

Abstract Background: Incidence of patients having brain metastasis of breast cancer has increased due to enhanced treatment for peripheral disease. Breast cancer brain metastasis is often fatal and occurs in 15-20% of patients diagnosed with stage IV breast cancer. Traditional chemotherapy delivery to the brain is limited by the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Low intensity focused ultrasound (LIFU) is a unique technique recently investigated to non-invasively disrupt these barriers and aid neuro-modulation. Herein, we demonstrate the use of a clinical LIFU device to modulate blood-tumor permeability and deliver combined chemotherapy in a novel preclinical model of brain metastases of breast cancer. Methods: Brain colonizing MDA-MB-231Br cells transfected with luciferase were injected into athymic Nu/Nu female mice by intracardiac injection into the left ventricle (per mouse 175,000 cells). Brain metastasis was allowed to develop, and tumor burden was monitored by bioluminescent imaging IVIS Spectrum CT (Perkin Elmer). Mice were randomized into sham or single treatment or combined treatment groups on Day 21 (Doxil nanoparticles 5.6 mg/kg or Paclitaxel 10mg/kg). Mice receiving LIFU were placed on a specially designed animal restraint with the skull partially immersed in degassed water. T1 weighted MRI was used to identify and localize sonication spots within the cortex, and mice were sonicated using 0.4uL/kg Definity© at 0.3 cavitation dose for 60sec. Treatment with LIFU was continued once weekly for three consecutive weeks. Survival and tumor burden was recorded until mice developed neurological symptoms. LIFU induced blood-brain barrier breakdown in healthy mice was also analyzed by in-situ brain perfusion with 14CSucrose, 3HGlucose, and 3HIvermectin to monitor BBB integrity and transporter function. Results: Mice receiving LIFU+paclitaxel+Doxil-NP showed significantly lower tumor burden (peak area 635.9) and higher median survival of 60 days as compared to vehicle (29d) and LIFU+vehicle (32d) groups. Analysis of BBB permeability in healthy mice demonstrated significantly higher passive permeability marker accumulation than non-sonicated contralateral hemisphere. Conclusion: Timing the combined chemotherapy with LIFU induced windows of maximal BBB opening resulted in slower tumor progression and increased survival in a preclinical model of breast cancer brain metastasis. Future directions aim to combine these results and evaluate underlying mechanisms of enhanced permeability post-LIFU. Citation Format: Tasneem A. Arsiwala, Kathryn E. Blethen, Samuel A. Sprowls, Ross A. Fladeland, Brooke Keilkowski, Morgan Glass, Trenton Pritt, Peng Wang, Ali Rezai, Paul R. Lockman. Focused ultrasound induced blood-tumor barrier permeability of combinatorial chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3461.

Transcriptomic Analysis of Rat Cerebral Cortex Reveals the Potential Mechanism of Electroacupuncture Opening Blood Brain Barrier.

Therapeutic treatment options for central nervous system (CNS) diseases are greatly limited by the blood-brain barrier (BBB). Electroacupuncture (EA) can be used to induce an increase in BBB permeability on rats, providing a potential approach for the delivery of drugs from the systemic circulation into the brain. However, there remains a large gap in our knowledge regarding the impact of EA on brain gene expression. This work is focused on investigating the transcriptional changes of rat cerebral cortex following EA and expression changes in genes and bioinformatic analysis was performed. We found that the potential mechanism of EA opening BBB involves receptor-mediated/carrier-mediated endocytosis (RMT/CMT), and related genes include solute carrier (SLC) transporter genes and ATP-binding cassette (ABC) transporter genes. The results also suggested that EA may affect the expression of tight junction (TJ) proteins in endothelial cells by affecting integrin binding, autophagy pathway and calcium signaling pathway, thus further affecting the permeability of blood-brain barrier. Our results provide a valuable resource that will guide mechanism research of EA opening BBB and other ways to mediate drug delivery into the brain.

DDRE-49. TRANSIENT OPENING OF THE BLOOD-BRAIN BARRIER BY VASOACTIVE PEPTIDES TO INCREASE CNS DRUG DELIVERY: REALITY VERSUS WISHFUL THINKING?

Abstract BACKGROUND Drug delivery to treat neurologic disease and cancers of the central nervous system (CNS) is severely limited by the blood-brain barrier (BBB). Vasoactive peptides (VAPs) such as regadenoson, adenosine, and labradimil have been shown in animal studies to transiently open the BBB, and regadenoson is currently under investigation in humans to determine if it might improve CNS drug delivery. There is currently limited information regarding the potential for other VAPs to open the BBB transiently. METHODS We performed a review of the literature evaluating the physiologic effects of vasoactive peptides on the vasculature of the brain and systemic organs. To assess the likelihood that a vasoactive peptide might transiently disrupt the BBB, we devised a four-tier classification system to organize data available in the literature which factors in alterations in BBB integrity and effects on normal brain vasculature and systemic blood vessels. This data was further sorted based on whether it comes from humans, animals, or in vitro systems. RESULTS We identified 38 VAPs with potential BBB permeability-altering properties. To date, none of these has been shown to open the BBB in humans. Thirteen VAPs increased BBB permeability in rodents. The remaining 25 had favorable physiologic effects on blood vessels but lack specific information on permeability changes to the BBB. We ranked VAPs in a four-tier ranking system related to their known physiologic actions. CONCLUSION Rodent studies document that analogs of bradykinin and adenosine transiently disrupt the BBB leading to higher chemotherapy concentrations in the CNS. VAPs remain an understudied class of drugs with the potential to increase drug delivery to the CNS. Dozens of VAPs have yet to be formally evaluated for this important clinical effect. This retrospective review summarizes the available data on VAPs highlighting agents that deserve further in vitro and in vivo investigations.

Hepatic Ischemia-Reperfusion Impairs Blood-Brain Barrier Partly Due to Release of Arginase From Injured Liver.

Aim: Hepatic ischemia-reperfusion (HIR) induces remote organs injury, including the brain. The homeostasis of the brain is maintained by the blood-brain barrier (BBB); thus, we aimed to investigate whether HIR impaired BBB and attempted to elucidate its underlying mechanism. Methods: Cell viability of human cerebral microvascular endothelial cells (hCMEC/D3) was measured following 24 h incubation with a serum of HIR rat undergoing 1 h ischemia and 4 h reperfusion, liver homogenate, or lysate of primary hepatocytes of the rat. The liver homogenate was precipitated using (NH4)2SO4 followed by separation on three columns and electrophoresis to identify the toxic molecule. Cell activity, apoptosis, proliferation, cell cycle, and expressions of proteins related to cell cycle were measured in hCMEC/D3 cells incubated with identified toxic molecules. HIR rats undergoing 1 h ischemia and 24 h reperfusion were developed to determine the release of an identified toxic molecule. BBB function was indexed as permeability to fluorescein and brain water. Endothelial cell proliferation and expressions of proteins related to the cell cycle in cerebral microvessels were measured by immunofluorescence and western blot. Results: Toxic molecule to BBB in the liver was identified to be arginase. Arginase inhibitor nor-NOHA efficiently attenuated hCMEC/D3 damage caused by liver homogenate and serum of HIR rats. Both arginase and serum of HIR rats significantly lowered arginine (Arg) in the culture medium. Arg addition efficiently attenuated the impairment of hCMEC/D3 caused by arginase or Arg deficiency, demonstrating that arginase impaired hCMEC/D3 via depriving Arg. Both arginase and Arg deficiency damaged hCMEC/D3 cells by inhibiting cell proliferation, retarding the cell cycle to G1 phase, and downregulating expressions of cyclin A, cyclin D, CDK2, and CDK4. HIR notably increased plasma arginase activity and lowered Arg level, increased the BBB permeability accompanied with enhanced brain water, and decreased the proliferative cells (marked by Ki67) in cerebral microvessels (marked by CD31) and protein expressions of cyclin A, cyclin D, CDK2 and CDK4 in isolated brain microvessels. Oral supplement of Arg remarkably attenuated these HIR-induced alterations. Conclusion: HIR leads to substantial release of arginase from the injured liver and then deprives systemic Arg. The Arg deficiency further impairs BBB via inhibiting the proliferation of brain microvascular endothelial cells by cell cycle arrest.

Angiogenesis and Blood-Brain Barrier Permeability in Vascular Remodeling after Stroke.

Angiogenesis, the growth of new blood vessels, is a natural defense mechanism helping to restore oxygen and nutrient supply to the affected brain tissue following an ischemic stroke. By stimulating vessel growth, angiogenesis may stabilize brain perfusion, thereby promoting neuronal survival, brain plasticity, and neurologic recovery. However, therapeutic angiogenesis after stroke faces challenges: new angiogenesis-induced vessels have a higher than normal permeability, and treatment to promote angiogenesis may exacerbate outcomes in stroke patients. The development of therapies requires elucidation of the precise cellular and molecular basis of the disease. Microenvironment homeostasis of the central nervous system is essential for its normal function and is maintained by the blood-brain barrier (BBB). Tight junction proteins (TJP) form the tight junction (TJ) between vascular endothelial cells (ECs) and play a key role in regulating the BBB permeability. We demonstrated that after stroke, new angiogenesis-induced vessels in peri-infarct areas have abnormally high BBB permeability due to a lack of major TJPs in ECs. Therefore, promoting TJ formation and BBB integrity in the new vessels coupled with speedy angiogenesis will provide a promising and safer treatment strategy for improving recovery from stroke. Pericyte is a central neurovascular unite component in vascular barriergenesis and are vital to BBB integrity. We found that pericytes also play a key role in stroke-induced angiogenesis and TJ formation in the newly formed vessels. Based on these findings, in this article, we focus on regulation aspects of the BBB functions and describe cellular and molecular special features of TJ formation with an emphasis on role of pericytes in BBB integrity during angiogenesis after stroke.

Quantifying Glioblastoma Drug Response Dynamics Incorporating Treatment Sensitivity and Blood Brain Barrier Penetrance From Experimental Data.

Many drugs investigated for the treatment of glioblastoma (GBM) have had disappointing clinical trial results. Efficacy of these agents is dependent on adequate delivery to sensitive tumor cell populations, which is limited by the blood-brain barrier (BBB). Additionally, tumor heterogeneity can lead to subpopulations of cells with different sensitivities to anti-cancer drugs, further impacting therapeutic efficacy. Thus, it may be important to evaluate the extent to which BBB limitations and heterogeneous sensitivity each contribute to a drug's failure. To address this challenge, we developed a minimal mathematical model to characterize these elements of overall drug response, informed by time-series bioluminescence imaging data from a treated patient-derived xenograft (PDX) experimental model. By fitting this mathematical model to a preliminary dataset in a series of nonlinear regression steps, we estimated parameter values for individual PDX subjects that correspond to the dynamics seen in experimental data. Using these estimates as a guide for parameter ranges, we ran model simulations and performed a parameter sensitivity analysis using Latin hypercube sampling and partial rank correlation coefficients. Results from this analysis combined with simulations suggest that BBB permeability may play a slightly greater role in therapeutic efficacy than relative drug sensitivity. Additionally, we discuss recommendations for future experiments based on insights gained from this model. Further research in this area will be vital for improving the development of effective new therapies for glioblastoma patients.

Alterations in Expression and Function of ABC Family Transporters at Blood-Brain Barrier under Liver Failure and Their Clinical Significances.

Liver failure is often associated with hepatic encephalopathy, due to dyshomeostasis of the central nervous system (CNS). Under physiological conditions, the CNS homeostasis is precisely regulated by the blood-brain barrier (BBB). The BBB consists of brain microvessel endothelial cells connected with a junctional complex by the adherens junctions and tight junctions. Its main function is to maintain brain homoeostasis via limiting the entry of drugs/toxins to brain. The brain microvessel endothelial cells are characterized by minimal pinocytotic activity, absent fenestrations, and highly expressions of ATP-binding cassette (ABC) family transporters (such as P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated proteins). These ABC transporters prevent brain from toxin accumulation by pumping toxins out of brain. Accumulating evidences demonstrates that liver failure diseases altered the expression and function of ABC transporters at The BBB, indicating that the alterations subsequently affect drugs’ brain distribution and CNS activity/neurotoxicity. ABC transporters also mediate the transport of endogenous substrates across the BBB, inferring that ABC transporters are also implicated in some physiological processes and the development of hepatic encephalopathy. This paper focuses on the alteration in the BBB permeability, the expression and function of ABC transporters at the BBB under liver failure status and their clinical significances.

221 Functional Modulation of the Blood Brain Barrier

Abstract INTRODUCTION Delivery of therapeutic agents to the brain is constrained by the blood-brain barrier (BBB). Previous work (Yarnitsky) suggested BBB permeability was increased with stimulation of the sphenopalatine ganglion (SPG). However, their model looked at FITC-dextran signal in CSF superfusate, a reflection of epithelial tight junctions at the blood-CSF barrier, and quantified BBB permeability using Evans blue, a marker insufficient for this role (Saunders). METHODS Experiments were conducted in Sprague-Dawley rats using 70 kDa FITC-dextran as a marker to quantify BBB permeability. Once anesthetized, the right femoral vein was exposed and catheterized. Next, SPG fibers were exposed behind the right eye and an electrode was hooked around those fibers. Stimulation occurred in blocks of 90 seconds of “on” time at 5 volts and 10 Hz followed by 60 seconds of “off” time. Injection of 0.1 mL of a 100 mg/mL concentration of FITC coincided with each “on” stimulation cycle; a total of 1.0 mL was injected per animal. Control animals were not stimulated, but had the same injection protocol as test animals. 90 seconds after the final injection, a blood sample was collected and the cerebrovasculature was flushed. Each brain was removed, equally divided, and homogenized. Post-centrifugation supernatant from blood and tissue homogenates was analyzed using fluorescent spectrometry. FITC concentrations were calculated using known standards. An uptake ratio was calculated by dividing sample FITC concentration by blood FITC concentration. RESULTS >Data from 4 control animals and 4 test animals demonstrated a significantly greater uptake ratio in test brains compared with control brains (P = 0.001). A significantly increased uptake ratio was also observed in both right and left hemispheres of test animals compared with controls (right, P = 0.03; left, P = 0.01). CONCLUSION Stimulation of SPG fibers significantly increased BBB permeability in both cerebral hemispheres of test animals when compared with controls.

Connexin Channels at the Glio-Vascular Interface: Gatekeepers of the Brain.

Neuronal survival, electrical signaling and synaptic activity require a well-balanced micro-environment in the central nervous system. This is achieved by the blood-brain barrier (BBB), an endothelial barrier situated in the brain capillaries, that controls near-to-all passage in and out of the brain. The endothelial barrier function is highly dependent on signaling interactions with surrounding glial, neuronal and vascular cells, together forming the neuro-glio-vascular unit. Within this functional unit, connexin (Cx) channels are of utmost importance for intercellular communication between the different cellular compartments. Connexins are best known as the building blocks of gap junction (GJ) channels that enable direct cell-cell transfer of metabolic, biochemical and electric signals. In addition, beyond their role in direct intercellular communication, Cxs also form unapposed, non-junctional hemichannels in the plasma membrane that allow the passage of several paracrine messengers, complementing direct GJ communication. Within the NGVU, Cxs are expressed in vascular endothelial cells, including those that form the BBB, and are eminent in astrocytes, especially at their endfoot processes that wrap around cerebral vessels. However, despite the density of Cx channels at this so-called gliovascular interface, it remains unclear as to how Cx-based signaling between astrocytes and BBB endothelial cells may converge control over BBB permeability in health and disease. In this review we describe available evidence that supports a role for astroglial as well as endothelial Cxs in the regulation of BBB permeability during development as well as in disease states.

Acute effects of focused ultrasound-induced increases in blood-brain barrier permeability on rat microvascular transcriptome

Therapeutic treatment options for central nervous system diseases are greatly limited by the blood-brain barrier (BBB). Focused ultrasound (FUS), in conjunction with circulating microbubbles, can be used to induce a targeted and transient increase in BBB permeability, providing a unique approach for the delivery of drugs from the systemic circulation into the brain. While preclinical research has demonstrated the utility of FUS, there remains a large gap in our knowledge regarding the impact of sonication on BBB gene expression. This work is focused on investigating the transcriptional changes in dorsal hippocampal rat microvessels in the acute stages following sonication. Microarray analysis of microvessels was performed at 6 and 24 hrs post-FUS. Expression changes in individual genes and bioinformatic analysis suggests that FUS may induce a transient inflammatory response in microvessels. Increased transcription of proinflammatory cytokine genes appears to be short-lived, largely returning to baseline by 24 hrs. This observation may help to explain some previously observed bioeffects of FUS and may also be a driving force for the angiogenic processes and reduced drug efflux suggested by this work. While further studies are necessary, these results open up intriguing possibilities for novel FUS applications and suggest possible routes for pharmacologically modifying the technique.

Angiopoietin-2-induced blood-brain barrier compromise and increased stroke size are rescued by VE-PTP-dependent restoration of Tie2 signaling.

The homeostasis of the central nervous system is maintained by the blood–brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angiogenesis, vascular stability, vascular permeability and lymphatic integrity. However, the precise role of angiopoietin/Tie2 signaling at the BBB remains unclear. We investigated the influence of Ang-2 on BBB permeability in wild-type and gain-of-function (GOF) mice and demonstrated an increase in permeability by Ang-2, both in vitro and in vivo. Expression analysis of brain endothelial cells from Ang-2 GOF mice showed a downregulation of tight/adherens junction molecules and increased caveolin-1, a vesicular permeability-related molecule. Immunohistochemistry revealed reduced pericyte coverage in Ang-2 GOF mice that was supported by electron microscopy analyses, which demonstrated defective intra-endothelial junctions with increased vesicles and decreased/disrupted glycocalyx. These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes. In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated. In mice, Ang-2 GOF resulted in increased infarct sizes and vessel permeability upon experimental stroke, implicating a role of Ang-2 in stroke pathophysiology. Increased permeability and stroke size were rescued by activation of Tie2 signaling using a vascular endothelial protein tyrosine phosphatase inhibitor and were independent of VE-cadherin phosphorylation. We thus identified Ang-2 as an endothelial cell-derived regulator of BBB permeability. We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1551-3) contains supplementary material, which is available to authorized users.

A Review of the Mechanisms of Blood-Brain Barrier Permeability by Tissue-Type Plasminogen Activator Treatment for Cerebral Ischemia.

Cerebrovascular homeostasis is maintained by the blood-brain barrier (BBB), which forms a mechanical and functional barrier between systemic circulation and the central nervous system (CNS). In patients with ischemic stroke, the recombinant tissue-type plasminogen activator (rt-PA) is used to accelerate recanalization of the occluded vessels. However, rt-PA is associated with a risk of increasing intracranial bleeding (ICB). This effect is thought to be caused by the increase in cerebrovascular permeability though various factors such as ischemic reperfusion injury and the activation of matrix metalloproteinases (MMPs), but the detailed mechanisms are unknown. It was recently found that rt-PA treatment enhances BBB permeability not by disrupting the BBB, but by activating the vascular endothelial growth factor (VEGF) system. The VEGF regulates both the dissociation of endothelial cell (EC) junctions and endothelial endocytosis, and causes a subsequent increase in vessel permeability through the VEGF receptor-2 (VEGFR-2) activation in ECs. Here, we review the possibility that rt-PA increases the penetration of toxic molecules derived from the bloodstream including rt-PA itself, without disrupting the BBB, and contributes to these detrimental processes in the cerebral parenchyma.

Utilizing Ultrasound to Transiently Increase Blood-Brain Barrier Permeability, Modulate of the Tight Junction Proteins, and Alter Cytoskeletal Structure.

The central nervous system is protected by the blood-brain barrier (BBB). The tight junction (TJ) proteins claudin-5 and zonula occludens-1 (ZO-1) as well as the cytoskeletal component F-actin play key roles in maintaining homeostasis of the BBB. Increases in BBB permeability may be beneficial for the delivery of pharmacological substances into the brain. Therefore, here, we assessed the use of ultrasound to induce transient enhancement of BBB permeability. We used fluorescein isothiocyanate (FITC)-dextran 40 to detect changes in the membrane permeability of bEnd.3 cells during ultrasound treatment. Ultrasound increased FITC-dextran 40 uptake into bEnd.3 cells for 2-6 h after treatment; however, normal levels returned after 24 h. An insignificant increase in lactate dehydrogenase (LDH) leakage also occurred 3 and 6 h after ultrasound treatment, whereas at 24 h, LDH leakage was indistinguishable between the control and treatment groups. Expression of claudin-5, ZO-1, and F-actin at the messenger RNA (mRNA) and protein levels was assessed with real-time polymerase chain reaction and western blotting. Ultrasound induced a transient decrease in claudin-5 mRNA and protein expression within 2 h of treatment; however, no significant changes in ZO-1 and F-actin expression were observed. Claudin-5, ZO-1, and F-actin immunofluorescence demonstrated that the cellular structures incorporating these proteins were transiently impaired by ultrasound. In conclusion, our ultrasound technique can temporarily increase BBB permeability without cytotoxicity to exposed cells, and the method can be exploited in the delivery of drugs to the brain with minimal damage.

Immune responses to non-tumor antigens in the central nervous system.

The central nervous system (CNS), once viewed as an immune-privileged site protected by the blood–brain barrier (BBB), is now known to be a dynamic immunological environment through which immune cells migrate to prevent and respond to events such as localized infection. During these responses, endogenous glial cells, including astrocytes and microglia, become highly reactive and may secrete inflammatory mediators that regulate BBB permeability and recruit additional circulating immune cells. Here, we discuss the various roles played by astrocytes, microglia, and infiltrating immune cells during host immunity to non-tumor antigens in the CNS, focusing first on bacterial and viral infections, and then turning to responses directed against self-antigens in the setting of CNS autoimmunity.

‘À la Carte’ Peptide Shuttles: Tools to Increase Their Passage across the Blood–Brain Barrier

Noninvasive methods for efficient drug delivery to the brain is an unmet need. Molecular access to the brain is regulated by the blood-brain barrier (BBB) established by the endothelial cells of brain vessels. Passive diffusion is one of the main mechanisms that organic compounds use to travel through these endothelial cells. This passage across the BBB is determined mainly by certain physicochemical properties of the molecule such as lipophilicity, size, and the presence of hydrogen bond donors and acceptors. One emerging strategy to facilitate the passage of organic compounds across the BBB is the use of peptide shuttles.1 In using this approach the permeability in front the BBB is, clearly, determined by the combined physicochemical properties of both the cargo and the shuttle. Herein we report the synthesis of a series of variations of one of the more efficient peptide shuttles, (N-MePhe)n . These include diverse structural features such as various backbone stereochemistries or the presence of non-natural amino acids, including halogenated residues. In several cases, we assessed the BBB permeability of both the shuttles alone and linked to a few cargos. Our results show how factors such as stereochemistry or halogen content influences the passage across the BBB and, more importantly, opens the way to a strategy of peptide shuttles 'à la carte', in which a particular fine-tuned shuttle is used for each specific cargo.

Targeting metabolic inflammation in Parkinson’s disease: Implications for prospective therapeutic strategies

1. Parkinson's disease (PD) is one of the most common neurodegenerative disorders and is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Although the aetiology of PD has not been clarified as yet, it is believed that ageing, diet, diabetes and adiposity are associated with PD. 2. Type 2 diabetes and lipid abnormalities share multiple common pathophysiological mechanisms with PD. In particular, inflammation plays a critical role in the destruction of both pancreatic islet β-cells and dopaminergic neurons in the substantia nigra. Emerging evidence indicates that dysfunctions of energy metabolism evoke metabolic inflammation, which differs to the narrow concept of inflammation, participating in systemic pathological processes such as neurodegenerative disease and diabetes. 3. The brain is considered an immunologically privileged organ, free from immune reactions, because it is protected by the blood-brain barrier (BBB). However, studies have shown that there is gradual impairment of neurovascular function with ageing and in neurodegenerative disorders, resulting in abnormal states, including increased BBB permeability. Consequently, harmful elements that would not normally be able to cross the BBB, such as pro-inflammatory factors, reactive oxygen species and neurotoxins, infiltrate into the brain, triggering neural injury. 4. Currently, the drugs available for the treatment of PD only ameliorate the symptoms of the disease. Therapeutic strategies aimed at stopping or modifying disease progression are still being sought. Most recent studies suggest that both central and peripheral inflammation may be dysregulated in PD. Therefore, therapeutic strategies aimed at modulating systemic inflammatory reactions or energy metabolism may represent a goal in neuroprotection in PD.