Testing of a hypothesis for osmotic opening of the blood-brain barrier.

Human induced pluripotent stem cell-derived neural stem cells (hiPS-NSCs) represent an exciting therapeutic strategy for traumatic spinal cord injury (SCI) as they can replace lost neural circuits, remyelinate denuded axons and provide local trophic support. Unfortunately, most patients are in the chronic phase of their injuries where dense chondroitin sulfate proteoglycan (CSPG) scarring significantly impairs neurite outgrowth and regenerative cell migration. Several scarmodifying enzymes have been shown to synergistically enhance NSCmediated recovery, however, nonspecific intrathecal administration can produce off-target effects. We aimed to generate a geneticallyengineered line of hiPS-NSCs, termed Spinal Microenvironment Modifying and Regenerative Therapeutic (SMaRT) cells, uniquely capable of expressing a scar-modifying enzyme within the host environment to enhance functional recovery. A proprietary enzyme was non-virally integrated into hiPS-NSCs and a monocloncal line of SMaRT cells was generated and extensively characterized. The expressed enzyme rapidly degrades CSPGs on biochemical assays and allows neurons to extend into CSPG-rich regions in vitro. Furthermore, unlike wild-type hiPS-NSC media, conditioned SMaRT cell media can degrade post-injury rodent CSPGs in ex vivo injured cord cryosections. T-cell deficient rats (N = 60) with translationallyrelevant chronic C6-7 clip-contusion injuries have been randomized to receive: (1) vehicle, (2) hiPS-NSCs, (3) SMaRT cells, or (4) sham surgery (laminectomy). While blinded sensorimotor behavioural assessments and rehabilitation are ongoing with a long-term 32-week endpoint, interim histologic analysis shows that grafted human cells are extending remarkably long ( 20,000 lm) axons along host white matter tracts in the rostral and caudal directions. This work provides exciting proof-of-concept data that genetically-engineered SMaRT cells can degrade CSPGs in vitro and that human NSC transplants can grow long axons in chronic cervical SCI to potentially form a bridge for sensorimotor signal transmission. This work is generously supported by the Canadian Institutes of Health Research, Phillip and Peggy DeZwirek, OIRM, and the Krembil Foundation.

AT THE END OF THE CENTURY before the last, a young graduate student performed an experiment that would forever change the way we look at the microvasculature of the brain. He injected basic dyes into the blood and noted that nearly every tissue of the body was colored by the dye except for the central nervous system (CNS). This experiment and the observation that bile salts could cause seizures when injected into the brain but not intravenously were the seminal 19th century observations giving rise to the concept of a blood-brain barrier (BBB). The young graduate student, Paul Ehrlich, would get many things right in his career, winning a Nobel prize in 1908 for his work on antibiotics. But the BBB he got wrong. Ehrlich thought the brain did not stain because the dye was not taken up by brain tissue, not because a barrier prevented the dye from reaching the brain. Indeed, that a BBB really existed and that it resided at the level of the capillary bed and the choroid plexus were not finally established until the late 1960s. Elegant physiological experiments by Davson and Segal (10) and electron microscopy studies by Reese and Karnovsky (24) demonstrated the basis of the BBB: the capillary bed of adult mammals is modified to exclude the production of a plasma ultrafiltrate. The major modifications include a greatly decreased rate of pinocytosis, a lack of intracellular pores and fenestrae, and obliteration of the intercellular space between brain endothelial cells by tight junctions that essentially “cement” apposing endothelial cells together. The lack of production of an ultrafiltrate means that circulating proteins such as albumin do not exude from blood into brain. This is the basis for Ehrlich’s 120-year-old observation: basic dyes bind to albumin so tightly that they are a visible proxy for plasma

Blood-Brain Barrier Breakdown and Blood-Brain Communication in Neurological and Psychiatric Diseases

To the Editor: We read with interest the paper from G. D. Sysyn et al. ([2][1]), who published their observations regarding the effects of postnatal corticosteroid (intramuscular dexamethasone injections, doses: 0.01, 0.25, and 0.5 mg/kg) on regional blood-brain barrier permeability (transfer

Objective To explore the role of MMP-9 in blood-brain barrier (BBB) disruption and to promote rMSCs migration.Methods To investigate the effect of MMP-9 on the permeability of BBB,an in vitro model of BBB was established and performed Transwell experiments.To observe the pathologic changes of the cerebral cortex,rat brains from Sham treated group,HIBD group,MMP-9 treated group and TIMP-1 (intervention) treated group at different time points (0.5,1,3,7,14 d) were prepared and stained with Hematoxylin-Eosin and quantified the brain water content.Permeability of BBB examined by EB values measurement.MMP-9 protein level of rat cortex was detected by Western Blot.The number of Brdu labeled rMSCs in the rat cerebral cortex of each group was quantified by Immunohistochemistry (IHC).Results Transwell experiments results showed that migration of rMSCs increased remarkably in hypoxic condition compared to that of normal control (P＜0.01).The number of rMSCs migrated in the MMP-9 treated group was much more than that of negative control group and TIMP-1 group (P＜0.01).The results of pathology showed that compared to HIBD group,brain water content and the permeability of the BBB were increased in MMP-9 treated group but reduced in TIMP-1 treated one.The expression of MMP-9 protein in MMP-9 treated group was reached the peak at 3 d point,which was higher than that of HIBD group and TIMP-1 group (P＜0.05,P＜0.01).The quantity of Brdu labeled rMSCs crossed BBB in MMP-9 treated group was extremely higher than that of HIBD group (P＜0.05,P＜0.01).Conclusions The expression level of MMp-9 protein was improved after hypoxic-ischemic.MMP-9 could facilitate the migration of rMSCs through vitro BBB and control the opening of BBB,which indicate that MMP-9 may facilitate the migration of rMSCs through BBB into brain. Key words: Mesenchymal stem cells; Matrix metalloproteinases-9; Hypoxia-ischemia; Blood-brain barrier

Increased brain microvascular permeability and disruption of blood-brain barrier (BBB) function are among hallmarks of ischemic stroke. Recently, peptidase neurlysin (Nln) has been identified as a compensatory and cerebroprotective mechanism in the post-stroke brain that functions to process a diverse group of extracellular neuropeptides, including bradykinin (BK), neurotensin (NT) and substance P (SP). A number of studies suggest involvement of BK, NT and SP in BBB impairment and edema formation after stroke, however there is paucity of data in regards to the direct effects of these peptides on the brain microvascular endothelial cells (BMECs) and BBB. The purpose of this study was to evaluate the direct effects of BK, NT and SP on permeability of BBB in an in vitro model based on human, induced pluripotent stem cell (iPSC)-derived BMECs. Our data indicate that all three peptides increase BBB permeability in a concentration-dependent manner in an in vitro model formed from two different iPSC lines (CTR90F and CTR65M) and widely used hCMEC/D3 human BMECs. Combination of BK, NT and SP at a sub-effective concentration also resulted in increased BBB permeability in the iPSC-derived model. Furthermore, we observed abrogation of BK, NT and SP effects with pretreatment of pharmacological blockers targeting their specific receptors or in presence of recombinant neurolysin (Nln). This is the first experimental study to document increased permeability of BBB in response to direct action of NT in an in vitro model. In addition, our study confirms the expected but not well-documented, direct effect of SP on BBB permeability and adds to the well-recognized actions of BK on BBB. Lastly, we demonstrate that peptidase Nln can neutralize the effects of these peptides on BBB, suggesting potential therapeutic implications.

Overexpression of Mfsd2a attenuates blood brain barrier dysfunction via Cav-1/Keap-1/Nrf-2/HO-1 pathway in a rat model of surgical brain injury

Stroke Literature Synopses: Basic Science.

The integrity of the blood-brain barrier may play an important pathophysiological role during postischemic reperfusion. To determine the factors that lead to exacerbation of brain injury by reperfusion, we investigated changes in cerebral blood flow, blood-brain barrier permeability, edema formation, and infarction in permanent or temporary middle cerebral artery occlusion in rats and studied the relation between local cerebral blood flow and blood-brain barrier disruption. Middle cerebral artery occlusion was performed with the rat suture model, allowing either permanent (6 hours) or temporary occlusion (3 hours of occlusion and 3 hours of reperfusion). We measured brain water, ion contents, and infarct volumes and determined cerebral blood flow using laser Doppler flowmetry and blood-brain barrier permeability with [3H] alpha-aminoisobutyric acid. During occlusion, cerebral blood flow was reduced to 7% to 15% (permanent) and 10% to 17% (temporary) of the baseline. During 3 hours of reperfusion, it returned to 47% to 80% (lateral cortex) and 78% to 98% (medial cortex) of the baseline. Compared with the contralateral hemisphere, the water content in the ischemic area increased in both permanent and temporary groups (P < .05, P < .01). Both infarct volume and blood-brain barrier disruption were greater in the reperfusion group compared with the permanent occlusion group (P < .05). Blood-brain barrier disruption correlated with cerebral blood flow during reperfusion (P < .05). These findings demonstrate that brain infarct and blood-brain barrier disruption are exacerbated after reperfusion in this model of focal ischemia. Blood-brain barrier disruption may relate to the degree of cerebral blood flow recovery. Thus, although early reperfusion in focal ischemia may preserve penumbra tissue, late reperfusion may increase the tissue injury.

Background: One major issue related to carotid artery revascularization procedures is their potential for inducing disturbances in blood brain barrier (BBB) permeability, which is considered to play a critical role in the pathophysiology of hyperperfusion syndrome. In this study, our aim was to evaluate the predictors of impaired BBB permeability and its topographic distribution among patients undergoing carotid artery stenting. Methods: Clinical information (including demographic data, cardiovascular risk factors, degree of stenosis, indication and timing of the procedure), together with pre- and immediate post- (&lt;24 hours) procedural magnetic resonance imaging (MRI) data was prospectively collected in a consecutive series of patients. BBB integrity was specifically evaluated on post-contrast FLAIR sequences. Regions of contrast extravasation were semi-automatically outlined and co-registered to a standard template in order to determine the topography of BBB disruption. Results: Evidence for BBB disruption was observed in 11 out of 43 patients (26%) prior to stenting, a feature that was solely related to a recent history of ischemic stroke (&lt;30 days) in multivariate analysis (p=0.033). New-onset or increased BBB disruption following the procedure was seen in 22 patients (51%); increased permeability was primarily observed in the ipsilateral cortical watershed territories (Figure) and was inversely related to the duration elapsed between carotid revascularization and MRI (p=0.036). Hyperperfusion syndrome developed in 7% of our patients, all of whom also having evidence of increased BBB permeability on post-stenting MRI. Conclusion: Our findings highlight that BBB disruption in the peri-stenting setting is a frequently common observation, with specific spatial and temporal characteristics. Its predictive utility for predicting hyperperfusion syndrome and unfavorable clinical course, on the other hand, is fairly low.

The effects of 17β-estradiol on blood brain barrier integrity in the absence of the estrogen receptor alpha; an in-vitro model

Increased brain microvascular permeability and disruption of blood-brain barrier (BBB) function are among hallmarks of several acute neurodegenerative disorders, including stroke. Numerous studies suggest the involvement of bradykinin (BK), neurotensin (NT) and substance P (SP) in BBB impairment and oedema formation after stroke; however, there is paucity of data in regard to the direct effects of these peptides on the brain microvascular endothelial cells (BMECs) and BBB. The present study aimed to evaluate the direct effects of BK, NT and SP on the permeability of BBB in an in vitro model based on human induced pluripotent stem cell (iPSC)-derived BMECs. Our data indicate that all three peptides increase BBB permeability in a concentration-dependent manner in an in vitro model formed from two different iPSC lines (CTR90F and CTR65M) and widely used hCMEC/D3 human BMECs. The combination of BK, NT and SP at a sub-effective concentration also resulted in increased BBB permeability in the iPSC-derived model indicating potentiation of their action. Furthermore, we observed abrogation of BK, NT and SP effects with pretreatment of pharmacological blockers targeting their specific receptors. Additional mechanistic studies indicate that the short-term effects of these peptides are not mediated through alteration of tight-junction proteins claudin-5 and occludin, but likely involve redistribution of F-actin and secretion of vascular endothelial growth factor. This is the first experimental study to document the increased permeability of the BBB in response to direct action of NT in an in vitro model. In addition, our study confirms the expected but not well-documented, direct effect of SP on BBB permeability and adds to the well-recognised actions of BK on BBB. Lastly, we demonstrate that peptidase neurolysin can neutralise the effects of these peptides on BBB, suggesting potential therapeutic implications.

The blood-brain barrier (BBB) is critically important for brain health by regulating molecular exchanges between blood and brain. Hypertension (HTN) induces breakdown of the BBB which may contribute to its deleterious effect on the brain, but the cellular bases of the BBB opening remain to be established. We used a model of angiotensin II (AngII) HTN (600ng/kg/min x 2 weeks; n&gt;5/group) to investigate the mechanisms of the BBB opening. BBB permeability was assessed spectrophotometrically in C57BL/6J mice using 3000 MW FITC-dextran as a tracer. HTN increased BBB permeability in Ang II HTN (29.8 ± 0.9 vs 18.1 ± 0.5 ng/g in control, p&lt;0.01), an effect partially ameliorated by the angiotensin type 1 receptor (AT1R) antagonist losartan in the drinking water (22.9 ± 0.6 ng/g) but not by hydralazine + hydrochlorothiazide (27.4 ± 0.7 ng/g), suggesting involvement of AT1R and not elevated blood pressure. Next, we sought to identify the mechanisms of the breakdown of the BBB in HTN. First, we used electron microscopy to examine the ultrastructure of endothelial tight junctions (TJ) and assess vesicular transport, key components of the BBB. HTN reduced the length (-25%) and complexity (-11%) of TJ, and increased the number of endothelial vesicles (2.22 vs 1.42 vesicles/endothelial area, p&lt;0.05). The TJ remodeling was associated with a reduction in occludin (-17%, p&lt;0.01) and claudin-5 (-30%, p&lt;0.05) mRNA in microvascular preparations. Additionally, the expression of Mfsd2a, a lipid transporter that also suppresses vesicular transcytosis, was markedly attenuated (-43%, p&lt;0.01). Taken together, these data suggest a strong effect of Ang II on cerebral endothelial cells to induce BBB opening. Since perivascular macrophages (PVM) mediate cerebral endothelial dysfunction in HTN (J Clin Invest 2016;126:4674), we tested their involvement in the BBB opening. PVM depletion with icv clodronate or deletion of AT1 receptors in PVM partially attenuated the BBB opening in Ang II HTN (p&lt;0.05). Thus, we conclude that AT1R in cerebral endothelial cells and PVM mediate the BBB opening by targeting both paracellular and vesicular transport. Such increase in BBB permeability to circulating agents may contribute to the cerebrovascular and cognitive dysfunction associated with HTN.

Blood-Brain Barrier Permeability in Aging and Alzheimer's Disease.

BackgroundEndothelial cell (EC) released microvesicles (EMVs) can affect various target cells by transferring carried genetic information. Astrocytes are the main components of the blood brain barrier (BBB) structure in the brain and participate in regulating BBB integrity and blood flow. The interactions between ECs and astrocytes are essential for BBB integrity in homeostasis and pathological conditions. Here, we studied the effects of human brain microvascular ECs released EMVs on astrocyte functions. Additionally, we investigated the effects of EMVs treated astrocytes on regulating BBB function and cerebral ischemic damage.ResultsEMVs prepared from ECs cultured in normal condition (n-EMVs) or oxygen and glucose deprivation (OGD-EMVs) condition had diverse effects on astrocytes. The n-EMVs promoted, while the OGD-EMVs inhibited the proliferation of astrocytes via regulating PI3K/Akt pathway. Glial fibrillary acidic protein (GFAP) expression (marker of astrocyte activation) was up-regulated by n-EMVs, while down-regulated by OGD-EMVs. Meanwhile, n-EMVs inhibited but OGD-EMVs promoted the apoptosis of astrocytes accompanied by up/down-regulating the expression of Caspase-9 and Bcl-2. In the BBB model of ECs-astrocytes co-culture, the n-EMVs, conversely to OGD-EMVs, decreased the permeability of BBB accompanied with up-regulation of zonula occudens-1(ZO-1) and Claudin-5. In a transient cerebral ischemia mouse model, n-EMVs ameliorated, while OGD-EMVs aggravated, BBB disruption, local cerebral blood flow (CBF) reduction, infarct volume and neurological deficit score.ConclusionsOur data suggest that EMVs diversely modulate astrocyte functions, BBB integrity and CBF, and could serve as a novel therapeutic target for ischemic stroke.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0243-1) contains supplementary material, which is available to authorized users.