The Protective Mechanism for the Blood–Brain Barrier Induced by Aminoguanidine in Surgical Brain Injury in Rats

This study was performed to investigate the mechanism of blood–brain barrier (BBB) permeability change, which was induced by aminoguanidine (AG) after surgical brain injury (SBI) in rats. Compared to control group, AG (150 mg/kg, i.p.) significantly reduced Evans blue extravasation into brain tissue at 24 h after surgical resection, it also induced a 32% decrease of malondialdehyde (MDA) values and a 1.1-fold increase of the glutathione (GSH) levels at 12 h after injury. The expression of inducible nitric oxide synthase (iNOS) reached the peak value at 24 h after SBI, which was significantly attenuated after AG treatment. In addition, ZO-1 protein was up-regulated by AG (150 mg/kg) treatment at 24 h after SBI. Our results indicated that AG could protect the BBB after SBI, which could be correlated with antioxidative property, the down-regulation of iNOS and up-regulation of tight junction protein expression.     

Enhanced Therapeutic Potential of Nano-Curcumin Against Subarachnoid Hemorrhage-Induced Blood–Brain Barrier Disruption Through Inhibition of Inflammatory Response and Oxidative Stress

Curcumin and nano-curcumin both exhibit neuroprotective effects in early brain injury (EBI) after experimental subarachnoid hemorrhage (SAH). However, the mechanism that whether curcumin and its nanoparticles affect the blood–brain barrier (BBB) following SAH remains unclear. This study investigated the effect of curcumin and the poly(lactide-co-glycolide) (PLGA)-encapsulated curcumin nanoparticles (Cur-NPs) on BBB disruption and evaluated the possible mechanism underlying BBB dysfunction in EBI using the endovascular perforation rat SAH model. The results indicated that Cur-NPs showed enhanced therapeutic effects than that of curcumin in improving neurological function, reducing brain water content, and Evans blue dye extravasation after SAH. Mechanically, Cur-NPs attenuated BBB dysfunction after SAH by preventing the disruption of tight junction protein (ZO-1, occludin, and claudin-5). Cur-NPs also up-regulated glutamate transporter-1 and attenuated glutamate concentration of cerebrospinal fluid following SAH. Moreover, inhibition of inflammatory response and microglia activation both contributed to Cur-NPs’ protective effects. Additionally, Cur-NPs markedly suppressed SAH-mediated oxidative stress and eventually reversed SAH-induced cell apoptosis in rats. Our findings revealed that the strategy of using Cur-NPs could be a promising way in improving neurological function in EBI after experimental rat SAH.     

Phospholipid transfer protein (PLTP) deficiency impaired blood–brain barrier integrity by increasing cerebrovascular oxidative stress

Phospholipid transfer protein (PLTP) regulates lipid metabolism and plays an important role in oxidative stress. PLTP is highly expressed in blood–brain barrier (BBB), but the role of PLTP in BBB integrity is not clear. In this study, BBB permeability was detected with in vivo multiphoton imaging and Evans blue assay. We found that PLTP deficient mice exhibited increased BBB permeability, as well as decreased expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Cerebrovascular oxidative stress increased in PLTP deficient mice, including increased levels of reactive oxygen species (ROS) and lipid peroxidation marker 4-hydroxy-2-nonenal (HNE) and reduced superoxide dismutase (SOD) activity. Dietary supplementation of antioxidant vitamin E increased BBB integrity and tight junction proteins expression via reducing cerebrovascular oxidative stress. These findings indicated an essential role of PLTP in maintaining BBB integrity, possibly through its ability to transfer vitamin E, and modulate cerebrovascular oxidative stress.     

Matrix metalloproteinases inhibition provides neuroprotection against hypoxia-ischemia in the developing brain

The present study was designed to investigate the role of matrix metalloproteinases (MMPs) in the immature brain and the long term effects of early MMPs inhibition after hypoxic-ischemic (HI) injury. HI was induced by unilateral ligation of the right carotid artery followed by hypoxia (8% O₂ for 2 h) in P7 rat pups. GM6001, a broad spectrum MMPs inhibitor, was injected (50 mg/kg or 100 mg/kg) intraperitoneally at 2 h and 24 h after HI injury. Blood-brain barrier (BBB) integrity, brain edema, MMP-2/-9 activity, TIMP-1/-2 and tight junction protein (TJP) level were evaluated using IgG staining, Evan's blue extravasation, brain water content, zymography and western blot. Doxycycline, another MMPs inhibitor, was injected (10 mg/kg or 30 mg/kg) intraperitoneally at 2 h after HI, then BBB integrity and brain edema were measured at 48 h post-HI using brain water content measurement and IgG staining. The long-term effects of early MMPs inhibition (GM6001, 100 mg/kg) were evaluated by neurobehavioral tests, body weight, and brain atrophy measurement. GM6001 attenuated brain edema and BBB disruption at the dosage of 100 mg/kg. MMP-2 activity increased at 24 h and peaked at 48 h after HI, whereas MMP-9 activity peaked at 24 h and tapered by 48 h after HI. MMP-9/-2 activities were significantly attenuated by GM6001 at 24 h and 48 h after HI. The degradation of TJPs (ZO-1 and occludin) at 48 h after HI was reversed by GM6001 treatment. Early MMPs inhibition had long-term effects that attenuated ipsilateral brain tissue loss, and improved neurobehavioral outcomes after HI. These results suggest that early MMPs inhibition with a broad-spectrum inhibitor provides both acute and long-term neuroprotection in the developing brain by reducing TJPs degradation, preserving BBB integrity, and ameliorating brain edema after neonatal HI injury.     

Dendritic cell-derived exosomal miR-3064-5p inhibits SIRT6/PCSK9 to protect the blood-brain barrier after subarachnoid hemorrhage

The protection of the blood-brain barrier (BBB) is the key direction to improving subarachnoid hemorrhage (SAH). Therefore, developing appropriate targeted drugs and therapies has become an urgent task for SAH patients. In this study, we investigated the role of dendritic cells (DCs) exosomal miR-3064-5p in repairing the BBB, providing a new basis for treating SAH. We detected the expression of miR-3064-5p in exosomes secreted by DCs (DCs-exo). An SAH rat model was constructed by intravascular perforation and characterized by HE and TUNEL-IF staining. We found that overexpression of miR-3064-5p in SAH rats suppressed iNOS expression and promoted the accumulation of tight junction proteins (Occludin, Claudin-3, ZO-1), whereas knockdown of miR-3064-5p exerted the opposite effect. Dual-LUC assay confirmed that miR-3064-5p could target and inhibit SIRT6. Knockdown of SIRT6 inhibited inflammatory cytokine (IL-6, IL-1β, IFN-γ, and TGF-β1) levels and apoptosis. The results of the co-IP assay showed that SIRT6 interacted with PCSK9, and knockdown of SIRT6 suppressed the expression of PCSK9. Moreover, DCs-exo reduced brain edema, upregulated miR-3064-5p and downregulated SIRT6 and PCSK9 in SAH rats. DCs-exo reduced inflammatory factors and increased tight junction proteins in SAH rats. Overexpression of miR-3064-5p enhanced the protective effect of DCs-exo, while overexpression of SIRT6 partially counteracted the effect. This study confirmed that DCs could secrete miR-3064-5p to ameliorate BBB damage after SAH. Mechanistically, miR-3064-5p alleviated BBB damage by targeting and inhibiting SIRT6/PCSk9 signaling pathway.     

Mice lacking insulin or insulin-like growth factor 1 receptors in vascular endothelial cells maintain normal blood-brain barrier

The blood-brain barrier (BBB) is created by a combination of endothelial cells with tight junctions and astrocytes. One of the key tight junction proteins, zona occludens-1 (ZO-1), has been reported to be stimulated in its expression by insulin and IGF-1. To assess the role of insulin and IGF-1 in endothelial cells in the BBB we have utilized mice with a vascular endothelial cell-specific knockout of the insulin receptor (VENIRKO) and IGF-1 receptor (VENIFARKO). Both of these mice show a normal BBB based on no increase in leakage of Evans blue dye in the brain of these mice basally or after cold injury. Furthermore, the structural integrity of the BBB and blood-retinal barrier (BRB) was intact using the vascular markers lectin B-4 and ZO-1, and both proteins were properly co-localized in both brain and retinal vascular tissue of these mice. These observations indicate that neither insulin nor IGF-1 signaling in vascular endothelial cells is required for development and maintenance of BBB or BRB.     

Blood-Brain Barrier Breakdown after Embolic Stroke in Rats Occurs without Ultrastructural Evidence for Disrupting Tight Junctions

The term blood-brain barrier (BBB) relates to the ability of cerebral vessels to hold back hydrophilic and large molecules from entering the brain, thereby crucially contributing to brain homeostasis. In fact, experimental opening of endothelial tight junctions causes a breakdown of the BBB evidenced as for instance by albumin leakage. This and similar observations led to the conclusion that BBB breakdown is predominantly mediated by damage to tight junction complexes, but evidentiary ultrastructural data are rare. Since functional deficits of the BBB contribute to an increased risk of hemorrhagic transformation and brain edema after stroke, which both critically impact on the clinical outcome, we studied the mechanism of BBB breakdown using an embolic model of focal cerebral ischemia in Wistar rats to closely mimic the essential human pathophysiology. Ischemia-induced BBB breakdown was detected using intravenous injection of FITC-albumin and tight junctions in areas of FITC-albumin extravasation were subsequently studied using fluorescence and electron microscopy. Against our expectation, 25 hours after ischemia induction the morphology of tight junction complexes (identified ultrastructurally and using antibodies against the transcellular proteins occludin and claudin-5) appeared to be regularly maintained in regions where FITC-albumin massively leaked into the neuropil. Furthermore, occludin signals along pan-laminin-labeled vessels in the affected hemisphere equaled the non-affected contralateral side (ratio: 0.966 vs. 0.963; P = 0.500). Additional ultrastructural analyses at 5 and 25 h after ischemia induction clearly indicated FITC-albumin extravasation around vessels with intact tight junctions, while the endothelium exhibited enhanced transendothelial vesicle trafficking and signs of degeneration. Thus, BBB breakdown and leakage of FITC-albumin cannot be correlated with staining patterns for common tight junction proteins alone. Understanding the mechanisms causing functional endothelial alterations and endothelial damage is likely to provide novel protective targets in stroke.     

Attenuation of Acute Phase Injury in Rat Intracranial Hemorrhage by Cerebrolysin that Inhibits Brain Edema and Inflammatory Response

The outcome of intracerebral hemorrhage (ICH) is mainly determined by the volume of the hemorrhage core and the secondary brain damage to penumbral tissues due to brain swelling, microcirculation disturbance and inflammation. The present study aims to investigate the protective effects of cerebrolysin on brain edema and inhibition of the inflammation response surrounding the hematoma core in the acute stage after ICH. The ICH model was induced by administration of type VII bacterial collagenase into the stratum of adult rats, which were then randomly divided into three groups: ICH + saline; ICH + Cerebrolysin (5 ml/kg) and sham. Cerebrolysin or saline was administered intraperitoneally 1 h post surgery. Neurological scores, extent of brain edema content and Evans blue dye extravasation were recorded. The levels of pro-inflammatory factors (IL-1β, TNF-α and IL-6) were assayed by Real-time PCR and Elisa kits. Aquaporin-4 (AQP4) and tight junction proteins (TJPs; claudin-5, occludin and zonula occluden-1) expression were measured at multiple time points. The morphological and intercellular changes were characterized by Electron microscopy. It is found that cerebrolysin (5 ml/kg) improved the neurological behavior and reduced the ipsilateral brain water content and Evans blue dye extravasation. After cerebrolysin treated, the levels of pro-inflammatory factors and AQP4 in the peri-hematomal areas were markedly reduced and were accompanied with higher expression of TJPs. Electron microscopy showed the astrocytic swelling and concentrated chromatin in the ICH group and confirmed the cell junction changes. Thus, early cerebrolysin treatment ameliorates secondary injury after ICH and promotes behavioral performance during the acute phase by reducing brain edema, inflammatory response, and blood–brain barrier permeability.     

Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats

Background

Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood–brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection.

Methods

The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure.The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies.

Results

The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats.

Conclusions

We demonstrated that the BBB permeability was altered in a rat model with rabies virus inoculation. Delivery of neutralizing mAb to the infected site in brain combined with effective breakdown of BBB could be an aggressive but feasible therapeutic mode in rabies when the CNS infection has been established.     

Rho-kinase inhibitor hydroxyfasudil protects against HIV-1 Tat-induced dysfunction of tight junction and neprilysin/Aβ transfer receptor expression in mouse brain microvessels

HIV-1 transactivator protein (Tat) induces tight junction (TJ) dysfunction and amyloid-beta (Aβ) clearance dysfunction, contributing to the development and progression of HIV-1-associated neurocognitive disorder (HAND). The Rho/ROCK signaling pathway has protective effects on neurodegenerative disease. However, the underlying mechanisms of whether Rho/ROCK protects against HIV-1 Tat-caused dysfunction of TJ and neprilysin (NEP)/Aβ transfer receptor expression have not been elucidated. C57BL/6 mice were administered sterile saline (i.p., 100 μL) or Rho-kinase inhibitor hydroxyfasudil (HF) (i.p., 10 mg/kg) or HIV-1 Tat (i.v., 100 μg/kg) or HF 30 min before being exposed to HIV-1 Tat once a day for seven consecutive days. Evans Blue (EB) leakage was detected via spectrophotometer and brain slides in mouse brains. The protein and mRNA levels of zonula occludens-1 (ZO-1), occludin, NEP, receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP1) in mouse brain microvessels were, respectively, analyzed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Exposure of the mice to HIV-1 Tat increased the amount of EB leakage, EB fluorescence intensity, blood–brain barrier (BBB) permeability, as well as the RAGE protein and mRNA levels, and decreased the protein and mRNA levels of ZO-1, occludin, NEP, and LRP1 in mouse brain microvessels. However, these effects were weakened by Rho-kinase inhibitor HF. Taken together, these results provide information that the Rho/ROCK signaling pathway is involved in HIV-1 Tat-induced dysfunction of TJ and NEP/Aβ transfer receptor expression in the C57BL/6 mouse brain. These findings shed some light on potentiality of inhibiting Rho/Rock signaling pathway in handling HAND.

     

Immunolocalization of tight junction proteins in blood vessels in human germinal matrix and cortex

Brain development occurs in a specialized environment maintained by a blood–brain barrier (BBB). An important structural element of the BBB is the endothelial tight junction (TJ). TJs are present during the embryonic period, but BBB impermeability accrues over an extended gestational interval. In studies of human premature infants, we used immunomicroscopy to determine if amounts of the TJ proteins ZO-1, claudin and occludin increase with gestational age in vessels of germinal matrix (GM) and cortex. By 24 weeks postconception (PC), TJ proteins were present in both GM and cortical vessels, but immunoreactivity in the GM of the youngest subjects was less than in older subjects. At 24 weeks PC, TJ protein immunoreactivity in GM vessels was less than in cortical vessels suggesting that TJ maturation progresses along a superficial to deep brain axis. This concept correlates with conclusions from previous analyses of the expression of brain endothelial cell alkaline phosphatase (AP) activity. AP appears in cortical vessels before appearing in deep white matter and GM vessels. Together, these data indicate that differentiation of some functional specializations is still in progress in GM vessels during the third trimester. This maturation could relate to the pathogenesis of germinal matrix hemorrhage–intraventricular hemorrhage.     

Blood-Brain Barrier Permeability Is Exacerbated in Experimental Model of Hepatic Encephalopathy via MMP-9 Activation and Downregulation of Tight Junction Proteins

The present study was designed to investigate the mechanisms involved in blood-brain barrier (BBB) permeability in bile duct ligation (BDL) model of chronic hepatic encephalopathy (HE). Four weeks after BDL surgery, a significant increase was observed in serum bilirubin levels. Masson trichrome staining revealed severe hepatic fibrosis in the BDL rats. ^99mTc-mebrofenin retention was increased in the liver of BDL rats suggesting impaired hepatobiliary transport. An increase in permeability to sodium fluorescein, Evans blue, and fluorescein isothiocyanate (FITC)-dextran along with increase in water and electrolyte content was observed in brain regions of BDL rats suggesting disrupted BBB. Increased brain water content can be attributed to increase in aquaporin-4 mRNA and protein expression in BDL rats. Matrix metalloproteinase-9 (MMP-9) mRNA and protein expression was increased in brain regions of BDL rats. Additionally, mRNA and protein expression of tissue inhibitor of matrix metalloproteinases (TIMPs) was also increased in different regions of brain. A significant decrease in mRNA expression and protein levels of tight junction proteins, viz., occludin, claudin-5, and zona occluden-1 (ZO-1) was observed in different brain regions of BDL rats. VCAM-1 mRNA and protein expression was also found to be significantly upregulated in different brain regions of BDL animals. The findings from the study suggest that increased BBB permeability in HE involves activation of MMP-9 and loss of tight junction proteins.     

Cerebral ischemia enhances tyrosine phosphorylation of occludin in brain capillaries

Cerebral ischemia induces disruption of the blood-brain barrier (BBB), and this disruption can initiate the development of brain injuries. Although the molecular structure of tight junctional complexes in the BBB has been identified, little is known about alterations of tight junctional proteins after cerebral ischemia. Therefore, we investigated alterations of tight junctional proteins, i.e., occludin and zonula occludens (ZO)-1, in isolated rat brain capillaries after microsphere-induced cerebral embolism. We demonstrated that the levels of occludin and ZO-1 had decreased after the embolism. The embolism also resulted in a marked increase in tyrosine phosphorylation of occludin, which was coincident with an increase in the activity of c-Src. These results suggest that a decrease in the levels of occludin and ZO-1, and an increase in tyrosine phosphorylation of occludin may play an important role in the disruption of tight junctions, which may lead to dysfunction of the BBB after cerebral ischemia.     

Eupatilin alleviates inflammatory response after subarachnoid hemorrhage by inhibition of TLR4/MyD88/NF-κB axis

Early brain injury (EBI) is associated with the adverse prognosis of subarachnoid hemorrhage (SAH) patients. The key bioactive component of the Chinese herbal medicine Artemisia asiatica Nakai (Asteraceae) is eupatilin. Recent research reports that eupatilin suppresses inflammatory responses induced by intracranial hemorrhage. This work is performed to validate whether eupatilin can attenuate EBI and deciphers its mechanism. A SAH rat model was established by intravascular perforation in vivo. At 6 h after SAH in rats, 10 mg/kg eupatilin was injected into the rats via the caudal vein. A Sham group was set as the control. In vitro, BV2 microglia was treated with 10 μM Oxyhemoglobin (OxyHb) for 24 h, followed by 50 μM eupatilin treatment for 24 h. The SAH grade, brain water content, neurological score, and blood-brain barrier (BBB) permeability of the rats were measured 24 h later. The content of proinflammatory factors was detected via enzyme-linked immunosorbent assay. Western blot analysis was conducted to analyze the expression levels of TLR4/MyD88/NF-κB pathway-associated proteins. In vivo, eupatilin administration alleviated neurological injury, and decreased brain edema and BBB injury after SAH in rats. Eupatilin markedly reduced the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), and suppressed the expression levels of MyD88, TLR4, and p-NF-κB p65 in the SAH rats' cerebral tissues. Eupatilin treatment also reduced the levels of IL-1β, IL-6, and TNF-α, and repressed the expression levels of MyD88, TLR4, and p-NF-κB p65 in OxyHb-induced BV2 microglia. Additionally, pyrrolidine dithiocarbamate or resatorvid enhanced the suppressive effects of eupatilin on OxyHb-induced inflammatory responses in BV2 microglia. Eupatilin ameliorates SAH-induced EBI via modulating the TLR4/MyD88/NF-κB pathway in rat model.     

Hydrogen sulfide protects blood–brain barrier integrity following cerebral ischemia

By using two structurally unrelated hydrogen sulfide (H₂S) donors 5‐(4‐methoxyphenyl) ‐3H‐1, 2‐dithiole‐3‐thione (ADT) and sodium hydrosulfide (NaHS), this study investigated if H₂S protected blood–brain barrier (BBB) integrity following middle cerebral artery occlusion (MCAO). ICR mice underwent MCAO and received H₂S donors at 3 h after reperfusion. Infarction, neurological scores, brain edema, Evans blue (EB) extravasation, and tight junction protein expression were examined at 48 h after MCAO. We also investigated if ADT protected BBB integrity by suppressing post‐ischemic inflammation‐induced Matrix Metalloproteimase‐9 (MMP9) and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). ADT increased blood H₂S concentrations, decreased infarction, and improved neurological deficits. Particularly, ADT reduced EB extravasation, brain edema and preserved expression of tight junction proteins in the ischemic brain. NaHS also increased blood H₂S levels and reduced EB extravasation following MCAO. Moreover, ADT inhibited expression of pro‐inflammatory markers induced Nitric Oxide Synthase (iNOS) and IL‐1β while enhanced expression of anti‐inflammatory markers arginase 1 and IL‐10 in the ischemic brain. Accordingly, ADT attenuated ischemia‐induced expression and activity of MMP9. Moreover, ADT reduced NOX‐4 mRNA expression, NOX activity, and inhibited nuclear translocation of Nuclear Factor Kappa‐B (NF‐κB) in the ischemic brain. In conclusion, H₂S donors protected BBB integrity following experimental stroke possibly by acting through NF‐κB inhibition to suppress neuroinflammation induction of MMP9 and NOX4‐derived free radicals.

     

Matrix metalloproteinase-2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins

Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia.     

Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins

A readily obtainable in vitro paradigm of the blood-brain barrier (BBB) would offer considerable benefits. Toward this end, in this study, we describe a novel method for purifying murine brain microvascular endothelial cells (BMEC) for culture. The method uses limited collagenase-dispase digestion of enriched brain microvessels, followed by immunoisolation of digested, microvascular fragments by magnetic beads coated with antibody to platelet-endothelial cell adhesion molecule-1. When plated onto collagen IV-coated surfaces, these fragments elaborated confluent monolayers of BMEC that expressed, as judged by immunocytochemistry, the adherens junction-associated proteins, VE-cadherin and beta-catenin, as well as the tight junction (TJ)-associated proteins, claudin-5, occludin, and zonula occludin-1 (ZO-1), in concentrated fashion along intercellular borders. In contrast, cultures of an immortalized and transformed line of murine brain capillary-derived endothelial cells, bEND.3, displayed diffuse cytoplasmic localization of occludin and ZO-1. This difference in occludin and ZO-1 staining between the two endothelial cell types was also reflected in the extent of association of these proteins with the detergent-resistant cytoskeletal framework (CSK). Although both occludin and ZO-1 largely partitioned with the CSK fraction in BMEC, they were found predominantly in the soluble fraction of bEND.3 cells, and claudin-5 was found associated equally with both fractions in BMEC and bEND.3 cells. Moreover, detergent-extracted cultures of the BMEC retained pronounced immunostaining of occludin and ZO-1, but not claudin-5, along intercellular borders. Because both occludin and ZO-1 are thought to be functionally coupled to the detergent-resistant CSK and high expression of TJs is considered a seminal characteristic of the BBB, these results impart that this method of purifying murine BMEC provides a suitable platform to investigate BBB properties in vitro.     

Reproductive hormones regulate the selective permeability of the blood-brain barrier

Reproductive hormones have been demonstrated to modulate both gap and tight junction protein expression in the ovary and other reproductive tissues, however the effects of changes in reproductive hormones on the selective permeability of the blood-brain barrier (BBB) remain unclear. Age-related declines in BBB integrity correlate with the loss of serum sex steroids and increase in gonadotropins with menopause/andropause. To examine the effect of reproductive senescence on BBB permeability and gap and tight junction protein expression/localization, female mice at 3 months of age were either sham operated (normal serum E2 and gonadotropins), ovariectomized (low serum E2 and high serum gonadotropins) or ovariectomized and treated with the GnRH agonist leuprolide acetate (low serum E2 and gonadotropins). Ovariectomy induced a 2.2-fold increase in Evan's blue dye extravasation into the brain. The expression and localization of the cytoplasmic membrane-associated tight junction protein zona occludens 1 (ZO-1) in microvessels was not altered among groups indicating that the increased paracellular permeability was not due to changes in this tight junction protein. However, ovariectomy induced a redistribution of the gap junction protein connexin-43 (Cx43) such that immunoreactivity relocalized from along the extracellular microvascular endothelium to become associated with endothelial cells. An increase in Cx43 expression in the mouse brain following ovariectomy was suppressed in ovariectomized animals treated with leuprolide acetate, indicating that serum gonadotropins rather than sex steroids were modulating Cx43 expression. These results suggest that elevated serum gonadotropins following reproductive senescence may be one possible cause of the loss of selective permeability of the BBB at this time. Furthermore, these findings implicate Cx43 in mediating changes in BBB permeability, and serum gonadotropins in the cerebropathophysiology of age-related neurodegenerative diseases such as stroke and Alzheimer's disease.     

The protective effect of alpha lipoic acid against traumatic brain injury in rats

Traumatic brain injury (TBI) was induced by a weight-drop device using 300 g–1 m weight-height impact. The study groups were: control, alpha-lipoic acid (LA) (100 mg/kg, po), TBI, and TBI + LA (100 mg/kg, po). Forty-eight hours after the injury, neurological scores were measured and brain samples were taken for histological examination or determination of thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) levels, myeloperoxidase (MPO) and Na⁺-K⁺ ATPase activities, whereas cytokines (TNF-α, IL-1β) were determined in blood. Brain oedema was evaluated by wet–dry weight method and blood–brain barrier (BBB) permeability was evaluated by Evans Blue (EB) extravasation. As a result, neurological scores mildly increased in trauma groups. Moreover, TBI caused a significant decrease in brain GSH and Na⁺-K⁺ ATPase activity, which was accompanied with significant increases in TBARS level, MPO activity and plasma proinflammatory cytokines. LA treatment reversed all these biochemical indices as well as histopathological alterations. TBI also caused a significant increase in brain water content and EB extravasation which were partially reversed by LA treatment. These findings suggest that LA exerts neuroprotection by preserving BBB permeability and by reducing brain oedema probably by its anti-inflammatory and antioxidant properties in the TBI model.     

Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly

The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.