Induce Blood-brain Barrier Breakdown Research Articles

Suppression of cerebral ischemia injury induced blood brain barrier breakdown by dexmedetomidine via promoting CCN1.

Blood-brain barrier (BBB) could aggravate cerebral ischemia injury. Dexmedetomidine (Dex) has been believed to play a protective role in cerebral ischemia injury-induced BBB injury. Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD) models were established to simulate cerebral ischemia injury. Animal experiments included 4 groups, Sham, MCAO, MCAO+Dex, MCAO+Dex+sh-CCN1. Generally applicable gene set enrichment analysis was performed to analyze gene expression difference. Total collagen content and Evans blue staining were performed to measure infarct ratio and BBB breakdown, respectively. The cell apoptosis, mRNA and protein expression were measured through flow cytometry, PCR, and western blotting, respectively. The levels of IL-1β, TNF-α, and IL-6 in serum were measured with commercial ELISA kits. Dex greatly promoted the expression level of CCN1. Dex suppressed cerebral ischemia injury, increased tight junction protein expression, improved the memory ability and neurological function of MCAO rats through targeting CCN1. The significant increase of inflammatory factors in the serum of MCAO rats were suppressed by Dex. Dex suppressed OGD induced increase of HRP permeability and promoting tight junction protein expression in vitro through regulating CCN1. The neurological function evaluation was performed with Neurological Severity Score (NSS) and Longa Score Scale. Dex could remarkably alleviate cerebral ischemia injury by inhibiting BBB breakdown, inflammatory response, and promoting neurological function and tight junction protein expression via up-regulating CCN1. This study might provide a novel therapeutic target for the prevention and treatment of cerebral ischemia injury-induced BBB.

Understanding the role of the blood brain barrier and peripheral inflammation on behavior and pathology on ongoing confined cortical lesions

BackgroundInflammation in the Central Nervous System (CNS) is associated with blood brain barrier (BBB) breakdown during the early stages of Multiple Sclerosis (MS), indicating a facilitated entry of waves of inflammatory cells from the circulation to the CNS. In the progressive forms of MS, as the lesion becomes chronic, the inflammation remains trapped within the CNS compartment forming the slow evolving lesion, characterized by low inflammation and microglia activation at the lesions edges. The chronic expression of interleukin 1β (IL-1β) in the cortex induces BBB breakdown, demyelination, neurodegeneration, microglial/macrophage activation and impaired cognitive performance. The latter can be improved, as long as the BBB recovers and the lesion presents low inflammation. Here, we study the effects of peripheral inflammation on cortical central lesions after the restoration of the BBB, in order to elucidate the role of the peripheral inflammation on these lesions with intact BBB, as it occurs in the progressive forms of MS. Materials and methodsCortical lesions and peripheral inflammation were induced by the chronic expression of IL-1β using an adenovector. We performed histological, immunohistochemistry on brain tissue and behavioural analyses. ResultsThe effects of the chronic expression of IL-1β in the cortex resolved within 56 days. However, peripheral and sustained inflammation re-opened the BBB, allowing the reappearance of the neuroinflammatory processes within the cortical lesions, increased demyelination and neurodegeneration, and an increase of the behavioral symptoms, such as cognitive impairment and anxiety-like symptoms. ConclusionsThe early treatment of peripheral inflammatory processes should be considered in order to protect the brain from exacerbation of the ongoing neurodegenerative process.

Pathologic sequelae of vascular cognitive impairment and dementia sheds light on potential targets for intervention.

Vascular contributions to cognitive impairment and dementia (VCID) is one of the leading causes of dementia along with Alzheimer's disease (AD) and, importantly, VCID often manifests as a comorbidity of AD(Vemuri and Knopman 2016; Schneider and Bennett 2010)(Vemuri and Knopman 2016; Schneider and Bennett 2010). Despite its common clinical manifestation, the mechanisms underlying VCID disease progression remains elusive. In this review, existing knowledge is used to propose a novel hypothesis linking well-established risk factors of VCID with the distinct neurodegenerative cascades of neuroinflammation and chronic hypoperfusion. It is hypothesized that these two synergistic signaling cascades coalesce to initiate aberrant angiogenesis and induce blood brain barrier breakdown trough a mechanism mediated by vascular growth factors and matrix metalloproteinases respectively. Finally, this review concludes by highlighting several potential therapeutic interventions along this neurodegenerative sequalae providing diverse opportunities for future translational study.

Abstract P111: Systemic TLR4 Blockade Ameliorates Neuroinflammation & Blood-Brain Barrier Disruption In Hypertensive Rats

Hypertension is linked to blood-brain barrier (BBB) disruption and neuroinflammation in areas such as the hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM). We have shown that Angiotensin II (AngII) induces BBB breakdown via type 1 receptors (AT1r) and activates hypothalamic microglia ex vivo through AT1r and Toll-like receptor 4 (TLR4). We hypothesized that TLR4 activation is a key contributor to AngII-mediated BBB disruption, neuroinflammation, and autonomic dysfunction. Spontaneously hypertensive rats (SHR) were treated with Losartan (AT1r blocker; 20 mg/kg; oral; 4 weeks; SHR-Los) or TAK-242 (TLR4 blocker; 2 mg/kg; i.p. ; 2 weeks; SHR-TAK), and age-matched to control SHR and Wistar Kyoto rats (WKY). Indirect tail-cuff mean arterial pressure (MAP) was normalized in SHR-Los (104 vs 100 mmHg in WKY) and reduced in SHR-TAK (129 vs 154 mmHg in SHR). During conscious baroreflex assessment, TAK-242 rescued the responsiveness of SHR baroreflex gain (ΔHR/ΔMAP; phenylephrine: WKY: -2.56; SHR: -1.47; SHR-TAK: -2.20; nitroprusside: WKY: -2.39; SHR: -0.91; SHR-TAK: -2.06). Sympathetic nervous system activity (indirect; hexamethonium) was elevated in SHR (-65 ΔMAP) versus WKY (-47 ΔMAP) and SHR-TAK (-43 ΔMAP). Microglial activation, indexed by morphological analysis of IBA1 immunofluorescence (IF), was enhanced in SHR ( PVN: end-points [EP]: -36.1%, branch length [BL]: -26.8%; RVLM: EP: -41.6%, BL: -33.4% vs WKY) and normalized in SHR-Los and SHR-TAK. Elevated TNF-α IF densities in SHR (PVN: +57.9; RVLM: +58.0% vs WKY) were abolished with AT1r or TLR4 blockade. Both treatments normalized PVN IL-6 IF (SHR: +46.6% vs WKY) and reduced RVLM IL-6 (SHR: +77.4%; SHR-Los: +32.4%; SHR-TAK: +33.9% vs WKY). Increased BBB permeability in SHR, quantified by leakage of a low MW dextran-conjugated dye, was apparent in the PVN (3.619% area), RVLM (3.700% area), and nucleus tractus solitarius (NTS; 3.588% area) compared to WKY (PVN: 1.558%, RVLM: 1.572%, NTS: 1.800% area), while TAK-242 restored the barrier’s integrity in each region. Taken together, these data support a significant contribution of TLR4 activation to AngII-driven autonomic dysfunction, neuroinflammation, and BBB disruption in hypertension.

DRES-12. QUANTIFYING INDIVIDUALIZED ABT-414 SENSITIVITY AND BLOOD-BRAIN BARRIER PENETRANCE FROM SERIAL IMAGING OF PATIENT-DERIVED XENOGRAFTS MODELS OF GLIOBLASTOMA

Abstract INTRODUCTION Glioblastoma (GBM) is an aggressive primary brain tumor, known for its poor prognosis. Due to its diffuse invasiveness into normal-appearing brain, localized treatments such as surgical resection and radiotherapy are typically supplemented with chemotherapy. However, to reach invading tumor cells, such antineoplastic drugs must cross the blood-brain barrier (BBB). That is, while angiogenesis induces BBB breakdown in dense tumor regions, the BBB remains rather intact for invading GBM cells. As a result, it is unclear whether BBB-impermeable drugs are delivered at a sufficient level to be effective. METHODS In order to study heterogeneity in BBB breakdown, experiments were conducted using both flank and intracranial patient-derived xenografts (PDXs) treated with the EGFR-targeted monoclonal antibody drug conjugate, depatuxizumab mafodotin (ABT-414). Time-series bioluminescence imaging (BLI) data was used to develop a differential equation model of tumor growth for three PDX cell lines. Data from untreated PDXs, both in flank and intracranially, were used to parameterize tumor proliferation rates. Flank PDX data were used to parameterize individual sensitivity to ABT-414, whereas intracranial PDX data were used to determine the proportion of drug exposed to the tumor. RESULTS Each PDX line differed in response to the study drug ABT-414. As expected, such heterogeneous responses can primarily be attributed to differences in both drug sensitivity and the proportion of drug that reached the tumor. Notably, the estimated proportion of drug that reached the tumor was highest in the PDX line with the longest survival times, despite also having higher estimates of resistance. This suggests that PDXs with greater overall BBB breakdown may respond better to this agent. CONCLUSIONS Although more cell lines are needed to validate our approach, parameterizing this model for PDXs gives valuable insight into the extent of BBB breakdown in patient GBMs and may aid in determining optimal therapies for individual patients.

A novel model of cerebral hyperperfusion with blood-brain barrier breakdown, white matter injury, and cognitive dysfunction.

Cerebral hyperperfusion (CHP) is associated with considerable morbidity. Its pathophysiology involves disruption of the blood-brain barrier (BBB) with subsequent events such as vasogenic brain edema and ischemic and/or hemorrhagic complications. Researchers are trying to mimic the condition of CHP; however, a proper animal model is still lacking. In this paper the authors report a novel surgically induced CHP model that mimics the reported pathophysiology of clinical CHP including BBB breakdown, white matter (WM) injury, inflammation, and cognitive impairment. Male Sprague-Dawley rats were subjected to unilateral common carotid artery (CCA) occlusion and contralateral CCA stenosis. Three days after the initial surgery, the stenosis of CCA was released to induce CHP. Cortical regional cerebral blood flow was measured using laser speckle flowmetry. BBB breakdown was assessed by Evans blue dye extravasation and matrix metalloproteinase-9 levels. WM injury was investigated with Luxol fast blue staining. Cognitive function was assessed using the Barnes circular maze. Other changes pertaining to inflammation were also assessed. Sham-operated animals were prepared and used as controls. Cerebral blood flow was significantly raised in the cerebral cortex after CHP induction. CHP induced BBB breakdown evident by Evans blue dye extravasation, and matrix metalloproteinase-9 was identified as a possible culprit. WM degeneration was evident in the corpus callosum and corpus striatum. Immunohistochemistry revealed macrophage activation and glial cell upregulation as an inflammatory response to CHP in the striatum and cerebral cortex. CHP also caused significant impairments in spatial learning and memory compared with the sham-operated animals. The authors report a novel CHP model in rats that represents the pathophysiology of CHP observed in various clinical scenarios. This model was produced without the use of pharmacological agents; therefore, it is ideal to study the pathology of CHP as well as to perform preclinical drug trials.

β-Caryophyllene protects invitro neurovascular unit against oxygen-glucose deprivation and re-oxygenation-induced injury.

β-Caryophyllene (BCP) mediates neuroprotection in cerebral ischemic animals. The neurovascular unit (NVU) acts as an intricate network to maintain the neuronal homeostatic microenvironment. However, the effects exerted by BCP on NVU remain unclear. Therefore, we established an invitro NVU model to investigate the effects of BCP on oxygen-glucose deprivation and re-oxygenation (OGD/R)-induced injury. This model involved the co-culture of brain microvascular endothelial cells, neurons, and astrocytes. BCP (10μmol/L) was applied for 24h prior to OGD/R and maintained throughout OGD/R. Blood-brain barrier (BBB) integrity and neuronal apoptosis were analyzed. BCP pre-treatment prior to the initiation of OGD/R significantly (i) decreased BBB permeability and neuronal apoptosis, (ii) mitigated oxidative stress damage and the release of inflammatory cytokines, (iii) down-regulated Bax expression, metalloproteinase-9 activity and expression, and (iv) up-regulated claudin-5, occludin, ZO-1, growth-associated protein-43 and Bcl-2 expression. Thus, BCP pre-treatment exerted multiple protective effects on NVU in the context of OGD/R-induced injury. These protective effects potentially occur via reductions in oxidative stress damage and inflammatory cytokines that induce BBB breakdown, subsequently resulting in reduced neuronal apoptosis. The NVU serves as putative therapeutic targets for cerebral ischemia, and the results of this study provide new insights for the application of BCP as a neuroprotective agent.

A combination of an iron chelator with an antioxidant effectively diminishes the dendritic loss, tau-hyperphosphorylation, amyloids-β accumulation and brain mitochondrial dynamic disruption in rats with chronic iron-overload

Iron-overload can cause cognitive impairment due to blood–brain barrier (BBB) breakdown and brain mitochondrial dysfunction. Although deferiprone (DFP) has been shown to exert neuroprotection, the head-to-head comparison among iron chelators used clinically on brain iron-overload has not been investigated. Moreover, since antioxidant has been shown to be beneficial in iron-overload condition, its combined effect with iron chelator has not been tested. Therefore, the hypothesis is that all chelators provide neuroprotection under iron-overload condition, and that a combination of an iron chelator with an antioxidant has greater efficacy than monotherapy. Male Wistar rats (n=42) were assigned to receive a normal diet (ND) or a high-iron diet (HFe) for 4months. At the 2nd month, HFe-fed rats were treated with a vehicle, deferoxamine (DFO), DFP, deferasirox (DFX), n-acetyl cysteine (NAC) or a combination of DFP with NAC, while ND-fed rats received vehicle. At the end of the experiment, rats were decapitated and brains were removed to determine brain iron level and deposition, brain mitochondrial function, BBB protein expression, brain mitochondrial dynamic, brain apoptosis, tau-hyperphosphorylation, amyloid-β (Aβ) accumulation and dendritic spine density. The results showed that iron-overload induced BBB breakdown, brain iron accumulation, brain mitochondrial dysfunction, impaired brain mitochondrial dynamics, tau-hyperphosphorylation, Aβ accumulation and dendritic spine reduction. All treatments, except DFX, attenuated these impairments. Moreover, combined therapy provided a greater efficacy than monotherapy. These findings suggested that iron-overload induced brain iron toxicity and a combination of an iron chelator with an antioxidant provided a greatest efficacy for neuroprotection than monotherapy.

Endothelial β-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System Homeostasis.

The blood-brain barrier (BBB) formed by brain endothelial cells interconnected by tight junctions is essential for the homeostasis of the central nervous system. Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. Using a mouse model with tamoxifen-inducible endothelial cell-restricted disruption of ctnnb1 (iCKO), we show here that endothelial β-catenin signaling is essential for maintaining BBB integrity and central nervous system homeostasis in adult mice. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and central nervous system inflammation, and all had postictal death. Disruption of endothelial β-catenin induced BBB breakdown and downregulation of the specific tight junction proteins claudin-1 and -3 in adult brain endothelial cells. The clinical relevance of the data is indicated by the observation of decreased expression of claudin-1 and nuclear β-catenin in brain endothelial cells of hemorrhagic lesions of hemorrhagic stroke patients. These results demonstrate the prerequisite role of endothelial β-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective β-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity, and central nervous system inflammation.

Hypersensitivity Responses in the Central Nervous System.

Immune-mediated tissue damage or hypersensitivity can be mediated by autospecific IgG antibodies. Pathology results from activation of complement, and antibody-dependent cellular cytotoxicity, mediated by inflammatory effector leukocytes include macrophages, natural killer cells, and granulocytes. Antibodies and complement have been associated to demyelinating pathology in multiple sclerosis (MS) lesions, where macrophages predominate among infiltrating myeloid cells. Serum-derived autoantibodies with predominant specificity for the astrocyte water channel aquaporin-4 (AQP4) are implicated as inducers of pathology in neuromyelitis optica (NMO), a central nervous system (CNS) demyelinating disease where activated neutrophils infiltrate, unlike in MS. The most widely used model for MS, experimental autoimmune encephalomyelitis, is an autoantigen-immunized disease that can be transferred to naive animals with CD4+ T cells, but not with antibodies. By contrast, NMO-like astrocyte and myelin pathology can be transferred to mice with AQP4–IgG from NMO patients. This is dependent on complement, and does not require T cells. Consistent with clinical observations that interferon-beta is ineffective as a therapy for NMO, NMO-like pathology is significantly reduced in mice lacking the Type I IFN receptor. In MS, there is evidence for intrathecal synthesis of antibodies as well as blood–brain barrier (BBB) breakdown, whereas in NMO, IgG accesses the CNS from blood. Transfer models involve either direct injection of antibody and complement to the CNS, or experimental manipulations to induce BBB breakdown. We here review studies in MS and NMO that elucidate roles for IgG and complement in the induction of BBB breakdown, astrocytopathy, and demyelinating pathology. These studies point to significance of T-independent effector mechanisms in neuroinflammation.

Endothelial NOS activation induces the blood–brain barrier disruption via ER stress following status epilepticus

The blood–brain barrier (BBB) maintains the unique brain microenvironment, which is separated from the systemic circulating system. Since the endoplasmic reticulum (ER) is an important cell organelle that is responsible for protein synthesis, the correct folding and sorting of proteins contributing to cell survivals, ER stress is a potential cause of cell damage in various diseases. Therefore, it would be worthy to explore the the relationship between the ER stress and BBB disruption during vasogenic edema formation induced by epileptogenic insults. In the present study, we investigated the roles of ER stress in vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced status epilepticus (SE). SE-induced eNOS activation induces BBB breakdown via up-regulation of GRP78 expression and dysfunction of SMI-71 (an endothelial BBB marker) in the piriform cortex (PC). In addition, caveolin-1 peptide (an eNOS inhibitor) effectively attenuated GRP78 expression and down-regulation of SMI-71. Taken together, our findings suggest that eNOS-mediated ER stress may participate in SE-induced vasogenic edema formation. Therefore, the modulation of ER stress may be a considerable strategy for therapy in impairments of endothelial cell function.

Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity

Blood brain barrier (BBB) breakdown is not only a consequence of, but also contributes to many neurological disorders, including stroke and Alzheimer’s disease. How the basement membrane (BM) contributes to the normal functioning of the BBB remains elusive. Here we use conditional knockout mice and an acute adenovirus-mediated knockdown model to show that lack of astrocytic laminin, a brain-specific BM component, induces BBB breakdown. Using functional blocking antibody and RNAi, we further demonstrate that astrocytic laminin, by binding to integrin α2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, and thus maintains the integrity of BBB. Additionally, loss of astrocytic laminin decreases aquaporin-4 (AQP4) and tight junction protein expression. Altogether, we report a critical role for astrocytic laminin in BBB regulation and pericyte differentiation. These results indicate that astrocytic laminin maintains the integrity of BBB through, at least in part, regulation of pericyte differentiation.

A proposed role of human defensins in Helicobacter pylori-related neurodegenerative disorders

Cationic host defence peptides (CHDPs), also known as antimicrobial peptides (AMPs), are essential components of the innate immunity with antimicrobial and pleiotropic immunomodulatory properties. In mammals the two major families of CHDPs are defensins and cathelicidins that comprise an arsenal of innate regulators of principal importance in the host tissues. Research in the last decade has demonstrated that defensins are crucial effectors of both innate and adaptive immunity. Defensins can modulate immune responses, either by stimulation or suppression, thereby controlling inflammatory processes and infections. Currently only few data, mostly hypothetical, focus on the role of defensins in central nervous system (CNS) physiopathology and neurodegeneration. Defensins may function as an initial line of defense within the CNS either as an antimicrobial, immunomodulator, or both. A dysregulation of brain expression of specific defensins might either exacerbate or ameliorate the inflammatory response within the CNS depending upon which extracellular conditions predominate. It is proposed that reduction or abnormal elevation of AMP expression by cerebral microglia, astrocytes or choroid plexus epithelium might contribute to loss of AMP-induced regulation of immune responses, thereby promoting neuronal cell injury and death observed in Alzheimer’s disease and possibly in other neurodegenerative disorders. Nevertheless, whether certain AMPs play a crucial role in the onset or promotion of the neuroinflammatory process and neurodegeneration is currently unknown, thereby emphasizing the necessity of further investigation into the regulatory mechanisms that control innate and adaptive immunity within the brain. Recent data indicate that Helicobacter pylori (H. pylori) induces defensins’ release associated with chronic inflammatory tissue damage. However, it remains unclear whether and how H. pylori evades the attack by defensins. Moreover, recent evidence indicates that H. pylori infection might contribute to the pathogenesis of neurodegenerative diseases, by releasing several inflammatory mediators that could induce blood–brain barrier breakdown, thereby being involved in the pathogenesis of neurodegeneration. However, currently there are no data regarding the potential impact of human defensins on H. pylori-related neurodegenerative disorders. We herein propose that human defensins might contribute to the pathophysiology of H. pylori-related neurodegenerative disorders by modulating variably innate and adaptive immune system responses. Better understanding of the mechanisms regarding human defensins’ possible involvement in H. pylori-induced neurodegeneration might help develop novel therapeutic strategies against H. pylori-related neurodegenerative disorders.

Peroxiredoxin triggers cerebral post-ischemic inflammation

Post-ischemic inflammation is an essential step in the progression of ischemic stroke. However, it has not been sufficiently clarified how post-ischemic inflammation begins. Brain is a sterile organ, but Toll-like receptor (TLR) 2 and TLR4 have a pivotal role in the initiation of inflammation. Some endogenous danger associated molecular patterns (DAMPs) are released from injured brain cells. Among them, high mobility group box 1 (HMGB1) and peroxiredoxin (Prx) family proteins are key initiators of post-ischemic inflammation. HMGB1 induces blood brain barrier breakdown at the hyperacute phase; on the other hand, Prxs directly activate infiltrating macrophage through TLR2/4 and induce inflammatory cytokines production at the acute phase. Thus, there is a time lag as well as functional differences between HMGB1 and Prxs. Novel neuroprotective treatment could be developed through more detailed elucidation of the regulation of post-ischemic inflammation. Rec.12/10/2012, Acc.1/29/2013, pp150-155

Immune mechanisms in epileptogenesis.

Epilepsy is a chronic brain disorder that affects 1% of the human population worldwide. Immune responses are implicated in seizure induction and the development of epilepsy. Pre-clinical and clinical evidence have accumulated to suggest a positive feedback cycle between brain inflammation and epileptogenesis. Prolonged or recurrent seizures and brain injuries lead to upregulation of proinflammatory cytokines and activated immune responses to further increase seizure susceptibility, promote neuronal excitability, and induce blood–brain barrier breakdown. This review focuses on the potential role of innate and adaptive immune responses in the pathogenesis of epilepsy. Both human studies and animal models that help delineate the contributions of brain inflammation in epileptogenesis will be discussed. We highlight the critical role of brain-resident immune mediators and emphasize the contribution of brain-infiltrating peripheral leukocytes. Additionally, we propose possible immune mechanisms that underlie epileptogenesis. Several proinflammatory pathways are discussed, including the interleukin-1 receptor/toll-like receptor signaling cascade, the pathways activated by damage-associated molecular patterns, and the cyclooxygenase-2/prostaglandin pathway. Finally, development of better therapies that target the key constituents and processes identified in these mechanisms are considered, for instance, engineering antagonizing agents that effectively block these pathways in an antigen-specific manner.

Phloretin ameliorates 2-chlorohexadecanal-mediated brain microvascular endothelial cell dysfunction in vitro

2-Chlorohexadecanal (2-ClHDA), a chlorinated fatty aldehyde, is formed via attack on ether-phospholipids by hypochlorous acid (HOCl) that is generated by the myeloperoxidase–hydrogen peroxide–chloride system of activated leukocytes. 2-ClHDA levels are elevated in atherosclerotic lesions, myocardial infarction, and neuroinflammation. Neuroinflammatory conditions are accompanied by accumulation of neutrophils (an ample source of myeloperoxidase) in the brain. Microvessel damage by inflammatory mediators and/or reactive oxidants can induce blood–brain barrier (BBB) dysfunction, a pathological condition leading to cerebral edema, brain hemorrhage, and neuronal death. In this in vitro study we investigated the impact of 2-ClHDA on brain microvascular endothelial cells (BMVEC), which constitute the morphological basis of the BBB. We show that exogenously added 2-ClHDA is subject to rapid uptake and metabolism by BMVEC. Using C16 structural analogues of 2-ClHDA we found that the cytotoxic potential decreases in the following order: 2-ClHDA>hexadecanal>palmitic acid>2-ClHDA-dimethylacetal. 2-ClHDA induces loss of barrier function, mitochondrial dysfunction, apoptosis via activation of caspase 3, and altered intracellular redox balance. Finally we investigated potential protective effects of several natural polyphenols on in vitro BBB function. Of the compounds tested, phloretin almost completely abrogated 2-ClHDA-induced BMVEC barrier dysfunction and cell death. These data suggest that 2-ClHDA has the potential to induce BBB breakdown under inflammatory conditions and that phloretin confers protection in this experimental setting.

Involvement of oxidative stress in hypoxia-induced blood–brain barrier breakdown

The blood–brain barrier (BBB) is a cellular barrier formed by specialized brain endothelial cells under the influence of astrocytes and pericytes. Among the several stress factors known to induce BBB breakdown, hypoxia is probably the most represented but also the least understood. Recent evidence of oxidative stress occurring during hypoxia/ischemia situation raises its possible contribution to barrier breakdown. In this study, we investigated the relevance of oxidative stress in hypoxia-induced barrier disruption.Prolonged hypoxic exposure induced reactive oxygen species (ROS) formation and induced glutathione oxidation. Such effects were accentuated under extreme O2 deprived environment. Pro-oxidant treatment significantly disrupted barrier function under normal conditions, whereas anti-oxidant treatment contributed to maintain better barrier function and cell survival in an O2-reduced environment. In addition, the endothelial response to oxidative stress appeared modulated by the presence of astrocytes and pericytes, thus explaining some of the beneficial contribution of these cells as previously described.Taken together, this study highlights the importance of oxidative stress signaling at the barrier. In addition, cells of the neurovascular compartment differentially modulate ROS levels and also regulate barrier function. Thus, use of reactive oxygen scavengers may be useful to support barrier function following stroke injury.

Human Blood-Brain Barrier Disruption by Retroviral-Infected Lymphocytes: Role of Myosin Light Chain Kinase in Endothelial Tight-Junction Disorganization

The blood-brain barrier (BBB), which constitutes the interface between blood and cerebral parenchyma, has been shown to be disrupted during retroviral associated neuromyelopathies. Human T cell leukemia virus (HTLV-1)-associated myelopathy/tropical spastic paraparesis is a slowly progressive neurodegenerative disease, in which evidence of BBB breakdown has been demonstrated by the presence of lymphocytic infiltrates in the CNS and plasma protein leakage through cerebral endothelium. Using an in vitro human BBB model, we investigated the cellular and molecular mechanisms involved in endothelial changes induced by HTLV-1-infected lymphocytes. We demonstrate that coculture with infected lymphocytes induces an increase in paracellular endothelial permeability and transcellular migration, via IL-1alpha and TNF-alpha secretion. This disruption is associated with tight junction disorganization between endothelial cells, and alterations in the expression pattern of tight junction proteins such as zonula occludens 1. These changes could be prevented by inhibition of the NF-kappaB pathway or of myosin light chain kinase activity. Such disorganization was confirmed in histological sections of spinal cord from an HTLV-1-associated myelopathy/tropical spastic paraparesis patient. Based on this BBB model, the present data indicate that HTLV-1-infected lymphocytes can induce BBB breakdown and may be responsible for the CNS infiltration that occurs in the early steps of retroviral-associated neuromyelopathies.

Breakdown of the blood-brain barrier and neuropathological changes induced by Phoneutria nigriventer spider venom

The blood-brain barrier (BBB) is responsible for selective flux of substances between blood and brain. The selective permeability of the BBB is crucial for the maintenance of the brain microenvironment homeostasis, and alterations in the barrier may be involved in many pathophysiological processes. Phoneutria nigriventer armed spider venom produces excitatory signals and symptoms in humans, and its recognized neurotoxic action suggests a potential ability to alter BBB permeability. The aim of the present study was to investigate the capacity of P. nigriventer venom (PNV) in promoting BBB breakdown in adult rats. After intravenous injection of 850 micro g/kg of the whole venom, BBB lesions were evaluated after 18 h to 9 days by ultrastructural methods using the extracellular tracer lanthanum nitrate. Clinical signs and symptoms of rats showed acute neurotoxicity, with some of the animals presenting convulsions, but which were clinically resolved by 12 h post-envenoming. The results showed that PNV is able to increase BBB permeability, particularly in the hippocampus. Changes were first detected in arterioles and post-capillary venules 18 h to 5 days after venom inoculation. The increased permeation of the extracellular tracer peaked on day 1, representing about 42% of the examined vessels (P<0.01). This appeared to occur by both transendothelial and intercellular routes, i.e., by pinocytic transport and through interendothelial junctions. Concomitantly, the surrounding tissue showed vasogenic edema and swollen astrocytic processes, without inflammatory infiltrates. The peak of the edema occurrence was observed on day 3, in about 60% of the vessels (P<0.001). Enhanced capillary permeability was observed on day 9, and affected 36% of all capillaries (P<0.05). The affected capillaries were characterized by increased number of pinocytotic vesicles, which, in addition, were filled with the extracellular tracer, but without visible transport through the interendothelial pathway. This study demonstrates that systemic PNV inoculation induces BBB breakdown through trans- and paracellular routes. It is concluded that BBB breakdown is an event not associated with the acute neurotoxicity exhibited by the rats.

Evaluation of brain toxicity following near infrared light exposure after indocyanine green dye injection

Indocyanine green (ICG) has excellent safety records and is widely used in medical diagnosis. Recently, a new method has been developed to estimate cerebral blood flow (CBF) using ICG in combination with near-infrared spectroscopy (NIRS). The new technique may be of wide clinical interest, as it is noninvasive and easy to perform at the bedside in stroke patients. Additionally, ICG with the use of specific wavelength lasers is documented to be effective in photodynamic therapy (PDT). Under normal conditions ICG does not cross the intact blood brain barrier (BBB). However, in patients with brain injuries where the BBB may be disturbed, ICG could accumulate in brain parenchyma and in combination with NIR-light exposure, phototoxicity could occur. The aim of the present study was to examine the possible toxicity of ICG in combination with NIRS in a specific setting for CBF measurements. In five rats with mannitol induced BBB breakdown no traces of ICG were found during spectrophotometric analysis of the brain cell suspensions. In ten rats with disrupted BBB there were no significant increases of brain temperature or histological signs of brain damage following 1 h NIR-light exposure after ICG injection. The existing literature concerning the application of ICG in combination with NIR light is reviewed.