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Involvement of oxidative stress in hypoxia-induced blood-brain barrier breakdown
Abstract
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 O(2) 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 O(2)-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.
... PDGFRβ is a pericyte-shedded marker in response to hypoxia [59][60][61][62] and is associated with blood-brain barrier (BBB) permeability [60,61,[63][64][65]. Disruption of the BBB is a central process involved in PD pathophysiology [66] and therefore, acute effects of hypoxia therapy on PDGFRβ would give insight in the influence that hypoxia might have on BBB integrity in the long term [65]. ...
... PDGFRβ is a pericyte-shedded marker in response to hypoxia [59][60][61][62] and is associated with blood-brain barrier (BBB) permeability [60,61,[63][64][65]. Disruption of the BBB is a central process involved in PD pathophysiology [66] and therefore, acute effects of hypoxia therapy on PDGFRβ would give insight in the influence that hypoxia might have on BBB integrity in the long term [65]. ...
Background
Parkinson’s disease (PD) is a neurodegenerative disease, for which no disease-modifying therapies exist. Preclinical and clinical evidence suggest that hypoxia-based therapy might have short- and long-term benefits in PD. We present the contours of the first study to assess the safety, feasibility and physiological and symptomatic impact of hypoxia-based therapy in individuals with PD.
Methods/Design
In 20 individuals with PD, we will investigate the safety, tolerability and short-term symptomatic efficacy of continuous and intermittent hypoxia using individual, double-blind, randomized placebo-controlled N-of-1 trials. This design allows for dose finding and for including more individualized outcomes, as each individual serves as its own control. A wide range of exploratory outcomes is deployed, including the Movement Disorders Society Unified Parkinson’s Disease Rating scale (MDS-UPDRS) part III, Timed Up & Go Test, Mini Balance Evaluation Systems (MiniBES) test and wrist accelerometry. Also, self-reported impression of overall symptoms, motor and non-motor symptoms and urge to take dopaminergic medication will be assessed on a 10-point Likert scale. As part of a hypothesis-generating part of the study, we also deploy several exploratory outcomes to probe possible underlying mechanisms of action, including cortisol, erythropoietin and platelet-derived growth factor β. Efficacy will be assessed primarily by a Bayesian analysis.
Discussion
This evaluation of hypoxia therapy could provide insight in novel pathways that may be pursued for PD treatment. This trial also serves as a proof of concept for deploying an N-of-1 design and for including individualized outcomes in PD research, as a basis for personalized treatment approaches.
Trial registration
ClinicalTrials.gov Identifier: NCT05214287 (registered January 28, 2022).
... However, CCH also occurs in the normally aging brain (Iadecola, 2013). CCH induced by a decrease in cerebral blood flow leads to hypoxia, in turn leading to neuronal cell death, glial activation, immune cell infiltration, and energy depletion (Al Ahmad et al., 2012;Fiala et al., 2002;Saggu et al., 2016). Such changes cause oxidative stress, neuroinflammation, glutamatergic excitotoxicity, and disruption of the blood-brain barrier (BBB), resulting in a progressive decline in cognitive and executive function (Gorelick et al., 2011;Iadecola, 2013;Kalaria, 2016). ...
... Damage to blood vessels caused by CCH activates astrocytes and microglia, leading to gliosis and neuroinflammation, in turn leading to neuronal damage and a decrease in cognitive function. Several studies have reported that the destruction of the BBB in the cerebral vasculature may play an essential role in the pathogenesis of vascular dementia (Al Ahmad et al., 2012;Iadecola, 2013). Previous studies related to EE have focused on the neuroprotective effect . ...
Vascular dementia (VaD) is one of the most common types of dementia followed by Alzheimer's disease (AD). Recent studies showed that approximately 30%–35% of patients with AD at post-mortem exhibited vascular pathologies, which suggested that mixed dementia may be the most common type of dementia. Permanent bilateral common carotid artery occlusion (2VO) is a well-characterized method for investigating cognitive functions and the histopathological consequences of chronic cerebral hypoperfusion (CCH) in rats. In the present study, we investigated the effects of environmental enrichment (EE) on cognitive impairment after CCH, as well as the effects of CCH-induced neurovascular damage on cognitive function. Wistar rats were randomly allocated to a sham group, a 2VO group, and a 2VO + EE group. The 2VO procedure was performed at 12 weeks, while EE was performed for 8 weeks before and 6 weeks after 2VO. The effect of EE on cognitive functions in 2VO rats was investigated using the radial-arm maze and Morris Water Maze tests. Neurovascular integrity was assessed based on immunoreactivity for glial fibrillary acidic protein (GFAP), morphological changes in microvessels, and the expression of matrix metalloproteinase-9 (MMP-9) and zonula occludens-1 (ZO-1) in the motor cortex and hippocampus. EE ameliorated microvessel fragmentation by sustaining the tight junction through increases of ZO-1 expression after CCH, resulting in preserving the neurovascular unit. In summary, EE mitigated cognitive impairment by restoring neurovascular integrity. These findings suggest that EE can be a valuable and meaningful environmental intervention for patients with cognitive impairment.
... However, the once established neural vascular barrier in adults is also under the dynamic control in response to various kinds of stress. In neural diseases, such as cerebral infarction, diabetic retinopathy and so forth, it is known that the decrease in oxygen concentration, hypoxia, of affected tissues give rise to the disruption of neural vascular barrier and consequently accelerates their disease progression [11][12][13]. Therefore, the analyses of mechanisms underlying hypoxia-induced disruption of neural vascular barrier would lead to the establishment of new therapies for intractable neural diseases. ...
... It is highly suggested that the hypoxia-induced disruption of neural vascular barrier is mediated by intracellular induction of Fe(II) ion and subsequent production of hydroxyl radicals which are the well-known ROS harmful for organisms [33,34]. It is generally accepted that disruption of neural vascular barrier occurs in various intractable neural diseases and accelerates their disease progression [11,13,35]. Therefore, the procedure to repair the pathologically opened vascular barrier would lead to the establishment of new effective therapies for intractable neural diseases. ...
Neural vascular barrier maintains the optimal tissue microenvironment of central nervous system in which neural cells can function normally. In various neural diseases, the decrease in oxygen concentration, hypoxia, of affected tissues is known to accelerate the disease progression through disruption of neural vascular barrier. Therefore, the clarification of mechanisms underlying hypoxia-induced disruption of neural vascular barrier would definitely lead to the establishment of new effective therapies for intractable neural diseases. In the present study, we first found that hypoxia disrupts neural vascular barrier through pathways independent of HIF-1α and HIF-2α. Then, with a specific fluorescence probe for ferrous, Fe(II) ion, we have obtained the interesting data showing that hypoxia increased the intracellular level of Fe(II) ion in endothelial cells of our in vitro model for neural vascular barrier, and that hypoxia-induced disruption of neural vascular barrier could be inhibited by chelating Fe(II) ion in endothelial cells. Furthermore, in the presence of a reducing reagent for reactive oxygen species (ROS), hypoxia could not disrupt the neural vascular barrier despite that the hypoxic increase in intracellular level of Fe(II) ion was confirmed in endothelial cells. These results indicate that hypoxia-triggered increase in the level of intracellular Fe(II) ion and subsequent production of ROS, probably through Fenton reaction, are the essential pathway mediating the disruption of neural vascular barrier under hypoxia.
... (b) Oxidative stress disrupts the blood-brain barrier (Al Ahmad, Gassmann, & Ogunshola, 2012;Blasig, Mertsch, & Haseloff, 2002;Enciu, Gherghiceanu, & Popescu, 2013;Haorah et al., 2007;Hurst, Heales, Dobbie, Barker, & Clark, 1998;Katsu et al., 2010;Lochhead et al., 2010;Nittby et al., 2009;Salford, Brun, Sturesson, Eberhardt, & Persson, 1994;Sirav & Seyhan, 2009Takemori, Murakami, Kometani, & Ito, 2013;Tang et al., 2015). ...
... Oxidative stress contributes to auxiliary mechanisms of radiation injury, such as mitochondrial dysfunction. Oxidative stress contributes to multiple documented auxiliary mechanisms of RF/MW damage that likely contribute to health effects in subsets, including membrane alterations-cell membranes (Benderitter, Vincent-Genod, Pouget, & Voisin, 2003) and mitochondrial membranes (Shonai et al., 2002;Thomas, Gebicki, & Dean, 1989;Vayssier-Taussat et al., 2002;Wang et al., 2002), blood-brain barrier disruption (Al Ahmad et al., 2012;Barichello et al., 2011;Freeman & Keller, 2012;Gasche, Copin, Sugawara, Fujimura, & Chan, 2001;Haorah, Knipe, Leibhart, Ghorpade, & Persidsky, 2005;Haorah et al., 2007;Hurst et al., 1998;Lochhead et al., 2010;Nittby et al., 2009;Salford et al., 1994;Zehendner et al., 2013), effects on voltage gated calcium channels (Cui et al., 2012) affected by and affecting oxidative stress- (Nishiyama, Nakano, & Hitomi, 2010;Pall, 2015)-but also on voltage-gated anion channels that are an important part of the outer mitochondrial membrane (Ferrer, 2009) potentially contributing to mitochondrial impairment and amplification of oxidative stress, EEG spiking (Naziroglu, Celik et al., 2012), impaired mitochondrial function (Aitken, Bennetts, Sawyer, Wiklendt, & King, 2005;-bidirectionally related to oxidative stress (Houston, Nixon, King, De Iuliis, & Aitken, 2016;Mancuso, Coppede, Migliore, Siciliano, & Murri, 2006;Wei & Lee, 2002)-and protected by melatonin (Tan, Manchester, Qin, & Reiter, 2016), impaired blood flow-e.g., via oxidative stress-driven endothelial dysfunction (Engin, Sepici-Dincel, Gonul, & Engin, 2012;Indik, Goldman, & Gaballa, 2001;Jarasuniene & Simaitis, 2003;Loscalzo, 2002), autoantibodies (Ahsan, Ali, & Ali, 2003;Fiorini et al., 2013;Gilgun-Sherki, Melamed, & Offen, 2004;Kirkham et al., 2011;Kumagai et al., 2003;Maes et al., 2013;Ryan, Nissim, & Winyard, 2014), and apoptosis (Aoki et al., 2001;Bresgen et al., 2003;Espino et al., 2010;Filomeni, Cardaci, Da Costa Ferreira, Rotilio, & Ciriolo, 2011;France-Lanord, Brugg, Michel, Agid, & Ruberg, 1997;Li et al., 2015;Li et al., 2008;Salido & Rosado, 2009;Yalcinkaya et al., 2009;Zhang, Zhang, Rabbani, Jackson, & Vujaskovic, 2012)-programmed cell death, which in turn triggers inflammation and coagulation activation (Reutelingsperger & van Heerde, 1997). Laboratory correlates for some of these were reported in ES participants in the French study: about 15% of those with ES had elevated markers of blood-brain barrier permeability; 29% in those with ES (23% in those with ES and multiple chemical sensitivity, MCS) had antibodies to O-myelin (Belpomme et al., 2015). ...
Importance: A mystery illness striking U.S. and Canadian diplomats to Cuba (and now China) “has confounded the FBI, the State Department and US intelligence agencies” (Lederman, Weissenstein, & Lee, 2017). Sonic explanations for the so-called health attacks have long dominated media reports, propelled by peculiar sounds heard and auditory symptoms experienced. Sonic mediation was justly rejected by experts. We assessed whether pulsed radiofrequency/microwave radiation (RF/MW) exposure can accommodate reported facts in diplomats, including unusual ones.
Observations: (1) Noises: Many diplomats heard chirping, ringing or grinding noises at night during episodes reportedly triggering health problems. Some reported that noises were localized with laser-like precision or said the sounds seemed to follow them (within the territory in which they were perceived). Pulsed RF/MW engenders just these apparent “sounds” via the Frey effect. Perceived “sounds” differ by head dimensions and pulse characteristics and can be perceived as located behind in or above the head. Ability to hear the “sounds” depends on high-frequency hearing and low ambient noise. (2) Signs/symptoms: Hearing loss and tinnitus are prominent in affected diplomats and in RF/MW-affected individuals. Each of the protean symptoms that diplomats report also affect persons reporting symptoms from RF/MW: sleep problems, headaches, and cognitive problems dominate in both groups. Sensations of pressure or vibration figure in each. Both encompass vision, balance, and speech problems and nosebleeds. Brain injury and brain swelling are reported in both. (3) Mechanisms: Oxidative stress provides a documented mechanism of RF/MW injury compatible with reported signs and symptoms; sequelae of endothelial dysfunction (yielding blood flow compromise), membrane damage, blood-brain barrier disruption, mitochondrial injury, apoptosis, and autoimmune triggering afford downstream mechanisms, of varying persistence, that merit investigation. (4) Of note, microwaving of the U.S. embassy in Moscow is historically documented.
Conclusions and relevance: Reported facts appear consistent with pulsed RF/MW as the source of injury in affected diplomats. Nondiplomats citing symptoms from RF/MW, often with an inciting pulsed-RF/MW exposure, report compatible health conditions. Under the RF/MW hypothesis, lessons learned for diplomats and for RF/MW-affected civilians may each aid the other.
... RBE4 exposed to hypoxia showed increased ROS formation providing EC disintegration. This phenomenon led Ahmad et al. to inhibit ROS generation by diphenyliodiunium and verify, whether it leads to maintaining BBB function, demonstrating promising results of using ROS inhibitors for supporting BBB function during stroke or cerebrovascular injury (Al Ahmad et al. 2012). ...
The blood–brain barrier (BBB) is responsible for maintaining homeostasis within the central nervous system (CNS). Depending on its permeability, certain substances can penetrate the brain, while others are restricted in their passage. Therefore, the knowledge about BBB structure and function is essential for understanding physiological and pathological brain processes. Consequently, the functional models can serve as a key to help reveal this unknown. There are many in vitro models available to study molecular mechanisms that occur in the barrier. Brain endothelial cells grown in culture are commonly used to modeling the BBB. Current BBB platforms include: monolayer platforms, transwell, matrigel, spheroidal, and tissue-on-chip models. In this paper, the BBB structure, molecular characteristic, as well as its dysfunctions as a consequence of aging, neurodegeneration, or under hypoxia and neurotoxic conditions are presented. Furthermore, the current modelling strategies that can be used to study BBB for the purpose of further drugs development that may reach CNS are also described.
... Oxidative stress plays a major role in the changes of cerebral vascular structure [28][29][30] . Cerebral blood flow changes with changes in the structure of cerebral vessels, which is affected in a complex way. ...
... Hypoxia is one of the most common causes of BBB degradation, although its processes are poorly understood. Long-term exposure to hypoxia increases reactive oxygen species (ROS) levels and induces glutathione oxidation (16). ROS are important signaling molecules activated in response to oxidative stress (17). ...
Objectives:
Intracerebroventricular (ICV) injections of mesenchymal stem cells (MSCs) may improve the function and structure of blood-brain barrier (BBB), possibly by preserving the BBB integrity. This study examined the impact of Wharton's jelly (WJ)-MSCs on cognitive dysfunction and BBB disruption following a protracted hypoxic state.
Materials and methods:
Twenty-four male Wistar rats were randomly studied in four groups: Control (Co): Healthy animals, Sham (Sh): Rats were placed in the cage without hypoxia induction and with ICV injection of vehicle, Hypoxic (Hx)+vehicle: Hypoxic rats with ICV injection of vehicle (5 μl of PBS), and Hx+MSCs: Hypoxic rats with ICV injection of MSCs. Spatial learning and memory were evaluated one week after WJ-MSCs injection, and then animals were sacrificed for molecular research.
Results:
Hypoxia increased latency and lowered the time and distance required reaching the target quarter, according to the findings. Furthermore, hypoxic rats had lower gene expression and protein levels of hippocampus vascular endothelial (VE)-cadherin, claudin 5, and tricellulin gene expression than Co and Sh animals (P<0.05). Finally, administering WJ-MSCs after long-term hypoxia effectively reversed the cognitive deficits and prevented the BBB breakdown via the upregulation of VE-cadherin, claudin 5, and tricellulin genes (P<0.05).
Conclusion:
These findings suggest that prolonged hypoxia induces spatial learning and memory dysfunction and increases BBB disruption, the potential mechanism of which might be via reducing VE-cadherin, claudin 5, and tricellulin genes. Hence, appropriate treatment with WJ-MSCs could reverse ischemia adverse effects and protect the BBB integrity following prolonged hypoxia.
... These findings suggest that BCCAO-induced destruction of vessels demonstrated an increase in the number of fragmented microvessels in the cerebral cortex, and that AA extract had protective effects against these damages (Zlokovic, 2010). This can induce the appropriate supply of CBF so that AA provides resilience to nerve damage through neurovascular protection and preserves BBB integrity to prevent the development of VaD (Al Ahmad et al., 2012). ...
Background:
Vascular dementia (VaD) is the second most common type of dementia after Alzheimer's disease. Currently, no FDA-approved drugs are available for the treatment of VaD. Artemisia annua Linné (AA) is known to have antioxidant properties, but its effects and mechanisms of action on cognitive impairment are still unknown.
Purpose:
In this study, the improvement in cognitive impairment by AA in terms of protection against oxidative stress, neuroinflammation, and preservation of the integrity of the neurovascular unit (NVU) was assessed in an animal model of VaD with bilateral common carotid artery occlusion (BCCAO).
Methods:
Eight-week-old male Wistar rats were allowed to adapt for four weeks, and BCCAO was induced at 12 weeks of age. The rats were randomly assigned into four groups, with seven rats in each group: sham group without BCCAO, VaD group that received oral administration of distilled water after BCCAO surgery, and two AA groups that received oral administration of 150 mg/kg or 750 mg/kg AA after BCCAO surgery for 8 weeks. Nine weeks after BCCAO surgery, the cognitive function of the rats was evaluated and accumulated oxidative stress was assessed by immunohistochemistry, immunofluorescence, and western blotting. Damage to the components of the NVU was evaluated, and sirtuin (Sirt) 1 and 2 expression and nuclear factor-erythrocyte 2-associated factor 2 (Nrf2)/Kelch-like ECH-associated protein1 (Keap1) activation were investigated to assess the reduction in cell signaling and antioxidant pathways.
Results:
BCCAO-induced cerebral perfusion decreased memory function and induced neuroinflammation and oxidative stress. But AA treatment mitigated cognitive impairment and reduced neuroinflammation and oxidative stress caused by chronic cerebral hypoperfusion. AA extracts activated the Nrf2/Keap1/activating antioxidant response elements pathway and maintained Sirt 1 and 2, subsequently leading to the maintenance of neurons, improved construct of microvessels, increased platelet-derived growth factor receptor beta, and platelet-endothelial cell adhesion molecule-1 associated with the blood-brain barrier integrity.
Conclusion:
AA is effective in alleviating BCCAO-induced cognitive decline and its administration may be a useful therapeutic approach for VaD.
... BBB is a cellular barrier formed by brain endothelial cells under the action of astrocytes and pericytes, [96] which plays a crucial role in maintaining homeostasis. Studies have shown that TSF, a TMP derivative, can increase the expression of tight junction protein junction adhesion molecule-1 and occludin in I/R rats, and reduce the expression of MMP-9 and aquaporin AQP4, thus significantly reducing BBB permeability and brain oedema, [97] which suggests that TMP may have the potential to treat I/R. ...
Objectives:
The role and mechanism of tetramethylpyrazine (TMP) in cardio-cerebrovascular diseases (CCVDs), as well as the research of its new formulations are reviewed, which provides a new strategy for the clinical application of TMP.
Methods:
We searched the databases including PubMed, Web of Science, Google Scholar and CNKI for relevant literature from 1991 to 2021 by searching for the keywords "TMP", "ligustrazine", "cardiovascular disease" and "nanoformulation". The inclusion criteria are as follows: (1) the literature is an experimental article, (2) the article studies cardiovascular and cerebrovascular-related diseases and (3) the article also includes the pharmacy research of TMP. A total of 160 articles were screened.
Key findings:
TMP has various pharmacological effects in the treatment of many CCVDs, such as atherosclerosis, myocardium, cerebral ischemia, reperfusion injury and hypertension. Its protective effects are mainly related to its anti-platelet activity, protection of endothelial cells, and anti-inflammation, anti-oxidant and anti-apoptotic effects. In addition to pharmacological activity studies, the information of the new formulations is also significant for the further development and utilization of TMP.
Conclusions:
Above all, TMP can protect cardio-cerebro vessels, and preparing new formulations can improve its bioavailability, indicating that TMP has broad prospects in the treatment of CCVDs.
... The BBB is intrinsically dynamic, hence it has important functional implications during development, the physiological aging process and age-associated neurodegenerative diseases and cognitive performance. Therefore, it is unsurprising that increased BBB permeability is observed in VaD patients [10,52,[78][79][80][81][82][83]. An increased BBB permeability allows for uncontrolled movement of substances between the blood and brain parenchyma, which may include entry of immune cells and pathogens into the brain. ...
The prevalence of cerebrovascular disease increases with age, placing the elderly at a greater lifetime risk for dementia. Vascular cognitive impairment (VCI) encompasses a spectrum of cognitive deficits from mild cognitive impairment to dementia. VCI and its most severe form, vascular dementia (VaD), is becoming a major public health concern worldwide. As growing efforts are being taken to understand VCI and VaD in animal models and humans, the pathogenesis of the disease is being actively explored. It is postulated that chronic cerebral hypoperfusion (CCH) is a major cause of VCI. CCH activates a molecular and cellular injury cascade that leads to breakdown of the blood brain barrier (BBB) and neurodegeneration. The BBB tightly regulates the movement of substances between the blood and the brain, thereby regulating the microenvironment within the brain parenchyma. Here we illustrate how BBB damage is causal in the pathogenesis of VCI through the increased activation of pathways related to excitotoxicity, oxidative stress, inflammation and matrix metalloproteinases that lead to downstream perivascular damage, leukocyte infiltration and white matter changes in the brain. Thus, CCH-induced BBB damage may initiate and contribute to a vicious cycle, resulting in progressive neuropathological changes of VCI in the brain. This review outlines the molecular and cellular mechanisms that govern BBB breakdown during CCH and highlights the clinical evidence in identifying at-risk VCI patients.
... the stress on the 2 physiological functions of the endothelium (ie, the barrier and fluid-pump functions), which maintain stromal deturgescence, 20 has not been examined to date except for indirect evidence in our recent study on hypothermia. 48 Based on the knowledge that oxidative stress induces a breakdown of the barrier function in vascular endothelial cells, [52][53][54][55][56] we investigated the effects of oxidative stress in cultured corneal endothelial cells. We were also motivated by our previous finding that microtubule disassembly triggered by (TNF-a)-induced oxidative stress also elicits a breakdown of PAMR. ...
Purpose: To investigate the impact of oxidative stress, which is a hallmark of Fuchs dystrophy, on the barrier function of the corneal endothelial cells. Methods: Experiments were carried out with cultured bovine and porcine corneal endothelial cells. For oxidative stress, cells were supplemented with riboflavin (Rf) and exposed to UV-A (15-30 min) to induce Type-1 photochemical reactions that release H2O2. The effect of the stress on the barrier function was assayed by transendothelial electrical resistance (TER) measurement. In addition, the associated changes in the organization of the microtubules, perijunctional actomyosin ring (PAMR), and ZO-1 were evaluated by immunocytochemistry, which was also repeated after direct exposure to H2O2 (100 μM, 1 h). Results: Exposure to H2O2 led to the disassembly of microtubules and the destruction of PAMR. In parallel, the contiguous locus of ZO-1 was disrupted, marking a loss of barrier integrity. Accordingly, a sustained loss in TER was induced when cells in the Rf-supplemented medium were exposed to UV-A. However, the addition of catalase (7,000 U/mL) to rapidly decompose H2O2 limited the loss in TER. Furthermore, the adverse effects on microtubules, PAMR, and ZO-1 were suppressed by including catalase, ascorbic acid (1 mM; 30 min), or pretreatment with p38 MAP kinase inhibitor (SB-203580; 10 μM, 1 h). Conclusions: Acute oxidative stress induces microtubule disassembly by a p38 MAP kinase-dependent mechanism, leading to the destruction of PAMR and loss of barrier function. The response to oxidative stress is reminiscent of the (TNF-α)-induced breakdown of barrier failure in the corneal endothelium.
... The mammalian brain is susceptible to changes in cellular homeostasis as a result of environmental or physiological insults. Neurological diseases constitute one of the most challenging and devastating pathologies [1]. Neonatal hypoxia-ischemia (HI) induces brain injury and causes neurological impairment, including cognitive impairment, motor dysfunction, and seizure [2]. ...
Regulation of microglial activation and neuroinflammation are critical factors in the pathogenesis of ischemic brain injury. Interest in protease-activated receptor 2 (PAR2) as a pharmaceutical target for various diseases is creasing. However, it is unclear the expression and functions of PAR2 in hypoxia-ischemic (HI) brain injury. Mice with HI and cells with oxygen–glucose deprivation and reoxygenation (OGD/R) were studied. Immunoblot and qRT-PCR were used to study the differential gene expression in cultured microglia and neurons. Immunofluorescent staining was used to study the expression pattern of PAR2 in the HI brain and phagocytotic activity of microglia after OGD/R. In neonatal mice brain after HI, we found PAR2 expression was abundant in neurons, but barely in microglia from the contralateral side of cortex and hippocampus. Conversely, PAR2 expression was barely in neurons while significantly increased in activated microglia from the ipsilateral side of cortex and hippocampus. The activations of PAR2 were increased in both microglia and neuron in a cell model of OGD/R. PAR2 activation mediated the cross-talk between microglia and neurons including the following: microglial PAR2 mediated inflammatory responses that induced neuronal damage; neuronal PAR2 regulated chemokines that recruited activated microglia to damage area; microglia PAR2 controlled the phagocytosis of degenerating neurons. These data suggested differential expression and distinct roles of PAR2 in microglia and neurons after HI injury; thereby, interventions targeting PAR2 may provide insights into the inflammatory-related diseases.
... The effects of hypoxia on the mammalian BBB have primarily been examined in humans in the context of either high altitude exposure (mountain sickness) or ischemia induced by disorders such as stroke, cardiac arrest, and respiratory distress [62,63]. Moreover, most studies report the effects of artificially induced hypoxia in laboratory rodents, with the assumption that the results can be generalized to all mammals [64,65]. Chronic exposure to low oxygen levels in mice causes the BBB to become leaky, and the depletion of microglia further exacerbates the hypoxia-induced leakiness [66]. ...
The need to protect neural tissue from toxins or other substances is as old as neural tissue itself. Early recognition of this need has led to more than a century of investigation of the blood-brain barrier (BBB). Many aspects of this important neuroprotective barrier have now been well established, including its cellular architecture and barrier and transport functions. Unsurprisingly, most research has had a human orientation, using mammalian and other animal models to develop translational research findings. However, cell layers forming a barrier between vascular spaces and neural tissues are found broadly throughout the invertebrates as well as in all vertebrates. Unfortunately, previous scenarios for the evolution of the BBB typically adopt a classic, now discredited ‘scala naturae’ approach, which inaccurately describes a putative evolutionary progression of the mammalian BBB from simple invertebrates to mammals. In fact, BBB-like structures have evolved independently numerous times, complicating simplistic views of the evolution of the BBB as a linear process. Here, we review BBBs in their various forms in both invertebrates and vertebrates, with an emphasis on the function, evolution, and conditional relevance of popular animal models such as the fruit fly and the zebrafish to mammalian BBB research.
... Hypoxia-induced oxidative stress contributes to BBB breakdown. The different cell types at the NVU exhibit distinct sensitivity to oxygen deprivation; BECs are markedly more sensitive than pericytes or astrocytes, and pericytes more than astrocytes (Ahmad et al. 2012). Although the pathophysiology of BBB disruption related to environmental stress is not fully understood, it is recognized as multifactorial, involving hypoxia and oxidative stress. ...
The blood–brain barrier is a dynamic structure, collectively referred to as the neurovascular unit. It is responsible for the exchange of blood, oxygen, ions, and other molecules between the peripheral circulation and the brain compartment. It is the main entrance to the central nervous system and as such critical for the maintenance of its homeostasis. Dysfunction of the blood–brain barrier is a characteristic of several neurovascular pathologies. Moreover, physiological changes, environmental factors, nutritional habits, and psychological stress can modulate the tightness of the barrier. In this contribution, we summarize our current understanding of structure and function of this important component of the brain. We also describe the neurological deficits associated with its damage. A special emphasis is placed in the effect of the exposure to xenobiotics and pollutants in the permeability of the barrier. Finally, current protective strategies as well as the culture models to study this fascinating structure are discussed.
... The global deletion of ZIPK in an animal model of middle cerebral artery occlusion (MCAO) significantly attenuates BBB dysfunction by inhibiting EC contraction, as proven by reduced infarct and edema volume . Oxidative stress after stroke can also induce endothelial cell apoptosis, which is suggested to have detrimental consequences on BBB integrity, subsequently leading to brain edema (Rizzo and Leaver, 2010;Al Ahmad et al., 2012). Protecting ECs from apoptosis after stroke is beneficial to the integrity of BBB and the reduction of brain edema (Park et al., 2004;Zhang et al., 2016;Yang et al., 2017). ...
Brain edema is a severe stroke complication that is associated with prolonged hospitalization and poor outcomes. Swollen tissues in the brain compromise cerebral perfusion and may also result in transtentorial herniation. As a physical and biochemical barrier between the peripheral circulation and the central nervous system (CNS), the blood–brain barrier (BBB) plays a vital role in maintaining the stable microenvironment of the CNS. Under pathological conditions, such as ischemic stroke, the dysfunction of the BBB results in increased paracellular permeability, directly contributing to the extravasation of blood components into the brain and causing cerebral vasogenic edema. Recent studies have led to the discovery of the glymphatic system and meningeal lymphatic vessels, which provide a channel for cerebrospinal fluid (CSF) to enter the brain and drain to nearby lymph nodes and communicate with the peripheral immune system, modulating immune surveillance and brain responses. A deeper understanding of the function of the cerebral lymphatic system calls into question the known mechanisms of cerebral edema after stroke. In this review, we first discuss how BBB disruption after stroke can cause or contribute to cerebral edema from the perspective of molecular and cellular pathophysiology. Finally, we discuss how the cerebral lymphatic system participates in the formation of cerebral edema after stroke and summarize the pathophysiological process of cerebral edema formation after stroke from the two directions of the BBB and cerebral lymphatic system.
... Additionally, the massive release of inflammatory factors can cause neuroinflammation, thereby continuously leading to neuronal damage or even death. The nervous impairments combined with the persistent decline of CBF increase the area of cerebral infarction, deteriorate cognitive abilities, and eventually lead to VaD [65][66][67] Correspondingly, many studies have found that some treatments (such as simvastatin [68], omega-3 PUFAs [69], and acupuncture [70]) can remarkably attenuate WM damage and even cognitive impairments in VaD mice by inhibiting inflammation. ...
Vascular dementia (VaD) is the second most common form of dementia worldwide. It is caused by cerebrovascular disease, and patients often show severe impairments of advanced cognitive abilities. Nitric oxide synthase (NOS) and nitric oxide (NO) play vital roles in the pathogenesis of VaD. The functions of NO are determined by its concentration and bioavailability, which are regulated by NOS activity. The activities of different NOS subtypes in the brain are partitioned. Pathologically, endothelial NOS is inactivated, which causes insufficient NO production and aggravates oxidative stress before inducing cerebrovascular endothelial dysfunction, while neuronal NOS is overactive and can produce excessive NO to cause neurotoxicity. Meanwhile, inflammation stimulates the massive expression of inducible NOS, which also produces excessive NO and then induces neuroinflammation. The vicious circle of these kinds of damage having impacts on each other finally leads to VaD. This review summarizes the roles of the NOS/NO pathway in the pathology of VaD and also proposes some potential therapeutic methods that target this pathway in the hope of inspiring novel ideas for VaD therapeutic approaches.
... Queste entità molecolari sono dei prodotti intermedi dei processi cellulari di riduzione dell'ossigeno e hanno un'azione sulla permeabilità della barriera emato-encefalica, incrementandola. Una maggior permeabilità della barriera emato-encefalica permette l'entrata di linfociti e altre cellule del sistema immunitario, tra cui anche le citochine infiammatorie presumibilmente responsabili del mal di testa (Al Ahmad, Gassmann, & Ogunshola, 2012). ...
The placebo effect refers to a clinical improvement that could be observed in an individual following the intake of an inert substance. This improvement deals with several elements of the psycho-social context in which the placebo therapy is administered and with the doctor-patient relationship itself. All these elements meet the patient's clinical experience, consisting of memories of previous medical experiences, personal beliefs, emotions and expectations. The psychological mechanisms that underlie the placebo effect are anxiety modulation processes, classical conditioning and social learning processes. The neurobiological underpinnings of placebo effect are made up of different neurotransmitter systems such as the opioid, the endocannabinoid and the dopaminergic systems. The nocebo effect instead indicates the psychobiological effects that occur in the patient's brain after taking a nocebo. Following negative verbal suggestions of increased pain perception or symptomatic worsening, the individual is induced into a psychological state of anticipatory anxiety, a state which activates two independent biochemical pathways: the cholecystochinergic system and the hypothalamic-pituitary-adrenal axis, responsible for symptomatic worsening. In this dissertation a new and very promising paradigm for the investigation on the effects of placebo and nocebo phenomenon on physiological functions will be introduced: hypoxia (insufficient oxygen supply). While hypobaric hypoxia is a condition that occurs naturally at high altitude, normobaric hypoxia could be experimentally induced. In an innovative experimental project, we tried to better understand the influence of the nocebo effect, obtained through negative expectations and 'cognitive conditioning', on the performance of dry apnea (holding the breath) in a sample of healthy volunteers. We measured both behavioral performance parameters (apnea duration) and physiological parameters such as heart rate (HR) and peripheral oxygen saturation level in the blood (SpO2). The results about behavioral variables (breath holding duration) showed the effectiveness of suggestions and cognitive conditioning on apnea performance: subjects belonging to the experimental group Nocebo (N), in fact, showed a worsening in apnea duration after 4 sessions. Experimental group N had a worse performance of 5.5% in the last session, the evocation phase, compared to the one obtained during the basal test, unlike the Natural History control group (NH), which even showed an improvement in apnea times 21.7% compared to the baseline. The physiological results showed that the Nocebo group had higher peripheral oxygen saturation levels already from the first session (baseline) compared to the NH group, both during apnea and during the recovery phase. Furthermore, the HR levels in the N group were generally higher than those in the NH group, particularly as regards the recovery phase, also in this case already from the first experimental session. These results showed that hypoxia conditions could be a promising paradigm for investigating the influences of placebo and nocebo effects on physical performance and physiological parameters.
... In the cerebral capillaries, the monolayer endothelium is an important channel for the exchange of nutrients and metabolites between the blood and brain, and also functions as a barrier against blood neurotoxicity (3,6). Because of the presence of crucial tight junction complexes and efflux transporters, these Ecs play a crucial role as doormen, regulating central nervous system homeostasis (6,7). Brain microvessel endothelial cells (BMECs) are the cells of the cellular interface which function to separate the blood and its constituents from the brain extracellular fluid (6,(8)(9)(10). ...
Background:
Under hypoxic conditions, the brain can undergo irreversible damage. The present study aimed to explore new higher-oxygen-content carbamide peroxide (CP) compounds and the effect of their oxygen-releasing property on human brain microvascular endothelial cell (EC) proliferation under in vitro hypoxic conditions.
Methods:
Two different additives including alpha-terpineol and sorbic acid were added to the reaction system to obtain the carbamide peroxide of CP-I and CP-II.
Results:
We evaluated the oxygen generation capabilities of CP samples by using a portable dissolved oxygen meter. Consequently, alpha-terpineol as a stabilizer exhibited a unique effect on the oxygen generation of CP. CP-I was uniquely able to promote cell proliferation ability at 10 µg·L-1 for hypoxic conditions, with the proliferation rates being 36.2% compared with the control group. The safety of CP to cells was further verified by calcein-AM/PI staining. Under hypoxic conditions, CP-I at 10 µg·L-1 promoted the migration rate, and the migration rate being 32.37%.
Conclusions:
These compounds have the advantages of simple synthesis, long storage time, low cost, and rich oxygen content. Used spectrophotometry, oxygen electrode test, and indicator titration for testing the oxygen production rate and oxygen production. The results indicate that alpha-terpineol is the best additive. CP-I exhibited the highest oxygen content and a superior effect on the cell phenotype than CP-II, especially under hypoxia. This study is the first to report the effects of CP on cells, and provides new therapeutic insights into cerebrovascular injury repair.
... These two conditions allowed a significant increase in ROS (Fig. 7). Our results are in agreement with the literature: an increase in ROS in acute hypoxic conditions in the endothelial cells of BBB [50] or in other tissues such as nasal polyp-derived fibroblasts [51]. The same observation is found in conditions of IH in the BBB [52][53][54]. ...
Background/aims:
Obstructive sleep apnea (OSA) is characterized by repeated episodes of complete or partial obstruction of the upper airways, leading to chronic intermittent hypoxia (IH). OSA patients are considered at high cerebrovascular risk and may also present cognitive impairment. One hypothesis explored is that disturbances may be linked to blood-brain barrier (BBB) dysfunction. The BBB is a protective barrier separating the brain from blood flow. The BBB limits the paracellular pathway through tight and adherens junctions, and the transcellular passage by efflux pumps (ABC transporters). The aims of this study were to evaluate the impact of IH and sustained hypoxia (SH) on a validated in vitro BBB model and to investigate the factors expressed under both conditions.
Methods:
Exposure of endothelial cells (HBEC-5i) in our in vitro model of BBB to hypoxia was performed using IH cycles: 1% O2-35 min/18% O2-25 min for 6 cycles or 6 h of SH at 1% O2. After exposure, we studied the cytotoxicity and the level of ROS in our cells. We measured the apparent BBB permeability using sodium fluorescein, FITC-dextran and TEER measurement. Whole cell ELISA were performed to evaluate the expression of tight junctions, ABC transporters, HIF-1α and Nrf2. The functionality of ABC transporters was evaluated with accumulation studies. Immunofluorescence assays were also conducted to illustrate the whole cell ELISAs.
Results:
Our study showed that 6 h of IH or SH induced a BBB disruption marked by a significant decrease in junction protein expressions (claudin-5, VE-cadherin, ZO-1) and an increase in permeability. We also observed an upregulation in P-gp protein expression and functionality and a downregulation in BCRP. Hypoxia induced production of ROS, Nrf2 and HIF-1α. They were expressed in both sustained and intermittent conditions, but the expression and the activity of P-gp and BCRP were different.
Conclusion:
Understanding these mechanisms seems essential in order to propose new therapeutic strategies for patients with OSA.
... Several mediators, including NO, calcium influx, release of inflammatory cytokines, and hemodynamic alterations, may be responsible for this alteration [91]. Moreover, hypoxia-induced oxidative stress also contributes to BBB breakdown [92]. The different cell types at the NVU exhibit distinct sensitivity to oxygen deprivation: brain ECs are markedly more sensitive than pericytes or astrocytes, and pericytes more sensitive than astrocytes. ...
The blood–brain barrier (BBB) is a dynamic platform for exchange of substances between the blood and the brain parenchyma, and it is an essential functional gatekeeper for the central nervous system (CNS). While it is widely recognized that BBB disruption is a hallmark of several neurovascular pathologies, an aspect of the BBB that has received somewhat less attention is the dynamic modulation of BBB tightness to maintain brain homeostasis in response to extrinsic environmental factors and physiological changes. In this review, we summarize how BBB integrity adjusts in critical stages along the life span, as well as how BBB permeability can be altered by common stressors derived from nutritional habits, environmental factors and psychological stress.
... At this early stage of stroke, hyperglycemia also causes BBB disruption, again mediated by MMP-2/9 extracellular degradation, caveolin-1-mediated intracellular translocation, and autophagy-lysosome-mediated degradation of ZO-1 protein (94). Hypoxia is another insult that alters BBB integrity, increasing its permeability through MMP-9-dependent loss of tight junctions with disrupted continuity of occludin and ZO-1 (95) and through the generation of reactive oxygen species (96). Hyperthermia is another significant and independent contributor to BBBP (95,97). ...
The blood–brain barrier (BBB) is a dynamic interface responsible for maintaining the central nervous system homeostasis. Its unique characteristics allow protecting the brain from unwanted compounds, but its impairment is involved in a vast number of pathological conditions. Disruption of the BBB and increase in its permeability are key in the development of several neurological diseases and have been extensively studied in stroke. Ischemic stroke is the most prevalent type of stroke and is characterized by a myriad of pathological events triggered by an arterial occlusion that can eventually lead to fatal outcomes such as hemorrhagic transformation (HT). BBB permeability seems to follow a multiphasic pattern throughout the different stroke stages that have been associated with distinct biological substrates. In the hyperacute stage, sudden hypoxia damages the BBB, leading to cytotoxic edema and increased permeability; in the acute stage, the neuroinflammatory response aggravates the BBB injury, leading to higher permeability and a consequent risk of HT that can be motivated by reperfusion therapy; in the subacute stage (1–3 weeks), repair mechanisms take place, especially neoangiogenesis. Immature vessels show leaky BBB, but this permeability has been associated with improved clinical recovery. In the chronic stage (>6 weeks), an increase of BBB restoration factors leads the barrier to start decreasing its permeability. Nonetheless, permeability will persist to some degree several weeks after injury. Understanding the mechanisms behind BBB dysregulation and HT pathophysiology could potentially help guide acute stroke care decisions and the development of new therapeutic targets; however, effective translation into clinical practice is still lacking. In this review, we will address the different pathological and physiological repair mechanisms involved in BBB permeability through the different stages of ischemic stroke and their role in the development of HT and stroke recovery.
... The blood brain barrier (BBB) is disrupted in the course of chronic cerebral hypoperfusion (Rosenberg, 2012). Indeed, brain hypoxia is known to damage endothelial cells, pericytes, and astrocytes (Jiang et al., 2018) and cause increased leakage from the BBB (Al Ahmad et al., 2012), as demonstrated in vivo in a rat model of brain hypoperfusion produced by bilateral stenosis of the carotids (Ueno et al., 2002). Furthermore, breakdown of the BBB causes extravasation of plasma proteins, such as immunoglobulins, fibrinogen, and complement, all potent proinflammatory molecules, and increases the production of free radicals (Morgan et al., 1997;Yoshida et al., 2002;Davalos and Akassoglou, 2012;Crehan et al., 2013). ...
Neurons have been long regarded as the basic functional cells of the brain, whereas astrocytes and microglia have been regarded only as elements of support. However, proper intercommunication among neurons-astrocytes-microglia is of fundamental importance for the functional organization of the brain. Perturbation in the regulation of brain energy metabolism not only in neurons but also in astrocytes and microglia may be one of the pathophysiological mechanisms of neurodegeneration, especially in hypoxia/ischemia. Glial activation has long been considered detrimental for survival of neurons, but recently it appears that glial responses to an insult are not equal but vary in different brain areas. In this review, we first take into consideration the modifications of the vascular unit of the glymphatic system and glial metabolism in hypoxic conditions. Using the method of triple-labeling fluorescent immunohistochemistry coupled with confocal microscopy (TIC), we recently studied the interplay among neurons, astrocytes, and microglia in chronic brain hypoperfusion. We evaluated the quantitative and morpho-functional alterations of the neuron-astrocyte-microglia triads comparing the hippocampal CA1 area, more vulnerable to ischemia, to the CA3 area, less vulnerable. In these contiguous and interconnected areas, in the same experimental hypoxic conditions, astrocytes and microglia show differential, finely regulated, region-specific reactivities. In both areas, astrocytes and microglia form triad clusters with apoptotic, degenerating neurons. In the neuron-astrocyte-microglia triads, the cell body of a damaged neuron is infiltrated and bisected by branches of astrocyte that create a microscar around it while a microglial cell phagocytoses the damaged neuron. These coordinated actions are consistent with the scavenging and protective activities of microglia. In hypoxia, the neuron-astrocyte-microglia triads are more numerous in CA3 than in CA1, further indicating their protective effects. These data, taken from contiguous and interconnected hippocampal areas, demonstrate that glial response to the same hypoxic insult is not equal but varies significantly. Understanding the differences of glial reactivity is of great interest to explain the differential susceptibility of hippocampal areas to hypoxia/ischemia. Further studies may evidence the differential reactivity of glia in different brain areas, explaining the higher or lower sensitivity of these areas to different insults and whether glia may represent a target for future therapeutic interventions.
... This protocol allows for the differentiation of astrocytes and neurons using adherent cells rather than floating neurospheres, which eases the differentiation protocol. iPSC-derived astrocytes showed expression of several cell markers considered typical for human astrocytes (GFAP+, S100β+, Nestin-), were able to induce barrier properties in iPSC-derived BMECs monolayers, and responded to environmental injury (e.g., hypoxia/ischemia) [52] similar to primary astrocytes monocultures Estimating Brain Permeability Using In Vitro Blood-Brain Barrier Models or co-cultured with BMECs [53][54][55][56]. We will also describe one of the most common platforms of in vitro models, "Transwell" filters, in this protocol. ...
The blood-brain barrier (BBB) is a vital biological interface that regulates transfer of different molecules between blood and brain and, therefore, maintains the homeostatic environment of the CNS. In order to perform high-throughput screening of therapeutics in drug discovery, specific properties of the BBB are investigated within in vitro BBB platforms. In this chapter, we detail the process and steps for the iPSC to BMEC and astrocyte differentiation as well as TEER and permeability measurement in Transwell platform of in vitro BBB model. Also, advanced microfluidic iPSCs-derived BMECs on chip and permeability measurement within this model have been elucidated.
... Various stress factors could affect the BBB. For example, oxidative stress can induce BBB breakdown (Al Ahmad et al., 2012). Psychosocial stress also induces BBB perturbation (Lehmann et al., 2018). ...
The microbiota and microbiome and disruption of the gut-brain axis were linked to various metabolic, immunological, physiological, neurodevelopmental, and neuropsychiatric diseases. After a brief review of the relevant literature, we present our hypothesis that intestinal serotonin, produced by intestinal enterochromaffin cells, picked up and stored by circulating platelets, participates and has an important role in the regulation of membrane permeability in the intestine, brain, and other organs. In addition, intestinal serotonin may act as a hormone-like continuous regulatory signal for the whole body, including the brain. This regulatory signal function is mediated by platelets and is primarily dependent on and reflects the intestine's actual health condition. This hypothesis may partially explain why gut dysbiosis could be linked to various human pathological conditions as well as neurodevelopmental and neuropsychiatric disorders.
... The involvement of oxidative stress has been recognized as a causative factor in stroke-induced BBB dysfunction in the hypertensive rat model, 85 permanent rat MCAO 86 and in vitro O 2 deprivation-reoxygenation model. 87 Increased oxidative stress may increase neuroinflammatory response 88 and lead to degradation of TJ protein in the BBB. 89 Further understanding of its mechanisms will certainly bring more therapeutic targets for clinical intervention. ...
With the emergence of novel technologies and ground breaking concepts in BBB basic research in the past decades, the researchers’ attention on the critical role of blood brain barrier (BBB) integrity after stroke is surging. The main novelty of this review is that it not only highlights recent findings of the signaling pathways mediating ischemic BBB disruption, such as vascular permeability, lactate transportation failure, mitochondrial crisis and ion channel and water channel disability, endothelial actin polymerization, junctional proteins disassembly and endothelial transcytosis. Evolutional breakthroughs in identification of novel molecules that orchestrates the neuro-vascular unit restoration, immune cells of distinct phenotype that play dualistic roles in the evolution of ischemic brain injury are also included in this review. The aim of this review is to provide intriguing therapeutic targets to protect the BBB after cerebral ischemia.
... Various stress factors could affect the BBB. For example, oxidative stress can induce BBB breakdown (Al Ahmad et al., 2012). Psychosocial stress also induces BBB perturbation (Lehmann et al., 2018). ...
The microbiota and microbiome and disruption of the gut-brain axis have been linked to various metabolic, immunological, physiological, neurodevelopmental and neuropsychiatric diseases. After a brief review of the relevant literature we present our hypothesis that intestinal serotonin, produced by intestinal enterochromaffin cells picked up and stored by circulating platelets participates and has an important role in the regulation of membrane permeability in the intestine, brain and other organs. In addition, intestinal serotonin may act as a hormone-like continuous regulatory signal for the whole body, including the brain. This regulatory signal function is mediated by platelets and is primarily dependent on and reflects the intestine's actual health condition. This hypothesis may partially explain why gut dysbiosis could be linked to various human pathological conditions as well as neurodevelopmental and neuropsychiatric disorders.
... BBB disruption is a canonical feature of ischemic stroke 18 . Oxidation and inflammation exacerbate the loss of tight junction proteins such as claudin-5 and occludin in the microvasculature, and further increase BBB permeability 19,20 . Our data showed that OPCs transplantation significantly attenuated infarct volume and brain Fig. 3 OPCs transplantation maintained the integrity of the BBB after MCAO. a IgG staining showed that IgG protein leaked into brain tissue in Sham, Stroke + PBS and Stroke + OPC groups. ...
Blood–brain barrier damage is a critical pathological feature of ischemic stroke. Oligodendrocyte precursor cells are involved in maintaining blood–brain barrier integrity during the development. However, whether oligodendrocyte precursor cell could sustain blood–brain barrier permeability during ischemic brain injury is unknown. Here, we investigate whether oligodendrocyte precursor cell transplantation protects blood–brain barrier integrity and promotes ischemic stroke recovery. Adult male ICR mice (n = 68) underwent 90 min transient middle cerebral artery occlusion. After ischemic assault, these mice received stereotactic injection of oligodendrocyte precursor cells (6 × 105). Oligodendrocyte precursor cells transplantation alleviated edema and infarct volume, and promoted neurological recovery after ischemic stroke. Oligodendrocyte precursor cells reduced blood–brain barrier leakage via increasing claudin-5, occludin and β-catenin expression. Administration of β-catenin inhibitor blocked the beneficial effects of oligodendrocyte precursor cells. Wnt7a protein treatment increased β-catenin and claudin-5 expression in endothelial cells after oxygen–glucose deprivation, which was similar to the results of the conditioned medium treatment of oligodendrocyte precursor cells on endothelial cells. We demonstrated that oligodendrocyte precursor cells transplantation protected blood–brain barrier in the acute phase of ischemic stroke via activating Wnt/β-catenin pathway. Our results indicated that oligodendrocyte precursor cells transplantation was a novel approach to the ischemic stroke therapy.
... Hypoxia/ischemia is an important environmental stress factor involved in several diseases associated with a disruption of the BBB such as cerebral ischemia (Kim et al. 2018a, b;Lu et al. 2018;Haley and Lawrence 2017;Merali et al. 2017;Turner and Sharp 2016;Choi et al. 2016;Suzuki et al. 2016;Kassner and Merali 2015;Prakash and Carmichael 2015;O'Donnell 2014;Kuntz et al. 2014;Yang et al. 2013;Fernandez-Lopez et al. 2012), cerebral amyloid angiopathy (Freeze et al. 2019;Daulatzai 2017;Ghiso et al. 2014), high-altitude cerebral edema (Lafuente et al. 2016;Ogunshola and Al-Ahmad 2012;Natah et al. 2009;Hackett and Roach 2004;Mark and Davis 2002;Schoch et al. 2002), or neonatal hypoxic/ischemic encephalitis (Lee et al. 2017;Ma et al. 2017;Gussenhoven et al. 2019). Disruption of the BBB following hypoxic/ischemic event has been documented with a loss of tight junction complexes (Kokubu et al. 2017;Zhang et al. 2016;Page et al. 2016;Engelhardt et al. 2015Engelhardt et al. , 2014aEngelhardt et al. , 2014bAl Ahmad et al. 2012;Ogunshola and Al-Ahmad 2012;Zhu et al. 2012;Al Ahmad et al. 2011;Bauer et al. 2010;Haarmann et al. 2010;Al Ahmad et al. 2009), resulting in cerebral edema formation, a potentially fatal condition. At the BBB, brain microvascular endothelial cells (BMECs) constitute the first cell type of the neurovascular unit to respond to hypoxic/ischemic injury, via the activation of the hypoxia-inducible factor (HIF) pathway . ...
The blood-brain barrier (BBB) plays an important role in brain homeostasis. Hypoxia/ischemia constitutes an important stress factor involved in several neurological disorders by inducing the disruption of the BBB, ultimately leading to cerebral edema formation. Yet, our current understanding of the cellular and molecular mechanisms underlying the BBB disruption following cerebral hypoxia/ischemia remains limited. Stem cell-based models of the human BBB present some potentials to address such issues. Yet, such models have not been validated in regard of its ability to respond to hypoxia/ischemia as existing models. In this study, we investigated the cellular response of two iPSC-derived brain microvascular endothelial cell (BMEC) monolayers to respond to oxygen-glucose deprivation (OGD) stress, using two induced pluripotent stem cells (iPSC) lines. iPSC-derived BMECs responded to prolonged (24 h) and acute (6 h) OGD by showing a decrease in the barrier function and a decrease in tight junction complexes. Such iPSC-derived BMECs responded to OGD stress via a partial activation of the HIF-1 pathway, whereas treatment with anti-angiogenic pharmacological inhibitors (sorafenib, sunitinib) during reoxygenation worsened the barrier function. Taken together, our results suggest such models can respond to hypoxia/ischemia similarly to existing in vitro models and support the possible use of this model as a screening platform for identifying novel drug candidates capable to restore the barrier function following hypoxic/ischemic injury.
... Such disruption will eventually lead to a vascular leakage. The mechanisms by which H/I impacts barrier function have been extensively studied in rodents and non-human primates [9][10][11][12][13][14][15][16][17], yet the literature showing similar outcomes at the human BBB remains unclear. ...
Cerebral hypoxia/ischemia (H/I) is an important stress factor involved in the disruption of the blood-brain barrier (BBB) following stroke injury, yet the cellular and molecular mechanisms on how the human BBB responds to such injury remains unclear. In this study, we investigated the cellular response of the human BBB to chemical and environmental H/I in vitro.
METHODS:
In this study, we used immortalized hCMEC/D3 and IMR90 stem-cell derived human brain microvascular endothelial cell lines (IMR90-derived BMECs). Hypoxic stress was achieved by exposure to cobalt chloride (CoCl2) or by exposure to 1 % hypoxia and oxygen/glucose deprivation (OGD) was used to model ischemic injury. We assessed barrier function using both transendothelial electrical resistance (TEER) and sodium fluorescein permeability. Changes in cell junction integrity were assessed by immunocytochemistry and cell viability was assessed by trypan-blue exclusion and by MTS assays. Statistical analysis was performed using one-way analysis of variance (ANOVA).
RESULTS:
CoCl2 selectively disrupted the barrier function in IMR90-derived BMECs but not in hCMEC/D3 monolayers and cytotoxic effects did not drive such disruption. In addition, hypoxia/OGD stress significantly disrupted the barrier function by selectively disrupting tight junctions (TJs) complexes. In addition, we noted an uncoupling between cell metabolic activity and barrier integrity.
CONCLUSIONS:
In this study, we demonstrated the ability of IMR90-derived BMECs to respond to hypoxic/ischemic injury triggered by both chemical and environmental stress by showing a disruption of the barrier function. Such disruption was selectively targeting TJ complexes and was not driven by cellular apoptosis. In conclusion, this study suggests the suitability of stem cell-derived human BMECs monolayers as a model of cerebral hypoxia/ischemia in vitro.
... At 9, 15, 30, 45 and 60 min aliquots were taken from the bottom compartment and tracer flux was determined with a fluorescence plate reader (FLx800, Biotek Instruments, Winooski, VT). A clearance slope established from the measurements obtained at the different time points was used to calculate the permeability coefficient (Pe) [30]. ...
Hypoxic blood-brain barrier (BBB) dysfunction is a common feature of CNS diseases however mechanisms underlying barrier disturbance are still largely unknown. This study investigated the role of transforming growth factor β (TGFβ), a cytokine known to induce expression of the proprotein convertase Furin, in hypoxia-mediated barrier compromise.We show that exposure of brain endothelial cells (ECs) to hypoxia (1% O2) rapidly stimulates their migration. Additional exogenous TGFβ (0.4nM) exposure potentiated this effect and increased Furin expression in a TGFβ type I receptor activin-like kinase 5 (ALK5) - dependent manner (prevented by 10μM SB431542). Furin inhibition prevented hypoxia-induced EC migration and blocked TGFβ-induced potentiation suggesting existence of a feedback loop. TGFβ and Furin were also critical for hypoxia-induced BBB dysfunction. TGFβ treatment aggravated hypoxia-induced BBB permeability but ALK5 or Furin blockade reversed injury-induced permeability changes. Thus during insult Furin compromises endothelial integrity by mediating the effects of TGFβ. Targeting the Furin or ALK5 pathway may offer novel therapeutic strategies for improving BBB stability and CNS function during disease.
... Hypoxia can also occur without a reduction of CBF, through other mechanisms such as reduced blood oxygenation, occurring in acute lung injury, or a decreased oxygen transport in anemia. Among the variety of molecular mechanisms, which may be involved in producing the tissue alterations following brain hypoperfusion/ischemia, it is worth mentioning a number of changes affecting the small cerebral vessels both in structure and density, with concomitant alterations in the blood brain barrier (BBB) integrity and impairment of the neurovascular unit [51][52][53][54][55]. When CBH occurs naturally or during experimental conditions, the BBB is altered [56][57][58] and neurovascular coupling is depressed. ...
Autophagy primarily works to counteract nutrient deprivation that is strongly engaged during starvation and hypoxia, which happens in hypoperfusion. Nonetheless, autophagy is slightly active even in baseline conditions, when it is useful to remove aged proteins and organelles. This is critical when the mitochondria and/or proteins are damaged by toxic stimuli. In the present review, we discuss to that extent the recruitment of autophagy is beneficial in counteracting brain hypoperfusion or, vice-versa, its overactivity may per se be detrimental for cell survival. While analyzing these opposite effects, it turns out that the autophagy activity is likely not to be simply good or bad for cell survival, but its role varies depending on the timing and amount of autophagy activation. This calls for the need for an appropriate autophagy tuning to guarantee a beneficial effect on cell survival. Therefore, the present article draws a theoretical pattern of autophagy activation, which is hypothesized to define the appropriate timing and intensity, which should mirrors the duration and severity of brain hypoperfusion. The need for a fine tuning of the autophagy activation may explain why confounding outcomes occur when autophagy is studied using a rather simplistic approach.
... Ly-6C hi CCR2 + monocytes initiate infiltration into the ischemic brain after the destruction of the BBB starting several hours after the onset of stroke. Ischemic insult and oxidized stress directly induce disruption of the BBB [52,53], while high-mobility group box 1 (HMGB1) is one of the key DAMPs in the ischemic brain which triggers post-ischemic inflammation by promoting BBB breakdown. HMGB1 is a nuclear protein bound with DNA/RNA that is released into the extracellular space from dying brain cells a few hours after stroke onset. ...
Inflammatory responses play a multifaceted role in regulating both disability and recovery after ischemic brain injury. In the acute phase of ischemic stroke, resident microglia elicit rapid inflammatory responses by the ischemic milieu. After disruption of the blood-brain barrier, peripheral-derived neutrophils and mononuclear phagocytes infiltrate into the ischemic brain. These infiltrating myeloid cells are activated by the endogenous alarming molecules released from dying brain cells. Inflammation after ischemic stroke thus typically consists of sterile inflammation triggered by innate immunity, which exacerbates the pathologies of ischemic stroke and worsens neurological prognosis. Infiltrating immune cells sustain the post-ischemic inflammation for several days; after this period, however, these cells take on a repairing function, phagocytosing inflammatory mediators and cellular debris. This time-specific polarization of immune cells in the ischemic brain is a potential novel therapeutic target. In this review, we summarize the current understanding of the phase-dependent role of innate myeloid cells in ischemic stroke and discuss the cellular and molecular mechanisms of their inflammatory or repairing polarization from a therapeutic perspective.
... 58 Several studies have shown that oxidative stress mediates BBB disruption in ischemia/reperfusion (I/R) injury. 59,60 Increased oxidative stress during cerebral ischemia is also reported to enhance direct MMP activation and reduce the level of tissue inhibitors of MMPs. 61 ...
... Activation of apoptosis is initiated by EGL-1 which binds and inhibits the function of CED-9 eventually cleaving the CED-4 (activator of cell death) from a complex of protein CED-9/CED-4. Moreover, Activation of BH3-only proteins also occurs un-der stressed conditions [34]. The detailed expression of pro-tein in hypothalamus after induced microgravity is listed in Table 1. ...
Space travelling has emerged as a hot issue in the world for various purposes. The man has been trying to establish space stations and these efforts have turned into a reality. Longtime stay in space is very much challenging due to various limiting environmental problems and health issues. Among many, one is microgravity that generates uneven flow of fluids in the body which results in oxidative stress. This stress markedly interrupts cerebral activity of astronauts both structurally and functionally. This oxidative stress produced by microgravity distort many essential protein signaling pathways that intricate various homeostatic behaviors within the body. The current knowledge of mi-crogravity effects on human brain especially the hypothalamus is expanding; whereas the comprehension of the basic components of this phenomenon is restricted. Various literature reports already outlined many key phenomena distorted by earth-based simulated microgravity. Therefore, an effort is being made to summarize this bulk of knowledge in a simple way for the convenience of space research community. This review will elucidate the effect of microgravity on the expression of key proteins involved in structural, morphological, functional and molecular functions
... Hypoxia is the subnormal concentration of oxygen in cells. Acute hypoxia can increase blood-brain barrier per-meability [7] and can breakdown the BBB by the reason of involvement of oxidative stress [8]. Decreased oxygenation or hypoxia is a characteristic of brain disorders that increases barrier permeability [9]. ...
Sodium thiopental is rapid-acting and intravenous anesthetic as non-opioid agents. Hypoxia is the subnormal concentration of oxygen in cells. Recent years, researchers discuss on hypoxia: Friend or Foe? The present study was conducted to examine the effect of intermittent hypoxia on thiopental anesthesia.
... Furthermore, the rise of ROS signaling leads to BBB breakdown. EC integrity is disrupted by increasing oxidative stress [238]. Oxidative stress is in turn able to activate MMPs. ...
Among age-related diseases, cardiovascular and cerebrovascular diseases are major causes of death. Vascular dysfunction is a key characteristic of these diseases wherein age is an independent and essential risk factor. The present work will review morphological alterations of aging vessels in-depth, which includes the discussion of age-related microvessel loss and changes to vasculature involving the capillary basement membrane, intima, media, and adventitia as well as the accompanying vascular dysfunctions arising from these alterations.
Glucose is a major source of energy for the brain. At the blood–brain barrier (BBB), glucose uptake is facilitated by glucose transporter 1 (GLUT1). GLUT1 Deficiency Syndrome (GLUT1DS), a haploinsufficiency affecting SLC2A1 , reduces glucose brain uptake. A lot of effort has been made to characterize GLUT1DS at the BBB, but the impact on astrocytes remains unclear. In this study, we investigated the impact of GLUT1DS on astrocyte differentiation and function in vitro, using human induced pluripotent stem cells GLUT1DS (GLUT1DS‐iPSCs) differentiated into astrocyte‐like cells (iAstros). GLUT1 expression is decreased during the differentiation of iPSCs into astrocytes, with neural progenitor cells showing the lowest expression. The presence of a truncated GLUT1 did not compromise the differentiation of iPSCs into iAstros, as these cells could express several key markers representative of the astrocyte lineage. GLUT1DS‐iAstros failed to express full‐length GLUT1 at protein levels while showing no signs of impaired GLUT4 expression. However, GLUT1DS‐iAstros showed decreased glucose uptake and lactate production compared to control‐iAstros, reduced glycolysis, and mitochondrial activity as well as ATP deficit. In addition to reduced energy production, astrocytes displayed a reduced extracellular glutamate release. As previously observed, one iAstros clone (C7) showed the most severe phenotype from all groups. Our study provides an insightful view of the contribution of GLUT1 in astrocytes' energetic metabolism and raises the possible contribution of these cells in the astrocyte–neuron metabolic coupling. Our future direction is to understand better how GLUT1DS impacts astrocytes and neurons within their metabolic coupling.
The blood-brain barrier (BBB) is formed by microvascular endothelial cells which are ensembled with pericytes, astrocytes, microglia and neurons in the neurovascular unit (NVU) that is crucial for neuronal function. Given that the NVU and the BBB are highly dynamic and regulated structures, their integrity is continuously challenged by intrinsic and extrinsic factors. Herein, factors from peripheral organs such as gonadal and adrenal hormones may influence vascular function also in CNS endothelial cells in a sex- and age-dependent manner. The communication between the periphery and the CNS likely takes place in specific areas of the brain among which the circumventricular organs have a central position due to their neurosensory or neurosecretory function, owing to physiologically leaky blood vessels. In acute and chronic pathological conditions like liver, kidney, pulmonary disease, toxins and metabolites are generated that reach the brain via the circulation and may directly or indirectly affect BBB functionality via the activation of the immunes system. For example, chronic kidney disease (CKD) currently affects more than 840 million people worldwide and is likely to increase along with western world comorbidities of the cardio-vascular system in continuously ageing societies. Toxins leading to the uremic syndrome, may further lead to neurological complications such as cognitive impairment and uremic encephalopathy. Here we summarize the effects of hormones, toxins and inflammatory reactions on the brain vasculature, highlighting the urgent demand for mechanistically exploring the communication between the periphery and the CNS, focusing on the BBB as a last line of defense for brain protection.
Upregulation of reactive oxygen species (ROS) levels is a principal feature observed in the brains of neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). In these diseases, oxidative stress can disrupt the blood–brain barrier (BBB). This disruption allows neurotoxic plasma components, blood cells, and pathogens to enter the brain, leading to increased ROS production, mitochondrial dysfunction, and inflammation. Collectively, these factors result in protein modification, lipid peroxidation, DNA damage, and, ultimately, neural cell damage. In this review article, we present the mechanisms by which oxidative damage leads to BBB breakdown in brain diseases. Additionally, we summarize potential therapeutic approaches aimed at reducing oxidative damage that contributes to BBB disruption in neurodegenerative diseases.
Changes to blood–brain barrier structure and function may affect the delivery of drugs into the brain. It is worthwhile to exploring more study on how the blood–brain barrier changes in structure and function and how that affects drug transport in high-altitude hypoxic environment. The DIA high-throughput sequencing technique indicate that the rats blood–brain barrier has been identified to have 7252 proteins overall and 8 tight junction proteins, among which Claudin-7 was a plateau-specific tight junction protein under high-altitude hypoxia, and based on the interaction network study, 2421 proteins are found to interact with one another, with ZO-1 being the primary target. The results of the projected gene function analysis demonstrated that changes in tight junction proteins are related to the control of TRP channels by inflammatory mediators, the wnt signaling pathway, the ABC transporter system, and drug metabolism-CYP450 enzyme regulation. Additionally, the electron microscopy, the Evans blue combination with confocal laser scanning microscopy, and the Western Blot and RT-qPCR revealed that high-altitude hypoxic environment induces blood–brain barrier tight junctions to open, blood–brain barrier permeability increases, ZO-1, Occludin, Claudin-5 protein and mRNA expression decreased. Our research implies that structural and functional alterations in the blood–brain barrier induced by high altitude hypoxia may impact drug transport inside the central nervous system, and that drug transporters and drug-metabolizing enzymes may be key players in this process.
The alteration of tight junction proteins and mechanisms under high-altitude hypoxic environment
Cerebral small vessel disease (CSVD) is considered as one of the main causes of cognitive decline and dementia. However, despite extensive research, the pathogenesis of CSVD and the mechanisms through which CSVD leads to its clinical manifestations remain largely unclear. The challenging in vivo quantification of CSVD hampers progress in further unraveling the pathogenesis and pathophysiology of CSVD. Currently, markers of CSVD are mainly brain abnormalities attributed to CSVD, but these are limited in reflecting morphological and functional changes of the microvasculature. We describe aspects of CSVD that are reflected by currently used techniques and those that are still insufficiently captured.
The blood-brain barrier is essential for maintaining the stability of the central nervous system and is also crucial for regulating drug metabolism, changes of blood-brain barrier's structure and function can influence how drugs are delivered to the brain. In high-altitude hypoxia, the central nervous system's function is drastically altered, which can cause disease and modify the metabolism of drugs in vivo. Changes in the structure and function of the blood-brain barrier and the transport of the drug across the blood-brain barrier under high-altitude hypoxia, are regulated by changes in brain microvascular endothelial cells, astrocytes, and pericytes, either regulated by drug metabolism factors such as drug transporters and drug-metabolizing enzymes. This article aims to review the effects of high-altitude hypoxia on the structure and function of the blood-brain barrier as well as the effects of changes in the blood-brain barrier on drug metabolism. We also hypothesized and explore the regulation and potential mechanisms of the blood-brain barrier and associated pathways, such as transcription factors, inflammatory factors, and nuclear receptors, in regulating drug transport under high-altitude hypoxia.
Vascular cognitive impairment (VCI) and depression frequently coexist in geriatric populations and reciprocally increase disease risks. We assert that a shared pre-disease state of the psycho-immune-neuroendocrine (PINE) network model mechanistically explains bidirectional associations between VCI and depression. Five pathophysiological sub-networks are identified that are shared by VCI and depression: neuroinflammation, kynurenine pathway imbalance, hypothalamic-pituitary-adrenal (HPA) axis overactivity, impaired neurotrophic support and cerebrovascular dysfunction. These do not act independently, and their complex interactions necessitate a systems biology approach to better define disease pathogenesis. The PINE network is already established in the context of non-communicable diseases (NCDs) such as depression, hypertension, atherosclerosis, coronary heart disease and type 2 diabetes mellitus. We build on previous literature to specifically explore mechanistic links between MDD and VCI in the context of PINE pathways and discuss key mechanistic commonalities linking these comorbid conditions and identify a common pre-disease state which precedes transition to VCI and MDD. We expand the model to incorporate bidirectional interactions with biological ageing. Diathesis factors for both VCI and depression feed into this network and the culmination of shared mechanisms (on an ageing substrate) lead to a critical network transition to one or both disease states. A common pre-disease state underlying VCI and depression can provide clinicians a unique opportunity for early risk assessment and intervention in disease development. Establishing the mechanistic elements and systems biology of this network can reveal early warning or predictive biomarkers together with novel therapeutic targets. Integrative studies are recommended to elucidate the dynamic networked biology of VCI and depression over time.
For the pharmacotherapy of neurodegenerative diseases, drugs must pass the blood–brain barrier (BBB). The BBB seems to play an important role in disease initiation and or progression, and many changes in BBB properties in neurodegeneration have been reported. In vivo studies including measurements of unbound drug concentrations in plasma and brain are needed for insight into BBB transport, intra-brain and target site distribution, and specific changes related to neurodegenerative conditions. However, it is surprising that only a limited number of such studies have been performed to date. This chapter summarizes the published work on these in vivo studies and provides a perspective on what is needed to advance and foster more understanding in the future . Though it is generally thought that the BBB is compromised in neurodegenerative disorders, quantitative studies indicate that this is not necessarily always the case. It is recommended to increase in vivo studies that can integrate the impact of neurodegenerative processes, to complement studies on neurodegenerative components in isolation, and to improve our understanding of target site distribution of drugs intended to treat the disease condition. As in vivo studies on human brain sampling are ethically restricted, we must rely on animal models and translational mathematical approaches to infer relevance for clinical work.
In the Gulf toadfish (Opsanus beta), the serotonin (5-HT) transporter (SERT) is highly expressed in the heart, and the heart and gill both demonstrate the capacity for SERT-mediated uptake of 5-HT from the circulation. Because 5-HT is a potent vasoconstrictor in fish, we hypothesized that hypoxia exposure may increase 5-HT uptake by these tissues—and increase excretion of 5-HT—to prevent branchial vasoconstriction that would hamper gas exchange. Spot sampling of blood, bile, and urine revealed that fish exposed to chronic hypoxia (1.83 ± 0.12 mg·L⁻¹ O2 for 24–26 h) had 41% lower plasma 5-HT in the ventral aorta (immediately following the heart) than in the hepatic vein (immediately before the heart), suggesting enhanced cardiac 5-HT uptake during hypoxia. 5-HT concentrations in the bile were greater than those in the urine, but there were no effects of acute (1.31 ± 0.06 mg·L⁻¹ O2 for 25 min) or chronic hypoxia on 5-HT levels in these fluids. In 5-HT radiotracer experiments, the presence of tracer in the bile decreased upon hypoxia exposure, but, surprisingly, neither acute nor chronic hypoxia-induced changes in [³H]5-HT uptake in the heart, gill, or other tissues. Given the likely impact of the hypoxia exposure on metabolic rate, future studies should examine the effects of a milder hypoxia exposure on 5-HT uptake into these tissues and the role of 5-HT degradation.
Cerebral amyloid angiopathy (CAA), Alzheimer’s disease (AD) and ischaemic stroke are diseases that often co-occur in the ageing population. Emerging evidence suggests that ischaemia and amyloid β (Aβ) interact in a mutually detrimental way, though the exact mechanisms remain unclear. With the blood-brain barrier (BBB) as a major constituent of cerebrovascular function and Aβ clearance, this work focuses on changes of BBB transporters and tight junctions (TJs) during ageing, AD/CAA and ischaemia in order to broaden the understanding of these diseases’ development and progression. Two ATP-binding cassette (ABC) transporters that have been implicated in Aβ clearance are p-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp). Using intact isolated capillaries as an ex-vivo model of the BBB, age- and sex-dependent changes in P-gp and Bcrp activity were observed in APP23 mice, a transgenic model of AD/CAA. In wild-type (WT) mice, P-gp activity declined with age in both sexes (3 versus 20−22 months), in agreement with current literature. However, while Bcrp activity was also lower in old male mice, an up-regulation was noted for females. Protein expression reflected the functional results found in females. This sex-specific difference at the BBB might explain some of the disparities observed between men and women in age-associated diseases such as AD and stroke. Since P-gp and Bcrp are both linked to Aβ transport, age-associated decline in their activity may contribute to the onset of AD/CAA. Indeed, it was found that transport of Aβ also decreased with age. In APP23 mice, P-gp and Bcrp activity was elevated in young and middle-aged males, with the old mice showing non-significant differences (age range: 2−5, 10−16 and 23−24 months). In contrast, P-gp and Bcrp activity was down-regulated in female APP23 mice at all ages. The tendency for elevated P-gp and Bcrp activity in male mice could point to a stronger compensatory action in response to elevated Aβ levels and emphasize the importance of separating by sex when studying AD/CAA. No difference was found in Aβ transport between old WT and APP23 mice in either males or females, although both P-gp and Bcrp were down-regulated in female APP23 mice. This may be linked to the presence of compensatory actions by other Aβ transport mechanisms. Although there is some evidence of disruption of BBB integrity during ageing and AD/CAA, western blot analysis of capillary TJs claudin 5 (Cldn-5), occludin (Ocln) and zona occludens 1 (ZO-1) generally showed increased expression in brain capillary for both conditions in middle-aged mice (age range: 10−14.5 months). Potentially, TJ disruption either sets in during later stages of ageing and AD/CAA, or is mitigated by factors other than protein expression, such as localisation. To probe the interaction of AD/CAA and ischaemia at the BBB directly, primary mouse brain microvascular endothelial cell (BMEC) cultures were treated with Aβ and oxygen-glucose deprivation (OGD), followed by a reoxygenation period to simulate reperfusion. Aβ1-40 was used at 1 and 12 nM, while OGD intervals were 12 and 24 h, with 0, 2, 4, 6 and 18 h of reoxygenation. qPCR and western blot analysis of P-gp, Bcrp, glucose transporter 1 (Glut-1), TJs and hypoxia-inducible factor 1α (HIF-1α) showed that their expression is highly dependent on concentration, order of treatment, and the duration of treatment and reoxygenation periods. The combination of OGD and Aβ often resulted in a non-additive effect with the response differing between WT and APP23 BMECs, revealing an intricate relationship between ischaemia and Aβ. Interestingly, the addition of Aβ during OGD in WT BMECs tended to counteract the down-regulation brought on by OGD when followed by either 0 or 2 h of reoxygenation, supporting previous studies suggesting possible beneficial roles for Aβ. Overall, the findings of this work help to identify age-dependent changes at the BBB that may contribute to AD/CAA, as well as denote the presence of important sex-dependent differences. Additionally, the complex interaction between OGD and Aβ shown at the BBB suggests the need for more systematic studies in order to fully understand the relationship between ischaemic stroke and AD/CAA.
Ischemic stroke is a leading cause of death and disability worldwide and is in urgent need of new treatment options. The only approved treatment for stroke restores blood flow to the brain, but much of the tissue damage occurs during the subsequent reperfusion. Antioxidant therapies that directly address ischemia-reperfusion injury have shown promise in preclinical results. In this review, we discuss that reformulating antioxidant therapies as nanomedicine can potentially overcome the barriers that have kept these therapies from succeeding in the clinic. We begin by reviewing the pathophysiology of ischemic stroke with a focus on the effects of reperfusion injury. Next, we review nanotherapeutic systems designed to treat the disease with a focus on those addressing reperfusion injury. Mechanisms of passive and active transport required to traverse a blood-brain barrier are discussed. Finally, we conclude by outlining design parameters for potentially successful nanomedicines as front-line therapeutics for ischemic stroke.
Objective:
To explore the effects of N-butylphthalide on the expressions of ZO-1 and claudin-5 in blood-brain barrier (BBB) in rats with acute carbon monoxide (CO) poisoning.
Methods:
A total of 144 adult healthy male Sprague-Dawley (SD) rats were randomly divided into normal control group, CO poisoning group, and NBP treatment group, with 48 rats in each group. The acute CO poisoning model was reproduced in hyperbaric oxygen chamber, and all model rats were given hyperbaric oxygen therapy once daily. The rats in the normal control group were free to breathe fresh air. The rats in NBP treatment group were administered orally NBP 60 mg/kg twice a day at 2 hours after poisoning until death. The rats in normal control group and CO poisoning group were treated with equal amount of pure olive oil. Four rats were sacrificed from each group at 1, 3, 7, 14 days after model reproducing, respectively. The changes in ultrastructure of BBB were observed under transmission electron microscope. The expressions of ZO-1 and claudin-5 proteins were determined by immunofluorescence staining and Western Blot. The localization of the two target proteins was observed by immunofluorescence double staining. The correlation between the two proteins was analyzed by linear regression.
Results:
The ultrastructure of BBB was normal in normal control group, some ZO-1 and a large number of claudin-5 positive cells were observed. The ultrastructure of BBB was seriously injured, ZO-1 and claudin-5 positive cells in brain tissue were significantly decreased, and the expressions of ZO-1 and claudin-5 proteins in brain tissue at 1 day after poisoning in CO poisoning group were significantly lower than those of normal control group (ZO-1 protein: 3.38±0.30 vs. 24.50±5.62, claudin-5 protein: 11.38±0.93 vs. 46.35±6.88, both P < 0.05), and although gradually restored, they were maintained at relatively lower levels until 14 days as compared with those in normal control group (ZO-1 protein: 10.35±0.80 vs. 24.63±3.57, claudin-5 protein: 32.35±3.11 vs. 46.43±7.20, both P < 0.05). NBP treatment could significantly alleviate the ultrastructure injury of BBB induced by acute CO poisoning, the amount of ZO-1 and claudin-5 positive cells in brain tissue were significantly increased, as well as the expressions of ZO-1 and claudin-5 proteins were significantly increased, which were significantly higher than those of CO poisoning group from 1 day and 3 days on, respectively (1-day ZO-1 protein: 7.57±0.69 vs. 3.38±0.30, 3-day claudin-5 protein: 20.46±1.42 vs. 11.43±0.86, both P < 0.05), and which showed an increase tendency with time prolongation. The results of immunofluorescence double staining showed that ZO-1 and claudin-5 proteins could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed the positive correlation between the expressions of ZO-1 and claudin-5 proteins in brain tissue of rats with acute CO poisoning (R2 = 0.917, P = 0.022).
Conclusions:
NBP could markedly improve the ultrastructure and functional integrity of BBB through up-regulating the expressions of ZO-1 and claudin-5 proteins, and then reduce brain damage caused by CO poisoning.
Adaptive homeostasis is defined as the transient expansion or contraction of the homeostatic range following exposure to subtoxic, non-damaging, signaling molecules or events, or the removal or cessation of such molecules or events (Mol. Aspects Med. (2016) 49, 1-7). Adaptive homeostasis allows us to transiently adapt (and then de-adapt) to fluctuating levels of internal and external stressors. The ability to cope with transient changes in internal and external environmental stress, however, diminishes with age. Declining adaptive homeostasis may make older people more susceptible to many diseases. Chronic oxidative stress and defective protein homeostasis (proteostasis) are two major factors associated with the etiology of age-related disorders. In the present paper, we review the contribution of impaired responses to oxidative stress and defective adaptive homeostasis in the development of age-associated diseases.
Disruption of the blood-brain barrier (BBB) has an important part in cellular damage in neurological diseases, including acute and chronic cerebral ischemia, brain trauma, multiple sclerosis, brain tumors, and brain infections. The neurovascular unit (NVU) forms the interface between the blood and brain tissues. During an injury, the cascade of molecular events ends in the final common pathway for BBB disruption by free radicals and proteases, which attack membranes and degrade the tight junction proteins in endothelial cells. Free radicals of oxygen and nitrogen and the proteases, matrix metalloproteinases and cyclooxgyenases, are important in the early and delayed BBB disruption as the neuroinflammatory response progresses. Opening of the BBB occurs in neurodegenerative diseases and contributes to the cognitive changes. In addition to the importance of the NVU in acute injury, angiogenesis contributes to the recovery process. The challenges to treatment of the brain diseases involve not only facilitating drug entry into the brain, but also understanding the timing of the molecular cascades to block the early NVU injury without interfering with recovery. This review will describe the molecular and cellular events associated with NVU disruption and potential strategies directed toward restoring its integrity.
Hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular adaptation to hypoxia and has been suggested as a potent therapeutic target in cerebral ischemia. Here we show in an ischemic stroke model of rats that inhibiting HIF-1 and its downstream genes by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) significantly increases mortality and enlarges infarct volume evaluated by MRI and histological staining. Interestingly, the HIF-1 inhibition remarkably ameliorates ischemia-induced blood-brain barrier (BBB) disruption determined by Evans blue leakage although it does not affect brain edema. The result demonstrates that HIF-1 inhibition has differential effects on ischemic outcomes and BBB permeability. It indicates that HIF-1 may have different functions in different brain cells. Further analyses show that ischemia upregulates HIF-1 and its downstream genes erythropoietin (EPO), vascular endothelial growth factor (VEGF), and glucose transporter (Glut) in neurons and brain endothelial cells and that YC-1 inhibits their expression. We postulate that HIF-1-induced VEGF increases BBB permeability while certain other proteins coded by HIF-1's downstream genes such as epo and glut provide neuroprotection in an ischemic brain. The results indicate that YC-1 lacks the potential as a cerebral ischemic treatment although it confers certain protection to the cerebral vascular system.
Diabetes mellitus causes cerebral microvasculature deterioration and cognitive decline. The specialized endothelial cells of cerebral microvasculature comprise the blood-brain barrier, and the pericytes (PC) that are in immediate contact with these endothelial cells are vital for blood-brain barrier integrity. In diabetes, increased mitochondrial oxidative stress is implicated as a mechanism for hyperglycemia-induced PC loss as a prerequisite leading to blood-brain barrier disruption. Mitochondrial carbonic anhydrases (CA) regulate the oxidative metabolism of glucose and thus play an important role in the generation of reactive oxygen species and oxidative stress. We hypothesize that the inhibition of mitochondrial CA would reduce mitochondrial oxidative stress, rescue cerebral PC loss caused by diabetes-induced oxidative stress, and preserve blood-brain barrier integrity. We studied the effects of pharmacological inhibition of mitochondrial CA activity on streptozotocin-diabetes-induced oxidative stress and PC loss in the mouse brain. At 3 wk of diabetes, there was significant oxidative stress; the levels of reduced glutathione were lower and those of 3-nitrotyrosine, 4-hydroxy-2-trans-nonenal, and superoxide dismutase were higher. Treatment of diabetic mice with topiramate, a potent mitochondrial CA inhibitor, prevented the oxidative stress caused by 3 wk of diabetes. A significant decline in cerebral PC numbers, at 12 wk of diabetes, was also rescued by topiramate treatment. These results provide the first evidence that inhibition of mitochondrial CA activity reduces diabetes-induced oxidative stress in the mouse brain and rescues cerebral PC dropout. Thus, mitochondrial CA may provide a new therapeutic target for oxidative stress related illnesses of the central nervous system.
Disruptions of the blood-brain barrier (BBB) and edema formation both play key roles in the development of neurological dysfunction in acute and chronic cerebral ischemia. Animal studies have revealed the molecular cascades that are initiated with hypoxia/ischemia in the cells forming the neurovascular unit and that contribute to cell death. Matrix metalloproteinases cause reversible degradation of tight junction proteins early after the onset of ischemia, and a delayed secondary opening during a neuroinflammatory response occurring from 24 to 72 hours after. Cyclooxygenases are important in the delayed opening as the neuroinflammatory response progresses. An early opening of the BBB within the 3-hour therapeutic window for tissue-type plasminogen activator can allow it to enter the brain and increase the risk of hemorrhage. Chronic hypoxic hypoperfusion opens the BBB, which contributes to the cognitive changes seen with lacunar strokes and white matter injury in subcortical ischemic vascular disease. This review will describe the molecular and cellular events associated with BBB disruption and potential therapies directed toward restoring the integrity of the neurovascular unit.
The blood–brain barrier (BBB) has a critical role in central nervous system homeostasis. Intercellular tight junction (TJ) protein complexes of the brain microvasculature limit paracellular diffusion of substances from the blood into the brain. Hypoxia and reoxygenation (HR) is a central component to numerous disease states and pathologic conditions. We have previously shown that HR can influence the permeability of the BBB as well as the critical TJ protein occludin. During HR, free radicals are produced, which may lead to oxidative stress. Using the free radical scavenger tempol (200 mg/kg, intraperitoneal), we show that oxidative stress produced during HR (6% O2 for 1 h, followed by room air for 20 min) mediates an increase in BBB permeability in vivo using in situ brain perfusion. We also show that these changes are associated with alterations in the structure and localization of occludin. Our data indicate that oxidative stress is associated with movement of occludin away from the TJ. Furthermore, subcellular fractionation of cerebral microvessels reveals alterations in occludin oligomeric assemblies in TJ associated with plasma membrane lipid rafts. Our data suggest that pharmacological inhibition of disease states with an HR component may help preserve BBB functional integrity.
Blood-brain barrier (BBB) disruption, resulting from loss of tight junctions (TJ) and activation of matrix metalloproteinases (MMPs), is associated with edema formation in ischemic stroke. Cerebral edema develops in a phasic manner and consists of both vasogenic and cytotoxic components. Although it is contingent on several independent mechanisms, involving hypoxic and inflammatory responses, the single effect of prolonged hypoxia on BBB integrity in vivo was not addressed so far. Exposing mice to normobaric hypoxia (8% oxygen for 48 h) led to a significant increase in vascular permeability associated with diminished expression of the TJ protein occludin. Immunofluorescence studies revealed that hypoxia resulted in disrupted continuity of occludin and zonula occludens-1 (Zo-1) staining with significant gap formation. Hypoxia increased gelatinolytic activity specifically in vascular structures and gel zymography identified MMP-9 as enzymatic source. Treatment with an MMP inhibitor reduced vascular leakage and attenuated disorganization of TJ. Inhibition of vascular endothelial growth factor (VEGF) attenuated vascular leakage and MMP-9 activation induced by hypoxia. In conclusion, our data suggest that hypoxia-induced edema formation is mediated by MMP-9-dependent TJ rearrangement by a mechanism involving VEGF. Therefore, inhibition of MMP-9 might provide the basis for therapeutic strategies to treat brain edema.
While numerous lines of evidence point to increased levels of oxidative stress playing a causal role in a number of neurodegenerative conditions, our current understanding of the specific role of oxidative stress in the genesis and/or propagation of neurodegenerative diseases remains poorly defined. Even more challenging to the "oxidative stress theory of neurodegeneration" is the fact that many antioxidant-based clinical trials and therapeutic interventions have been largely disappointing in their therapeutic benefit. Together, these factors have led researchers to begin to focus on understanding the contribution of highly localized structures, and defined anatomical features, within the brain as the sites responsible for oxidative stress-induced neurodegeneration. This review focuses on the potential for oxidative stress within the cerebrovascular architecture serving as a modulator of neurodegeneration in a variety of pathological settings. In particular, this review highlights important implications for vascular-derived oxidative stress in the initiating and promoting pathophysiology in the brain, identifying new roles for cerebrovascular oxidative stress in a variety of brain disorders. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.
The neurovascular unit (NVU) comprises brain endothelial cells, pericytes or vascular smooth muscle cells, glia and neurons. The NVU controls blood-brain barrier (BBB) permeability and cerebral blood flow, and maintains the chemical composition of the neuronal 'milieu', which is required for proper functioning of neuronal circuits. Recent evidence indicates that BBB dysfunction is associated with the accumulation of several vasculotoxic and neurotoxic molecules within brain parenchyma, a reduction in cerebral blood flow, and hypoxia. Together, these vascular-derived insults might initiate and/or contribute to neuronal degeneration. This article examines mechanisms of BBB dysfunction in neurodegenerative disorders, notably Alzheimer's disease, and highlights therapeutic opportunities relating to these neurovascular deficits.
Baicalin is one of the principal flavonoids isolated from the dried root of Scutellariae Baicalensis Georgi and has been widely used as a traditional herbal medicine to suppress brain edema and reduce cerebral ischemic damage. However, the effects of baicalin on the blood-brain barrier (BBB) are poorly understood.
To explore the effects of baicalin on the permeability of the BBB under ischemic conditions in vitro with regard to changes in the tight junctions(TJ) proteins claudin-5 and zonula occludens-1 (ZO-1).
Brain microvascular endothelial cells(BMVECs) from Bal b/c mice were cultured to establish an in vitro BBB model. Oxygen and glucose deprivation (OGD) was applied to simulate ischemia. The experiment consisted of a normal control group, a model group and baicalin-treated groups (high-dose group, moderate-dose group and low-dose group). Transendothelial electrical resistance (TEER) and permeability to HRP were used as indicators of changes in BBB permeability. A real-time fluorescent quantitative assay was utilized to monitor the transcriptional changes in claudin-5 and ZO-1, and western blotting was used to detect the changes in protein expression of claudin-5, ZO-1 and PKC.
OGD led to a significant increase of permeability in this in vitro BBB model. Baicalin effectively decreased the permeability of the BBB, promoted transcription and expression of TJ proteins (claudin-5 and ZO-1) and reduced the levels of PKC.
We propose that baicalin is capable of restoring the barrier function of the BBB under ischemic conditions and this beneficial effect may be linked to the decreased expression of TJ proteins.
Proper understanding of blood-brain barrier (BBB) regulation is crucial to reduce/prevent its disruption during injury. Since high brain complexity makes interpretation of in vivo data challenging BBB studies are frequently performed using simplified in vitro models. Although such models represent an important and frequently employed alternative for investigation of BBB function and alterations, our ability to translate in vitro findings to in vivo situation remains sub-optimal. Consequently, despite the fact that our knowledge of the cellular and molecular mechanisms underlying BBB physiology and pathophysiology is constantly increasing, our ability to modulate barrier function remains virtually non-existent. Classical in vitro model systems have provided a wealth of knowledge until now, but it is now evident that newer in vitro models that are more representative of the in vivo situation are needed to further our understanding of barrier physiology. This paper will provide an overview of the BBB cellular components and the most frequently used in vitro BBB model systems. I will discuss their advantages and disadvantages, as well as highlight recently developed models that more closely mimic the BBB in vivo.
The authors investigated the mechanisms by which hypoxia regulates glutathione (GSH) in lung epithelial cells, and specifically whether the mitogen-activated protein kinase (MAPK) system is involved in the response to hypoxia. Hypoxia decreased cellular GSH content and appeared to decrease the effect of N-acetylcysteine on repletion of GSH after hypoxia. Hypoxia decreased 2 key enzyme activities that regulate GSH synthesis, glutamate cysteine ligase (GCL) (E.C. 6.3.2.2) and glutathione synthase (GS) (E.C. 6.3.2.3). No hypoxia-dependent change occurred in GCL or GS protein expression on Western blots. When epithelial cells were transfected with an adenoviral vector that caused over expression of human catalase protein (Ad.Cat or Ad.mCat), GCL and GS activities did not decrease in hypoxia. Inhibition of p38(MAPK) (using SB203580) or extracellular signal-regulated kinase (ERK; PD98059) prevented the hypoxia-dependent decrease in GCL and GS activity. To seek in vivo correlation, the authors assayed total glutathione in lungs and livers from MK2(-/-) (homozygous knockout) mice. MK2(-/-) mice are presumably unable to phosphorylate heat shock protein 27 (Hsp27) normally, because of absent kinase (MK2) activity. Liver GSH content (expressed per mg protein) was 20% less in MK2(-/-) mice than in nontransgenic Black 6 controls. Down-regulation of lung GSH content in hypoxia depends on peroxide tone of the cell and the p38(MAPK) system.
For mammals, oxygen sensing is fundamental to survive. An adequate response to reduced oxygen tension, herein termed hypoxia, requires an instantaneous adaptation of the respiratory and the circulatory systems. While the glomus caroticum as well as the pulmonary and systemic vasculature and potentially also the airway chemoreceptors enable a corresponding response within seconds, changes in gene expression require minutes to hours. Hypoxia-induced gene expression depends on the activation of several transcription factors. Hypoxia-inducible factor-1 (HIF-1) has been identified as the key transcription factor complex that coordinates gene expression during hypoxia. To understand how abundance and activation of HIF-1 is regulated is of fundamental importance as it may open new therapeutic avenues to treat ischemic diseases or cancer where HIF-1 appears to be a key component of the pathophysiology.
Hypoxia inducible factor 1 (HIF-1) regulates the transcription of vascular endothelial growth factor (VEGF), which plays important roles in angiogenesis. We investigated the redox effect of glutathione (GSH) on the hypoxic induction of HIF-1 in a human oral squamous cell carcinoma (HSC-2) cell line. The maximal induction of HIF-1 in HSC-2 cells was observed 30 h after hypoxia, and VEGF mRNA was expressed after 36 h under hypoxia. GSH ethyl ester (GSHee, a membrane permeable analog of GSH) and N-acetyl-L-cysteine (NAC, a membrane permeable precursor of GSH) reduced HIF-1 binding activity in a dose-dependent manner. Further, HIF-1 dependent promoter activity was similarly reduced by GSHee and NAC. However, ebselen, which increases glutathione peroxidase activity and oxidizes GSH, negated the effect of GSHee on HIF-1 dependent promoter activity. The inhibitory effect of GSHee and NAC on HIF-1 binding activity was reversed by bis (2-chlorethyl)-nitrosourea, an oxidized glutathione (GSSG) reductase inhibitor which increases the concentration of GSSG. GSSG methyl ester (GSSGme), a membrane permeable analog of GSSG, enhanced HIF-1 dependent promoter activity and exhibited a bell-shaped concentration-dependant activity curve. The increasing effect of GSSGme on HIF-1 induction was also observed under chemically-induced hypoxia obtained using cobalt chloride. These results suggest that changes in the intracellular GSSG/GSH ratio may regulate HIF-1 induction during hypoxia.
The blood-brain barrier (BBB), consisting of specialized endothelial cells surrounded by astrocytes and pericytes, plays a crucial role in brain homeostasis. Many cerebrovascular diseases are associated with BBB breakdown and oxygen (O(2)) deprivation constitutes a critical factor that onsets its disruption. We investigated the impact of astrocytes and pericytes on brain endothelial cell permeability and survival during different degrees of O(2) deprivation. Prolonged exposure to 1% O(2) caused barrier breakdown and exposure to 0.1% O(2) dramatically accelerated disruption and induced cell death, mediated at least in part via caspase-3 activation. Reoxygenation allowed only cells exposed to 1% O(2) to re-establish barrier function. Notably co-culture with astrocytes and pericytes substantially enhanced barrier function under normoxic conditions, and produced differential responses during O(2) deprivation. At 1% O(2) astrocytes partially maintained barrier integrity whereas pericytes accelerated its disruption in the short-term, having positive effects only after prolonged exposure. Unexpectedly, at 0.1% O(2) pericytes were more effective than astrocytes in preserving barrier function although the protection afforded by both cells involved inhibition of caspase-3 pathways. Furthermore, cell-specific regulation of auto- and paracrine VEGF signaling pathways were also in part responsible for the differential modulation of barrier function. Our data suggests that cellular cross-talk within the neurovascular unit is crucial for preservation of barrier integrity and that pericytes, not astrocytes, play a significant role during severe and prolonged O(2) deprivation.
Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII-related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen-coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the blood-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (gamma GTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel-related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed gamma GTP and ALP activities. Such structures, associated with high levels of gamma GTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood-brain barrier-related gamma GTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood-brain barrier.
The immortalized rat brain microvessel endothelial cell line RBE4 was used to investigate the in vitro regulation of two blood-brain barrier specific enzymes, gamma-glutamyl transpeptidase (GTP) and alkaline phosphatase (ALP). The effects of bFGF, astroglial factors, and retinoic acid (a cell differentiation agent) on GTP and ALP activities were separately or simultaneously studied in order to define optimal culture conditions for induction of these two specific enzymes of the blood-brain barrier. In the present study, a phenotypically distinct subpopulation of endothelial cells has been shown to develop from confluent cobblestone monolayers of RBE4 immortalized cerebral endothelial cells. These distinct cells were present within multicellular aggregates and specifically exhibited GTP and ALP activities. Addition of bFGF, astroglial factors, or retinoic acid induced the formation of these three-dimensional structures and in consequence an increase in GTP and ALP activities. For retinoic acid and astroglial factors, this increase could also be explained by the stimulation of either GTP or ALP expression in the phenotypically distinct positive cells associated with aggregates. Simultaneous treatment with retinoic acid and astroglial factors had a synergistic effect on GTP and ALP expression and thus may allow these distinct cells to evolve toward a more differentiated state. Since such results were also obtained with physiological concentrations of retinoic acid, we suggest that addition of this agent might contribute to greater differentiation of cells in in vitro blood-brain barrier models where endothelial cells are cocultured with astrocytes.
Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1. 5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (rho0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) rho0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in rho degrees cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.
Astrocytes (AC) induce blood-brain barrier (BBB) properties in brain endothelial cells (EC). As antioxidative activity (AOA) is assumed to be a BBB characteristic, we tested whether AC improve AOA of EC. Monocultivated AC showed higher AOA [manganese superoxide dismutase (SOD), catalase (Cat), glutathione peroxidase (GPx)] than EC. Cocultivation elevated AOA in EC (MnSOD, CuZnSOD, Cat, GPx), and AC (MnSOD, CuZnSOD, GPx). Hypoxia increased radical-induced membrane lipid peroxidation in monocultivated, but not in cocultivated EC. Thus, EC/AC cocultivation intensifies AOA in both cell types, protects the EC, and therefore, the BBB against oxidative stress. The high AOA is regarded as an essential property of the BBB, which is induced by AC.
Adaptation to oxidative and nitrosative stress occurs in cells first exposed to a nontoxic stress, resulting in the ability to tolerate a toxic challenge of the same or a related oxidant. Adaptation is observed in a wide variety of cells including endothelial cells on exposure to nitric oxide or oxidized lipids, and lung epithelial cells exposed to air-borne pollutants and toxicants. This acquired characteristic has been related to the regulation of a family of stress responding proteins including those that control the synthesis of the intracellular antioxidant glutathione. The focus of this article, which includes a review of recent results along with new data, is the regulation and signaling of glutathione biosynthesis, especially those relating to adaptive mechanisms. These concepts are illustrated with examples using nitric oxide and oxidized low density lipoprotein mediated adaptation to oxidative stress. These data are discussed in the context of other adaptive mechanisms relating to glutathione synthesis including those from dietary constituents such as curcumin.
Occludin and zonular occludens (ZO)-1 in tight junctions (TJs) and actin play an important role in maintaining blood-brain barrier (BBB) endothelial ion and solute barriers. Malfunction of BBB by reactive oxygen species (ROS) has been attributed to the disruption of TJs. This study examined H2O2 effects on changes of paracellular permeability, actin, and TJ proteins (occludin and ZO-1) using primary culture of bovine brain microvessel endothelial cells. The BBB permeability, measured as transendothelial electrical resistance (TER), decreased in a dose- and time-dependent manner when treated with H2O2. Cytotoxicity test revealed that H2O2 did not cause cell death at 0.01, 0.1, and 1.0 mM H2O2. H2O2 caused increased protein expression of occludin (1.17- to 1.29-fold) and actin (1.2- to 1.3-fold). ZO-1 maintained steady state levels of expression. H2O2 caused rearrangement of occludin and ZO-1 at tight junctions and formation of actin stress fiber. Although ZO-1 did not show significant change in protein expression, permeability changes shown in the current study correlate with alterations in expression and localization of occludin, actin, and ZO-1. These data suggest that H2O2 induces increased paracellular permeability of BBB that is accompanied with redistribution of occludin and ZO-1 and increased protein expression of occludin and actin.
We examined whether hypoxia alone could produce changes in the permeability of brain capillary endothelial cells (EC) and whether a stimulation of hypoxic status alters the gene expression of occludin and glucose transporter 1 (GLUT1). Exposure of EC to hypoxia resulted in increased permeability, with the greatest decrease in transendothelial electrical resistance (TER) at 40 h. Moreover, hypoxia alone induced the expression of both mRNA in EC. Furthermore, we found that interleukin-1 (IL-1)beta, glutamate, hydrogen peroxide (H2O2), and sodium nitroprusside (SNP) induced the expression of mRNA for occludin and GULT1 under normoxic condition. The decrease in TER due to hypoxia was inhibited on addition of an anti-IL1 antibody and nitric oxide synthase (NOS) inhibitor in EC. These results indicate that the expression of occludin and GLUT1 mRNA is sensitive to exposure to hypoxia and that the changes of permeability in EC are associated with IL-1beta and NO.
Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis in the mammalian eye, suggesting that loss of PEDF is implicated in the pathogenesis of proliferative diabetic retinopathy. However, a role for PEDF in early diabetic retinopathy remains to be elucidated. Since oxidative stress is thought to be involved in pericyte loss and dysfunction, one of the changes characteristic of early diabetic retinopathy, we investigated whether and how PEDF could protect cultured retinal pericyte against oxidative stress injury. High glucose (30 mM) increased intracellular reactive oxygen species (ROS) generation in pericytes, which was completely blocked by PEDF. High glucose or H2O2 was found to induce growth retardation and apoptotic cell death of pericytes. PEDF completely restored these cytopathic effects on pericytes. An increased ratio of bax to bcl-2 mRNA level with subsequent activation of caspase-3 was observed in high-glucose- or H2O2-exposed pericytes, which was also completely prevented by PEDF. PEDF significantly increased glutathione peroxidase (GPx) mRNA levels and activity in pericytes. Further, PEDF was found to completely inhibit high-glucose- or H2O2-induced increase in a mRNA ratio of angiopoietin-2 to angiopoietin-1 and up-regulation of VEGF mRNA levels in pericytes. PEDF mRNA levels themselves were down-regulated in high-glucose- or H2O2-exposed pericytes. These results demonstrate that PEDF protects against high-glucose- or H2O2-induced pericyte apoptosis and dysfunction through its anti-oxidative properties via GPx induction. Our present study suggests that substitution of PEDF proteins might be a promising therapeutic strategy for treatment of patients with early diabetic retinopathy.
Reduction in oxygenation induces inhibition of Na+/K+ ATPase in a number of cells and tissues, including hepatocytes. When not reversed, decrease in Na+/K+ pump activity leads to a gradual Na+ accumulation, cell swelling and death. However, when accompanied by suppression of dissipative cation pathways, it has also been shown to be a beneficial adaptive strategy used by some hypoxia-tolerant species to reduce ATP consumption during prolonged periods of anoxia. This study aims to investigate acute hypoxic responses of the Na+/K+ ATPase in primary cultures of trout hepatocytes. Gradual decrease in oxygenation was followed by an instantaneous transient dose-dependent downregulation of the Na+/K+ ATPase transport activity, but was without an effect on hydrolytic function of the enzyme. Hypoxia-induced inhibition of active K+ influx was reversed spontaneously when hypoxic incubation time exceeded 20 min. The stimulating effect of prolonged hypoxic exposure on the Na+/K+ pump is most probably secondary to hypoxia-induced activation of the Na+/H+ exchanger with the following Na+ accumulation leading to Na+/K+ ATPase activation. Hypoxia-induced inhibition of the Na+/K+ pump was not caused by ATP depletion or global oxidative stress. However, local controlled production of reactive oxygen species seems to play an important role in hypoxia-induced regulation of the Na+/K+ ATPase. Treatment of cells with mercaptopropionyl glycine (MPG), a scavenger of OH*-, abolished hypoxia-induced inhibition of the Na+/K+ ATPase. Earlier on we have shown that activation of Na+/H+ exchanger under hypoxic conditions can be opposed by MPG treatment as well. Taken together our results suggest that regulation of both oxygen-sensitive transporters may be accomplished by local changes in free radical production.
According to the free radical theory, aging can be considered as a progressive, inevitable process partially related to the accumulation of oxidative damage into biomolecules -- nucleic acids, lipids, proteins or carbohydrates -- due to an imbalance between prooxidants and antioxidants in favor of the former. More recently also the pathogenesis of several diseases has been linked to a condition of oxidative stress. In this review we focus our attention on the evidence of oxidative stress in aging brain, some of the most important neurodegenerative diseases -- Alzheimer's disease (AD), mild cognitive impairment (MCI), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD) -- and in two common and highly disabling vascular pathologies--stroke and cardiac failure. Particular attention will be given to the current knowledge about the biomarkers of oxidative stress that can be possibly used to monitor their severity and outcome.
Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K(+) influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an "optimal redox potential range" to higher or lower levels cause suppression of the Na-K pump activity.
Vascular endothelial growth factor (VEGF), a potent mediator of endothelial proliferation and migration, has an important role also in brain edema formation during hypoxia and ischemia. VEGF binds to the tyrosine kinase receptors Flt-1 and Flk-1. Yet, their relative importance for hypoxia-induced hyperpermeability is not well understood. We used an in vitro blood-brain barrier (BBB) model consisting of porcine brain microvascular endothelial cells (BMEC) to determine the role of Flt-1 in VEGF-induced endothelial cell (EC) barrier dysfunction. Soluble Flt-1 abolished hypoxia/VEGF-induced hyperpermeability. Furthermore, selective antisense oligonucleotides to Flt-1, but not to Flk-1, inhibited hypoxia-induced permeability changes. Consistent with these data, addition of the receptor-specific homolog placenta-derived growth factor, which binds Flt-1 but not Flk-1, increased endothelial permeability to the same extent as VEGF, whereas adding VEGF-E, a viral VEGF molecule from the orf virus family activating Flk-1 and neuropilin-1, but not Flt-1, did not show any effect. Using the carcinoma submandibular gland cell line (CSG), only expressing Flt-1, it was demonstrated that activation of Flt-1 is sufficient to induce hyperpermeability by hypoxia and VEGF. Hyperpermeability, induced by hypoxia/VEGF, depends on activation of phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), nitric oxide synthase (NOS) and protein kinase G (PKG). The activation of the PI3-K/Akt pathway by hypoxia was confirmed using an in vivo mice hypoxia model. These results demonstrate that hypoxia/VEGF-induced hyperpermeability can be mediated by activation of Flt-1 independently on the presence of Flk-1 and indicate a central role for activation of the PI3-K/Akt pathway, followed by induction of NOS and PKG activity.
The antineoplastic drug paclitaxel is known to block cells in the G2/M phase of the cell cycle through stabilization of microtubules. The development of paclitaxel resistance in tumors is one of the most significant obstacles to successful therapy. Vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) are important regulators of neovascularization. HIF-1 regulates VEGF expression at the transcriptional level. Here, we investigated whether paclitaxel treatment affects VEGF expression for the development of paclitaxel resistance. Paclitaxel treatment induced dose-dependent cell death and increased VEGF expression. Paclitaxel also induced nuclear factor-kappaB activation and stabilized HIF-1alpha, which stimulated luciferase activity of HIF-1alpha response element on VEGF gene. As paclitaxel treatment produced reactive oxygen species (ROS), VEGF expression was increased by H2O2 treatment and reduced by various ROS scavengers such as N-acetyl-L-cysteine, pyrrolidine dithiocarbamate and diphenylene iodonium. Paclitaxel-induced cell death was aggravated by incubation with those ROS scavengers. Collectively, this suggests that paclitaxel-induced VEGF expression could be mediated by paclitaxel-induced ROS production through nuclear factor-kappaB activation and HIF-1alpha stabilization, which could affect resistance induction to antitumor therapeutics during cancer treatment.
Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation
- J J Lochhead
Lochhead, J. J., et al., 2010. Oxidative stress increases blood-brain barrier permeability
and induces alterations in occludin during hypoxia-reoxygenation. J Cereb Blood
Flow Metab. 30, 1625-36.