Search

With the recent advances in medicine, human life expectancy is increasing; however, the extra years of life are not necessarily spent in good health or free from disability, resulting in a significantly higher incidence of age-associated pathologies. Among these disorders, neurodegenerative diseases have a significant impact. To this end, the presence of the protective blood-brain barrier (BBB) represents a formidable obstacle to the delivery of therapeutics. Thus, this makes it imperative to define strategies to bypass the BBB in order to successfully target the brain with the appropriate drugs. It has been demonstrated that targeting the BBB by ultrasound (US) can transiently make this anatomical barrier permeable and in so doing, allow the delivery of therapeutics. Thus, our aim was to carry out an in depth in vitro molecular and morphological study on the effects of US treatment on the BBB. The rat brain endothelial (RBE4) cell line was challenged with exposure to 12 MHz diagnostic US treatment for 10, 20, and 30 min. Cell viability assays, Western blotting analysis on the endoplasmic reticulum (ER), and oxidative stress marker evaluation were then performed, along with cytological and immunofluorescence staining, in order to evaluate the effects of US on the intercellular spaces and tight junction distribution of the brain endothelial cells. We observed that the US treatment exerted no toxic effects on either RBE4 cell viability or the upregulation/dislocation of the ER and oxidative stress marker (GRP78 and cytochrome C, respectively). Further, we observed that the application of US induced an increase in the intercellular spaces, as shown by Papanicolaou staining, mainly due to the altered distribution of the tight junction protein zonula occludens -1 (ZO-1). This latter US-dependent effect was transient and disappeared 20 min after the removal of the stimulus. In conclusion, our results show that US induces a transient alteration of the BBB, without altering the intracellular signaling pathways such as the ER and oxidative stress that could potentially be toxic for endothelial cells. These results suggested that US treatment could represent a potential strategy for improving drug delivery to the brain.

In recent years, alcohol abuse has dramatically grown with deleterious consequence for people's health and, in turn, for health care costs. It has been demonstrated, in humans and animals, that alcohol intoxication induces neuroinflammation and neurodegeneration thus leading to brain impairments. Furthermore, it has been shown that alcohol consumption is able to impair the blood-brain barrier (BBB), but the molecular mechanisms underlining this detrimental effect have not been fully elucidated. For this reason, in this study we investigated the effects of alcohol exposure on a rat brain endothelial (RBE4) cell line, as an in vitro-validated model of brain microvascular endothelial cells. To assess whether alcohol caused a concentration-related response, the cells were treated at different times with increasing concentrations (10-1713 mM) of ethyl alcohol (EtOH). Microscopic and molecular techniques, such as cell viability assay, immunofluorescence and Western blotting, were used to examine the mechanisms involved in alcohol-induced brain endothelial cell alterations including tight junction distribution, apoptosis, and reactive oxygen species production. Our findings clearly demonstrate that alcohol causes the formation of gaps between cells by tight junction disassembly, triggered by the endoplasmic reticulum and oxidative stress, highlighted by GRP78 chaperone upregulation and increase in reactive oxygen species production, respectively. The results from this study shed light on the mechanisms underlying alcohol-induced blood-brain barrier dysfunction and a better understanding of these processes will allow us to take advantage of developing new therapeutic strategies in order to prevent the deleterious effects of alcohol.

Cadmium (Cd) is a highly toxic environmental pollutant released from the smelting and refining of metals and cigarette smoking. Oral exposure to cadmium may result in adverse effects on a number of tissues, including the central nervous system (CNS). In fact, its toxicity has been related to neurological disorders, as well as neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Under normal conditions, Cd barely reaches the brain in adults because of the presence of the blood-brain barrier (BBB); however, it has been demonstrated that Cd-dependent BBB alteration contributes to pathogenesis of neurodegeneration. However, the mechanism underlying Cd-dependent BBB alteration remain obscure. Here, we investigated the signaling pathway of Cd-induced tight junction (TJ), F-actin, and vimentin protein disassembly in a rat brain endothelial cell line (RBE4). RBE4 cells treated with 10 μM cadmium chloride (CdCl 2 ) showed a dose- and time-dependent significant increase in reactive oxygen species (ROS) production. This phenomenon was coincident with the alteration of the TJ zonula occludens-1 (ZO-1), F-actin, and vimentin proteins. The Cd-dependent ROS increase elicited the upregulation of GRP78 expression levels, a chaperone involved in endoplasmic reticulum (ER) stress that induces caspase-3 activation. Further signal profiling by the pannexin-1 (PANX1) specific inhibitor 10 Panx revealed a PANX1-independent increase in ATP spillage in Cd-treated endothelial cells. Our results point out that a ROS-dependent ER stress-mediated signaling pathway involving caspase-3 activation and ATP release is behind the BBB morphological alterations induced by Cd.

Cadmium (Cd) is a well-known occupational and environmental pollutant worldwide, and its toxicity is widely recognised. Cd is reported to increase the permeability of the blood–brain barrier (BBB) and to penetrate and accumulate in the brain. Although many lines of evidence show that Cd toxicity is induced by different mechanisms, one of the best known is the Cd-dependent production of reactive oxygen species (ROS). Zinc is a trace element known as coenzyme and cofactor for many antioxidant proteins, such as metallothioneins and superoxide dismutase enzymes. To date, very little is known about the role of Zn in preventing Cd-induced blood–brain barrier (BBB) alterations. The goal of this study was to test the Zn antioxidant capacity against Cd-dependent alterations in a rat brain endothelial cell line (RBE4), as an in vitro model for BBB. In order to mimic acute Cd poisoning, RBE4 cells were treated with CdCl2 30 µM for 24 h. The protective role of ZnCl2 (50 µM) was revealed by evaluating the cell viability, reactive oxygen species (ROS) quantification, cytochrome C distribution, and the superoxide dismutase (SOD) protein activity. Additionally, the effectiveness of Zn in counteracting the Cd-induced damage was investigated by evaluating the expression levels of proteins already known to be involved in the Cd signalling pathway, such as GRP78 (an endoplasmic reticulum (ER) stress protein), caspase3 pro- and cleaved forms, and BAX. Finally, we evaluated if Zn was able to attenuate the alterations of zonula occludens-1 (ZO-1), one of the tight-junction (TJ) proteins involved in the formation of the BBB. Our data clearly demonstrate that Zn, by protecting from the SOD activity impairment induced by Cd, is able to prevent the triggering of the Cd-dependent signalling pathway that leads to ZO-1 dislocation and downregulation, and BBB damage. [ABSTRACT FROM AUTHOR]

The article focuses on studying the blood-brain barrier (BBB) and the crucial role of astrocytes in its formation and function, highlighting their influence on the expression and correct distribution of tight junction proteins like claudin 5 in brain endothelial cells.

The article offers information on morphological features of rat brain vascular pericytes (RBVP) during toxic insult and their role in blood-brain barrier characterization. Topics include information on pericytes which are fibroblast like cells; information on extensive cytoplasmic elongation; and different cell types of central nervous system.

C Cadmium is a toxic heavy metal known as environmental pollutant world-wide distributed. Its toxicity, predominantly due to its long half-life of 15-20 years, affects many organs and tissues including kidney, lung, liver, heart, bone, muscles and the central nervous system (CNS) as well [1]. Our research group previously demonstrated that cadmium is able to alter the blood-brain barrier by modifying the tight junction sealing morphology and to induce oxidative stress, thus leading to neuronal degeneration mainly by oxidative stress [2]. Among the cellular population of the CNS, glial cells own a pivotal role in maintaining the physiological brain homeostasis and their alteration has been demonstrated to indirectly affect neurons [3]. The aim of the present research is to evaluate cadmium toxicity in two glial cell lines (astrocyte DITNC1 and microglia BV-2), at different time and increasing concentration of metal exposure. Cadmium increasing concentrations (0.1-100 µM) were administered in starvation medium for 8, 24 and 48 hours. Cell viability assay, western blotting analysis, and immunofluorescent staining were employed in order to evaluate the molecular and morphological alterations induced by the heavy metal. Our results clearly demonstrated that Cd is able to affect the cell viability even at the lowest concentrations and in a time-dependent manner, by altering the oxidative balance (ROS production, cytochrome C expression) and inducing an endoplasmic reticulum stress (GRP78 upregulation). Moreover, Cd is able to induce glial morphological alterations as well as an overexpression of activation glial markers, such as GFAP, S100ß, CD86, and Iba1. In conclusion, the data obtained clearly demonstrated that Cd toxicity occurs even at low doses and for very short exposure times Further experiments should be performed in order to evaluate the mechanisms underlying the Cd-dependent activation of glial cells and how this, in turn, induces a neuronal homeostasis alteration. [ABSTRACT FROM AUTHOR]

Cadmium (Cd), a category I human carcinogen, is a well-known widespread environmental pollutant. Chronic Cd exposure affects different organs and tissues, such as the central nervous system (CNS), and its deleterious effects can be linked to indirect reactive oxygen species (ROS) generation. Since Cd is predominantly present in +2 oxidation state, it can interplay with a plethora of channels and transporters in the cell membrane surface in order to enter the cells. Mitochondrial dysfunction, ROS production, glutathione depletion and lipid peroxidation are reviewed in order to better characterize the Cd-elicited molecular pathways. Furthermore, Cd effects on different CNS cell types have been highlighted to better elucidate its role in neurodegenerative disorders. Indeed, Cd can increase blood–brain barrier (BBB) permeability and promotes Cd entry that, in turn, stimulates pericytes in maintaining the BBB open. Once inside the CNS, Cd acts on glial cells (astrocytes, microglia, oligodendrocytes) triggering a pro-inflammatory cascade that accounts for the Cd deleterious effects and neurons inducing the destruction of synaptic branches. [ABSTRACT FROM AUTHOR]

Degenerative diseases of the central nervous system are significant causes of mortality among aging population in industrialized countries, as well as expensive for the national health systems and caregivers [1]. Nowadays, pharmacological therapies are still not decisive because they are hindered by the blood brain barrier (BBB) [2]. Although in the last decades many researchers have conducted in vivo experiments to better identify a therapeutic approach of ultrasound (US) in neurodegenerative diseases, little is known about their role in increasing the BBB permeability [3,4]. The present study aims to identify, the effect of focused US on a rat brain endothelial cell line (RBE4). After ultrasound stimulation (10-20-30 min.), MTT and western blotting assay were conducted to demonstrate the non-toxic effects of US. Furthermore, immunostaining of stimulated cells was performed to detect changes in cytoskeletal F-actin fibers and Zonula occludens-1 (ZO1) tight juntion. BBB opening was evaluated by measuring the extent of the intercellular space, in Papanicolaou stained cells. The results evidenced an ultrasonic-dependent mechanical action on F-actin fibers that altered their distribution within the cells showing the formation of numerous stress fibers. F-actin alterations were accompanied by an alteration of the ZO-1 distribution, exhibiting a "zipper-like" staining pattern and holes that became visible between cells. Papanicolaou staining confirm the opening of the BBB evidencing many wider areas of intercellular space. All these structural changes on RBE4 cell line occurred without significant alterations in metabolic activity as well as in absence of apoptotic or endoplasmic reticulum stress markers. In conclusion, these results confirm and highlight the potential role of ultrasound in the permeabilization of the BBB, thus suggesting new ways for drugs administration. [ABSTRACT FROM AUTHOR]