Dora Brites

Looking at the blood–brain barrier: Molecular anatomy and possible investigation approaches

The blood-brain barrier (BBB) is a dynamic and complex interface between blood and the central nervous system that strictly controls the exchanges between the blood and brain compartments, therefore playing a key role in brain homeostasis and providing protection against many toxic compounds and pathogens. In this review, the unique properties of brain microvascular endothelial cells and intercellular junctions are examined. The specific interactions between endothelial cells and basement membrane as well as neighboring perivascular pericytes, glial cells and neurons, which altogether constitute the neurovascular unit and play an essential role in both health and function of the central nervous system, are also explored. Some relevant pathways across the endothelium, as well as mechanisms involved in the regulation of BBB permeability, and the emerging role of the BBB as a signaling interface are addressed as well. Furthermore, we summarize some of the experimental approaches that can be used to monitor BBB properties and function in a variety of conditions and have allowed recent advances in BBB knowledge. Elucidation of the molecular anatomy and dynamics of the BBB is an essential step for the development of new strategies directed to maintain or restore BBB integrity and barrier function and ultimately preserve the delicate interstitial brain environment.

Neurovascular Unit: a Focus on Pericytes

The blood–brain barrier (BBB) is a highly specialized system that controls the exchanges between the blood and the central nervous system (CNS). This barrier shields the CNS from toxic substances in the blood and provides nutrients to CNS, thus playing an essential role in the maintenance of homeostasis. The anatomical basis of the BBB is formed by the endothelial cells of brain microvasculature, with elaborated tight and adherens junctions, which together with pericytes, the basement membrane, and astrocytes, as well as neurons, microglia and oligodendrocytes form the neurovascular unit. The interaction between all these components guarantees a proper environment for neural function and a restricted permeability and transport. Pericytes were initially reported by Rouget in 1873 and since then they have been recognized as an important component of the BBB, despite the difficulty of their identification. Diverse functions have been assigned to pericytes, including a role in BBB properties, hemostasis, and angiogenesis, as well as a contractile, immune, and phagocytic function. These cells are also seen like multipotent cells and so with a great potential for therapy. Here, we review the neurovascular unit composition and the interplay between the diverse components, addressing pericytes with a particular detail.

Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier

We describe a method for generating primary cultures of human brain microvascular endothelial cells (HBMVECs). HBMVECs are derived from microvessels isolated from temporal tissue removed during operative treatment of epilepsy. The tissue is mechanically fragmented and size filtered using polyester meshes. The resulting microvessel fragments are placed onto type I collagen-coated flasks to allow HBMVECs to migrate and proliferate. The overall process takes less than 3 h and does not require specialized equipment or enzymatic processes. HBMVECs are typically cultured for approximately 1 month until confluent. Cultures are highly pure (∼97% endothelial cells; ∼3% pericytes), are reproducible, and show characteristic brain endothelial markers (von Willebrand factor, glucose transporter-1) and robust expression of tight and adherens junction proteins as well as caveolin-1 and efflux protein P-glycoprotein. Monolayers of HBMVECs show characteristically high transendothelial electric resistance and have proven useful in multiple functional studies for in vitro modeling of the human blood-brain barrier.

Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxycholic acid but prevented by ursodeoxycholic acid.

BACKGROUND/AIMS Unconjugated bilirubin (UCB) can be neurotoxic in jaundiced neonates and in patients with Crigler-Najjar syndrome. UCB toxicity may culminate in cell death, however, the occurrence of apoptosis has never been investigated. Ursodeoxycholic acid (UDCA) is a strong modulator of the apoptotic threshold in both hepatic and nonhepatic cells. The aims of this study were to determine whether apoptosis plays a role in neural cell death induced by UCB, and to investigate the ability of UDCA to prevent cell death. METHODS Cultured rat astrocytes were incubated with UCB (17 and 86 microM) plus albumin (5.7 and 28.7 microM) for 4-22 h. In addition, astrocytes and neurones were treated with either UCB, 50 microM UDCA, or their combination for 4 h. Cultures were scored for nonviable cells by trypan blue dye exclusion. Apoptosis was assessed by Hoechst staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling assay. RESULTS UCB induced a concentration- and time-dependent decrease in astrocyte viability. Apoptosis was 4- and 7-fold increased after 4 h exposure to 17 and 86 microM UCB, respectively (P < 0.01). UDCA reduced apoptosis to <7%, which represents a appoximately 60% protection (P < 0.01). Cholic acid was not protective, and chenodeoxyholic acid aggravated UCB toxicity (P < 0.05). Finally, neurones showed a 1.5-fold greater sensitivity than astrocytes to UCB, while UDCA was still protective. CONCLUSIONS UCB is toxic to both astrocytes and neurones, causing cell death through an apoptotic process. Moreover, UDCA inhibits UCB-induced apoptosis in neural cells and this could not be mimicked by other bile acids.

Inflammatory signalling pathways involved in astroglial activation by unconjugated bilirubin

During neonatal hyperbilirubinaemia, astrocytes activated by unconjugated bilirubin (UCB) may contibute to brain toxicity through the production of cytokines. As a first step in addressing the signal transduction cascades involved in the UCB‐induced astroglial immunological response, we tested whether tumour necrosis factor (TNF)‐α receptor 1 (TNFR1), mitogen‐activated protein kinase (MAPK) and nuclear factor κB (NF‐κB) would be activated in astrocytes exposed to UCB, and examined the profile of cytokine production. Astrocyte cultures stimulated with UCB showed a rapid rise in TNFR1 protein levels, followed by activation of the MAPKs p38, Jun N‐terminal kinase1/2 and extracellular signal‐regulated kinase1/2, and NF‐κB. Interestingly, the induction of these signal effectors preceded the early up‐regulation of TNF‐α and interleukin (IL)‐1β mRNAs, and later secretion of TNF‐α, IL‐1β and IL‐6. Treatment of astrocytes with UCB also induced cell death, with levels comparable to those obtained after exposure of astrocytes to recombinant TNF‐α and IL‐1β. Moreover, loss of cell viability and cytokine secretion were reduced when the NF‐κB signal transduction pathway was inhibited, suggesting a key role for NF‐κB in the astroglial response to UCB. These results demonstrate the complexity of the molecular mechanisms involved in cell injury by UCB during hyperbilirubinaemia and provide a basis for the development of novel therapeutic strategies.

Beneficial effect of ursodeoxycholic acid on alterations induced by cholestasis of pregnancy in bile acid transport across the human placenta

BACKGROUND/AIMS The existence of impairment in bile acid transport across the placenta during intrahepatic cholestasis of pregnancy and the effect of ursodeoxycholic acid treatment (1 g/day) were investigated. METHODS Kinetic parameters were calculated from experiments carried out on membrane vesicles obtained from basal (TPMb, fetal-facing) and apical (TPMa, maternal-facing) trophoblast plasma membranes. Bile acid uptake was measured using varying concentrations of [14C]-glycocholate and a rapid filtration technique. RESULTS The maximal velocity of transport (Vmax), the apparent affinity constant (Kt) and the efficiency (Ef) of transport (Vmax/Kt) of the anion:bile acid exchanger located at the TPMb were reduced in intrahepatic cholestasis of pregnancy. Ursodeoxycholic acid induced a reversal of Vmax, Kt and Ef to normal values. Owing to the 3-fold increase in Vmax, with no change in Kt, intrahepatic cholestasis of pregnancy induced an enhancement in Ef of ATP-independent bile acid transport across TPMa. Both Vmax and Ef were restored to normal values by ursodeoxycholic acid. Finally, in ATP-dependent bile acid transport across TPMa, a reduction in the Ef due to an increase in Vmax together with a more pronounced increase in Kt was found. This impairment was also reversed by ursodeoxycholic acid. CONCLUSIONS These results suggest that placenta bile acid transport systems are impaired in intrahepatic cholestasis of pregnancy. Moreover, together with the confirmed beneficial effect for intrahepatic cholestasis of pregnancy patients, such as the relief of pruritus and the improvement in biochemical markers of cholestasis, ursodeoxycholic acid treatment restores the ability of the placenta to carry out vectorial bile acid transfer.

Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin

High levels of unconjugated bilirubin (UCB) can be neurotoxic. Nevertheless, the mechanism of UCB interaction with neural cells is still unknown. This study investigates whether cultured rat neurons and astrocytes respond differently to UCB exposure. UCB toxicity was evaluated by lactate dehydrogenase release, induction of apoptosis, cytoskeleton degeneration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, and glutamate uptake. Primary cultures of rat brain astrocytes and neurons were incubated at 37°C with 85.5 μM UCB plus 28.5 μM albumin for 4 h. In assays of glutamate uptake, cells were exposed to 80–120 μM UCB plus 100 μM albumin for 15 min. The results showed that after incubation with 85.5 μM UCB, lactate dehydrogenase release was greater in neurons than in astrocytes (38%versus 14%, p < 0.05). Also, levels of apoptosis were markedly enhanced in neurons (29%versus 19%, p < 0.01). In accordance, neuronal cytoskeleton disassembly was evident during incubation with 85.5 μM UCB, whereas equivalent effects on astrocytes required as much as 171 μM. Conversely, inhibition of MTT metabolism and glutamate uptake by UCB was more pronounced in astrocytes than in neurons (74%versus 60%, p < 0.05 and 41% to 56%versus 25% to 33%, p < 0.05, respectively). In conclusion, the study demonstrates that astrocytes are more susceptible to inhibition of glutamate uptake and MTT reduction by UCB, whereas neurons are more sensitive to cell death by necrosis or apoptosis. These results suggest that UCB is toxic to both astrocytes and neurons, although through distinct pathways.

Correction of maternal serum bile acid profile during ursodeoxycholic acid therapy in cholestasis of pregnancy

BACKGROUND/AIMS Intrahepatic cholestasis of pregnancy is characterized by pruritus and increased levels of serum bile acids, and is often associated with premature delivery, fetal distress, and perinatal mortality. The aims of the present study were: (i) to better define the serum bile acid profile in intrahepatic cholestasis of pregnancy and its potential usefulness for differential diagnosis; (ii) to investigate the effect of ursodeoxycholic acid treatment on the bile acid pool; and (iii) to investigate possible adverse effects of therapy. METHODS Fifteen patients with intrahepatic cholestasis of pregnancy were enrolled in this study. Ursodeoxycholic acid (14 mg/kg body weight per day) was administered for 13 +/- 5 days. Twenty normal pregnant women served as controls. Serum bile acid profile was analyzed by high-performance liquid chromatography. RESULTS Patients with cholestasis of pregnancy showed significant alterations in the proportion of primary bile acids, with an increase in cholic acid (64.0 +/- 3.0% vs. 32.2 +/- 1.8%, p < 0.01), and a decrease in chenodeoxycholic acid (20.8 +/- 1.4% vs. 31.9 +/- 1.3%, p < 0.01), as compared to controls, resulting in a marked elevation in the cholic/chenodeoxycholic acid ratio (3.4 +/- 0.5 vs. 1.1 +/- 0.1, p < 0.01). The glycine/taurine ratio was reduced in cholestasis of pregnancy (0.8 +/- 0.1 vs. 1.4 +/- 0.1, p < 0.01). During ursodeoxycholic acid administration its proportion in serum increased from 1.4 +/- 0.6% (0.6 +/- 0.2 micromol/l) at baseline to 24.7 +/- 2.3% (5.9 +/- 1.9 micromol/l) with therapy (p < 0.01). This increment was accompanied by a significant decrease in the percentage of cholic acid (28.2 +/- 2.6%, p < 0.01) and an elevation in chenodeoxycholic acid proportion (25.0 +/- 1.9%, N.S.). Although lithocholic acid concentration in serum was maintained with treatment (1.2 +/- 0.2 micromol/l vs. 1.7 +/- 0.5 micromol/l), there was a significant increase in lithocholic acid proportion (p < 0.01) from 3.3 +/- 0.5% at baseline to 7.4 +/- 1.3% during therapy. The glycine/taurine ratio of serum bile acid pool returned to normal after ursodeoxycholic acid administration (1.7 +/- 0.3). CONCLUSIONS These results establish the importance of ursodeoxycholic acid treatment for the correction of maternal serum bile acid profile in cholestasis of pregnancy, indicating that ursodeoxycholic acid may improve fetal prognosis.

Microglia centered pathogenesis in ALS: insights in cell interconnectivity

Amyotrophic lateral sclerosis (ALS) is the most common and most aggressive form of adult motor neuron (MN) degeneration. The cause of the disease is still unknown, but some protein mutations have been linked to the pathological process. Loss of upper and lower MNs results in progressive muscle paralysis and ultimately death due to respiratory failure. Although initially thought to derive from the selective loss of MNs, the pathogenic concept of non-cell-autonomous disease has come to the forefront for the contribution of glial cells in ALS, in particular microglia. Recent studies suggest that microglia may have a protective effect on MN in an early stage. Conversely, activated microglia contribute and enhance MN death by secreting neurotoxic factors, and impaired microglial function at the end-stage may instead accelerate disease progression. However, the nature of microglial–neuronal interactions that lead to MN degeneration remains elusive. We review the contribution of the neurodegenerative network in ALS pathology, with a special focus on each glial cell type from data obtained in the transgenic SOD1G93A rodents, the most widely used model. We further discuss the diverse roles of neuroinflammation and microglia phenotypes in the modulation of ALS pathology. We provide information on the processes associated with dysfunctional cell–cell communication and summarize findings on pathological cross-talk between neurons and astroglia, and neurons and microglia, as well as on the spread of pathogenic factors. We also highlight the relevance of neurovascular disruption and exosome trafficking to ALS pathology. The harmful and beneficial influences of NG2 cells, oligodendrocytes and Schwann cells will be discussed as well. Insights into the complex intercellular perturbations underlying ALS, including target identification, will enhance our efforts to develop effective therapeutic approaches for preventing or reversing symptomatic progression of this devastating disease.

Neuroinflammation and Depression: Microglia Activation, Extracellular Microvesicles and microRNA Dysregulation

Patients with chronic inflammation are often associated with the emergence of depression symptoms, while diagnosed depressed patients show increased levels of circulating cytokines. Further studies revealed the activation of the brain immune cell microglia in depressed patients with a greater magnitude in individuals that committed suicide, indicating a crucial role for neuroinflammation in depression brain pathogenesis. Rapid advances in the understanding of microglial and astrocytic neurobiology were obtained in the past 15–20 years. Indeed, recent data reveal that microglia play an important role in managing neuronal cell death, neurogenesis, and synaptic interactions, besides their involvement in immune-response generating cytokines. The communication between microglia and neurons is essential to synchronize these diverse functions with brain activity. Evidence is accumulating that secreted extracellular vesicles (EVs), comprising ectosomes and exosomes with a size ranging from 0.1–1 μm, are key players in intercellular signaling. These EVs may carry specific proteins, mRNAs and microRNAs (miRNAs). Transfer of exosomes to neurons was shown to be mediated by oligodendrocytes, microglia and astrocytes that may either be supportive to neurons, or instead disseminate the disease. Interestingly, several recent reports have identified changes in miRNAs in depressed patients, which target not only crucial pathways associated with synaptic plasticity, learning and memory but also the production of neurotrophic factors and immune cell modulation. In this article, we discuss the role of neuroinflammation in the emergence of depression, namely dynamic alterations in the status of microglia response to stimulation, and how their activation phenotypes may have an etiological role in neurodegeneneration, in particular in depressive-like behavior. We will overview the involvement of miRNAs, exosomes, ectosomes and microglia in regulating critical pathways associated with depression and how they may contribute to other brain disorders including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD) and Parkinson’s disease (PD), which share several neuroinflammatory-associated processes. Specific reference will be made to EVs as potential biomarkers and disease monitoring approaches, focusing on their potentialities as drug delivery vehicles, and on putative therapeutic strategies using autologous exosome-based delivery systems to treat neurodegenerative and psychiatric disorders.