Concept: Blood-brain barrier
The glymphatic system is a recently defined brain-wide paravascular pathway for cerebrospinal fluid (CSF) and interstitial fluid (ISF) exchange that facilitates efficient clearance of solutes and waste from the brain. CSF enters the brain along para-arterial channels to exchange with ISF, which is in turn cleared from the brain along para-venous pathways. Because soluble amyloid β clearance depends on glymphatic pathway function, we proposed that failure of this clearance system contributes to amyloid plaque deposition and Alzheimer’s disease progression. Here we provide proof of concept that glymphatic pathway function can be measured using a clinically relevant imaging technique. Dynamic contrast-enhanced MRI was used to visualize CSF-ISF exchange across the rat brain following intrathecal paramagnetic contrast agent administration. Key features of glymphatic pathway function were confirmed, including visualization of para-arterial CSF influx and molecular size-dependent CSF-ISF exchange. Whole-brain imaging allowed the identification of two key influx nodes at the pituitary and pineal gland recesses, while dynamic MRI permitted the definition of simple kinetic parameters to characterize glymphatic CSF-ISF exchange and solute clearance from the brain. We propose that this MRI approach may provide the basis for a wholly new strategy to evaluate Alzheimer’s disease susceptibility and progression in the live human brain.
Astrocytes are the most populous glial subtype and are critical for brain function. Despite this, historically there have been few studies into the role that they may have in neurodegenerative diseases, such as Parkinson’s disease (PD). Recently, however, several studies have determined that genes known to have a causative role in the development of PD are expressed in astrocytes and have important roles in astrocyte function. Here, we review these recent developments and discuss their impact on our understanding of the pathophysiology of PD, and the implications that this might have for its treatment.
The blood-brain barrier (BBB) is composed of four cell populations, brain endothelial cells (BECs), pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs) into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs) were endowed with features consistent with BECs, including a high expression of nutrient transporters (CAT3, MFSD2A) and efflux transporters (ABCA1, BCRP, PGP, MRP5), and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development.
The acknowledgement of risks for traumatic brain injury in American football players has prompted studies for sideline concussion diagnosis and testing for neurological deficits. While concussions are recognized etiological factors for a spectrum of neurological sequelae, the consequences of sub-concussive events are unclear. We tested the hypothesis that blood-brain barrier disruption (BBBD) and the accompanying surge of the astrocytic protein S100B in blood may cause an immune response associated with production of auto-antibodies. We also wished to determine whether these events result in disrupted white matter on diffusion tensor imaging (DT) scans. Players from three college football teams were enrolled (total of 67 volunteers). None of the players experienced a concussion. Blood samples were collected before and after games (n = 57); the number of head hits in all players was monitored by movie review and post-game interviews. S100B serum levels and auto-antibodies against S100B were measured and correlated by direct and reverse immunoassays (n = 15 players; 5 games). A subset of players underwent DTI scans pre- and post-season and after a 6-month interval (n = 10). Cognitive and functional assessments were also performed. After a game, transient BBB damage measured by serum S100B was detected only in players experiencing the greatest number of sub-concussive head hits. Elevated levels of auto-antibodies against S100B were elevated only after repeated sub-concussive events characterized by BBBD. Serum levels of S100B auto-antibodies also predicted persistence of MRI-DTI abnormalities which in turn correlated with cognitive changes. Even in the absence of concussion, football players may experience repeated BBBD and serum surges of the potential auto-antigen S100B. The correlation of serum S100B, auto-antibodies and DTI changes support a link between repeated BBBD and future risk for cognitive changes.
The blood-brain barrier is essential for the proper homeostasis and function of the CNS, but its mechanism of function is poorly understood. Perivascular cells surrounding brain blood vessels are thought to be important for blood-brain barrier establishment, but their roles are not well defined. Here, we describe a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and scavenger behavior, these cells appear to be the zebrafish equivalent of cells variably characterized as Fluorescent Granular Perithelial cells (FGPs), perivascular macrophages, or ‘Mato Cells’ in mammals. Despite their macrophage-like morphology and perivascular location, zebrafish FGPs appear molecularly most similar to lymphatic endothelium, and our imaging studies suggest that these cells emerge by transdifferentiation from endothelium of the optic choroidal vascular plexus. Our findings provide the first report of a perivascular cell population in the brain derived from vascular endothelium.
Culture-based blood-brain barrier (BBB) models are crucial tools to enable rapid screening of brain-penetrating drugs. However, reproducibility of in vitro barrier properties and permeability remain as major challenges. Here, we report that self-assembling multicellular BBB spheroids display reproducible BBB features and functions. The spheroid core is comprised mainly of astrocytes, while brain endothelial cells and pericytes encase the surface, acting as a barrier that regulates transport of molecules. The spheroid surface exhibits high expression of tight junction proteins, VEGF-dependent permeability, efflux pump activity and receptor-mediated transcytosis of angiopep-2. In contrast, the transwell co-culture system displays comparatively low levels of BBB regulatory proteins, and is unable to discriminate between the transport of angiopep-2 and a control peptide. Finally, we have utilized the BBB spheroids to screen and identify BBB-penetrant cell-penetrating peptides (CPPs). This robust in vitro BBB model could serve as a valuable next-generation platform for expediting the development of CNS therapeutics.
Although blood-based liquid biopsies have emerged as a promising non-invasive method to detect biomarkers in various cancers, limited progress has been made for brain tumors. One major obstacle is the blood-brain barrier (BBB), which hinders efficient passage of tumor biomarkers into the peripheral circulation. The objective of this study was to determine whether FUS in combination with microbubbles can enhance the release of biomarkers from the brain tumor to the blood circulation. Two glioblastoma tumor models (U87 and GL261), developed by intracranial injection of respective enhanced green fluorescent protein (eGFP)-transduced glioblastoma cells, were treated by FUS in the presence of systemically injected microbubbles. Effect of FUS on plasma eGFP mRNA levels was determined using quantitative polymerase chain reaction. eGFP mRNA were only detectable in the FUS-treated U87 mice and undetectable in the untreated U87 mice (maximum cycle number set to 40). This finding was replicated in GL261 mice across three different acoustic pressures. The circulating levels of eGFP mRNA were 1,500-4,800 fold higher in the FUS-treated GL261 mice than that of the untreated mice for the three acoustic pressures. This study demonstrated the feasibility of FUS-enabled brain tumor liquid biopsies in two different murine glioma models across different acoustic pressures.
Astrocytes perform crucial supportive functions, including neurotransmitter clearance, ion buffering, and metabolite delivery. They can also influence blood flow and neuronal activity by releasing gliotransmitters in response to intracellular Ca(2+) transients. However, little is known about how astrocytes are engaged during different behaviors in vivo. Here we demonstrate that norepinephrine primes astrocytes to detect changes in cortical network activity. We show in mice that locomotion triggers simultaneous activation of astrocyte networks in multiple brain regions. This global stimulation of astrocytes was inhibited by alpha-adrenoceptor antagonists and abolished by depletion of norepinephrine from the brain. Although astrocytes in visual cortex of awake mice were rarely engaged when neurons were activated by light stimulation alone, pairing norepinephrine release with light stimulation markedly enhanced astrocyte Ca(2+) signaling. Our findings indicate that norepinephrine shifts the gain of astrocyte networks according to behavioral state, enabling astrocytes to respond to local changes in neuronal activity.
The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm(2) (± 0.9 Ω.cm(2)) and 28.2 Ω.cm(2) (± 1.3 Ω.cm(2)) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm(2). Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm(2). This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.
The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) and tight junction proteins by Western immunoblot in fetal sheep at 127days of gestation without ischemia, and 4, 24, or 48h after ischemia. The largest increase in K(i) (P<0.05) was 4h after ischemia. Occludin and claudin-5 expressions decreased at 4h, but returned toward control levels 24 and 48h after ischemia. Zonula occludens-1 and -2 decreased after ischemia. Inverse correlations between K(i) and tight junction proteins suggest that the decreases in tight junction proteins contribute to impaired blood-brain barrier function after ischemia. We conclude that impaired blood-brain barrier function is an important component of hypoxic-ischemic brain injury in the fetus, and that increases in quantitatively measured barrier permeability (K(i)) change as a function of the duration of reperfusion after ischemia. The largest increase in permeability occurs 4h after ischemia and blood-brain barrier function improves early after injury because the blood-brain barrier is less permeable 24 and 48 than 4h after ischemia. Changes in the tight junction molecular composition are associated with increases in blood-brain barrier permeability after ischemia.