Enhanced leptin transport across the blood–brain barrier by α1-adrenergic agents

Leptin is a 16 kDa protein secreted by fat cells which regulates body weight and thermogenesis at sites within the brain. Blood-borne leptin reaches those brain sites because of a saturable transport system located at the blood–brain barrier (BBB). Impaired transport occurs in obese rodents and likely underlies the resistance to the actions of peripheral leptin seen in obesity. Here, we show that leptin transport into the brain is enhanced 2–3-fold by epinephrine and other agents which are more specific for the α1 adrenergic receptor. Epinephrine had no effect on the transport across the BBB of insulin or tumor necrosis factor, on BBB integrity, or on the size of the vascular space of the brain. Dopamine, acetylcholine, histamine, serotonin, thyroid hormones, and phentolamine were without effect. Of several amino acids tested, only the catecholamine precursor tyrosine had an effect on leptin transport. Epinephrine was effective after intravenous or intraperitoneal injection, but neither epinephrine nor any of the other monoamines given by intracerebroventricular injection had an effect on leptin transport. These results show that epinephrine likely acts at a site on the luminal surface of the BBB. In conclusion, epinephrine works at an α1-like adrenergic, luminal side to enhance the transport of leptin across the BBB.     

Differential transport of rat and human interleukin-1alpha across the blood-brain barrier and blood-testis barrier in rats

Human interleukin-1alpha is transported across the murine blood-brain barrier (BBB) and blood-testis barrier (BTB) by a saturable transport system. Differences in the biological activity and binding of human IL-1 in mouse and rat brain raise the possibility of species differences in the transport of IL-1 across the BBB and BTB. We measured the transport of recombinant human 125I-IL-1alpha (I-huIL-1alpha) and rat 125I-IL-1alpha (I-ratIL-1alpha) across the rat BBB and BTB after intravenous injection using a sensitive in vivo technique and film autoradiography. I-ratIL-1alpha was found to cross the rat BBB and rat BTB at rates comparable to those reported previously for murine IL-1alpha in mice. Passage across the BBB was inhibited by the addition of unlabeled rat IL-1alpha, demonstrating saturable transport. In contrast, I-huIL-1alpha entered the brain of the rat much more slowly, and its entry was not inhibited by the addition of unlabeled human IL-1alpha. These results show that the rat interleukin-1 transporter, unlike the murine transporter, does not transport human IL-1alpha. This difference highlights the importance of species specificity in IL-1alpha transport and may partly explain the different physiological responses to exogenous human IL-1alpha among rodent species.     

Into rather unexplored terrain—transcellular transport across the blood–brain barrier

Efficient neuronal signaling in the central nervous system strictly depends on a well‐balanced microenvironment around glial cells, synapses, and axons. Unique features of the blood–brain barrier (BBB) endothelium largely determine the composition of this micro‐milieu and are dependent on the tight interplay with surrounding astrocytes and pericytes. BBB endothelial cells are endowed with a highly restrictive junctional complex that occludes the intercellular cleft, thereby preventing paracellular diffusion. The paracellular pathway is subject to extensive research as integrity loss of the junctional complex is associated with many neuropathologies, inflammation, and edema. Another important feature of the BBB endothelium is the low prevalence of nonspecific, transcytotic events, including (macro)pinocytosis, clathrin‐dependent and caveolin‐dependent endocytosis and the subsequent trafficking of vesicles to the opposite membrane. Although less studied, evidence is accruing that this pathway importantly contributes to increased BBB permeability, often when the junctional complex remains intact. Here, we review current knowledge on the contribution of the transcellular pathway to the BBB leak observed in different pathologic conditions. In addition, we hypothesize that nonselective, large pore connexin and pannexin channels may contribute to transcellular transport, either by providing a direct diffusion pathway across the endothelial monolayer, or indirectly, by exerting control over intracellular levels of the signaling ion Ca²⁺ that is involved in many steps of the vesicular pathway. We conclude that transcytotic events at the BBB, despite being less acknowledged, cannot be simply dismissed as done in the past, but actively contribute to BBB leakage in many different pathologies. GLIA 2016;64:1097–1123     

Effect of reduced flow on blood–brain barrier transport systems

Pathological states (i.e. stroke, cardiac arrest) can lead to reduced blood flow to the brain potentially altering blood–brain barrier (BBB) permeability and regulatory transport functions. BBB disruption leads to increased cerebrovascular permeability, an important factor in the development of ischemic brain injury and edema formation. In this study, reduced flow was investigated to determine the effects on cerebral blood flow (CBF), pressure, basal BBB permeability, and transport of insulin and K⁺ across the BBB. Anesthetized adult female Sprague–Dawley rats were measured at normal flow (3.1 ml min⁻¹), half flow (1.5 ml min⁻¹), and quarter flow (0.75 ml min⁻¹), using bilateral in situ brain perfusion for 20 min followed by capillary depletion analysis. Reduction in perfusion flow rates demonstrated a modest reduction in CBF (1.27–1.56 ml min⁻¹ g⁻¹), a decrease in pressure, and no significant effect on basal BBB permeability indicating that autoregulation remained functional. In contrast, there was a concomittant decrease in BBB transport of both insulin and K⁺ with reduced flow. At half and quarter flow, insulin transport was significantly reduced (R_Br%=17.2 and R_Br%=16.2, respectively) from control (R_Br%=30.4). Additionally, a significant reduction in [⁸⁶Rb⁺] was observed at quarter flow (R_Br%=2.5) as compared to control (R_Br%=4.8) suggesting an alteration in ion homeostasis as a result of low flow. This investigation suggests that although autoregulation maintains CBF, BBB transport mechanisms were significantly compromised in states of reduced flow. These flow alterations may have a significant impact on brain homeostasis in pathological states.     

Minocycline or iNOS inhibition block 3-nitrotyrosine increases and blood–brain barrier leakiness in amyloid beta-peptide-injected rat hippocampus

This work has examined levels of 3-nitrotyrosine (3-NT, a marker for peroxynitrite formation) and intactness of blood–brain barrier (BBB) in amyloid beta-peptide (Aβ_1–42)-injected rat hippocampus. Immunohistochemical analysis demonstrated 3-NT immunoreactivity in microglia/macrophages and astrocytes were significantly increased at 7 days post-Aβ_1–42 injection. Administration of the broad spectrum anti-inflammatory agent minocycline or the selective iNOS inhibitor 1400W markedly reduced 3-NT levels. Double immunofluorescence staining showed that 3-NT was prominently expressed in microglia/macrophages and astrocytes located in proximity to blood vessels. Additionally, Aβ_1–42 injection caused a marked increase in permeability of the BBB to immunoglobulin G (IgG); both minocycline and 1400W were highly effective in decreasing the leakiness of the BBB. Our results suggest the involvement of glial-derived reactive nitrogen species in mediating increased BBB permeability in Aβ_1–42 injected rat hippocampus.     

Biodegradation of α-MSH and derived peptides by rat brain extracts, and by rat and human serum

The peptides α-MSH and MSH/ACTH 4–10 were degraded by rat brain extracts and serum to yield free amino acids among the end-products. Breakdown of these two peptides was double that of a related synthetic hexapeptide Met (0)-Glu-His-Phe-D-Lys-Phe. No significant breakdown of the hexapeptide occurred after incubation with human serum; it also had almost negligible pigmentary effects in vivo and in vitro when compared to α-MSH. The patterns of amino acid release indicate possible endopeptidase cleavage at Phe-Arg in α-MSH followed by secondary exopeptidase action to release free amino acids. For the hexapeptide, the primary cleavage point occurred at the -His³-Phe⁴ bond. The stability of this analog in human sera, coupled with its lower rate of degradation in the CNS, may contribute to its more potent behavioral actions in vivo.     

The blood–brain barrier in health and disease

The blood–brain barrier (BBB) is a term used to describe a series of properties possessed by the vasculature of the central nervous system (CNS) that tightly regulate the movement of ions, molecules, and cells between the blood and the CNS. This barrier is crucial to provide the appropriate environment to allow for proper neural function, as well as protect the CNS from injury and disease. In this review, I discuss the cellular and molecular composition of the BBB and how the development and function of the BBB is regulated by interactions with the CNS microenvironment. I further discuss what is known about BBB dysfunction during CNS injury and disease, as well as methodology used to deliver drugs across the BBB to the CNS. ANN NEUROL 2012;72:648–672     

Recombinant interleukin-1β stimulates eicosanoid production in rat primary culture astrocytes

Astrocytes may play a prominent role in the initiation of immunoinflammatory responses in the central nervous system. They can be induced to synthesize eicosanoids but how immunologically relevant molecules modulate this process is not known. We examined the influence of recombinant interleukin-1 (rIL-1), an immunomodulating monokine on the release of arachidonic acid metabolites. IL-1 (1–30 U) induced a dose-related elaboration predominantly of the cyclo-oxygenation products prostaglandin E and throm☐ane B₂. Preincubation of rIL-1 with a specific antibody abrogated and heat-inactivation destroyed this activity. Both mepacrine and the isoquinolinesulfonamide H7 blocked the stimulatory effect dose-dependently, indicating involvement of protein kinase C in this novel biologic activity of IL-1. In central nervous system inflammation, IL-1-evoked release from astrocytes of arachidonic acid-derived metabolites may influence the severity of phlogistic responses and modulate local immune reactivity.     

Permeability of the blood–brain barrier to neurotrophins

To evaluate the feasibility of applying blood-borne neurotrophins to promote normal function of the central nervous system (CNS) and to rescue neuronal degeneration, we characterized the permeability of the blood–brain barrier (BBB) to neurotrophins. We report here that some members of the neurotrophin family (NGF, βNGF, NT3, and NT5) can cross the BBB of mice in vivo to arrive at the brain parenchyma. BBB permeability differed among individual neurotrophins in that NGF had the fastest influx rate (K_i) and NT3 the slowest, and that the entry rate of NGF was twice that of its smaller bioactive subunit βNGF. BBB permeability also differed at various CNS regions in that the cervical spinal cord had the greatest rate of influx, whereas brain had the lowest. Saturability of influx was suggested by self-inhibition studies for NT3 in vivo, and for NGF in an in situ brain perfusion system, indicating the presence of saturable transport systems. The results suggest that peripheral administration of neurotrophins could have therapeutic effects within the CNS.     

Mechanisms of glucose transport at the blood–brain barrier: an in vitro study

How the brain meets its continuous high metabolic demand in light of varying plasma glucose levels and a functional blood–brain barrier (BBB) is poorly understood. GLUT-1, found in high density at the BBB appears to maintain the continuous shuttling of glucose across the blood–brain barrier irrespective of the plasma concentration. We examined the process of glucose transport across a quasi-physiological in vitro blood–brain barrier model. Radiolabeled tracer permeability studies revealed a concentration ratio of abluminal to luminal glucose in this blood–brain barrier model of approximately 0.85. Under conditions where [glucose]_lumen was higher than [glucose]_ablumen, influx of radiolabeled 2-deoxyglucose from lumen to the abluminal compartment was approximately 35% higher than efflux from the abluminal side to the lumen. However, when compartmental [glucose] were maintained equal, a reversal of this trend was seen (approximately 19% higher efflux towards the lumen), favoring establishment of a luminal to abluminal concentration gradient. Immunocytochemical experiments revealed that in addition to segregation of GLUT-1 (luminal>abluminal), the intracellular enzyme hexokinase was also asymmetrically distributed (abluminal>luminal). We conclude that glucose transport at the CNS/blood interface appears to be dependent on and regulated by a serial chain of membrane-bound and intracellular transporters and enzymes.     

An improved low-permeability in vitro-model of the blood–brain barrier: transport studies on retinoids, sucrose, haloperidol, caffeine and mannitol

Primary cultures of porcine brain capillary endothelial cells grown on collagen coated polycarbonate membranes were used to build up an in vitro-model for the blood–brain barrier. Improved cultivation techniques allowed cell-storage and experiments under serum-free conditions. We employed this model to perform permeability studies in vitro with the radioactively labelled marker substances sucrose, retinoic acid, retinol, haloperidol, caffeine, and mannitol. Permeability values obtained with this blood–brain barrier model (1.0×10⁻⁶ cm/s for sucrose, 6.2×10⁻⁶ cm/s for retinoic acid, 4.8×10⁻⁶ cm/s for retinol, 49.5×10⁻⁶ cm/s for haloperidol, 62.4×10⁻⁶ cm/s for caffeine, and 1.8×10⁻⁶ cm/s for mannitol) show a good correlation to data which are already known from in vivo-experiments. As judged by the sucrose permeability our blood–brain barrier model is less permeable than numerous other models published so far. Therefore it represents a powerful tool for in vitro-prediction of blood–brain barrier permeability of drugs and offers the possibility to scan a large quantity of drugs for their potential to enter the brain.     

Imatinib preserves blood–brain barrier integrity following experimental subarachnoid hemorrhage in rats

Blood–brain barrier (BBB) disruption and consequent edema formation contribute to the development of early brain injury following subarachnoid hemorrhage (SAH). Various cerebrovascular insults result in increased platelet‐derived growth factor receptor (PDGFR)‐α stimulation, which has been linked to BBB breakdown and edema formation. This study examines whether imatinib, a PDGFR inhibitor, can preserve BBB integrity in a rat endovascular perforation SAH model. Imatinib (40 or 120 mg/kg) or a vehicle was administered intraperitoneally at 1 hr after SAH induction. BBB leakage, brain edema, and neurological deficits were evaluated. Total and phosphorylated protein expressions of PDGFR‐α, c‐Src, c‐Jun N‐terminal kinase (JNK), and c‐Jun were measured, and enzymatic activities of matrix metalloproteinase (MMP)?2 and MMP‐9 were determined in the injured brain. Imatinib treatment significantly ameliorated BBB leakage and edema formation 24 hr after SAH, which was paralleled by improved neurological functions. Decreased brain expressions of phosphorylated PDGFR‐α, c‐Src, JNK, and c‐Jun as well as reduced MMP‐9 activities were found in treated animals. PDGFR‐α inhibition preserved BBB integrity following experimental SAH; however, the protective mechanisms remain to be elucidated. Targeting PDGFR‐α signaling might be advantageous to ameliorate early brain injury following SAH. © 2014 Wiley Periodicals, Inc.     

Transport of prion protein across the blood-brain barrier

The cellular form of the prion protein (PrP^c) is necessary for the development of prion diseases and is a highly conserved protein that may play a role in neuroprotection. PrP^c is found in both blood and cerebrospinal fluid and is likely produced by both peripheral tissues and the central nervous system (CNS). Exchange of PrP^c between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications, but it is unknown whether PrP^c can cross the blood-brain barrier (BBB). Here, we found that radioactively labeled PrP^c crossed the BBB in both the brain-to-blood and blood-to-brain directions. PrP^c was enzymatically stable in blood and in brain, was cleared by liver and kidney, and was sequestered by spleen and the cervical lymph nodes. Circulating PrP^c entered all regions of the CNS, but uptake by the lumbar and cervical spinal cord, hypothalamus, thalamus, and striatum was particularly high. These results show that PrP^c has bidirectional, saturable transport across the BBB and selectively targets some CNS regions. Such transport may play a role in PrP^c function and prion replication.     

Intravascular infusions of soluble β-amyloid compromise the blood–brain barrier, activate CNS glial cells and induce peripheral hemorrhage

Vascular wall levels of soluble β-amyloid_1–40 (Aβ_1–40) are elevated in Alzheimer's disease (AD). Moreover, plasma Aβ levels are increased in familial AD, as well as in some cases of sporadic AD. To determine the histopathologic and behavioral consequences of elevated vascular Aβ levels, Aβ_1–40 (50 μg in distilled water) or vehicle was intravenously infused twice daily into 3-month old male Sprague–Dawley rats for 2 weeks. Intravenous Aβ infusions impaired blood–brain barrier integrity, as indicated by substantial perivascular and parenchyma IgG immunostaining within the brain. Also evident in Aβ-infused animals was an increase in GFAP immunostaining around cerebral blood vessels, and an enhancement of OX-42 microglial immunostaining in brain white matter. Gross pulmonary hemorrhage was noted in most Aβ-infused animals. All the observed changes occurred in the absence of Congo red birefringence. No significant cognitive deficits were present in Aβ-infused animals during water maze acquisition and retention testing, which was conducted during the second week of treatment. These results indicate that circulating Aβ can: (1) induce vessel dysfunction/damage in both the brain and the periphery without complex Aβ fibril formation/deposition, and (2) induce an activation of brain astrocytes and microglia. Taken together, our results suggest that if circulating Aβ is elevated in AD, it is likely to have a pathophysiologic role.     

Upregulation of intercellular adhesion molecule-1 expression on human endothelial cells by tumour necrosis factor-α in an in vitro model of the blood–brain barrier

Adhesion molecules on the endothelial surface of the blood–brain barrier (BBB) play an important role in the pathogenesis of many encephalopathies, including multiple sclerosis (MS) and cerebral malaria (CM). The expression of four surface molecules of relevance to MS and CM on the immortalized human umbilical vein endothelial cell line, ECV304, was investigated using immunofluorescence flow cytometry. We found that ECV304 cells express intercellular adhesion molecule-1 (ICAM-1) and low levels of CD36, but not vascular cell adhesion molecule-1 (VCAM-1) or E-selectin. This expression pattern was unaltered on ECV304 cells which were co-cultured with C6 glioma cells; conditions under which the endothelial cells display enhanced barrier formation. Tumour necrosis factor-α (TNF-α), which is elevated in MS and CM, decreased the integrity of the barrier in co-cultured endothelial cells and upregulated the expression of ICAM-1 nine-fold. The significance of elevated ICAM-1 expression in relation to the binding of parasitised erythrocytes at the BBB in CM is discussed.     

Stereospecific transport of Tyr-MIF-1 across the blood-brain barrier by peptide transport system-1

Previous studies have suggested that peptide transport system-1 (PTS-1), the saturable system that transports Tyr-MIF-1, the enkephalins, and related peptides out of the central nervous system (CNS), exhibits stereospecificity. In the present studies, we showed that 125I-L-Tyr-MIF-1, but not 131I-D-Tyr-MIF-1, was cleared from the CNS more rapidly than could be accounted for by nonspecific mechanisms. Such clearance was inhibited by a 1.0 nmol dose of L-Tyr-MIF-1, but not by D-Tyr-MIF-1. Neither L- nor D-Tyr-MIF-1 altered the much lower clearance of I-D-Tyr-MIF-1 from the brain. Radioactivity recovered from the vascular space after the injection of 125I-Tyr-MIF-1 into the lateral ventricle of the brain eluted by HPLC primarily as intact peptide, demonstrating that most of the Tyr-MIF-1 was not degraded during transport. By contrast, the nonsaturable unidirectional influx of Tyr-MIF-1 into the CNS did not distinguish between the isomers. These studies confirm and extend the observations that Tyr-MIF-1 is transported out of the CNS by a saturable, stereospecific transport system as an intact peptide while the influx into the CNS is by a nonsaturable mechanism that does not distinguish between the isomers.     

Molecular and cellular permeability control at the blood–brain barrier

The blood–brain barrier (BBB) is formed by brain capillary endothelial cells. These cells have at least three properties which distinguish them from their peripheral counterparts: (1) tight junctions (TJs) of extremely low permeability; (2) low rates of fluid-phase endocytosis; (3) specific transport and carrier molecules. In combination, these features restrict the nonspecific flux of ions, proteins, and other substances into the central nervous system (CNS) environment. The restriction protects neurons from harmful compositional fluctuations occurring in the blood and allows uptake of essential molecules. Breakdown of the BBB is associated with a variety of CNS disorders and results in aggravation of the condition. Restoration of the BBB is thus one strategy during therapy of CNS diseases. Its success depends on a precise knowledge of the structural and functional principles underlying BBB functionality. In this review we have tried to summarise the current knowledge of TJs, including information gained from non-neuronal systems, and describe selected mechanisms involved in permeability regulation.     

Bidirectional transport of interleukin-1 alpha across the blood-brain barrier

Circulating interleukin-1 alpha (IL-1 alpha) has multiple effects on the central nervous system. We investigated the ability of radioiodinated IL-1 alpha (rIL-1 alpha) to cross the rodent blood-brain barrier and found its entry rate to be 43.9 times greater than that predicted by leakage alone. The rIL-1 alpha entered multiple regions of the brain, with over 40% entering at the cortex. The hypothalamus had the highest entry rate on a weight basis but only accounted for 2% of total entry. In all experiments, the entry rate of rIL-1 alpha greatly exceeded that of simultaneously injected radiolabeled albumin. The half-time disappearance of rIL-1 alpha from the brain after central injection was 21.9 min, a time that exceeds the reabsorption rate of cerebrospinal fluid. Pretreatment of animals with aluminum decreased both entry and exit rates, which is compatible with a saturable component of transport. Thus, rIL-1 alpha has access to many regions of the brain with bidirectional transport rates across the blood-brain barrier exceeding those predicted by nonspecific mechanisms.