The Blood-brain Barrier Dr. Eszter Farkas- Dr. Ferenc Domoki http://brainwaves.corante.com/Vasculature.gif
The discovery of the blood- brain barrier (BBB) • Paul Ehrlich (1885): injection of colored dyes into the circulation → all organs were stained except for the central nervous system • Edwin Goldmann (1913): the dye was injected into the central nervous system → the brain got stained, but no peripheral organs ⇒ proof for the existence of the BBB: the brain is an isolated organ
The discovery of the blood-brain barrier: Paul Ehrlich and Edwin Goldman (1913) Dye transport studies (Evans Blue-albumin, Na-fluorescein) are still in use and are important techniques to study the BBB integrity in vivo .
The cerebral capillary network • Dense capillary network: average intercapillary distance: 40μm • Large surface: endothelial layer: 100cm 2 /g • Volume: the mass of endothelial cells constitutes 0.1% of the brain tissue http://www.teknat.uu.se/forskning/uu/bild.php?typ=forskningsprogram&id=225
The cerebral capillary Layers: 1. Endothelium 2. Basal membrane (with pericytes) 3. Astrocyte-endfeet The tight junctions have been found to be responsible for the blocking of dye diffusion across the barrier
The structure of the cerebral capillaries
The ultrastructure of the cerebral capillaries • Endothelial cell (en: nucleus; ep: plasma; em: mitochondria) • Basal membrane (bm) • Pericyte (p) • Astrocytic endfeet (a) Farkas & Luiten, Progr. Neurobiol. 2001
Molecular structure of the blood-brain barrier (BBB) Tight junction: Primary seal is formed homodimers of claudins and occludin that are responsible for the low paracellular permeability. Zonula occludens (ZO) proteins anchor the seal to the cytoskeleton. Adherens junction: JAM and VE cadherins provide mechanical cell-to cell adhesion. P. Ballabh et al. / Neurobiology of Disease 16 (2004) 1–13
Primary rat cerebromicrovascular endothelial cell and pericyte cultures A B E ZO-1 occludin C D β -actin EC PC EC PC EC PC EC PC Rat Piglet 50 µ m Domoki, F., et al.: Am J Physiol Reg Integr Comp Physiol 295:R1099-108, 2008.
Transendothelial electrical resistance – a measure of the paracellular barrier in cell cultures 250 250 200 200 Piglet CMVEC TEER ( Ω cm 2 ) Rat CMVEC TEER ( Ω cm 2 ) 150 150 100 100 50 50 0 0 1DIV 2DIV 3DIV 4DIV 5DIV 1DIV 2DIV 3DIV 4DIV 5DIV Domoki, F., et al.: Am J Physiol Reg Integr Comp Physiol 295:R1099-108, 2008.
„In vitro BBB” modelling Nakagawa S et al. Neurochem Res 54:253-263 (2009)
Transport throught the BBB: blood � brain (influx) Diffusion Receptor-mediated Absorption-mediated endocytosis endocytosis Simple Facilitated + + + + × + blood + + - - - - + + + - - - - - - - - + + - endothelial - + + + - - - - cell - - - - + + + + + brain Gases Glucose Ferro- Plasma proteins H 2 O Aminoacids transferin Ethanol Nucleosides
Transport throught the BBB: brain � blood (efflux) P-glycoprotein (MDR1, ABCB1) Glutamine secretion S glu S gln blood glu gln glu gln endothelial S cell N G A glu gln gln brain glu Pl. cancer glutamate drugs Na + gln glutamine
Endothelial enzyme and transport barrier: another feature of the blood-brain barrier • Many substances cannot pass the endothelial cells because they are either (1) degraded by enzymes located in the luminal membrane or (2) pumped back to the blood by multispecific active transporters. • This barrier impedes greatly the delivery of drugs into the central nervous system • The most important of such pumps are the P- glycoprotein (multidrug resistant protein MDR1), the MDR-related protein (MRP2) and the organic anion transporter protein 2 (OATP2), they belong to a transporter superfamily: the ABC family!
The ATP binding casette (ABC) transporter family NBD: nucleotide binding domain TMD: transmembrane domain ABCB1=PGP
ABCB1 (P glycoprotein) activity measurement - in vitro BBB Note that polarity of ECs is maintained in the presence of pericytes and glial cells! Nakagawa S et al. Neurochem Res 54:253-263 (2009)
The enzymatic barrier Enzymatic barrier: break-down of neuroactive substances Enzyme Function Alkaline phosphatase De-phosphorylation (purine and pirimidine metabolism) Monoamino oxidase (MAO) Cathecolamine inactivation Aminopeptidase A Angiotensin metabolism Endopeptidase Break-down of neuropeptides (e.g. bradikynin, dynorphin, neurotensin) γ -glutamil transpeptidase Leukotriene C4 → D4 conversion
Summary Tight junctions CSF Blood Brain parenchyma
Areas outside the BBB Circumventricular organs: – Pineal gland (3) – Median eminence – Neurohypophysis (5) – Subfornical organ (1) – Subcomissural organ (2) – Area postrema (4) Function: – Organum vasculosum – Hormone production of lamina terminalis (6) – Sensory function – Choroid plexus – Production of CSF
The opening of the blood brain barrier Paracellular: Transcellular: increased permeability pinocytic transport of tight junctions × blood endothelial endothelial cell cell brain Pl. inflammation, ischemia, trauma
The opening of the blood-brain barrier Structural Conditions Mediators correlate TNF- α , IL- β , histamin, Loosening of Hyperosmolarity, acidic pH, the tight encephalitis, multiple sclerosis, bradykinin, serotonin, junction ischaemia arachidonic acid, e.t.c. TNF- α , IL- β , histamin, Pinocytic Hypertension, microwave activity irradiation, trauma, seizures, bradykinin, serotonin, tumors arachidonic acid, e.t.c. Increased Solvents (ethanol, propanol, membrane buthanol, DMSO) fluidity Formation of Some antidepressants pores (chlorpromazine, notriptylin) Altered Diabetes, Alzheimer ’s disease, GLUT-1, ICAM-1 activation of stroke, obesitas, multiple transporters sclerosis
The blood brain barrier: applications for medicine 2 big problems: 1. Intact BBB function 2. Impaired BBB function
Ad 1: Drugs and the BBB The intact BBB hinders/blocks drug delivery to the brain. It poses a problem for the treatment of many central nervous system diseases. Potential solutions: – Increased lipid-solubility of the drug – Transient opening of the BBB (e.g. osmotic) – „Wrapping” drugs (liposomes) – Intranasal pathway
Increased lipid solubility • Dihydropyridines: Ca 2+ channel antagonists • For the treatment of hypertension • Nimodipine: increased lipid solubility: for the treatment of stroke
Osmotic opening of the blood-brain barrier • Intra-carotid infusion of hyperosmotic solutions (for example: mannitol) • Transient shrinkage of the endothelial cells → loosening and opening of the tight junctions • Defining variables: – Length of infusion – Osmolarity of the solution • Influencing physiological parameters: – Concentration of blood gases – Cardiac output • Therapeutical target: pl. chemotherapy of brain tumors
Application of liposomes • Known for 30 years (Bangham) • Small, artificial phospholipid vesicles • For medicines targeting the CNS • In stroke treatment: e.g. SOD
Intranasal treatment strategies • Sensory nerve endings: – n. olfactorius – n. trigeminalis • Nonisnvasive • NGF, IGF, FGF • Way of passage: • Intraneuronal: axonal transport, hours to days → for specified brain regions • Extraneuronal: perineuronal, minutes → brain parenchyma, CSF
Ad 2: Impaired blood-brain barrier function • Amyloid angiopathy – Alzheim er’s disease • Basal membrane thickening – aging, neurodegenerative disease, hypertension • (Atherosclerosis) – hypertension • Neuroinflammation (ischemia, trauma, infection)
Amyloid angiopathy • Amyloid precursor protein: wrong slicing → β -amyloid peptide • Deposition into vessel walls • Double ring: space between the t. intima & media • Alzheim er’s disease
Basal membrane thickening • Aging, dementia, hypertension • Hindered transport
Atherosclerosis • Large vessels: rigidity, decreased flow • Small vessels: hindered flow & transport
Inflammation at the BBB • causes: infection, trauma, necrosis (stroke) • Inflammatory mediators activate the contractile machinery of the endothelial cells severing the paracellular junctions. (signalling details next slide) • Permeability increases + cellular transmigration is made possible.
Cellular signalling of EC contraction
Abbreviations for the previous slide • TRPC1 – canonical transient receptor potential 1 ion channel (store-operated Ca2+ channel) • IP 3 , IP 3 R – inositol triphosphate, and its receptor (Ca2+ channel) • Gq, G12/13 – heterotrimeric G-proteins • PLC – phospholipase C • ER – endoplasmic reticulum • Rho – small G protein • RhoGEF – Rho guanin nucleotide exchange factor (activator of Rho) • ROK – Rho kinase • MYPT1 – myosine phosphatase targeting subunit 1 • PP1 – protein phosphatase 1 • CaM – calmodulin • MLC, MLCK – myosin light chain, MLC kinase
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