Blood Brain Barrier

Question 1

Describe visually what blood brain barrier damage of mice infected with virus by Evan’s blue dye exclusion tests

Answer 1

Inclusion of blue colour dye n the brain indicated the increase permeability or damage of BBB.

Question 2

Why do we need the BBB?

Answer 2

CNS neurons are intolerant to the fluctuations of metabolites, proteins and immune mediators present in the peripheral blood
Several amino acids such as glutamate are active as NTs and their conc in the extracellular space of the brain must be tightly regulated
Common plasma proteins such as serum albumin have been implicated in the provocation of epileptic seizures through the downstream alteration in astrocyte activity through downstream alteration in astrocyte activity induced by direct interaction with astrocyte TGFbeta Receptor-2 (TGFbR2)
Immune cells and inflammatory mediators may have destructive actions in the CNS

Question 3

What is the barrier?

Answer 3

Non-fenestrated barrier endothelium of the blood brain vessels

Question 4

Describe the lining of the BBB

Answer 4

Brain vessels are lined by continuous endothelial cells (ECs) that lack the small pores called fenestrated a found in vessels of many other tissues and organs
Importantly the tight junctions that join the ECs don’t permit leakage of even very small solute molecules, giving the ECs control of everything of everything that crosses the barrier

Question 5

What gives the endothelial cells control over everything that crosses the barrier

Answer 5

Tight junctions that join the ECs don’t permit leakage of even very small solute molecules

Question 6

Describe the choroidal capillary ultra structure

Answer 6

Fenestrated non-barrier endothelium typical of endocrine glands, small intestine and the renal glomerulus

Question 7

Describe the dealing of the barrier

Answer 7

‘Tight’ junctions seal the barrier
The effective sealing of the gap between contiguous endothelial cells is maintained by extremely impermeable zonular occludes e belt-like tight junctions that completely seal the space between the ECs

Question 8

Continuous endothelium and sealing tight junctions of the CNS blood vessels enables….

Answer 8

Them to act as. Continuous membrane

Question 9

The tight junctions between BBB endothelial cells leads to …..

Answer 9

High endothelial electrical resistance and low paracellular permeability
The electrical resistance is in the range of 1500-2000 ohms/cm2 (pail vessels) compared to 3-33 ohms/cm2 in other tissues

Question 10

Permeability of the BBB to different classes of small molecules is similar to that of….

Answer 10

An intact phospholipid membrane

Question 11

What type of molecules can get through

Answer 11

Hydrophobic eg o2, co2, n2, steroid hormones

Question 12

How do amino acids transported across

Answer 12

Active transport

Question 13

Glucose crosses by….

Answer 13

Facilitated diffusion on the Glut-1 transporter

Question 14

CNS endothelial cells have …… than non barrier vessels

Answer 14

Greater volume fraction of mitochrondria in their cytoplasm than those of non-barrier vessels, to meet the greater energy demands of the ATP driven active transport systems

Question 15

Specific macromolecules such as carrier proteins are …..

Answer 15

Selectively transferred by receptor-mediated endocytosis and transcytosis

Question 16

Where are Glut-1 and Glut 3

Answer 16

Glucose transporters
Glut-1 is present on the plasma membranes of retinal neurons, glia and the vascular endothelial cells at the Blood Retinal Barrier
Glut-3 is neuron-specific and absent from the endothelium

Question 17

Glut-1 is expressed by ….

Answer 17

Retinal neurons, glia and by vascular endothelial cells at the BRB

Question 18

What permits selective transport of carrier proteins

Answer 18

Receptor-mediated endocytosis

Question 19

Transcytosis can deliver..

Answer 19

Essential macromolecules such as carrier proteins, or therapeutic antibodies, across the BB in a biologically active state
In receptor-mediated transcytosis, macromolecules internalised by RME at the apical surface are re-routed from early recycling endoscopes to the base-lateral surface in biologically intact form

Question 20

Intravascular horseradish peroxidase MW 35-40K is…

Answer 20

Confined to the endosomal system of the endothelium of retinal capillary

Question 21

What are caveolae

Answer 21

pecialised cholesterol-rich microdomains of the plasma membrane, related to ‘lipid rafts’ in a wide variety of cells. However, when first described in endothelial cells by Palade et al over 40 years ago, they were interpreted as transport vesicles because of their spherical shape and the fact that due to their small size (60-80nM), their continuity with the cell surface was often not obvious. (see notes)
Caveolae often create apparent multi-chambered compartments at the luminal surface of vascular endothelial cells that have been associated with pathological activity of vascular endothelial growth factor (VEGF). However, they are present under normal conditions in retinal and choroidal vessels of the eye.

Question 22

Caveolae of ten create

Answer 22

Apparent multi-chambered compartments at the luminal surface of vascular endothelial cells that have been associated with pathological activity of vascular endothelial GF (VEGF). However, they are present under normal conditions in retinal and choroidal vessels of the eye

Question 23

Where are caveolae predominantly located

Answer 23

At the albuminal plasma membrane of the endothelium in the brain and retinal vessels, but if they operated transcytosis there should e equivalent numbers on each side of the cell. The tracer HRP within the vessel lumen in this type of preparation is washed out by perfusion fixation showing that caveolae do not internalise the protein

Question 24

Caveolae in an endothelial cell process show…

Answer 24

Tight clustering around a mitochondrion consistent with their role in locating ATP dependent ion pumps in a protected micro environment at the surface of constantly moving cells

Question 25

The entire albuminal surface of the vasculature in the CNS is enclosed by the

Answer 25

Foot-processes of astrocytes

Question 26

Arterial character is determined by

Answer 26

The type of mural cells (either SMC or pericytes and not by the luminal diameter) highly granites basement membrane

Question 27

Immature capillary shows

Answer 27

Scanty deposits of ECM material in the space of the developing Basement membrane

Question 28

What acute vulnerability happens only in the neonatal period

Answer 28

Acute vulnerability to hyperoxia and VEGF down-regulation

Question 29

Maturation is thought to be dependent on but is actually dependent on

Answer 29

Pericyte investment
But maturation later found to be dependent on pericyte-derived angioprotien - 1 signalling via the endothelial specific Tie-2 receptor

Question 30

Ang-1 is produced by

Answer 30

Both pericytes and astrocytes, both of which have direct access to the barrier endothelium

Question 31

Pericytes essential for

Answer 31

Induction of the BBB

Question 32

What is Norrie’s disease

Answer 32

A condition characterised by breakdown of the blood-brain barrier, was caused by mutations in either the unconventional Wnt ligand Norris, or its cognate receptor Frizzled-4

Question 33

What is Norris secreted by and where

Answer 33

The Muller glia in the retina but not n the rest of the CNS

Question 34

What fills in for Norrin in cerebral cortex

Answer 34

Alternative Wnt Lgands

Question 35

What is neuron-derived Wnt signalling essential for

Answer 35

Induction of essential BBB associated genes Glut-1 and Claudin-3 in the vascular endothelium

Question 36

Pathological conditions that are factors causing barrier-breakdown

Answer 36

Chronic hypoxia - vascular disease or altitude sickness
Inflammation and/or infection
Hyperglycaemia - in diabetes mellitus
Tumour - induces fenestration in vasculature

Question 37

Specific mediators that cause barrier breakdown

Answer 37

VEGF induced by hypoxia and/or inflammation
Angiopoietin-2 (non-signalling ligand for tie-2 receptor - blocks effects of ang-1) ang2 release is initiated by pro-inflammatory cytokines such as TNF-alpha and also by persistent hypoxia
Release of biogenic amines such as histamine and bradykinin during inflammation

Question 38

Ang-1 induces

Answer 38

Vascular stabilisation via Tie-2 receptor signalling, an effect reversed by ang-2

Question 39

Pericyte derived ang-1 induced …

Answer 39

VE-Catherine and associated cytoskeletal and survival signalling complexes

Question 40

Endothelial cell derived ang 2…..

Answer 40

Destabilises EC junctions and matrix adhesion by altering beta 1 integrity associations
Ang-2 also induces Pericyte detachment fro the EC tube or even apoptosis

Question 41

VEGF induced vascular permeability

Answer 41

VEGF induces rapid phosphorylation and redistribution of the tight junction proteins occluding and ZO-1, as in diabetes retinopathy

Question 42

What is an example of Trojan horse strategy

Answer 42

Transcytosis of therapeutic antibodies
The transferrin receptor (TfR) can be used as a shuttle for antibodies across the BBB if the antibody binds to the TfR in a reversible mode

Question 43

Describe Trojan-horse transcytosis of anti-amyloid-beta therapeutic antibodies

Answer 43

Binding sites for both the BBB carrier (transferrin receptor TfR) and the therapeutic target of the antibody (BACE1/beta-secretase) can be accommodated in a single engineered bispecific antibody. Lowering the affinity of the Ab for the transferrin receptor, facilitated the release f the Ab at the brain side of the barrier and increased its concentration and half life within neural tissue

Question 44

Describe transient open-barrier approaches for the BBB

Answer 44

1. Hyper-osmolar infusions of agents such as mannitol
2. Inflammatory mediators such as bradykinin or histamine (require higher cones for CNS vessels)
3. Growth factor signalling (VEGF) that cases phosphorylation of junctional proteins such as beta-catenin
4. Activators of cytoskeletal modulators: Rho family of small GTPases and associated kinases such as ROCK
5. Physical agents: focused ultrasound (shown benefit in treatment of Alzheimer’s where treatment effect appears to be mediated by transient opening of the BBB)