Blood Brain Barrier (DONE)

Question 1

What is the BBB?

Answer 1

A neurovascular unit or network of vessels that form a structural and chemical barrier between the brain and systemic circulation

Question 2

What are the main functions of the BBB?

Answer 2

Protects the brain against xenobiotics
Barrier to neuroactive pharmaceuticals
Maintains a stable environment for precise communication between the nerve cells

Question 3

What is the active barrier made up of?

Answer 3

Efflux transporters- limit access of xenobiotics into the brain and clear waste out of the brain
Influx transporters- supply the brain with glucose, amino acids and other nutrients
Metabolizing enzymes- form a second line of defense by degrading xenobiotics entering the brain

Question 4

Discovery of the BBB history

Answer 4

P Ehrlich 1885- intravenous acidic vital dyes stain all rabbits body except brain and spinal cord
EE Goldmann 1909- cerebral capillaries provide anatomical basis for physiological barrier between brain and the rest of the body
Intra-thecal trypan blue stained the brain and spinal cord but not the rest of the body

Question 5

Neurovascular unit

Answer 5

Capillary surface area ~ 100 cm2/g tissue with capillary volume of 1% of brain tissue volume (very dense)
Inter-capillary distance 40 micrometres
Humans: 12m2 surface area of capillaries in the brain (~400 miles in length)
Choroid plexus is ~1/1000th of surface area

Question 6

Differences between brain and general capillaries

Answer 6

General capillary: continuous (although some fenestrated), small solutes can diffuse through intercellular clefts, pinocytosis independent of molecular size (pass large molecules)
Brain capillary: continuous, no fenestra, astrocyte/pericyte signalling, tight junctions (overlap), reduced pinocytosis, efflux transporters

Question 7

Regions of brain not enclosed by BBB- circumventricular organ functions

Answer 7

Pineal gland- secretes melatonin
Subfornical organ- fluid regulation
Organum vasculosum of the lamina terminalis (OVLT)- regulates osmolarity of blood
Area postrema- vomiting centre, senses toxins in blood
Median eminence- neural hormones
Neurohypophysis (posterior pituitary)- oxytocin, ADH

Question 8

Cerebral ventricles

Answer 8

Four ventricles in brain (two lateral, third and fourth)
Membrane bound cavities lined with ependymal cells
Filled with CSF (10mmHg pressure)
CSF production: walls of lateral ventricles and thrid ventricle by choroid plexus

Question 9

Cerebrospinal fluid

Answer 9

Clear fluid present in the ventricles of the brain, the central canal of the spinal cord, and the subarachnoid space, normally has almost no blood cells and little protein

Question 10

Blood-CSF barrier

Answer 10

Choroid plexus- epithelial cells, polarised, columnar, present in each of the four ventricles
Secretes CSF (lateral and third), secretes proteins (e.g. prealbumin), removes waste products, serves as a barrier, active transport
Displays tight junctions limiting passive protein transport from blood to intraventricular space containing CSF

Question 11

CSF pathways of flow

Answer 11

CSF flows from the lateral ventricle to the third ventricle
The third ventricle and fourth ventricle are connected to each other by the cerebral aqueduct
CSF flows into the spinal canal and subarachnoid space
CSF drains back to cerebral blood via arachnoid sinus, or via spinal nerve roots, or via olfactory tracts

Question 12

Epidural injections

Answer 12

During childbirth etc. (inc. caesarean)
During some types of surgery (surgical anaesthesia): pelvic area or legs, remain awake and responsive, less nausea and vomiting, quicker recovery afterwards, reduce risk of DVT, at the end of an operation post-operative pain (epidural analgesia)
Steroid medication can also be given as an epidural injection to treat back or leg pain caused by sciatica or prolapsed disc
Injected into dura- the tissue that keeps the spinal fluid around the spinal cord and spinal nerves

Question 13

Challenges and advantages of epidural injections

Answer 13

Challenges- still outside the CSF with a barrier remaining to cross
Advantages- not violated CSF space and reduced risk of infection, less technicall demanding

Question 14

Intra-thecal drug administration indications

Answer 14

Chronic spasticity due to injury, multiple sclerosis and cerebral palsy e.g. baclofen
Management of cancer, chronic non-malignant or neuropathic pain e.g. morphine
Chemotherapy lymphomatous meningitis e.g. methotrexate, cytarabine
Antibiotic treatment adjuvant to systemic therapy in bacterial meningitis and other infections of the central nervous system e.g. gentamicin

Question 15

Intra-thecal drug administration

Answer 15

Intra-thecal drug administration involves the direct injection of the dug into the CSF within the intra-thecal space of the spinal column

Question 16

Challenges and advantages of intra-thecal drug administration

Answer 16

Challenges- more highly skilled procedure, greater risk of infection
Advantages- bypasses dura accessing directly into the CSF

Question 17

Intra-ventricular drug administration

Answer 17

Intra-ventricular catheter system that can be used for the aspiration of cerebrospinal fluid or for the delivery of drugs e.g. chemotherapy into the cerebrospinal fluid
Reservoir implanted subcutaneously with catheter in one lateral ventricle attached to a reservoir
Used to treat brain tumours, leukemia and lymphoma
In the palliative care of terminal cancer, an Ommaya reservoir can be inserted for intracerebroventricular (ICV) injection of morphine

Question 18

Convection enhanced drug administration to brain

Answer 18

Delivering a drug directly to the brain through one or more very small tubes which are surgically placed into the brain tumour
Not routine
Placement of guide tube under surgery with CT/MRI scans
Catheter remains in place over a 3-4 day course of drug administration, then removed, the guide tube is left in for further rounds of treatment as required with a new catheter
Diffusion and convection driven distribution, 1-20mm distance from site of administration
e.g. carboplatin for brain stem tumours

Question 19

Generic routes across the BBB

Answer 19

Cell migration.g. non polar solutes, lipid soluble
Passive diffusion
Carrier mediated efflux e.g. lipid soluble, amphiphilic, drugs
Carrier mediated influx e.g. glucose, amino acids, amines
Receptor mediated transcytosis e.g. transferrin, insulin, cytokines
Adsorptive mediated transcytosis e.g. histone, avidin
TJ modulation e.g. polar solutes

Question 20

Barrier permeability: passive mechanism

Answer 20

Molecule properties: steric, hydrophobicity, ionic

Processes: partitioning, diffusion

Question 21

Polar surface area

Answer 21

The PSA is defined as the surface sum over all polar atoms, usually oxygen and nitrogen, including attached hydrogens

Question 22

PSA and CNS access

Answer 22

CNS drugs that penetrate brain by passive transport have polar surface area below 70A^2
Most orally administered non-CNS can have larger values up to 120 A^2

Question 23

ABC transporters

Answer 23

48 human ABC transporters
Grouped by homology into 7 families- A to G
ABC B- peptides, phosphatidylcholine, bile salts, iron, drugs
ABC C- organic anions, anionic conjugates, nucleotides, nucleosides, bile salts, peptides
ABC G- sterols, lipids, drugs

Question 24

Pgp expression

Answer 24

Pgp expressed in the luminal membrane of BBB and apical membrane of choroid plexus transporting into CSF

Question 25

General features of Pgp substrates

Answer 25

Lipid solubility with some amphiphilic properties
Large planar molecules
If charged, mildly cationic
Possibly electron donor pair pattern

Question 26

LAT

Answer 26

Heterodimeric membrane transport protein that preferentially transports neutral branched and aromatic amino acids
Highly expressed in brain capillaries compared to other tissues
Found in luminal and abluminal surfaces of BBB

Question 27

Brain tumours

Answer 27

100 different types of brain tumours
Half of all brain tumours are gliomas
70-80% of primary brain tumours are high grade gliomas

Question 28

Glioma

Answer 28

Ependymoma develops from ependymal cells which line ventricles
Oligodendroglioma develops from oligodendrocytes which support and insulate axons In the CNS
Astrocytoma develops from astrocytes
Grade 4 astrocytoma is also known as glioblastoma multiforme

Question 29

BBB and brain tumours

Answer 29

BBB surrounding tumour mass generally viewed as leakier- increased extracellular space at basal surface of endothelial cells, down-regulation of TJ proteins and increased paracellular permeability
Angiogenesis
Micro-tumour sites often retain fully capable restrictive BBB

Question 30

In brain parenchyma

Answer 30

Raised interstitial fluid pressure
Fluid movement along white matter tracks
Steroid treatment for vasogenic oedema due to fluid leakage

Question 31

Consequences of VEGF released by tumour

Answer 31

Increased hyper-permeability and attracts monocytes/macrophages
Induces angiogenesis within tumour

Question 32

Bevacizumab

Answer 32

Single approved agent for brain tumours
Humanized monoclonal antibody which neutralises VEGF
Anti-angiogenic agents can transiently normalize the tumour vessels, leading to reduced permeability, better perfusion and delivery of drugs

Question 33

Resistance mechanisms

Answer 33

Other resistance mechanisms exist beyond reliance upon BBB:
P-gp within target cells
Repair mechanisms

Question 34

BBB disruptive approaches

Answer 34

Ultrasound disruption
Hypertonic BBB disruption
Selective bradykinin B2 receptor agonist

Question 35

Do antibodies enter brain via extracellular routes?

Answer 35

Filtrate across endothelium of CVO and access ventricular CSF across leaky ependymal cells, then conductive movement with CSF flow to access pial membrnaes and brain perenchyma
Filtrate at CPE
CSF flow through subarachnoid space and pial surface

Question 36

Functions of the CSF

Answer 36

Gives protection (mechanical and chemical), by circulating exchanges nutrients and wastes
Maintenance of a constant external environment for neurons and glia
Mechanical cushion to protect the brain provides buoyancy
Serves as a lymphatic system and a conduit for neuropeptides
pH of CSF regulates pulmonary ventilation and CBF

Question 37

Anti-epileptic drugs (AEDs)

Answer 37

Patients with epilepsy whose seizures do not successfully respond to AED therapy are considered to have drug resistant epilepsy, also referred to as intractable, medically refractory, or pharmaco-resistant
As many as 20-40% of patients with epilepsy are likely to have refractory epilepsy
Most AEDs are lipophilic. Some evidence from patients and animal models that refractory epilepsy associated with overexpression of Pgp

Question 38

Epilepsy patients

Answer 38

In-vivo PET imaging with Pgp substrate verapamil showed Pgp functional expression increased in capillary endothelial cells of epileptic tissues vs non epileptic tissues
Pgp expression in patients with recurrent seizures significantly higher than in seizure free patients, overexpression of multidrug transporters may be induced by recurrent seizures

Question 39

AED and BBB

Answer 39

Animal models of temporal lobe epilepsy (TLE) drug resistant animals showed:

• increased expression of Pgp and decreased expression of AED targets than did drug-sensitive responsive animals
• lower cerebral cortex extracellular fluid (ECF) concentrations for phenytoin than did responsive animals
• Pgp inhibitors to increase ECF phenytoin concentrations and restore anti-convulsants responsiveness to phenytoin and phenobarbitone

Question 40

BBB transporters

Answer 40

OATP- organic anion transporter polypeptide family- bile acids, cholate, oestrogen conjugates, digoxin
OAT- organic anion transporter family- broad substrate
ENT- equilibrative nucleoside transporter
CNT- concentrative nucleoside transporter- thymidine
Amino acid systems- LAT1 and LAT2
MCT- monocarboxylic acid transporter- statins

Question 41

Parkinson’s disease and LAT1 transporter

Answer 41

A neurodegenerative disorder characterized by progressive motor dysfunction
Signs and symptoms are due to the degeneration of dopaminergic neurons projecting from the substantia nigra to the striatum
Deficiency of dopamine allows relative cholinergic dominance
Carboxyl group important for substrate binding into LAT1

Question 42

Drugs used in the treatment of Parkinson’s- levodopa

Answer 42

Immediate precursor of dopamine, crosses the BBB by means of transporter for aromatic amino acids, activity due to conversion to dopamine in CNS
Bradykinesia and rigidity quickly reversed, reversal of tremor requires continual therapy, changes in mood associated with Parkinson’s are reversed

Question 43

Drugs used in the treatment of Parkinson’s- carbidopa

Answer 43

L-aromatic amino acid decarboxylase (LAAD) is responsible for the conversion of dopa to dopamine
LAAD activity causes 95% of a dose of dopa to be converted to dopamine before entering the CNS
Carbidopa is an inhibitor of this peripheral decarboxylase and allows greater amounts of dopa to enter the CNS

Question 44

Extent of blood derived solutes accessing brain as measured by CSF

Answer 44

80% of CSF proteins derived as a filtrate of blood
Human total CSF=150mL
Production=300-500mL/day, entire volume renewed every 8-12 hours
CSF lymphocytes and monocytes (5000 cells/mL) =0.1% of levels in serum
Serum/CSF albumin 200:1 =2%
Serum/CSF IgG 800:1 =0.1%

Question 45

Therapeutic antibodies and intact BBB

Answer 45

Target the enzyme BACE1
BACE1 contributes to the processing of APP into AB peptides including those molecular species that aggregate to form the amyloid plaques found in the brains of Alzheimers patients
Blocking the activity of BACE1 should reduce production of the aggregation prone AB peptides, thus decreasing amyloid plaque formation and slowing Azheimers progression
Active site of BACE1 is relatively large, as such small molecule inhibitors have limited efficacy or specificity i.e. bulky molecules may be better inhibitors

Question 46

Transcytosis across the intact BBB

Answer 46

Transferrin protein binds Fe3+ to become Holo-Tf, at neutral pH Holo-TfR binds to Tfr
Endocytosis of TfR and bound Holo-Tf
Endosomal acidification leads to release of bound iron in endosome, endosome Apo-Tf remains bound to TfR
Classically Apo-Tf recycled to original membrane where at physiologic pH it is released
Free Fe2+ in the endosome transported into cytoplasm by divalent metal transporter
Cytosolic Fe2+ transported across basolateral membrane via ferroportin-1 transporter

Question 47

Increased brain delivery by using bi-specific antibodies

Answer 47

Can exploit anti-TfR antibodies to deliver anti-BACE activity
Lower affinity anti-TfR antibodies able to deliver more effectively to the brain