11.1 Blood-brain Barrier Maintains the Constancy of the Brain's Internal Environment
The blood-brain barrier (BBB) protects the neural tissue from variations in blood composition and toxins. Elsewhere in the body the extracellular concentrations of hormones, amino acids and potassium undergo frequent fluctuations, especially after meals, exercise or stressful times. Since many of these molecules regulate neuronal excitability, a similar change in the composition of interstitial fluid in the CNS can lead to uncontrolled brain activity. The endothelial cells forming the blood-brain barrier are highly specialized to allow precise control over the substances that enter or leave the brain.
Discovery of blood-brain barrier. The discovery of the BBB dates back more than 100 years when, in the 1880s, Paul Ehrlich observed that intravenous administration of certain dyes (e.g. trypan blue) stained all organs except the brain and the spinal cord. He concluded that the dyes had a lower affinity for binding to the nervous system as compared to other tissues. In 1913, Edwin Goldman, an associate of Ehrlich, demonstrated the very same dyes, when directly injected into the cerebrospinal fluid (CSF), readily stained nervous tissue but not other tissues. The term “blood-brain barrier” was coined, however, by Lewandowsky in 1898, after he and his colleagues had performed experiments to demonstrate that neurotoxic agents affected brain function only when directly injected into the brain but not when injected into the vascular system. It took an additional 70 years until Reese and colleagues localized the barrier to the capillary endothelial cells within the brain by electron-microscopic studies.
Figure 11.1a |
Figure 11.1b |
Endothelial cells in brain capillaries are the site of the BBB. The BBB in adults consists of a complex cellular system of a highly specialized basal membrane, a large number of pericytes embedded in the basal membrane and astrocytic end feet. Whereas the endothelial cells form the barrier proper, the interaction with adjacent cells seems to be required for the development of the barrier. The brain endothelial cells differ from endothelial cells from other organs in two important ways. First, continuous tight junctions are present between brain endothelial cells. These tight junctions prevent paracellular movement of molecules. Second, there are no detectable transendothelial pathways such as intracellular vesicles. These properties of brain endothelial cells provide a barrier between the blood and the brain. Some of the key processes are depicted in Figure 11.1.
Figure 11.2 Components of the BBB and the transport of molecules across the barrier. |
- The continuous tight junctions that join the endothelial cells in the brain capillaries limit the diffusion of molecules across the BBB.
- The basement (basal) membrane provides structural support for the capillary and specific proteins present in the basement membrane have been proposed to be involved in the development of the BBB.
- Astrocytic foot processes release specific factors and are necessary for the development of the BBB. Astrocytic foot processes contain water channels (aquaporin-4) that allow water uptake and contribute to brain swelling.
- Transport carriers for glucose and essential amino acids facilitate the movement of these solutes into the brain. Since brain cells cannot synthesize these essential amino acids, it is taken up from the blood.
- Secondary transport systems appear to cause efflux of small molecules and nonessential amino acids from the brain to the blood.
- Sodium ion transporters on the luminal membrane and Na,K-ATPase on the anti-luminal membrane account for movement of sodium from the blood to the brain. The large number of mitochondrias present in the brain endothelial cells provide energy for the function of this Na,K-ATPase.
- The "enzymatic blood-brain-barrier": Metabolic processes within the brain capillary endothelial cells are important to blood-brain function and control the entry of neurotransmitters into the brain.
11.2 Molecular Components of Tight Junctions
Figure 11.3 |
The tight junctions between endothelial cells are responsible for the barrier function. Occludin was the first integral membrane protein found to be exclusively localized within the tight junctions. However, mice carrying the null mutation in the occludin gene develop morphologically normal tight junctions, indicating that occluding is not essential for proper tight junction formation. In contrast to occluding, claudins have been shown to be required for the formation of tight junctions. The integral membrane proteins of tight junctions are linked to the cytoskeleton via zone occluding-1 (ZO-1), ZO-2 and ZO-3. In addition, non-occludin adherens junctions are found intermingled with tight junctions. In adherens junctions, the endothelial specific, integral membrane proteins VE-cadherins are found. Furthermore, a family of proteins called junctional adhesion molecules (JAM), and recently discovered endothelial cell-selective adhesion molecules (ESAM), are localized in the tight junctions of the BBB. Their precise function in BBB integrity remains to be determined.
All areas of the brain do not have a blood-brain barrier. The structures located at strategic positions in the midline of the ventricular system and lack the BBB are collectively referred to as circumventricular organs (CVOs). In these non-barrier regions, the tight junctions between endothelial cells are discontinuous thus allowing entry of molecules. Many of these areas participate in hormonal control.
Areas of brain without a blood-brain barrier:
- Pituitary gland
- Median eminence
- Area postrema
- Preoptic recess
- Paraphysis
- Pineal gland
- Endothelium of choroid plexus
Figure 11.4 |
Substances with High Lipid Solubility May Move Across the BBB by Simple Diffusion. Diffusion is the major entry mechanism for most psychoactive drugs. Figure 11.2 shows that the rate of entry of compounds that diffuse into the brain depends on their lipid solubility. The lipid solubility is estimated by oil/water partition coefficient.
Figure 11.5a depicts how the oil/water partition coefficient is calculated. Figure 11.5b shows the relationship between oil/water partition coefficient and brain penetration of selected molecules.
Figure 11.5a |
Figure 11.5b |
Figure 11.6 |
Water. Water readily enters the brain. As a consequence of its high permeability, water moves freely into and out of the brain as the osmolarity of the plasma changes. This phenomenon is clinically useful, since the intravenous administration of poorly permeable compounds such as mannitol will osmotically dehydrate the brain and reduce intracranial pressure. This method is sometimes used in head trauma patients to reduce intracranial pressure.
Gases. Gases such as CO2, O2 and volatile anesthetics diffuse rapidly into the brain. As a consequence, the rate at which their concentration in the brain comes into equilibrium with plasma is limited primarily by the cerebral blood flow rate.
11.3 Transport of Glucose and Amino Acids
Figure 11.7 |
Carrier-mediated transport enables molecules with low lipid solubility to traverse the blood-brain barrier. Glucose from blood enters the brain by a transport protein. Glucose is the primary energy substrate of the brain. Glucose transport protein (GLUT-1) is highly enriched in brain capillary endothelial cells. These transporters carry glucose molecules through the blood brain barrier. Although rare, patients with Glut-1 deficiency (caused by genetic mutations) can have severe learning difficulties. Low glucose sugar levels in the cerebrospinal, but not in the blood, will identify the condition.
The essential amino acids cannot be synthesized by the brain and, therefore, must be supplied from protein breakdown and diet. Phenylalanine, leucine, tyrosine, isoleucine, valine, tryptophan, methionine and histidine, which are essential amino acids, and also the precursor of dopamine, L-DOPA, enter the brain as rapidly as glucose. These amino acids are transported into the brain by the leucine-preferring or the L-type transport proteins. These compounds compete with each other for entry into the brain. Therefore, an elevation of plasma level of one will inhibit uptake of the others. This competition may be important for certain metabolic diseases such as phenylketonuria (PKU), where high levels of phenylalanine in plasma reduce brain uptake of other essential amino acids.
Small neutral amino acids, such as alanine, glycine, proline and GABA (gamma-aminobutyric acid), are markedly restricted in their entry into the brain. These amino acids are non-essential amino acids and are transported by alanine-preferring or A-type transport protein. The A-type transport protein is not present on the luminal surface of the blood brain barrier. In contrast, these small neutral amino acids appear to be transported out of the brain across the blood-brain-barrier.
11.4 Protection of Brain from Blood-borne Neurotoxins and Drugs
P-glycoproteins are ATP-driven pumps which confer multi-drug resistance to cancer cells by pumping drugs out of the cells. These proteins are expressed in brain endothelial cells that can limit the BBB permeability of hydrophobic compounds, such as cyclosporin A and vinblastine, by pumping them from the endothelial cells back to the blood.
Metabolic processes within the brain capillary endothelial cells are important to blood-brain function. Most neurotransmitters present in the blood do not enter the brain because of their low lipid solubility and lack of specific transport carriers in the luminal membrane of the capillary endothelial cell (see Figure 11.1). In contrast, L-DOPA, the precursor for dopamine, has an affinity for the L-type transporter. Therefore, it enters the brain more easily from the blood than would be predicted based on its lipid solubility. Patients with Parkinson's disease are treated with L-DOPA rather than with dopamine because of this fact. However, the penetration of L-DOPA into the brain is limited by the presence of enzymes L-DOPA decarboxylase and monoamine oxidase within the capillary endothelial cells. This "enzymatic blood-brain barrier" limits passage of L-DOPA into the brain and explains the need for large doses of L-DOPA in the treatment of Parkinson's disease. Therapy is currently enhanced by concurrent treatment with an inhibitor of L-DOPA decarboxylase.
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Figure 11.8a |
Figure 11.8b |
Endothelial monoamine oxidase may also play a role in the inactivation of neurotransmitters released by neuronal activity. Monoamines show very little uptake when presented from the luminal side. The uptake systems for monoamines are present in the antiluminal surface of the brain capillary endothelial cells. The brain endothelial capillary also contains a variety of other neurotransmitter-metabolizing enzymes such as cholinesterases, GABA transaminases, aminopeptidase and endopeptidases. In addition, several drug and toxin metabolizing enzymes are also found in the brain capillaries. Thus the "enzymatic blood brain barrier" protects the brain not only from circulating neurotransmitters but also from many toxins.
Compromised BBB and Disease. BBB dysfunction can lead to neuronal damage and disturbed brain function. Diseases such as encephalitis, multiple sclerosis (MS), stroke or tumors induce deterioration of the BBB with devastating influence on neuronal function. These conditions decrease the production of the tight junction protein claudin. Brain tumors cause complete breakdown of the BBB that leads to peritumoral edema. Furthermore, tumor cells secrete specific factors [e.g. vascular endothelial growth factor (VEGF) that induces formation of new blood vessels (or angiogenesis)] which tend to be leaky.
Figure 11.9 |
Bypassing the BBB with drugs. A number of drugs of potential therapeutic value do not readily enter the brain because they have low lipid solubility and are not transported by specific carriers present in the BBB. To overcome this limitation, schemes have been developed to enhance drug entry into the brain. 1) One way to bypass the BBB is to deliver the drug directly into the CSF. This approach can be used to treat patients with meningitis or cancerous cells in the CSF. 2) Certain vasoactive compounds such as bradykinin and histamine, which do not alter BBB in normal people, can enhance permeability of BBB in pathological conditions. These compounds can be used to deliver chemotherapeutic agents into the brain. 3) Drugs can be synthesized with high BBB permeability to improve entry into the brain. Most neuroactive drugs are effective because they dissolve in lipids and easily enter the brain. For example, heroin and morphine are very similar in structure. However, heroin, which has two acetyl groups, is more lipid soluble. This greater lipid solubility of heroin explains its more rapid onset of action. Once within the brain, the acetyl group of heroin is removed enzymatically to produce morphine, which only slowly leaves the brain. An understanding of the transport process is crucial to development of the next generation of drugs useful in treating brain diseases.
11.5 Cerebral Metabolism and Blood Flow
Cerebral Metabolism
The brain is metabolically one of the most active of all organs in the body. The brain does not store excess energy and derives almost all of its energy needs from aerobic oxidation of glucose. Therefore, it requires a continuous supply of glucose and oxygen to meet its energy requirements. Most of the brain's energy consumption is used for active transport of ions to sustain and restore the membrane potentials discharged during the process of excitation and conduction. When blood flow to the brain stops and absence of oxygen and blood occurs, a loss of consciousness results in 5-10 seconds. If the blood flow is not resumed within several minutes, there is permanent brain damage. It is well known that during crises, such as cardiac arrest, damage to the brain occurs earliest and is most decisive in determining the degree of recovery. The absence of glucose is equally destructive, but the time course resulting in irreversible damage from hypoglycemia is longer because other substrates can be used.
Different regions of the brain have different energy requirements, which are related to the neuronal activity in these regions. Measurement of amounts of glucose used per minute in different brain regions of a normal conscious rat and monkey demonstrates glucose utilization varies widely throughout the brain. Moreover, the average value in the gray matter is approximately five times more than that in the white matter.
The amount of blood flow is directly related to brain activity. In a separate group of animals the amount of blood flow to brain areas was determined. The results show that more blood flows to the area of the brain with high metabolic activity.
Figure 11.10 Relationship between cerebral blood flow and glucose metabolism. |
Figure 11.10 shows there is an excellent correlation between the amount of glucose uses and local cerebral blood flow.
Regulation of blood flow to a brain area is achieved by control of dilation of cerebral vessels. The dilation of blood vessels is controlled by local factors such as nitric oxide (NO), PaCO2, PaO2 and pH. High NO, high PaCO2, low PaCO2 and low pH, which are produced as a result of brain activity, tend to dilate the blood vessels and increase blood flow. The rate of production of these chemicals is dependent of activity and rates of energy metabolism. Therefore, blood flow to a brain region is related to neuronal activity in that region.
Glucose utilization and brain imaging. Glucose metabolism is the major energy source for the brain. Glucose from the blood enters the brain with help of Glut-1 transport protein. Once inside a brain cell, it enters the glycolytic pathway, where it is converted to pyruvate and then metabolized through the Krebs cycle to generate ATP. A fraction of ATP molecules is used to generate high energy phosphocreatine molecules. Under conditions, aerobic metabolism of glucose is capable of providing the brain with sufficient energy from ATP and phosphocreatine to maintain normal function. When brain failure occurs, there is a loss of phosphocreatine initially, followed by ATP depletion, which generally signals severe damage to the brain.
Glucose deprivation can result in abnormal brain function. Hypoglycemia, which can result from excessive insulin, is associated with changes in mental state. These changes can be rapidly reversed by glucose administration. In certain circumstances, such as during starvation, the brain can use “ketone bodies” in place of glucose as substrates. Ketone bodies, acetoacetate and D-beta-hydroxybutyrate are formed from catabolism of fatty acids by the liver. The ketone bodies are metabolized to generate acyl-CoA which enters the tricarboxylic acid cycle (TCA) at a sufficient rate to meet the metabolic demand of the brain.
Measurement of local glucose utilization. The local energy metabolism is coupled to local functional activity. Using an autoradiography analog of glucose, 2-deoxyglucose (2-DG), has been employed to measure glucose metabolism in experimental animals.
Figure 11.11a |
Figure 11.11b |
Figure 11.4 illustrates the fundamental principle of radioactive deoxy glucose method for measuring the local cerebral glucose utilization. Glucose utilization begins with phosphorylation of glucose by hexokinase. The resulting glucose-6-phosphate is not retained in the tissues. Instead, it is metabolized further to products such as CO2 and H2O that leave the tissue. 2-deoxyglucose is an analog of glucose and is transported across the blood-brain barrier by the glucose carrier system. Inside brain cells, 2-deoxyglucose is phosphorylated by hexokinase to deoxyglucose-6-phosphate (DG-6-P) and cannot be further degraded into CO2 and H2O. Instead it is trapped and accumulates in the tissue quantitatively for a reasonable length of time. By putting a label on deoxyglucose (such as in [18F] fluoro-2-deoxy-D-glucose), it is possible to measure the rate of labeled deoxyglucose-6-phosphate formation. The amount of 18FDG-6-phosphate can be directly determined using positron emission tomography (PET). The 2-deoxyglucose method has been modified for human use with PET, with short lived positron emitting isotopes labeled to the 2-deoxyglucose.
11.6 Functional Activation of Energy Metabolism
Figure 11.12 |
Because of the coupling of metabolism to function, functional activation by specific stimuli tasks leads to regional increase in the glucose metabolism in corresponding cerebral structures. Movement of fingers and hands increases metabolism in the respective brain regions. In right handed volunteers, spontaneous speech increased metabolic activities in Broca's region. Presentation of visual images increases glucose utilization in the primary visual cortex.
Functional MRI. A variant of MRI called functional MRI (fMRI) is based on the increase in the blood flow to specific brain regions that accompanies neuronal activity. Increase in blood flow results in a local decrease in deoxyhemoglobin due to less oxygen extraction. Deoxyhemoglobin is paramagnetic and serves as the source for the signal in fMRI. Unlike PET, fMRI uses a signal intrinsic to the brain and has emerged as the technology of choice for probing brain function.
Figure 11.13 |
11.7 Brain Disorders and Metabolism
Convulsive disorders are functional disturbances of brain activity and lead to marked changes in brain metabolism and cerebral blood flow. The metabolic changes detected by PET can frequently complement electrophysiological recordings to locate epileptogenic foci. This information helps neurosurgeons to surgically remove the epileptogenic focus.
Metabolic measurements using PET can be used to determine the size of infarction following ischemic stroke. Brain tumors have high metabolic needs and are heavily vascularized. PET or fMRI can be used to locate the tumor and evaluate effectiveness of a therapy.
