# Blood-brain barrier

The selective physiological barrier between the fluid spaces of the bloodstream and the central nervous system is referred to as the blood-brain barrier , also blood-brain barrier , or blood-brain barrier .

This particular demarcation of the blood (intravascular) from the extravascular space in the brain and spinal cord is developed in all terrestrial vertebrates (Tetrapoda) and enables the nervous tissue to maintain its own environmental conditions ( homeostasis ). Essentially, this barrier is formed by endothelial cells , which are closely linked here in the capillary blood vessels via tight junctions .

The blood-brain barrier protects the brain from pathogens , toxins and messenger substances circulating in the blood . It is a highly selective filter through which the nutrients required by the brain are supplied and the metabolic products that are created are removed. The supply and disposal is guaranteed by a series of special transport processes .

On the other hand, this protective function of the brain complicates the drug treatment of a large number of neurological diseases , since many active substances cannot cross the blood-brain barrier either. The crossing of the blood-brain barrier is a current research topic in order to be able to treat these diseases. Only very few - extremely rare - diseases are directly related to the blood-brain barrier, while it can itself be affected by a significantly higher number of widespread diseases. A disturbance or damage to the blood-brain barrier caused in this way is a complication to be taken very seriously .

The first experiments that indicated the existence of this barrier were carried out by Paul Ehrlich in 1885. However, he misinterpreted his test results. The final proof of the blood-brain barrier was made in 1967 by electron microscopic examinations.

Transmission electron micrograph of a thin section through the telencephalon (endbrain) of an 11.5-day-old mouse embryo. In the upper half of the picture in white, the lumen of the capillary vessel. The endothelial cells are connected to one another via tight junctions (darker "channels"). Below are the Adherens Junctions. The image width corresponds to about 4.2 µm.
Schematic representation of the nerve tissue: 1)  ependyma , 2)  neuron , 3)  axon , 4)  oligodendrocyte , 5)  astrocyte , 6)  myelin , 7)  microglia , 8)  capillary vessel
A network of capillaries supplies brain cells with nutrients.

## Functions of the blood-brain barrier

In humans, the brain makes up around 2% of the body mass. However, the proportion of the nutrient requirement is around 20%. In contrast to other organs in the body, the brain has extremely limited reserves of nutrients or oxygen . The nerve cells are also unable to meet their energy requirements anaerobically , i.e. without elemental oxygen. An interruption in the blood supply to the brain leads to unconsciousness after ten seconds and the nerve cells die just a few minutes later. Depending on the activity of a brain area, its energy requirements and reserves can be very different. In order to be able to adapt the supply to the respective needs, these areas regulate their blood supply automatically.

The complex functions of the brain are tied to highly sensitive electrochemical and biochemical processes, which can only run largely without interference in a constant internal environment, the homeostat. For example, fluctuations in the blood pH value must not be passed on to the brain. Fluctuations in the potassium concentration would change the membrane potential of the nerve cells. The neurotransmitters circulating in the blood vessels must not get into the central nervous system, as they would significantly disrupt the flow of information in the synapses present there . In addition, the neurons are not capable of regeneration in the event of damage caused by a fluctuation in the environment. Ultimately, the brain, as a centrally controlling organ, must also be protected from the effects of foreign substances such as xenobiotics and pathogens . The extensive impermeability of the blood-brain barrier to pathogens , antibodies and leukocytes in the blood makes it an immunological barrier .

On the other hand, the high energy requirements of the brain - in comparison to other organs - produce above-average quantities of metabolic degradation products, which have to be removed again via the blood-brain barrier.

In order to guarantee all these functions (supply, disposal and homeostasis), the cerebral blood vessel system of vertebrates shows a number of structural and functional differences compared to the peripheral blood vessel system. This differentiation causes a large separation of the brain from the surrounding extracellular space and is of essential importance for the protection of the sensitive neural tissue as well as for the maintenance of a constant inner milieu.

Changes in the function of the blood-brain barrier cause changes in the state of the central nervous system, which in turn can lead to dysfunction or disease in the CNS. Accordingly, a number of neurological diseases are directly or indirectly related to the blood-brain barrier.

## Blood Brain Barrier Anatomy

The blood-brain barrier, from the brain to the tight junctions
Comparison between peripheral and cerebral capillaries
Schematic structure of the blood vessels in the brain from the pia artery via arterioles to capillaries.

The essential element of the blood-brain barrier are the endothelial cells with their tight junctions . However, two other cell types, the pericytes and the astrocytes , are of great importance for the function, structure and development of the blood-brain barrier . The cell-cell interactions between endothelial cells, pericytes and astrocytes are closer than in any other cell. These three cell types together form the blood-brain barrier in most vertebrates, the endothelial blood-brain barrier . The following anatomical information relates to the endothelial blood-brain barrier of vertebrates. The glial blood-brain barrier developed in some vertebrates and many invertebrates is listed separately at the end of this chapter.

### The endothelium

Electron microscope image of a cross section of a peripheral blood vessel. The diameter of the capillary is 7 to 8 µm. In the middle is an erythrocyte in black .

As in the peripheral blood vessels, the capillaries are formed by endothelial cells. The endothelium of peripheral capillaries has openings (fenestrations) of approx. 50 nm diameter and intercellular gaps 0.1 to 1 µm in width for the exchange of water and substances dissolved or suspended in it between the blood and the extracellular fluid of the surrounding tissue. In contrast, there are no fenestrations and no intracellular gaps between the endothelial cells in the brain, which is why one speaks of a continuous endothelium . The (luminal) membrane facing the interior of the capillary differs considerably in terms of the type of membrane proteins from the apical side facing the interstitium .

The number of pinocytotic vesicles that enable endocytosis of dissolved substances is very small in the endothelium of the brain.

Comparison of the permeability surface product of capillary vessels of different organs. The permeability of the capillaries for five different model substances of different sizes (semi-logarithmic representation).

In contrast, the number of mitochondria is about 5 to 10 times higher than in the peripheral capillaries. This is an indication of a high energy requirement of the endothelia - among other things for active transport processes - and a high metabolic activity . The blood-brain barrier is not only a physical, but also a metabolic or enzymatic barrier. In the cell membrane of the endothelia there are a number of enzymes in significantly higher numbers than, for example, in the cells of the parenchyma . These include γ-glutamyltransferase , alkaline phosphatase and glucose-6-phosphatase . Metabolizing enzymes such as catechol-O-methyltransferase , monoamine oxidase or cytochrome P450 are also active in the endothelia in relatively high concentrations. In this way, many substances are metabolized before or during their intracellular transport. The endothelial cells are extremely thin at 0.3 to 0.5 µm. In comparison, enterocytes , the epithelial cells of the intestine , are considerably higher at 17 to 30 µm. As with other endothelial cells, the ratio of cholesterol to phospholipids is 0.7. Pure diffusion processes through the cell membrane are therefore just as fast across the blood-brain barrier as with other endothelial cells. The endothelial cells have a large number of aquaporins on their cell membrane for regulating the water balance of the brain . These channels allow water molecules to diffuse freely both towards the brain and towards the blood.

The non-existent fenestrations and the lack of pinocytotic vesicles represent the passive barrier of the blood-brain barrier. This barrier effect can be quantified physically via its electrical resistance . In a healthy adult color rat , the resistance is around 1500 to 2000 Ω cm². In contrast, the value for the capillaries in muscle tissue is around 30 Ω cm².

The active transporter systems of the endothelium, which will be described in more detail later, form the active barrier of the blood-brain barrier.

### The tight junctions

Schematic representation of membrane proteins in tight junctions
Schematic representation of the tight junctions
 A lumen B  apical side C  basal side a  mucus b  apical side c  basolateral surface d  tight junctions f  paracellular space g  cell membrane e  protein complex 1  occludin 2  claudin -1 3  E- cadherin 4  ZO-1 5  JAM-1 6  catenins 7  cingulin 8  actin
Electron microscopic freeze fracture image of the tight junctions of the blood-brain barrier in a rat

The endothelial cells are connected to one another via firm cell-cell connections, the so-called tight junctions . These tight junctions represent dense connections between neighboring cells and make a significant contribution to the barrier function of the blood-brain barrier. This is because they seal the space between the cells and thus practically exclude paracellular transport , the passage of substances along the intercellular space between adjacent endothelial cells past them.

Several transmembrane proteins , such as occludin , various claudins and junctional adhesion molecules (JAM), link the endothelia in the apical area of ​​the lateral cell walls by circumferential protein bands in tight junctions , and thus largely prevent paracellular passage via the intercellular gap between two endothelial cells for larger molecules. The individual protein-protein connections act like fuses connected in parallel. Of the 24 known claudins, the endothelial cells of the blood-brain barrier express only claudin-5 and claudin-12. Claudin-5 is considered to be the most important cell adhesion protein of the blood-brain barrier. Switching off ( gene knockout ) the CLDN5 gene, which codes for claudin-5 , leads to the blood-brain barrier in mice becoming permeable for molecules with a molar mass of up to 800 g · mol −1 . The genetically modified animals died within a few hours of their birth.

The fixation of the endothelia ensures that they are aligned in two very different sides: the luminal side facing the blood and the abluminal side facing the brain.

### The basal lamina

The basal lamina of an epithelial cell

The endothelial cells are completely surrounded by a protein layer, the basal lamina. This double layer is about 40 to 50 nm thick and only visible under an electron microscope. It essentially consists of type IV collagen , heparin sulfate - proteoglycans , laminin , fibronectin and other extracellular matrix proteins . The basal lamina borders on the plasma membrane of the terminal feet of the astrocytes.

### The pericytes

Electron microscope image of a pericyte (right), the capillary with three erythrocytes (left)

Pericytes, especially in the older literature after their discoverer Charles Marie Benjamin Rouget (1824–1904) referred to as Rouget cells , are an important component of the blood-brain barrier. Three essential properties are in the foreground: their ability to contract, the regulation of the endothelia and their macrophage activity.

About 20% of the outer endothelial surface of the cerebral capillaries is covered by the relatively small, oval-shaped pericytes and every second to fourth endothelial cell is connected to a pericyte. The pericytes are preferably located at the contact point between two endothelial cells. Pericytes can be found on almost all arterioles, venules and capillaries - not just in the cerebral endothelium. The degree of coverage of the endothelium with pericytes correlates directly with the impermeability of the tight junctions . In fenestrated capillaries, for example in skeletal muscles , the ratio of endothelia to pericytes is 100: 1.

Like the endothelia, the pericytes are also surrounded by a continuous basal lamina.

#### The cell contact pericyte-endothelium

Schematic representation of the gap junctions.
These channel-forming protein complexes are one type of connection that connects the pericytes to the endothelial cells.

The pericytes are firmly anchored to the endothelial cells. At least three types of pericyte-endothelial contact form the connection between these two cell types. These are gap junctions (channel-forming protein complexes), focal adhesion ( adhesion plaques ) and peg-and-socket intussusception . The gap junctions connect the two cytoplasms directly and enable the exchange of ions and smaller molecules between the two cell types via channels. The pericytes are mechanically firmly anchored to the endothelia via the focal adhesion. The peg-and-socket invaginations consist of mutually interwoven protuberances of the two interconnected cells. This type of cell contact also obviously contributes to the mechanical anchoring of the pericytes on the endothelia.

Both cell types mutually influence one another with regard to their mitosis and presumably also with regard to their phenotypic expressions .

#### contraction

Pericytes have a high proportion of the contractile structural protein actin . This allows you to change the capillary diameter via their extensions and thus regulate the blood pressure locally.

#### Macrocytic Properties

Obviously, only the pericytes on the cerebral capillaries have a special property. There they act as macrophages . There are many lysosomes in the cytoplasm of the pericytes and in vivo they show the ability to take up soluble tracers that are administered intravenously or intraventricularly and are otherwise only taken up by macrophages. The ability of pericytes to phagocytose and to present antigens was demonstrated in tissue cultures .

These macrocytic properties are a "second line of defense " against neurotoxic molecules that have already passed the endothelial layer towards the brain. Pericytes therefore make a significant contribution to the immune system of the central nervous system. On the other hand, through these properties, they are also involved in the development of various diseases, especially autoimmune diseases . An indirect involvement in Alzheimer's disease is also discussed.

#### Other properties of the cerebral pericytes

The pericytes regulate cell division and the differentiation of the endothelia. In addition, they are precursor cells ( progenitor cells ) formed from pluripotent adult stem cells that can differentiate into osteoblasts , adipocytes , chondrocytes and fibroblast-like cells. Some authors therefore refer to pericytes as pluripotent.

Pericytes synthesize a number of vasoactive agonists and play an important role in the formation of new blood vessels ( angiogenesis ).

### The astrocytes

An astrocyte (green) in a cell culture
Interleukin-6, a messenger substance in the endothelia

Astrocytes are star-shaped, branched cells from the macroglia family that are significantly larger than the pericytes . They are assigned to the central nervous system and are still capable of dividing after birth. In higher vertebrates they have no direct barrier function, even if they cover about 99% of the capillaries in the brain with their terminal feet. However, they are in direct interaction with the endothelia. Astrocytes induce the function of the blood-brain barrier in the endothelia of the cerebral blood vessels. This has been proven by transplant trials. Cerebral blood vessels that were transplanted into peripheral organs behaved like the “normal” capillaries there and, for example, formed fenestrations. In the reverse experiment, peripheral capillaries that were transplanted into the central nervous system assumed the phenotype there with tight junctions. The influence of astrocytes on the phenotypic expression of the endothelia can also be seen in in-vitro tests. In co-cultures from astrocytes and endothelial cells, the endothelia are more dense than in pure endothelial cultures .

Astrocytes release a number of messenger substances that can modulate the permeability of the endothelium in the range of seconds to minutes. Conversely, the endothelial cells secrete leukemia-inhibiting factor (LIF), a cytokine of the interleukin-6 class, which induces the differentiation of astrocytes. The distance between the astrocyte feet and the endothelial cells and pericytes is only 20 nm.

The main task of the astrocytes, however, is the selective supply of the neurons with nutrients and the regulation of the extracellular ion concentrations. Most of the cholesterol in the brain is produced by astrocytes. Cholesterol cannot cross the blood-brain barrier, which is why it has to be synthesized locally within the brain. About 25% of the body's cholesterol is found in the brain; essentially in the myelin that coats the axons of the neurons.

Representation of the terminal feet of the astrocytes on the endothelial cells

The terminal feet of the astrocytes form a delicate rosette-like contact with the endothelial cells. This arrangement is important for the mutual influencing and communication of the two cell types. Free diffusion between the endothelium and the parenchyma of the brain is also possible.

Diseases that directly or indirectly affect the astrocytes, such as Alzheimer's disease or astrocytomas , can significantly impair the functioning of the blood-brain barrier due to the close interaction between astrocytes and endothelium.

### Areas of the brain without a blood-brain barrier

Lateral section through the brain
The ventricular system of the brain in humans

Not all capillaries in the brain are built up as blood-brain barriers. For example, areas of the brain that release neuroendocrine compounds ( hormones ) into the blood or that have a sensory function - for example for certain peptide hormones - are necessarily without a blood-brain barrier. This is the case with six circumventricular organs . The pineal gland , the eminentia mediana , the neurohypophysis , the area postrema , the organum vasculosum laminae terminalis and the organum subfornicale have fenestrated capillaries. For example, as the neurons of the can area postrema detect for the body of toxic substances and the vomiting center a nausea stimulate. In the organum subcommissurale , the blood-brain barrier is the only one of the circumventricular organs. To protect the surrounding brain tissue, the circumventricular organs are surrounded by tanycytes . These are special ependymal cells with very dense tight junctions.

### Blood-brain barrier data

A network of over 100 billion capillary vessels, the total length of which is approximately 600 km for an adult, runs through the entire brain. The average distance between these blood vessels is 40 µm. The different brain regions are supplied to different degrees. The density of capillary vessels is highest in the cerebral cortex ( cortex cerebri ) with 300 to 800 capillary cross-sections per mm² of tissue.

The total area of ​​the blood vessels in the brain is between 12 and 20 m². About 610 ml of blood flow through these vessels per minute, the mean flow rate being 1 mm / s. The mean blood pressure is in the range from 15 to 35  mmHg . The mean transit time ( mean transit time MTT) is 5 seconds. In comparison, it takes about 40 hours in the intestine, whose blood vessels have a surface area of ​​180 m², and 30 seconds in the liver - with 70 m².

### The development of the blood-brain barrier

Up until the end of the 20th century it was assumed that the blood-brain barrier in fetuses and newborns was not yet fully developed (“immature”) or even lacking. The reason for this opinion, which is still widespread today, are methodological inadequacies in earlier physiological experiments. In most cases, protein-binding dyes or proteins themselves were injected into animal fetuses as markers. The first attempts to do this were carried out as early as 1920. In these experiments the markers were detected in the brain or in the CSF of the fetuses, while they were not detectable in the adult animals. However, either volumes were injected, some of which reached the blood volume of the test animals, or the binding capacity of the plasma proteins was exceeded by a factor of two. In both cases, the osmotic pressure is so high that the sensitive embryonic capillary vessels can partially tear. In experiments with reduced marker volumes, no passage of the markers into the brain was found.

If one looks at the body's own marker molecules, such as albumin , α-1-fetoprotein or transferrin , which are found in high concentrations in the plasma even at a very early embryonic stage, these molecules cannot be detected in the extracellular space of the brain at any time. The efflux transporter P-glycoprotein is also already present in the embryonic endothelia. The blood-brain barrier is therefore already present in the prenatal stage. However, this does not rule out changes, especially at the tight junctions, in the course of embryonic development. The tight junctions themselves are present between the endothelial cells at an extremely early stage, but are subject to progressive development.

For small polar molecules such as inulin or sucrose , the permeability of the prenatal and neonatal blood-brain barrier is significantly higher than that of the adult. The same effect can also be observed with ions. The transport of amino acids across the embryonic blood-brain barrier is - obviously due to the great need for further development of the brain - also significantly increased. The same applies to the hormone insulin.

On the other hand, the embryonic brain forms additional morphological barriers that are no longer present in the adult brain. In the embryonic brain, for example, there are so-called strap junctions on the inner ependyma at the interface between liquor and brain tissue .

### The blood-brain barrier in the animal kingdom and in the course of evolution

Phylogenetic relationships of the jaws (Gnathostomata)

In the course of evolution, the neural tissue of invertebrates and vertebrates became larger, more complex and assumed an increasingly central role in the respective organism. As a result, the coordination of the body functions could be continuously improved. This in turn leads to an advantage in the selection. On the other hand, a brain that is becoming larger and more complex must also be better supplied with nutrients and freed from metabolic products. In the case of more highly developed invertebrates, for example from the order of the decapods or the class of the cephalopods , and all vertebrates, this is ensured by a widely ramified capillary network in the brain. The formation of a protective barrier against xenobiotics, toxins and other substances harmful to the neuronal system is another evolutionary advantage.

Many invertebrates do not have a blood-brain barrier. This means that the endothelia are usually only incompletely lined or with gaps. In invertebrates with a blood-brain barrier, such as insects , crayfish (Crustacea) and cephalopods (Cephalopoda), it is exclusively glial in nature. In these cases one speaks of a glial blood-brain barrier .

The model organism Drosophila , for example, forms a glial blood-brain barrier.

All vertebrates have a blood-brain barrier, and almost all of them form the endothelial blood-brain barrier described in detail in the previous chapters, in which the tight junctions of the endothelia make the essential contribution to the barrier effect. The barrier effect of the blood-brain barrier is only guaranteed by the perivascular astrocytes in the subclass of the slab gill (Elasmobranchii), which includes sharks and rays , as well as the family of sturgeon . It is assumed that, in the course of evolution, the endothelium, if it was dense enough, took over the barrier effect. The glial blood-brain barrier is therefore also regarded as the original form of the blood-brain barrier. In the family of the pike fish (Polypteridae) and the order of the lung fish , the blood-brain barrier is formed by the tight junctions of the endothelia. In contrast, in the real sturgeon it is formed by a complex covering of glial cells without any recognizable tight junctions.

Today found in some vertebrates elements of glial barrier, such as in the glial limiting membrane of the avascular (avascular) spinal cord of the lamprey and in mammals in certain ependymialen structures such as the Tanyzyten, the latter especially in the circumventricular organs, the choroid plexus and the pigment epithelium the retina (RPE). Astrocytes are in principle able to form tight junctions with each other, even in mammals. This can be demonstrated, among other things, in the olfactory envelope cells (OEC, English olfactory ensheathing cells ), which enclose growing axons.

From the structural differences in the phenotype of the glial blood-brain barrier in invertebrates, it can also be deduced that such barriers arose several times and independently of one another in the course of evolution . The endothelial barriers offer a significant advantage in selection - presumably due to the stricter separation of the function of endothelium and astrocytes. The main selection pressure probably came from the need for homeostasis. It is believed that the endothelial blood-brain barrier developed at least six times in the course of evolution and that all vertebrates 400 to 500 million years ago were provided with a glial blood-brain barrier.

## Blood-liquor barrier

In addition to the blood-brain barrier, the blood-liquor barrier is the second border between the blood circulation and the central nervous system . This blood-liquor barrier is formed by epithelial cells and tight junctions of the choroid plexus . The blood-liquor barrier also ensures the homeostasis of the central nervous system. Only low molecular weight substances can pass through them as ultrafiltrates from the intravascular space of the blood vessels into the liquor space or those that are actively absorbed and secreted by the ependymal cells of a choroid plexus. However, the contribution to the exchange of substances is small and can by no means meet the needs of the brain. The exchange area of ​​the plexus epithelia is about 5000 times smaller than that of the intracerebral capillary network that forms the blood-brain barrier.

In addition to these two barriers that are so important for the central nervous system, other similar, highly selective barriers can be found in the human body that control the exchange of substances via the blood. These include the blood-placenta barrier , the blood-testis barrier , the blood-urine barrier , the blood-eye barrier , the blood-thymus barrier and the blood-air barrier .

## Transport processes of the blood-brain barrier

The transport processes at the blood-brain barrier

Despite its function as a protective barrier, the blood-brain barrier must also ensure the transport of nutrients to the brain and the removal of metabolic products from the brain. Water-soluble nutrients and peptides cross the blood-brain barrier mainly through specific transporters or special channels in the cell membrane. Most other soluble compounds pass, if at all, this barrier by diffusion.

### Paracellular transport

In peripheral capillaries, substances are transported to organs and muscles mainly through fenestrations and intracellular fissures. In a healthy and intact cerebral endothelium, the individual endothelial cells are tightly connected to one another via the tight junctions. The capillary vessels of the brain therefore mainly allow only a transmembrane transport of substances, which can also be better regulated by the cells than the paracellular transport. Water, glycerine, and urea are examples of small polar compounds that can diffuse through the tight junctions of the blood-brain barrier.

### Free diffusion

Schematic representation of the diffusion processes on the cell membrane
Dome model of the plasma membrane with a kink that creates free space (green) for an ethanol molecule

The simplest form of transport through the blood-brain barrier is free diffusion . This exchange, also known as passive transport, can in principle take place both through the cell membrane of the endothelia and through the tight junctions . As with every diffusion, an equalization of the concentration or the equalization of an electrochemical gradient is sought. With free diffusion, no energy is required from the cell for membrane transport. The material flow is proportional to the concentration and cannot be regulated by the cell.

The lipophilic (“fat-friendly”) properties of the cell membrane and their close connection via tight junctions reduce the number of substances that can cross the blood-brain barrier by diffusion, however, considerably. The permeability of the blood-brain barrier for a particular molecule is directly related to its lipophilicity. With regard to the molar mass , it is inversely proportional. That is, the more lipophilic and smaller a compound is, the easier it can diffuse through the endothelium. A maximum size of 400 to 500 g · mol −1 with an intact blood-brain barrier is given as the limit value for the molar mass of a molecule . Molecules above this limit cannot diffuse across the blood-brain barrier. However, the blood-brain barrier should not be understood as a discrete barrier that completely retains a certain size of molecules and allows a smaller one to completely diffuse into the brain. The diffusion processes at the blood-brain barrier are dynamic equilibria. For a molecule with a cross-sectional area of ​​0.52 nm², which corresponds to a molar mass of about 200 g · mol −1 , the blood-brain barrier is 100 times more permeable than for a molecule with an area of ​​1.05 nm² (= 450 g mol −1 ).

Diagram showing log P (octanol / water) on the x-axis versus log Pc (the permeability coefficient of capillaries in rat brains in cm / s) for various substances. Above the diagonal there are substances that either pass through the channel / transporter or, due to their small molecule diameter, pass the blood-brain barrier disproportionately. Below the diagonal are the connections that are prevented from diffusing to the brain by means of efflux.

In 1971 the Göttingen biophysicist Hermann Träuble developed a theory about the transport of small molecules through the cell membrane. According to this, small cavities located between the fatty acid chains of the lipid bilayer are responsible for the diffuse transport. The cavities are created by transitions from the anti - to gauche - conformation ( trans-gauche-trans Kinks ) in the fatty acid residues of the phospholipids of the cell membrane. There are only very low energy thresholds between these conformational transitions. The Kinks (engl. Kink , bend ') are mobile and migrate with the molecule to be transported through the membrane. Träuble's theory was confirmed by NMR spectroscopy by Anna and Joachim Seelig in 1974 .

With regard to lipophilicity, the partition coefficient in octanol / water is an important indicator of the ability of a substance to diffuse through the blood-brain barrier. The partition coefficient is usually in logarithmic form as log P stated. A log P value of a substance of, for example, 3.8 means that this substance is distributed in octanol (lipophilic) in a concentration that is 10 3.8 times higher than in water (hydrophilic). If the log P value is 0, the substance is distributed equally in both phases; if it is negative, the substance is hydrophilic. In principle, lipophilic substances can most easily pass through the cells' plasma membrane, which is made up of fatty acids. If the log P value is above 3, this effect usually decreases again, since such molecules have a high binding affinity for plasma proteins .

The prediction of the ability of a substance to penetrate the brain is possible using various models and simulations ex vivo or in silico . Reliable in-vitro studies can be carried out on isolated endothelial cell cultures.

Lipophilicity and small molecule size are no guarantees of possible diffusion to the brain. For example, over 98% of all low molecular weight pharmaceuticals ( small molecules ) are unable to cross the blood-brain barrier. High molecular substances such as monoclonal antibodies , recombinant proteins , antisense RNA or aptamers are retained by the blood-brain barrier per se.

### Channel-mediated permeability

Schematic representation of a channel protein in the cell membrane
Side view of an aquaporin model. Water molecules can diffuse in and out of the endothelium through the middle of the protein.

Small polar molecules, such as water, can only diffuse through the endothelium to a very limited extent via the hydrophobic kinks . Nevertheless, the blood-brain barrier has a very high permeability for water. This was demonstrated by William Henry Oldendorf (1925–1992) in 1970 in experiments with tritium- labeled water.

For the diffusion of water there are special hydrophilic channel proteins in the cell membrane, the aquaporins . While non-cerebral endothelia very often express aquaporin-1 (AQP1), this gene is inactivated at the blood-brain barrier. The presence of the astrocytes inhibits AQP1 expression. The specialized cerebral endothelia mainly express aquaporin-4 (AQP4) and aquaporin-9 (AQP9).

The water balance of the brain is regulated via the aquaporins. They allow a high capacity for the diffusion of water in both directions, following the osmotic gradient. For glycerol, urea and monocarboxylates forming Aquaglyceroporin own channels in the plasma membrane. At the blood-brain barrier, this is essentially aquaporin-9, which is also responsible for water transport.

Channels are much faster than transporters for molecule transport. The ion channels can be activated or deactivated ( gating ) by voltage pulses or interacting hormones and other influencing factors .

### Facilitated diffusion

Schematic representation of the facilitated transport (the three right representations). On the left, in comparison, a canal transport.
Schematic representation of the energy pathways and energy stores of the central nervous system.

A special form of diffusion through the cell membrane of the endothelial cells is the facilitated diffusion (engl. Facilitated diffusion ). Vital nutrients such as glucose and many amino acids are too polar and too large to be made available to the brain in sufficient quantities via the blood-brain barrier on the previously described transport routes. There is a special transport system for these molecules in the cell membrane: the so-called carrier-mediated transport . For example, D- glucose is transported into the brain via the GLUT-1 transporter. The density of GLUT-1 transporters is four times higher on the abluminal side of the endothelia than on the luminal side, i.e. the side facing the blood. The transport is only made possible by a concentration gradient and does not require any energy itself.

In addition to the D- glucose required by the brain in relatively large quantities, there are a number of other special transporters in addition to GLUT-1. Many of these transporters are from the Solute Carrier (SLC) family, including MCT-1 and MCT-2, which transport a number of short-chain monocarboxylic acids - including lactate , pyruvate , mevalonate , various butyrates and acetate . SLC7 transports cationic amino acids ( arginine , lysine and ornithine ). To date, 307 SLC genes are known from the mouse model organism . Of these, over 200 are expressed in the choroid plexus and other brain areas. However, so far only 36 have been detected at the blood-brain barrier of the same species . About 70% of the 36 have gene products that are involved in the transport of nutrients, vitamins, hormones and trace elements. The thyroid transporters SLC16a2 and SLCO1c1, the sulfate transporter SLC13a4, the L - ascorbic acid transporter SLC23a2, the amino acid transporter SLC 38a3 and the folate transporter SLC19a1 are particularly strongly expressed at the blood-brain barrier . Ascorbic acid (vitamin C) is supplied to the brain in oxidized form via the glucose transporter.

The transporters can work as a uniporter (only in one direction), as a symporter in two directions and as an antiporter in the opposite direction.

### Active transportation

In the passive types of transport through the endothelium described above, the molecules get to the brain or away from the brain without any additional need for energy. They follow the respective concentration gradient. With active transporters, so-called "pumps", transport is also possible against a concentration gradient. However, this directly or indirectly requires energy in the form of adenosine triphosphate. If the active transport takes place from the blood to the brain, one speaks of influx ("inflow"). In the opposite direction, one speaks of efflux ("drainage").

At the blood-brain barrier there are active influx transporters for leu-enkephalin , arginine-vasopressin (AVP) and [D-penicillamin2, D-penicillamin5] -enkephalin (DPDPE).

Schematic representation of the efflux transports on the endothelium of the blood-brain barrier.

P-glycoprotein - the gene product of the MDR1 gene  - was the first efflux transporter to be identified at the blood-brain barrier. The multidrug resistance-related proteins , for example MRP1, which also belong to the class of ABC transporters , were later added . The Breast Cancer Resistance Protein ( Breast Cancer Resistance proteins , BCRP) is located together with the P-glycoprotein essentially on the blood directed towards the luminal side of the endothelium.

Some of the efflux transporters - like some of the influx transporters - work stereoselectively . This means that they only transport one enantiomer from the brain into the blood vessel system. D - aspartic acid is a precursor of N -methyl- D -aspartate (NMDA) in the brain and influences the secretion of various hormones, such as luteinizing hormone , testosterone or oxytocin . The L contrast aspartic belongs together with L - glutamic acid to the excitatory amino acids. The efflux transporter ASCT2 (alanine-serine-cysteine ​​transporter) of the blood-brain barrier only transports the L -enantiomer of aspartic acid, the accumulation of which would have neurotoxic effects. In contrast , the D enantiomer required for the construction of NMDA is not transported by ASCT2. The EAAT transporters ( excitatory amino acid transporters ) SLC1A3, SLC1A2 and SLC1A6, on the other hand, transport both isoforms of aspartic acid.

In epileptic tissue, P-glycoprotein is overexpressed in the endothelia and astrocytes of the blood-brain barrier and in the neurons.

Furthermore, the organic anion transporters (OAT and OATP) and organic cation transporters (OCT) are located in the cell membrane of the endothelia. The efflux transporter in particular can actively transport a large number of different substrates from the endothelium into the capillaries.

For a number of transport processes on the endothelium, it is still unclear whether they are active (ATP-consuming) or carrier-mediated processes.

### Vesicular transport

#### Receptor-mediated transcytosis

A comparison of phagocytosis, pinocytosis and receptor-mediated endocytosis

There is receptor-mediated transcytosis for the transport of selected large molecules . Special receptors that protrude through the cell membrane into the lumen are responsible for recognizing the substances to be absorbed. Thus, for example, passes the 75.2  kDa heavy and consisting of 679 amino acids, transferrin from the blood into the extracellular fluid of the brain. The receptors in the lumen are internalized after the transferrin has been attached, that is, they are introduced into the cell interior. It is then transported to the other side of the cell (abluminal side) and discharged via coated pits . The same mechanism enables the transport of low-density lipoprotein to the brain via the LDL receptor in order to be able to produce cholesterol there . Also, insulin and other peptide hormones and cytokines go on this way to the brain.

In adsorptive-mediated transcytosis ( AMT), electrostatic interactions between the cell surface negatively charged by glycoproteins and positively charged molecules ( cations ) cause transport through the cytoplasm of the endothelia. This form of transport is also known as cationic transport . Peptides and proteins, for example, whose isoelectric point is basic, have a positive charge . Cationic transcytosis through the endothelium of the blood-brain barrier enables a higher degree of substance transport than receptor-mediated transcytosis.

### The most important transporters at the blood-brain barrier

 Transporter Designation (english) gene Chromosome, gene locus family source Facilitated diffusion GLUT1 glucose transporter 1 SLC2A1 1 p35-p31.3 solute carrier LAT1 large neutral amino acid transporter 1 SLC7A5 16 q24.3 solute carrier CAT1 cationic amino acid transporter 1 SLC7A1 13 q12.3 solute carrier MCT1 monocarboxylic acid transporter 1 SLC16A1 1 p13.2-p12 solute carrier CNT2 concentrative nucleoside transporter 2 SLC28A2 15 q15 solute carrier CHT1 choline transporter 1 SLC5A7 2 q12 solute carrier NBTX nucleobase transporter ? ? Active efflux MDR1 P-glycoprotein ABCB1 7 q21.1 ATP-binding cassette MRP1 multidrug resistance protein 1 ABCC1 16 p13.1 ATP-binding cassette MRP3 multidrug resistance protein 3 ABCC3 17 q22 ATP-binding cassette MRP4 multidrug resistance protein 4 ABCC4 13 q32 ATP-binding cassette MRP5 multidrug resistance protein 5 ABCC5 3 q27 ATP-binding cassette BCRP breast cancer resistance protein ABCG2 4 q22 ATP-binding cassette OAT3 organic anion transporter 3 SLC22A8 11 solute carrier OATP-A organic anion transporter polypeptide A SLC21A3 12 p12 solute carrier OATP3A1 organic anion transporter polypeptides 3 SLCO1A2 15 q26 solute carrier EAAT1 excitatory amino acid transporter 5 SLC1A3 5 p13 solute carrier TAUT taurine transporter SLC6A6 3 p25-q24 solute carrier Receptor-mediated transport INSR Insulin receptor INSR 19 p13.2 TFR1 transferrin receptor TFRC 3 q29 IGF1R insuline-like growth factor 1 receptor IGF1R 15 q25-q26 LEPR leptin receptor LEPR 1 p31 FCGRT Fc fragment of IgC, receptor transporter FCGRT 19 q13.3 SCARB1 scavenger receptor class B 1 SCARB1 12 q24.31

The interaction of the diverse members of the Solute Carrier family and the ABC transporter is an extremely effective protective mechanism of the blood- brain barrier to prevent xenobiotics from entering the brain.

## Measurement and display of the permeability of the blood-brain barrier

In the development of new pharmaceuticals, the extent to which a substance can cross the blood-brain barrier is an important pharmacological parameter ( brain uptake ). This applies both to neuropharmaceuticals , which are supposed to have their effect mainly in the central nervous system, and to pharmaceuticals, which are only supposed to have an effect in the periphery. A number of different methods have been developed in the past to investigate whether, to what extent and with which mechanism a substance can cross the blood-brain barrier. The classic methods work with model organisms in vivo . Newer approaches use cell cultures ( in vitro ) and a very new method works via computer simulations ( in silico ). Due to the largely identical structure of the blood-brain barrier in mammals, the in vivo results can be easily transferred to humans.

### Physical basics

A simplified model based on a single capillary was established by Renkin (1959) and Crone (1965) for measurements of the permeability of the blood-brain barrier. Despite the simplifications, this model is the best approximation of the real conditions. The so-called permeability surface ( permeability surface area product ) PS is a measure of the permeability of the capillary bed. The permeability surface product, also known as the Crone-Renkin constant , is the product of the permeability coefficient of a certain substance and the available transfer area. The unit of measurement is ml · min −1 · g −1 (g wet tissue) and corresponds to that of perfusion (blood flow) Q. The one-sided ( unidirectional ) extraction fraction E, that is the proportion of a substance that passes through the brain is transported, then results from:

${\ displaystyle E = 1-e ^ {- {\ frac {PS} {Q}}}}$.

If the perfusion (Q) is greater than the permeability surface product (PS), the transfer of substances is perfusion-limited. If the situation is reversed, the mass transfer is limited by the diffusion.

In principle, the value of E for all substances is always less than 1, since the influx cannot be greater than the supply of substance. At values ​​less than 0.2 it is usually said that permeation is the limiting factor for the transport of substances to the brain. In the range from 0.2 to 0.8 the permeation is moderate.

### In-silico procedure

The simulation methods are mainly used in the very early phase of drug development, i.e. drug design . The calculation models are currently limited to passive transport by diffusion and some molecular descriptors , such as lipophilicity, charge, molar mass and number of hydrogen bonds .

### In vitro method

Cell culture incubator.
In cultivated endothelial cells, quantitative investigations can be carried out on the behavior of substances at the blood-brain barrier.

The simplest in vitro method is to use isolated, still living capillaries. This makes it possible to investigate the transport mechanisms at the cellular level. In some cases, capillaries of human origin from autopsies are also used. The capillaries are metabolically active even after isolation , even if the ATP supply in the cells is largely depleted. In this method, however, both the luminal and the abluminal side of the endothelia are exposed to the active substance to be examined. It is therefore not possible to differentiate between the two sides with regard to the intake of active substances. The spatial distribution of incubated capillaries can be analyzed using confocal fluorescence microscopy . For example, the uptake of fluorescence- labeled monoclonal antibodies at the transferrin receptor can be examined. The method can be refined so that semi-quantitative assays for the efflux of active ingredients on the luminal side of the endothelia are also possible.

With immortalized endothelial cell lines, quantitative statements can be made about the permeability of a substance. There are now a variety of different cell lines from different species . These cell lines are used for both basic research and drug development. As in the capillary vessels, the endothelial cells are grown in single layers ( monolayers ). The quality of these layers can be assessed, for example, via the electrical resistance of the endothelial layer. The transendothelial electrical resistance ( TEER ) should be as high as possible. In the living organism this value is above 2000  cm² and is essentially determined by the quality of the tight junctions. In vitro values ​​are usually an order of magnitude lower. These values ​​improve significantly when co-cultures of endothelia and astrocytes are used. The astrocytes have a positive effect on the gene expression of the endothelia to form tight junctions. In this way, values ​​of up to 800  cm² can be achieved. Similarly high values ​​can also be achieved without astrocytes if the cell culture contains cortisol . ${\ displaystyle \ mathrm {\ Omega}}$${\ displaystyle \ mathrm {\ Omega}}$

### In vivo method

Laboratory rats are frequently used model organisms for in vivo experiments at the blood-brain barrier.

The first method used by Paul Ehrlich to demonstrate the (im) permeability of the blood-brain barrier was the injection of a dye and the subsequent dissection of the test animal. Dyes that are able to cross the blood-brain barrier permanently stain the interstitium of the brain. The method is limited to dyes and only allows qualitative considerations. Dyes are still used today in studies aimed at opening the blood-brain barrier in a targeted manner. For the same reason, the method of fluorescent labeling, which is otherwise very often used in microbiology, is completely unsuitable for investigating the transport mechanisms of small molecules at the blood-brain barrier. Fluorescent labeling can only be used for very large molecules, such as polypeptides, in which the molecule of the fluorescent dye is relatively small in relation to the peptide.

A number of different in vivo methods have been developed in the past, some of which are still used in practice today. In general, the in vivo methods are the reference methods. The intravenous or intra-arterial application of an active ingredient under real physiological conditions in a model organism and subsequent analysis of the brain tissue cannot currently be replaced by any in-vitro methods or even by simulation calculations. These are the most sensitive methods in which the uptake of a substance in the brain can be determined over long periods of time and several passages through the blood-brain barrier.

#### Brain uptake index

The first method for quantifying the uptake of nutrients and active substances across the blood-brain barrier was developed in 1970 by William H. Oldendorf. A radioactively marked substance is injected together with a likewise radioactively marked reference sample directly and as quickly as possible into the external carotid artery of a test animal (usually a rat). About five seconds after the injection, the animal is decapitated , the brain is isolated and the concentration of the labeled substances it contains is determined from the radioactivity. The so-called brain uptake index (BUI) is calculated from the ratio of the dose of the injected substances and the dose measured in the isolated brain for test substance and reference sample .

${\ displaystyle {\ text {BUI}} = {\ frac {\ {\ frac {C _ {\ text {BrainT}}} {C _ {\ text {injectedT}}}} \} {\ {\ frac {C_ { \ text {BrainR}}} {C _ {\ text {injectedR}}}} \}}}$

Because of the very short time in which the applied substances are administered and can permeate, the method is only suitable for substances that quickly pass the blood-brain barrier. In contrast, hydrophilic compounds, such as many peptides, are only absorbed slowly. The method is unsuitable for such substances.

The compounds to be investigated are mostly tritium or 14 C labeled. Tritated water or 14 C-butanol , for example, serve as reference substances . The volume injected into the test animal is kept as small as possible. Volumes smaller than 10 µl are necessary in order to avoid flow and distribution artifacts , as these influence the measured BUI. In contrast, the BUI's dependence on the concentration of the injected substance is rather low.

The brain uptake index of various substances

 connection BUI [%] log P M [g / mol] Cytosine 1.2 −1.72 243.2 urea 1.4 −1.52 60.1 Mannitol 2 −2.11 182.2 Thiourea 5 −0.96 76.12 Ethylene glycol 18th −1.3 62.1 Acetamide 23 −1.1 59.1 Methanol 103 −0.52 32 Propylene glycol 27 −0.92 76.1 Ethanol 107 −0.18 46.1 Butanol 117 0.93 74.1 Benzyl alcohol 94 1.1 108.1 phenobarbital 56 1.42 232.2 Phenazone 83 0.38 188.2 caffeine 103 0.02 194.2 Phenytoin 71 2.4 252.3 Estradiol 94 2.61 272.4 testosterone 85 3.28 288.4 heroin 87 1.14 369.4 Morphine 2.6 −0.2 285.3 nicotine 120 0.7 162.2

according to:, data, unless otherwise indicated, from:

#### Brain Efflux Index

In this procedure, developed in 1995, the efflux of a substance from the brain across the blood-brain barrier into the blood is determined.

As with the brain uptake index, the radioactively marked test substance is microinjected directly into the brain of the test animal together with a reference compound that is also radioactively marked. However, a non-permeable tracer - mostly inulin - is used for the reference compound. Here, too, the test animal is decapitated after a certain time and the brain is isolated. The radioactivity is then measured on the same side of the brain ( ipsilateral ). The brain efflux index (BEI) is then calculated from the proportion of the test substance that crosses the blood-brain barrier and circulates in the blood and the proportion remaining in the brain:

${\ displaystyle {\ text {BEI}} = 1 - {\ frac {\ {\ frac {C _ {\ text {TestG}}} {C _ {\ text {ReferenceG}}}} \} {\ frac {C_ { \ text {TestI}}} {C _ {\ text {ReferenceI}}}}}}$ .

#### Brain perfusion

In the perfusion technique, the radioactively labeled substance to be examined is perfused into the external carotid artery of the test animal. The animal is then killed and the brain removed. The brain is homogenized and the radioactivity in it is measured. The advantage of this method is that a possible breakdown of the test substance in the blood, for example by the enzymes it contains, is largely prevented. Guinea pigs are particularly suitable as test animals because the carotid artery does not branch between the neck and the brain, as is the case, for example, with the rat. This makes it possible to increase the perfusion time up to 30 minutes, while in the rat only 20 seconds are possible. The operational effort of this method is extremely high. Therefore, it is essentially only used for substances that do not have sufficient stability in the plasma or only have a very low perfusion capacity to the brain.

An improved variant, the capillary depletion method (Engl. Capillary depletion method ). The capillaries are separated from the brain tissue by centrifugation . This allows a distinction to be made between transcytosis or diffusion, i.e. actual permeation into the brain, and possible endocytosis in the endothelium.

#### Indicator diffusion technique

In the indicator diffusion technique , the active ingredient to be examined is applied to the external carotid artery together with a reference substance that has no permeation capacity . Both substances do not have to be radioactively labeled. From the backflow of blood, for example in the internal jugular vein , the concentration of both substances can be determined by taking blood and analyzing the plasma. The permeation of the substance into the brain can be calculated from the decrease in the test substance in the blood reflux.

In the brain, the indicator diffusion technique is only suitable for substances that have a high permeation capacity for this organ.

Autoradiogram of a section of the brain of a rat embryo. The labeling was carried out with oligonucleotide sequences which were conjugated with 35 S -dATP ( deoxyadenosine triphosphate ) and which bind to GAD67 ( glutamate decarboxylase 67). The areas with high radioactivity (high marker concentration) are black. This is particularly the case in the subventricular zone (SVZ). The blood-brain barrier prevents the marker from passing into the brain tissue. The black scale bar corresponds to a length of 2 mm.

Quantitative autoradiography (QAR) was developed in the 1970s. The substance , which is usually radioactively labeled with 14 C, is administered intravenously. At a certain point in time, the organs are isolated and the dried frozen sections are placed on an X-ray film or a high-resolution scintigraphic detector plate. The degree of blackening or the detected radiation dose can be used to measure the absorption of the substance in the brain, but also in other organs, by means of comparative experiments with substances with very low permeability - for example with 125 I -labelled albumin . If the local perfusion is determined in a separate measurement, the permeability surface product can be calculated.

#### Intracerebral microdialysis

In intracerebral microdialysis , a semi-permeable membrane is implanted in the brain tissue. Substances can be perfused via a microdialysis catheter located inside the membrane . Samples of the perfusate can then be taken and analyzed using the same catheter.

A variety of examinations and experiments at the blood-brain barrier can be carried out in the test animal. An example of this is the opening of the blood-brain barrier using hypertonic solutions, as described below. The method is suitable for continuous fluid withdrawal. In this way, concentration-time profiles can be created for intravenously or orally administered substances.

A wide variety of experiments to research transport mechanisms at the blood-brain barrier can be carried out with intracerebral microdialysis.

In human medicine, intracerebral microdialysis is used for neurochemical monitoring of strokes .

#### Imaging procedures

With the help of imaging methods , a non-invasive display and measurement of the permeability of the blood-brain barrier with various substances is possible. Some of these methods are used in clinical practice in human medicine. The moreover be used positron emission tomography (PET), the magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI).

PET
PET of the brain after administration of 18 F-fluorodeoxyglucose (FDG). FDG is transported to the brain via GLUT-1 like normal glucose (passive transport). The areas with the highest glucose requirement (= highest intake of FDG) are shown in red.

With positron emission tomography, essentially the efflux processes of potential active substances with P-glycoprotein are shown to investigate the blood-brain barrier. Understanding the function and influence of P-glycoprotein at the blood-brain barrier is of great importance for the development of neuropharmaceuticals. These purely scientific investigations are carried out with substances that are marked with a positron emitter (beta plus decay (β +)). The isotope 11 C is mainly used for this. The short-lived isotope 18 F is preferred for fluorine-containing compounds . Because of the very short half-life of 20.39 for 11 C or 110 minutes at 18 F, such experiments can only be carried out at research facilities near which a cyclotron is located. Examples of appropriately marked and investigated compounds are verapamil , carazolol , loperamide and carvedilol . Verapamil is of particular pharmacological interest because it is able to inhibit P-glycoprotein .

PET is one of the few methods that enables a direct in vivo comparison between the model organisms used in the preclinical phase and humans with regard to the interaction between active ingredient and P-glycoprotein.

Magnetic resonance imaging

Magnetic resonance tomography (MRT) is too insensitive as an imaging method to show the passage of active substances into the brain. The situation is completely different with a damaged blood-brain barrier. In these cases, contrast- enhanced MRI plays an important role as a diagnostic method for various neurodegenerative diseases and cancers in the brain. This is described in more detail in the chapter on diseases indirectly associated with the blood-brain barrier .

Magnetic resonance spectroscopy
Magnetic resonance spectroscopy of a selected area in a patient's brain. The three MRI images show the measurement area framed in turquoise. Including the associated NMR spectra with the peak of glutathione (GSH). At the top right the signal-concentration diagram derived from it. The area (y-axis) of the corresponding GSH peak is plotted against the GSH concentration (x-axis).

The magnetic resonance spectroscopy (MRS) is a on the nuclear magnetic resonance -based method in a magnetic resonance imaging scanner to carry out a nuclear spin resonance spectroscopy allows. Certain substances or their metabolic products can be spectroscopically detected and quantified in the brain. Compared to magnetic resonance tomography based on the proton spin of water, other atomic nuclei are of interest here. These are in particular 19 F, 13 C, 31 P and non-aqueous protons. Compared to the protons of the water, which are present in much larger quantities, these nuclei, which are usually only present in traces, provide correspondingly weak signals. The spatial resolution corresponds to a volume element of approximately 1 cm³. MRS and MRT can be easily combined with one another. The MRT provides the anatomical structure and the MRS the corresponding spatially resolved analysis of the anatomy. With the MRS, for example, the pharmacokinetics of active ingredients containing fluorine , such as the neuroleptics trifluoperazine and fluphenazine , can be observed and measured in the human brain. With magnetic resonance spectroscopy, it is possible to quantitatively differentiate between the active substance and its metabolites. It is also possible to differentiate between free and bound active substance.

Disadvantages of the MRS are the long measurement times required due to the low sensitivity of the method and the low spatial resolution. The latter is particularly problematic in experiments with small animals.

## Concepts for overcoming the blood-brain barrier

see main article: Concepts for Overcoming the Blood-Brain Barrier

As was shown in the chapter on transport processes , only a few substances are able to cross the blood-brain barrier, which is why many potential neuropharmaceuticals ultimately fail at the blood-brain barrier. 98% of these substances cannot cross the blood-brain barrier.

For decades, intensive research has therefore been carried out on methods that are intended to enable the transport of active substances into the brain by bypassing or - ideally more selectively - opening the blood-brain barrier. A number of strategies to overcome the blood-brain barrier have been developed or are still in the development stage.

## Disorders of the blood-brain barrier

Main article: Disruption of the blood-brain barrier

Disorders of the blood-brain barrier can be caused by a number of different diseases. The blood-brain barrier itself can also be the starting point for some very rare neurological diseases that are genetic .

The disruption of the protective effect of the blood-brain barrier is a complication of many neurodegenerative diseases and brain injuries. Some peripheral diseases, such as diabetes mellitus or inflammation , have a detrimental effect on the functioning of the blood-brain barrier.

Other diseases, on the other hand, disrupt the function of the endothelia from “inside”, that is, the integrity of the blood-brain barrier is impaired by influences that come out of the extracellular matrix. An example of this is glioblastoma .

In contrast, a number of diseases in the brain manifest themselves in that certain pathogens can cross the blood-brain barrier. These include, for example, the HI virus, the human T-lymphotropic virus 1 , the West Nile virus and bacteria such as Neisseria meningitidis or Vibrio cholerae .

In the case of multiple sclerosis, the "pathogens" are cells of the body's own immune system that cross the blood-brain barrier. Likewise, in some non-cerebral tumors, metastatic cells cross the blood-brain barrier and can lead to metastases in the brain.

## Exogenous effects on the blood-brain barrier

### alcohol

Alcohol abuse is a major risk factor for neuroethological diseases, inflammatory diseases and susceptibility to bacterial infections. In addition, chronic alcohol consumption damages the blood-brain barrier, which is seen as a major influencing factor for the development of some neurodegenerative diseases. The damage to the blood-brain barrier has been proven by neuropathological studies of alcohol addicts as well as animal experiments.

In animal experiments, it was found that the enzyme myosin light chain kinase (MLCK) activated by alcohol consumption leads to the phosphorylation of several tight junction or cytoskeletal proteins in the endothelia , which affects the integrity of the blood-brain barrier is pulled. In addition, the oxidative stress caused by alcohol leads to further damage to the blood-brain barrier.

It is not the alcohol itself, but its metabolites that activate the MLCK enzyme in the endothelia. The dysfunction of the blood-brain barrier facilitates the migration of leukocytes into the brain, thereby promoting neuroinflammatory diseases.

### nicotine

Chronic nicotine abuse not only increases the risk of lung cancer, but also increases the risk of cardiovascular disease . There is, in turn, a direct relationship between cardiovascular risk factors and an increased risk of dementia . In several meta-studies it was found that smokers have a significantly higher risk of dementia from Alzheimer's disease than non-smokers. The risk of vascular dementia and mild cognitive impairment is not or only slightly increased. The chronic administration of nicotine changes both the function and the structure of the blood-brain barrier of the test animals in animal experiments. The model substance sucrose can pass the endothelia significantly more easily, which is essentially due to a changed distribution of the tight junction protein ZO-1 and a reduced activity of claudin-3. Furthermore, after chronic administration of nicotine, increased formation of microvilli , regulated Na + K + 2Cl - symporters and regulated sodium-potassium pumps were observed in the endothelium of the blood-brain barrier .

In epidemiological studies, smokers were found to have a significantly higher risk of developing bacterial meningitis than non-smokers. Nicotine alters the actin filaments of the endothelial cytoskeleton , which apparently facilitates the passage of pathogens such as E. coli to the brain.

For some diffusion-restricted compounds such as the nicotine antagonist methyllycaconitine (MLA), which binds to the nicotine acetylcholine receptor (nAChrs), the blood-brain passage is worsened with chronic nicotine consumption.

A vaccine based on a nicotine-specific immunoglobulin G is under development. The aim of this vaccine is to stimulate the formation of antibodies that specifically bind nicotine and thus prevent passage through the BBB.

The negative health effects of electromagnetic radiation in the megahertz to gigahertz range with high energy density have been proven. In contrast, the effects of the same with a lower energy density, as is mainly used in mobile and data radio, are controversially discussed. The effects on the blood-brain barrier are also an issue.

When the energy density of electromagnetic radiation is high, significant warming is observed in the affected body tissue. In the skull, this warming can influence the blood-brain barrier and make it more permeable. Such effects are also demonstrated by the action of heat sources on peripheral parts of the body. With the services used in mobile communications, the brain can be heated by a maximum of 0.1 K (15-minute mobile communications call  with maximum transmission power). A warm bath or physical exertion can heat the brain more without harm. In scientific studies since the beginning of the 1990s, in particular from the working group of the Swedish neurosurgeon Leif G. Salford at the University of Lund , results have been obtained that open the blood-brain barrier in the non-thermal range after exposure to GSM . Frequencies, describe. In-vitro experiments on cell cultures later came to similar results.

Other working groups cannot confirm Salford's results. Other working groups also question the methodology used.

## Human diagnostics

### Contrast enhanced magnetic resonance imaging

Gd-DTPA, due to its high hydrophilicity, cannot cross the healthy blood-brain barrier.
Coronal MRI with 20 ml Gd-DTPA of a glioblastoma. The area of ​​the tumor becomes visible through the inflow of the contrast medium over the defective blood-brain barrier in the right half of the brain (white areas, shown on the left in the picture).
Disruption of the blood-brain barrier after an ischemic cerebral infarction in the flow area of ​​the left cerebral artery. T1-weighted coronal sequences without contrast agent on the left and with contrast agent on the right

Shortly after the development of gadopentetate dimeglumine (Gd-DTPA), the first contrast agent for magnetic resonance tomography, in 1984, the potential of contrast-enhanced magnetic resonance imaging for the diagnosis of local disorders of the blood-brain barrier was recognized. As a highly polar molecule, Gd-DTPA is far too hydrophilic to be able to cross the healthy blood-brain barrier. Changes to the tight junctions, such as those triggered by glioblastomas, enable the paracellular transport of this contrast agent into the interstitium. There it intensifies the contrast through the interaction with the protons of the surrounding water and thus makes the defective areas of the blood-brain barrier visible. Since these blood vessels are responsible for supplying the tumor and are in close proximity to it, the extent of the tumor can be shown.

In the case of an acute ischemic stroke , the disruption of the blood-brain barrier can also be used to make a diagnosis using contrast-enhanced MRI.

By determining the relaxation time , the amount of Gd-DTPA in the interstitium can be quantified.

### Other imaging tests

Using radioactively labeled tracers, which normally do not pass the healthy blood-brain barrier, tests can also be carried out to check the function of the blood-brain barrier in humans. In principle, both single-photon emission computed tomography ( SPECT) and positron emission tomography (PET) can be used. For example, an increased uptake of 99m Tc-hexamethylpropylamine oxime (HMPAO) in the brain can be detected in patients with an acute stroke .

With the help of computed tomography (CT) defects of the blood-brain barrier can be quantified by the diffusion of suitable contrast media from the capillaries into the interstitium.

## Discovery story

Paul Ehrlich around 1900 in his office in Frankfurt
Max Lewandowsky
Charles Smart Roy and Charles Scott Sherrington in Cambridge in 1893

The first proof of the existence of the blood-brain barrier comes from the German chemist Paul Ehrlich . In 1885, when water-soluble acidic vital dyes were injected into the bloodstream of rats, he found that all organs except the brain and spinal cord were stained by the dye.

From this he drew the wrong conclusion in 1904 that a low affinity of the brain tissue for the injected dye was the cause of this finding.

Edwin Goldmann , a former colleague of Paul Ehrlich, injected intravenously in 1909 the di-azo dye trypan blue, which Paul Ehrlich had synthesized for the first time five years earlier . He noticed that the choroid plexus was noticeably stained in contrast to the surrounding brain tissue. In 1913 he injected the same dye directly into the spinal fluid of dogs and rabbits . The entire central nervous system (brain and spinal cord) was stained, but no other organ. Goldmann concluded that the cerebrospinal fluid and the choroid plexus play an important role in transporting nutrients to the central nervous system. Furthermore, he suspected a barrier function for neurotoxic substances.

In 1898 Artur Biedl and Rudolf Kraus carried out experiments with bile acids . When administered systemically into the bloodstream, these compounds were non-toxic. However, when injected into the brain, they were neurotoxic , with reactions up to coma .

In 1900 Max Lewandowsky used potassium hexacyanidoferrate (II) (yellow blood liquor salt) in comparable experiments and came to similar results as Biedl and Kraus. Lewandowsky used the term "blood-brain barrier" for the first time.

In 1890, Charles Smart Roy and the later Nobel Prize winner Charles Scott Sherrington postulated that the brain had an intrinsic mechanism in which the vascular supply corresponds to the local fluctuations in functional activity.

"The brain possesses an intrinsic mechanism by which its vascular supply can be varied locally in correspondence with local variations of functional activity."

- CS Roy, CS Sherrington : On the regulation of the blood supply of the brain. 1890.

Lina Stern , the first female member of the Russian Academy of Sciences , made significant contributions to the research of the blood-brain barrier, which she referred to as the haemato-encephalic barrier in 1921 .

The differentiation between blood-brain and blood-liquor barriers was made by Friedrich Karl Walter and Hugo Spatz in the 1930s . They postulated that the flow of liquor for gas exchange in the central nervous system alone was completely inadequate.

Although Goldmann and Ehrlich's attempts suggested the existence of a barrier between the bloodstream and the central nervous system, there were doubts about its existence until the 1960s. One point of criticism of Goldmann's experiment was that blood and liquor , the two liquids into which the dyes were injected, differ considerably and thus the diffusion behavior and the affinity for the nervous tissue are influenced. Understanding was made even more difficult by the experimental finding that basic azo dyes stained the nerve tissue, i.e. crossed the blood-brain barrier, while the acidic dyes did not. Ulrich Friedemann concluded that the electrochemical properties of the dyes are responsible for this. The cerebral capillaries are permeable to uncharged substances or charged substances that are positive at the pH value of blood and impermeable to acidic substances. This thesis turned out to be inadequate in the period that followed, when a large number of substances were examined for their permeability at the blood-brain barrier. In the following explanatory models, a large number of parameters, such as molar mass, molecule size, binding affinities, dissociation constants, fat solubility, electrical charge and a wide variety of combinations thereof, were discussed.

Today's understanding of the basic structure of the blood-brain barrier is based on electron microscopic images of mouse brains that were carried out at the end of the 1960s. Thomas S. Reese and Morris J. Karnovsky injected horseradish peroxidase intravenously into the test animals in their experiments. When examined with an electron microscope, they found the enzyme only in the lumen of the blood vessels and in micropinocytic vesicles within the endothelial cells. They found no peroxidase outside the endothelium, in the extracellular matrix. They suspected that tight junctions between the endothelial cells prevent the transition to the brain.

## literature

• Norman R. Saunders et al .: The rights and wrongs of blood-brain barrier permeability studies: a walk through 100 years of history . In: Frontiers in Neuroscience . tape 8 , December 16, 2014, doi : 10.3389 / fnins.2014.00404 , PMID 25565938 .
Commons : Blood-Brain Barrier  - Collection of images, videos and audio files

## Individual evidence

1. S. Wolf et al .: The blood-brain barrier: a special feature of the cerebral microcirculation system. In: Naturwissenschaften 83, 1996, pp. 302-311. doi: 10.1007 / BF01152211 .
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