Physio 20 – Blood Brain Barrier PDF

PHYSIO 20 – Blood brain barrier There are barriers are between blood and interstitial fluid of the brain. Th exchanges between the blood and the interstitial fluid of the brain can be monitered through blood tests of a number of parameters. Capillaries structure is not constent in the body: the way...

PHYSIO 20 – Blood brain barrier There are barriers are between blood and interstitial fluid of the brain. Th exchanges between the blood and the interstitial fluid of the brain can be monitered through blood tests of a number of parameters. Capillaries structure is not constent in the body: the way in which endothelial cells are bound to each other can change. The most common capillaries are fenestrated: they allow communication between blood and interstitial fluid for small molecules (such as ions) and bigger molecules need different types of transport like channels, carrier proteins and transcytosis – membranes are a barrier themselves. In some capillaries, endothelial cells are sealed and exchanges between interstitial fluid of the brain and blood are tightly controlled: the only molecules passing without control are the lipophilic molecules (as they diffuse freely through the plasma membrane). Ions, big molecules, proteins and polar molecules cannot pass freely. 1. Evidence of a BBB: The interstitial fluid of the brain is special. The first to understand the peculiarity of the brain’s interstitial fluid with respect to other organs were Paul Ehrlich and Edwin Goldmann, in 1885. They injected trypan blue ink in the blood circulation, and then they analyzed all the tissues to see if it went everywhere. The expectation was to find trypan blue in every tissue, but the CNS was not colored (the trypan did not get there). When they injected the ink in the ventricular system of the CNS (containing the cerebrospinal fluid - CSF), the CNS turned blue. They concluded the existance of a barrier between interstitial fluid and blood (blood brain barrier or BBB) but not between CSF and CNS’s interstitial fluid. The choroid plexus where the CSF is produced from the blood constitutes another barrier (the blood-CSF barrier or BCSFB). This system of barriers is coherent with the idea of the complete exclusion of the CNS from blood circulation: the access to the brain is denied both directly and indirectly.  2. Vascularisation of the brain 1 and 2 represent the two sites in which contact between blood and brain interstitial fluid takes place. In the picture the communication between CSF and the interstitial fluid of the brain is not shown. Blood is filtered in the choroid plexuses (distributed all over the ventricles), and circulates all over the ventricular system, in the third and fourth ventricle, in the subarachnoid space, to be finally reabsorbed in the venous system through the arachnoid granulations. The barriers present in the brain are: - The blood-brain barrier, between plasma and interstitial fluid: it is located at the level of the capillaries in the brain parenchyma. - The blood-cerebrospinal fluid barrier, made up by the choroid plexus, entirely covered by the choroid epithelium (which also covers the ventricular system). - The meningeal barrier: the two barriers previously mentioned are not enough as there has to be a barrier also for the meningeal vessels. This barrier separates the CSF in the subarachnoid space and the overlying structures. - The ventricular ependyma (synonym for the choroid epithelium covering the ventricular system) constitutes a sort of barrier between CSF in the ventricular space and the interstitial fluid. This epithelium, even if it is not so abundant, allows for free exchange: this is the reason why trypan blue injected in the ventricular system diffuses in the brain. Every barrier is going to be analyzed separately, as it is much more complex than just tight junctions between endothelial cells. 3. The blood brain barriers In addition to their susceptibility to oxygen and glucose deprivation, brain cells are at risk from toxins circulating in the bloodstream. The brain is specifically protected in this respect by the blood–brain barrier. The interface between the walls of capillaries and the surrounding tissue is important throughout the body, as it keeps vascular and extravascular concentrations of ions and molecules at appropriate levels in these two compartments. In the brain, this interface is especially significant. The restriction of large molecules such as Ehrlich’s dyes (and many smaller molecules) to the vascular space is the result of tight junctions between neighboring capillary endothelial cells in the brain. Such junctions are not found in capillaries elsewhere in the body, where the spaces between adjacent endothelial cells allow much more ionic and molecular traffic. Substances that traverse the walls of brain capillaries must move through the endothelial cell membranes. Accordingly, molecular entry into the brain should be determined by an agent’s solubility in lipids, the major constituent of cell membranes. Nevertheless, many ions and molecules not readily soluble in lipids do move quite readily from the vascular space into brain tissue. A molecule such as glucose, the primary source of metabolic energy for neurons and glial cells, is an obvious example. This paradox is explained by the presence of specific transporters in the endothelial plasma membrane for glucose and other critical molecules and ions. In addition to tight junctions, astrocytic end feet (the terminal regions of astrocytic processes) surround the outside of capillary endothelial cells. The reason for this endothelial–glial allegiance is unclear, but may reflect an influence of astrocytes on the formation and maintenance of the blood–brain barrier and/or the passage of cerebrospinal fluid from perivascular space through aqueous channels in the astrocytic end feet. The blood brain barrier, from vessel to brain parenchyma is made of: - Endothelial cells connected to one another with luminal tight junctions - Basement membrane of the endothelial cells - Pericytes embedded in the basement membrane: they surround and cover, even if not completely, the endothelial cells - End foot processes of astrocytes Pial artery intracerebral arteriole capillary  4. Blood-CSF fluid barrier The blood cerebrospinal fluid barrier is made of epithelial cells of the choroid plexus (connected by tight junctions). The fenestrated capillaries don’t constitute part of the barrier. Between blood and interstitial fluid right outside of capillaries is free communication, due to the fenestration of the capillaries. For the fluid to access the CSF, the choroid epithelium has to be overcome. The barrier is in between the CSF and the interstitial fluid inside the choroid plexus, communicating with blood. CSF is produced by the choroid plexuses located mainly in the lateral ventricle, but also in the third and in the fourth ventricle. (Recall that the CSF from its circulation in the ventricles, flows in the subarachnoid space of the brain and of the spinal cord. The CSF is going to be reabsorbed by the arachnoid villi or granulation, in the venous sinuses, especially in the superior sagittal sinus. Thus the circulation of the CSF is a closed one). 5. The meningeal barrier (between CSF in subarachnoid space and the overlaying structures) The dural vessels have a fenestrated endothelium (*). The free communication with the subarachnoid space filled with CSF is avoided by an internal layer of epithelial cells lining the subarachnoid space, the arachnoid memebrane. It of formed of epithelial cells are connected by tight junctions. At the level if the pia matter, exchanges between CSF and brain interstitial fluid are free. The trypan blue could pass from the ventricular system to the brain due to mainly because of this communication and partially through the ependyma. As CSF and the brain's interstitial fluid are in communication, exchanges between CSF and blood need to be controlled. CSF-filled spaces aren’t just a cushion for the CNS (mechanical function): they also have a fundamental metabolic function. 6. Ventricular ependyma (between the CSF in the ventricular space and the interstitial fluid) There is a possible exchange.  7. BBB and BCSFB are anatomical and functional barriers The presence of the two barriers, the blood brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB), allows for communication between cerebral arterial blood and the brain’s interstitial fluid (BBB) and the CSF (BCSFB). The BBB and the BCSFB restrict free diffusion from blood to brain interstitial fluid while also providing transport processes for essential nutrients, ions and metabolic waste products. Homeostasis of interstitial fluid is strictly monitored as it needs to be kept in a narrow range of values. A summary of the relationship between the intracranial fluid compartments and the BBB and BCSFB. The tissue elements indicated in brackets form the barriers and arrows indicate the direction of fluid flow under normal conditions.  I. BBB The BBB is an active interface between the circulation and the central nervous system. The endothelial cells carry out two functions: barrier and carrier. 1. Barrier function of the BBB The barrier function is achieved through a fourfold defense line. - The 1st line is the paracellular barrier formed by inter-endothelial tight junctions, restricting free movement of water-soluble compounds between two adjacent cells. - The 2nd line is the transcellular barrier: the low levels of endocytosis and transcytosis of the brain’s endothelial cells inhibit the transport of substances through the cytoplasm. - The 3rd line is the enzymatic barrier, provided by a complex set of enzymes capable of degrading different chemical compounds: these enzymes can alter the nature of the compounds before they enter into the brain. - 4th line is a large number of highly selective efflux transporters complete the fourfold defense line. The endothelial cells of the BBB have a peculiar feature, as they are equipped with tight junctions, normally present in epithelia. Endothelial cells of the BBB have a double nature: - they have endothelial-like features as they have enzymes which are normally expressed by the endothelium, - they also have epithelial-like features as they have tight junctions, a low pinocytosis level and a high trans-endothelial resistance Pericytes are engulfed by the basal membrane and cover nearly 30% of the endothelium. They are important in the barrier function and in the first stages of development (as well as in adulthood) as they are regulators of endothelial proliferation (angiogenesis). They also play a role in inflammatory processes: they aren’t just passive structures present to reinforce the barrier, they have active roles. Astrocytes/oligodendrocytes cover the blood vessels with their end feet and they induce BBB properties in endothelial cells. They are a source of important factors (interleukins for example) such as TGF-β, GDNF, βFGF, IL-6 which act on the vessels. This means they are there to reinforce the barrier but are also main characters in processes that can occur between the blood and the interstitial fluid such as inflammation.  2. Elements of BBB permeability The adherens junctions are needed to develop proper tight junctions. Tight junctions are physical barriers forcing the transcellular route and they have: - fence function: cause a polarisation of the cell. The tight junctions separate the membrane in two portions, one facing the interstitial fluid and the other facing the blood. The basal membrane and the abluminal membrane are not the same in terms of what they express (transporters, channels and enzymes, …) as the membrane has been polarized - gate function: restrict the paracellular pathway 3. Neurovascular unit Microglia, astrocytes and neurons form a functional unit: the neurovascular unit. The structure is complex and its role is not confined to building up a default restrictive communication between the blood and the brain: all the components interfere with the exchange (for example, they may modify the blood flow or alter the permeability of the BBB by acting on the vessels). Indeed, this structure is dynamic: depending on the mediators/substances locally released, the membrane’s permeability may be altered. Local pH has an important role in determining the permeability of the BBB: this allows for vascular autoregulation of the brain. The cell association at the BBB: The cerebral endothelial cells form tight junctions at their margins which seal the aqueous paracellular diffusional pathway between the cells. Pericytes are distributed discontinuously along the cerebral capillaries and partially surround the endothelium. Both the endothelial cells and the pericytes are enclosed by the local basement membrane which forms a distinctive perivascular extracellular matrix. Foot processes from astrocytes form a complex network surrounding the capillaries and this is important in induction and maintenance of the barrier properties. Axonal projections from neurons onto arteriolar smooth muscle contain vasoactive neurotransmitters and peptides and regulate local cerebral blood which can regulate BBB permeability. Microglia are the resident immunocompetent cells of the brain. The movement of solutes across the BBB is either passive, driven by a concentration gradient from plasma to brain with more lipid soluble substances entering more easily. The movement can be facilitated by passive or active transporters in the endothelial cell membranes and the efflux transporters limit the CNS penetration of a wide variety of solutes. 4. The carrier function of the BBB The carrier function plays its role in the transport of nutrients to the brain and in the removal of metabolites/waste products. - Lipid soluble molecules pass through this system by passive diffusion. The lipidic nature alone is not sufficient for a molecule to be able to move through the membrane as the permeability also depends on dimensions, weight, shape and nature. - facilitative transport proteins - energy-dependent transporters - ion channels - transcytosis (very limited). The graph shows the brain uptake rate of a given solute against oil/water partition coefficient, a permeability coefficient defining a substance’s prevalence as lipid soluble or water soluble. The higher the oil/water partition coefficient (the higher the lipid solubility) of the substrate, the higher is the uptake rate. For example the uptake of L-glucose and glycine is very LOW, while the uptake of nicotine, ethanol and diazepam is HIGH. The graph also includes non-physiological substrates such as morphine and penicillin. However, the uptake of D-glucose, L-leucine and L-DOPA is VERY HIGH, despite them being big water soluble molecules: this shows that there are transporters involved in their uptake. The system of transporters and carriers also defines the direction of solutes’ movement, which is polarized and organized, according to transporters’ representation on basal and abluminal membranes. 5. Solute carriers A complex system of polarized transporters p° and ion channels determines the specific movement of water-soluble compounds and ions across barrier endothelial cells. Transporters can: - Facilitate the movement of substrates down the concentration gradient: GLUT1, LAT1, MCT1 - Actively transport subsyrates vua energy-dependant mechanisms: Na+/K+ ATPase, ATA2 The cell also has enzymatic systems such as amino acid decarboxylase (AADC) and monoamine oxidase (MAO) who function as metabolic barriers within the cell by converting substances such as L-DOPA (L-dihydroxyphenylalanine). The BBB is an extremely complex metabolic barrier made up of an active group of cells working on permeability: indeed, the interstitial fluid of the brain is very delicate, requiring a complex system to maintain its homeostasis. Glucose transporters (GLUT1) consist of 12 transmembrane segments and are expressed at both the luminal and abluminal endothelial cell membranes. They are energy independent, facilitative, saturable and stereospecific transporters. More than 99% of the glucose that enters barrier endothelial cells is shuttled across to be used by neurons and glial cells. Glucose moves down its concentration gradient from the lumen to the brain’s extracellular space via facilitated diffusion. GLUT1 is expressed on both the luminal and abluminal membranes of the endothelial cells and so glucose moves into the extracellular space through them. GLUT1 is insulin independent, however, GLUT1 expression on membranes is upregulated or downregulated depending on blood glucose concentration: prolonged decrease in blood glucose results in an increase in GLUT1 expression, while a prolonged excess in blood glucose results in a decrease in GLUT1 expression. This is done in order to ensure a constant glucose uptake to fuel the brain. Then glucose enters into the neurons through GLUT3. Monocarboxylate transporters (MCTs) transport monocarboxylic acids such as β-hydroxybutyrate (β-HB) across the barrier and they are energy independent. MCT1 is a lactate transporter. The CNS is an obligate glucose consumer that depends almost entirely on the supply of glucose from the systemic circulation. However, several findings suggest that glial cells and neurons do not use glucose as a fuel to the same extent: astrocytes take up glucose that is transported across the Barrier and use it for the glycolysis, producing lactate that is released into the Interstitial Fluid and subsequently taken up by surrounding neurons. Proton-linked monocarboxylate transporters (MCTs) MCT4 is expressed in astrocytes and its main role is to export lactate produced during glycolysis into the Interstitial Fluid; from there lactate is transported into neurons by MCT2 Barrier cells express MCT1 (SLC16A1) at both luminal and abluminal membranes and also in intracellular organelles In humans, plasma lactate is below 1 mM under normal physiologic conditions while in the brain ISF it is above 3 mM. Under those conditions the MCT1 at the BBB probably pays a role in lactate removal from the brain ISF to the blood, to avoid its accumulation in the brain. However, during starvation, when following a ketogenic diet or under hypoxic conditions, plasma lactate and ketone bodies increase so the gradient across the BBB could change. It has been shown that diet induced ketosis in rats caused a substantial upregulation of MCT1 at the BBB, associated with an increased extraction of plasma ketone bodies by the brain. 6. Amino acid transport The amino acid transporters of the barrier endothelial cells are divided into three distinct carrier systems (L-system, A-system and ASC system) depending on the type of amino acid and they are characterized by different patterns and mechanisms of transport. The basic, acidic and neutral amino acids are transported by three different families of carriers and within each family there is a competition for the transporter (for example, neutral amino acids, transported by the neutral amino acid transport system, compete among the other neutral amino acids for transport). However, some amino acids such as glutamate and alanine are preferred and are under strict control as they are also neurotransmitters. - The L-system transports large neutral amino acids with branched or ringed side chains (leucine and valine). This Na+ independent, facilitative transport system is located at luminal and abluminal endothelial cell membranes. - The A-system preferentially transports glycine and neutral amino acids with short linear or polar side chains, such as alanine or serine. It is Na+ dependent and pumps amino acids down a Na+ gradient maintained by Na+ -K+ adenosine triphosphatase (ATPase). - The ASC-system is also an energy-dependent and Na+ dependent transporter that preferentially recognizes alanine, serine, and cysteine. The A-system and ASC-system expression and function are localized at the abluminal endothelial cell surface and they transport small neutral amino acids out of the brain. The excitatory amino acid transporters (EEAT) are special sodium dependent transporters for moving amino acids that are also neurotransmitters. They permit a net removal of glutamate from the brain and assure a constant glutamate concentration in the brain. Glutamate is the most used excitatory amino acid neurotransmitter in the brain. Glutamate concentration in blood is 50-100 mM while in the whole brain homogenate it exceeds 10 mM and in the brain ISF, it is normally kept below 2 mM. Glutamate can cause neurotoxicity if it accumulates in the brain ISF because through its action on metabotropic NMDA receptors it could lead to Ca2+ overload causing neuronal injury or death. Glutamate is released during neurotransmission but is normally rapidly taken up by neurons and neighboring astrocytes Ions: - Na+ enters from the blood either - Through a channel w Cl- - Through a exchanger w H+: exchange system that gets rid of the H+ obtained from the reaction between CO2 and H20 to form HCO3- 7. BBB interruption: circumventricular organs There are some peculiar places in the nervous system that are free from the barrier. It means that there are nervous structures that exchange with the blood basically with the same rules of systemic circulation. These are the circumventricular organs. These areas are used for the brain to release fluids and contents (peptide hormones). Allows the monitering of the parameters of the blood.  II. BSCFB Since the CSF is in communication w the ISF through the pia matter, a filtering of the blood to produce CSF is necessary. In the graph the ventricular system where the CSF is produced can be seen. The two lateral ventricles are the main producers of the CSF. They are in communication w the 3rd ventricle through the foramen of Moro. The 3rd is in communication w the 4th ventricle through the cerebral aqueduct. Foramen of Luschka and Megendie allow the entrance in the subarachnoid space. On the left of the graph the two very thick meninges, Falx cerebri and Tentorium cerebelli are shown. On the falx, the arachnoid granulation is found in the venous sinus where the CSF is reabsorbed, the superior sagittal sinus. There are also arachnoid granulations in the subarachnoid space. 1. BCSFB There are fenesrated capillaries between the blood and the ISF. Barrier us in between the ISF and the CSG through the choroid epithelium.  The concentrations of AA and p° are diminished between the ISF and the CSF. The concentration of ions is decreased too. 2. Solute carrier. The membrane of the choroid plexus is similar to the one in the BBB. There is an asymmetrical distribution of the transporters causing a polarisation. GLUT1 is present but much less than in the BBB. Specific transport of folate, vit. C, vit.B6 that is specific to the BCSFB. AA import. Ion exchange and channels. This is a representation of the choroid cell. There are similar channels in chain systems and pumps similar to the blood brain barrier. The interstitial fluid is very similar to the CSF and it seems to be an ultrafiltrate of the plasma. 3. CSF volume and turnover rate. Maintaining normal CSF volume is nec for the brain’s health. The total volume is of about 150mL with a production rate is of 500mL/day. The entire CSF volume is renewed 4 time/day. 4. CSF flow Choroidally secreted CSF flows down the neuraxis to the 4th ventricle and then out through hind-brain foramina into the cisterna magna and the subarachnoid space in basal regions. In addition to this classically described pathway, new evidence indicates that ventricular CSF also flows by another route to the basal and midbrain cisterns, i.e., into subarachnoid extensions of the velum interpositum (from dorsal 3rd ventricle) and superior medullary velum (rostral 4th ventricle). In order to maintain a flow, a pressure gradient is needed in the form of hydrostatic pressure: the pressure is higher ALO the sites of production and lower ALO of the sites of reabsorption. The contribution of the pulsatility of the arteries is small. 5. CSF clearance CSF clearance into lymph and blood involves diverse anatomical sites and physiological mechanisms - Arachnoidal outflow resistance - Arachnoid villi vs. olfactory drainage routes - Fluid reabsorption along spinal nerves - Reabsorption across capillary aquaporin channels The pressure at the level of arachnoidal villi is about 5 mmHg. If we have 10 mmHg at the production site and 5 at the villi it would be normal since the pressure gradient will push the CSF to the reabsorption site. When there is a lower pressure than 5 mmHg at villi there is a net absorption. when the pressure is higher than 5mmHg, the flow does not change way due to the fact that there are venous sinuses w valves. 6. Functions of the CSF he CSF performs several functions that are crucial in protecting the brain and maintaining a stable milieu within the CNS. Three major functions : MECHANICAL SUPPORT of the brain and spinal cord, and regulation of ambient pressure are probably the primary functions of CSF.  o The protective cushion of CSF prevents CNS structures from impacting on the bony skull and spinal column during movement, thus protecting against potentially injurious blows. Applied forces and acceleration and decelerations o Furthermore, suspension of the brain within fluid imparts buoyancy, resulting in the reduction of its effective weight from approximately 1.5 kg to 50 g. Any object, wholly or partially immersed in a fluid, is buoyed up by a force equal to the weight of the fluid displaced by the object.— Archimedes of Syracuse MAINTENANCE OF HOMEOSTASIS in the extracellular environment of neurons is another major function of CSF. CSF is in free communication with the extracellular fluid bathing the neurons and glia and thus indirectly regulates its composition. CSF acts as a sink for potentially harmful metabolites that can then be selectively absorbed by the choroid plexus or nonselectively removed by flow through the arachnoid villi. CSF acts as a sink for K+: CSF is involved in maintaining homeostasis of neurons in other ways. Potassium allowed into the CSF. When a structure of the brain is intensely working, action potentials are being formed and fired a lot so there will be an increase in extracellular concentration of potassium (as potassium ions go out of the neurons during hyperpolarisation). In this case there would be a change in the equilibrium potential of potassium (Nernst equation would give a new value) causing a change in resting membrane potential, making the cell more or less excitable. To fix this, the CSF is used. The CSF is present and communicates with interstitial fluid at the level of pia. Some glial cells in the brain have a resting membrane potential which is equal to the equilibrium potential of potassium. Hence, when there is an increase in potassium outside, the electrochemical gradient will reverse and force the entrance of potassium into the neurons which communicate with glial cells by gap junctions, and so the potassium will travel along a chain of glial cells and will end entering vessels or CSF. The CSF acts as a sink for ions (K+, H+) and iron.  CSF also serves as a POTENTIAL ROUTE FOR CHEMICAL MESSENGERS such as neuroactive hormones to be widely distributed to remote sites in the nervous system. 7. Oedemas and lymphatic function In systemic circulation, normally blood arrives from arterioles and into the capillary bed for there to be an exchange with interstitial fluid. The extent of size of largest molecules able to pass out capillaries is based on the type of endothelium of capillaries. There are combined pressures driving fluid out of blood at level of arteriole, and combined pressures which act at the level of venule for reabsorption of interstitial fluid back into the blood. It can be said that there is a continuous flushing of the interstitial fluid, and there is a continuous filtration of blood into interstitial fluid and reabsorption. Normally however, in the systemic circulation there is a residual excess of fluid in the interstitial fluid which is not reabsorbed (physiologically), without there being an oedema. This excess is removed by lymphatic vessels. Lymphatic vessels reabsorb the few proteins which can escape the filter, and these proteins allow passage of water into the lymphatics by osmotic pressure -hence reabsorbing the excess fluid. However, in the brain there are no lymphatic vessels. Blood brain barrier is a strict barrier where the exchange of water is controlled, however there may still be situations where there is excess of fluid building up. This excess of fluid is not tolerated as there are no lymphatic vessels, and surrounding the brain is the skull which does not tolerate any amount of fluid (unlike the skin). Hence, the brain is very delicate and oedema cannot be tolerated at all as in the brain any variation of pressure cannot be tolerated. An excess of fluid - no matter its size - will have an impact on the brain as the skull will act as a pressure vector onto the brain, hence why brain oedemas is a problem and must be treated. Therefore an excess of production of CSF is an issue as variations in amount of fluid in brain (interstitial fluid or CSF) cannot be tolerated. Aquaporins Aquaporins are important as the water transfer is important as it is not passive but is very controlled. There is the expression of different aquaporins in the blood-cerebrospinal interface and blood-brain interface. Water is strictly monitored when passing the BBB. 8. Barrier dysfunction Damage to the blood brain barrier either due to infections, tumours or other pathologies. Studies of the past two decades have provided insight into the molecular biology which underlines function of the two most important blood-brain fluid interfaces, the BBB and the BCSFB. Efficient homeostatic mechanisms established by those two barriers control composition of brain extracellular fluids, the ISF and CSF. These are vital to normal neuronal function and signal processing in the CNS. Two obvious functions that are common to the BBB and the BCSFB are : - the restriction of free diffusion  - the transport of nutrients, waste products, signalling molecules and ions between blood and brain extracellular fluids. However, those two structures show important differences in their respective roles that are underlined by differences in expression of cell junction proteins, transport proteins and ion channels. An important similarity between the two barriers is that they are both dynamic systems and are able to respond rapidly to changes in brain requirements. The molecular basis of this feature is that the BBB and the BCSFB could be regulated via a number of molecular mechanisms under normal physiological or pathological conditions 9. Lumbar punction The contents of CSF can be monitored by accessing it by means of Lumbar Puncture. Lumbar Puncture is performed at a precise position: the needle is inserted into the vertebral column at the level of the cauda equina – not where there is the spinal cord. They must be aware of the amount of CSF being withdrawn as removing too much CSF will reduce the pressure in the system.