Question 1: Why is mannitol, a sugar that does not cross the blood-brain barrier, used to treat patients with traumatic brain injury where the brain may swell?

*These posts are from coursework answers for my degree, but the Figures that are referred to in the text didn't scan well and have already been handed in. These long posts would probably not interest most people but if you enjoy quite in-depth reading of scientific problems then this may be for you.

Question 1: The brain is protected to some extent by the blood-brain barrier - a membrane between circulating blood and the brain that keeps certain damaging substances from reaching the brain tissue. However, the brain is still subject to trauma that can cause it to swell, much like an ankle swells with a sprain. Because the cranium (skull) is a cavity of fixed size, brain oedema (swelling) can rapidly lead to coma and death. Knowing what you do about movement of water across a membrane, can you explain why mannitol, a type of sugar that does not cross the blood-brain barrier, is commonly used to treat patients who have suffered serious head trauma?

Answer:

If a patient has suffered serious head trauma then their brain has already experienced swelling. Given the physical limitations of swelling due to the cranium’s lack of flexibility, treatment of the patient must involve the use of substances which do not further exacerbate the condition of brain oedema. If the swelling were to be increased, the brain could expand to the point of being pushed against the skull, leading the brain injury, or death. Thus, in treating a patient with brain oedema, it is necessary to use a substance which does not cause any further swelling of the brain.

Mannitol, being a sugar and therefore a molecule which affects the movement of water into a cell or membrane, would increase the retention of water by cells which contain it. This is due to mannitol’s ability to dissolve in water and thus increase the level of solute contained within the cytoplasm of the cell. Consequently, the concentration of water molecules within the cell compared to dissolved solute molecules is decreased. This decreases the pressure of the solution within the cytoplasm of the cell. As a result of this, water from the extracellular fluid, will move inward via passive diffusion, or osmosis. This happens because the pressure of a solution which has more solutes added to it, decreases. Then, water with less solutes dissolved in it (having a higher level of pressure), will diffuse into the solution with a higher solute concentration. This eventually causes the pressures within both solutions to balance and thus osmosis will conclude. The pressure that would need to be applied to the solution that has the higher solute concentration (and lower pressure) in order to prevent the influx of water from a neighbouring solution of higher pressure, is called the osmotic pressure. Thus, the higher the concentration of solutes that there are in a solution, then the greater the osmotic pressure which is required for this solution to not take passively take in water. There are three main types of solution, as described by their solute concentrations in relation to either another solution or cell or other entity placed within them. For the following examples, consider that the respective solutions are compared to a healthy animal cell which has been placed inside the solution.

A hypotonic solution is one in which there is a low concentration of solutes compared to a healthy animal cell. The result of this interaction would be that the animal cell would at least swell up, and at most would actually burst, or lyse apart. This is shown in Figure 1.1a. In this diagram there are multiple arrows representing the movement of H₂O (water), and all of these arrows are moving into the cell. We can thus consider that the surrounding fluid which the cell is in has a much higher concentration of water molecules (and lower solute concentration) than the cell itself. Thus, the cell has a lower pressure than the surrounding solution and water moves across its membrane until both internal and external pressures (relative to the cell’s semi-permeable membrane) equalise. If the surrounding solution is extremely hypotonic then the cell may take in so much water that it both dies and bursts.

An isotonic solution is one in which the concentrations of water and solutes is equal to that of a cell placed in the solution. The result of this is that some water may diffuse out of the cell, but water would also move into the cell at the same rate. So at any given time there may be a slight difference in concentration of water and solutes between cell and surrounding solution, but this produces a pressure gradient that quickly leads to re-emergence of the balance in pressure between both solutions (intracellular and extracellular fluid). This means that the net movement of water is equal in both directions, i.e.  going into and coming out of the cell. This is shown in Figure 1.1b, with 1 arrow coming into the cell representing the movement of water, and another arrow exiting.

Lastly, a hypertonic solution is one in which there is a high concentration of solutes (low concentration of water molecules) in the solution compared to that of the cell which is here being used as a reference point. As a consequence of this, water will diffuse out of the cell and into the neighbouring solution. This is shown in Figure 1.1c, with three arrows of water leaving the cell and none going back in. Thus, the cell loses a great deal of water and will become dehydrated. If the extracellular solution is sufficiently high in solutes, then the animal cell will become desiccated and could die from dehydration.

Another way of describing this is to explain it in terms of concentrations, as opposed to pressure. If a solution has a high concentration of solute molecules then it by definition has a low concentration of water molecules. Supposing that the solution in question is surrounded by a semi-permeable membrane which allows water molecules to move freely between this and a pure water solution, but solutes are kept within the aforementioned solution, then a concentration gradient is set up. Because the first solution has a low concentration of water molecules and the pure water solution has a high concentration of water molecules, water will move passively from the pure water solution into the solution with solutes. This can be explained by saying that water moves down the concentration gradient. This is passive because movement in this direction does not require energy, it happens spontaneously. The two different explanations come together in the end: as the pure water diffuses into the solution contained solutes, this second solution begins to rise vertically up the container which it is present in. This increases the pressure of the second solution. At the same time, the water level decreases in the pure water solution. Eventually, the solution containing solutes will rise to such an extent that its overall pressure exerted upon the membrane dividing both solutions will be sufficient to prevent further diffusion. Thus, the osmotic pressure has been reached. If we only took into consideration the concentrations of solutions then the pure water would diffuse freely into the solution contain solutes ad infinitum. This is because pure water will always have a higher concentration of water molecules than a solution containing solutes. This is what would in fact happen if the solution containing solutes were constantly being drained to reduce its overall pressure. However, because both solutions are present within a container and the overall pressure between the two always adds up to the same amount (albeit, with the pressure increasing on the side with the solution containing solutes and decreasing on the side containing pure water), then there will always eventually come a point where the pressures on each side of the membrane will be equivalent and further diffusion will no longer take place. This situation is illustrated in figures 1.2a and 1.2b.

In a situation where a cell is in a relatively hypotonic solution, takes in an osmotically active substance (by passive or active means) and then retains water, there is a gradual accumulation of water within the cell. The rationale for this being that the cell takes in water via the above mechanisms previously described, but does not lose as much water because it has a lower pressure than the surrounding environment, at least temporarily. In this situation, the water gradually accumulates and thus over time the difference in pressure between intracellular and extracellular fluids decreases. This encourages a reduced rate of diffusion over time until eventually both fluids equalise in pressure/concentration and diffusion rate can be considered negligible. This is the basis for Fick’s Second Law of diffusion, represented graphically in Figure 1.3. From this it can be seen that initially (when Time = 0), the rate of diffusion is at its peak. However, as time goes on, and water gradually builds up within the cell, the rate of diffusion decreases. This is due to the difference of pressure or concentration between internal and external fluids of cell beginning to equalise. At the very end of the graph, the rate of diffusion is almost zero. The graph is not drawn to the point where diffusion rate drops to zero because, while this may true in a hypothetical sense, there will always be some movement of substances between the cell and its environment, even if the net effect is that there is no overall difference in concentrations between the two. The point at which there is no difference in concentrations of substances between two points, and consequently the concentration gradient has disappeared, is referred to as a state of equilibrium (Pack, 2001, p. 26).

The term given to a substance which exerts an effect upon the osmotic gradient or osmotic pressure of a solution is; osmotically active. Much of the above information regarding water retention by cells containing mannitol and subsequent explanations on varying types of solution are widely known from A-level biology, but elaborated upon in the work of Coan (2008).

With this in mind, we turn our attention to animal cells in general. Animal cells are quite limited in their ability to deal with varying concentrations of solute in a surrounding fluid. Plant cells are protected by their cell wall which helps prevent excessive drying out and dangerous water intake. So, a plant cell in a solution of pure water will swell up to a certain degree, but is unlikely to be killed by cellular lysis as a result of taking in too much water, provided that it has a healthy membrane and cell wall, whereas an animal cell almost certainly would succumb to this. Consequently, the concentration of both water and solutes in blood and the extracellular fluids surrounding animal cells inside of a living body is under tight control of various homeostatic mechanisms. This ensures that the animal cells supplied by these mediums do not become overwhelmed with either water or solutes and either become excessively dehydrated, or break down /lyse from over-hydration. This means that the extracellular fluid or blood surrounding an animal cell provides a strong indication of the solute and water concentrations of the cell itself. Therefore, a change in the concentration of a solute or of water in the blood or extracellular fluid will have a noticeable impact on the cytosol of the cell which is supported by it (Evans, ed. 2008).

So we can see that if mannitol were taken up by the cells of the brain when it was injected into the blood stream, then these cells would take in the mannitol, experience an increase in solute concentration and therefore a decrease in pressure, and this would consequently result in an increase in water retention from the surrounding fluid. This would occur because the cell’s cytosol would retain the osmotically active mannitol, resulting in a decrease of cellular pressure and decrease in overall water concentration. Thus, neighbouring cells and fluid would then lose water molecules via osmosis, to the cell which contained more mannitol, provided that all other solutes within the cells in question remained constant. The fact that other solutes must remain constant in order for mannitol to have its effect is because the osmotic pressure that we are considering here is known as a colligative property. A colligative property is one in which the concentration of both solvent (here, water, the dissolving agent) and solute are taken into account, but the actual form of both solvent and solute don’t matter. So, sodium for instance, could exert the same effect upon the concentration of water molecules as mannitol, thus it must be ensured that all other osmotically active solutes remain constant, for the effect of water retention to be caused by mannitol. So, if brain cells took in mannitol by any means, they would also retain water. This water would be diverted from other cells in the body with lower solute concentrations and from the blood and extracellular fluids. The brain cells would increase in size as they gained this water, and this would worsen the condition of brain oedema. Thus, it is essential that mannitol not enter the brain cells, or at least not in any concentration capable of significantly increasing cellular size.

This is where the blood-brain barrier comes in. Because of the potential for brain damage due to harmful substances entering the brain, a highly specific barrier called the blood-brain barrier is present and aptly named, for its ability to regulate the movement of many chemicals between the body’s blood supply and the brain’s cells. The blood-brain barrier is capable of preventing most harmful substances from entering the brain, although if a substance is lipid-soluble then it has a greater chance of getting through. The relatively high lipid-solubility of alcohol (ethanol) allows it to diffuse into the brain and explains the rather rapid onset of cognitive impairment when consuming alcoholic drinks (Seeley, VanPutte, Regan and Russo, 2011, p. 457).

Regarding the actual structure of the blood-brain barrier, a simplified diagram is shown in figure 1.4. It is composed of tightly-knit capillary endothelial cells (primarily). These are attached to astrocytic foot processes, pericytes and a basal lamina. Macrophages also occur in this general area to prevent the movement of harmful materials and life-forms (as well as viruses) into the brain. The function of the astrocytic foot processes is to maintain the close proximity of the endothelial cells. This is necessary to ensure that many substances are incapable of diffusing freely into the brain. If this were allowed to occur then toxins could enter the brain and damage sensitive tissue. Thus, the vast majority of substances entering the brain must enter via active mechanisms, e.g. active uptake or any other route requiring energy in which the substance is selected for its necessity (or shape and therefore ability to bind to carrier or channel proteins on the surface of the endothelial cells) instead of traversing the blood-brain barrier without restriction. If the cells of the blood-brain barrier were to drift apart or decrease in size due to strong dehydration, then diffusion of more substances into the brain would be a possibility (Vries, et al., 1997).

With this in mind, the effects of mannitol on the alleviation of intracranial pressure following a serious brain injury can be due to one of the following main mechanisms.

Firstly, the mannitol can be used to encourage osmosis within the cells of the blood-brain barrier. If the cells of the blood-brain barrier come into contact with a hypertonic solution then they will lose water and shrivel up, i.e. their size will decrease. This could occur by having a high concentration of mannitol in the blood circulating the entire body, or by injecting mannitol into the bloodstream at an area close to the blood-brain barrier. Consequently, water from other areas of the brain and also from the blood, once the blood has had a chance to travel to the kidneys and other bodily areas where the mannitol content will be reduced and overall tonicity will be reduced, will act to move water back into the cells of the blood-brain barrier which have been affected by the mannitol, via the process of osmosis. Neglecting the blood, this effect will mean that water moving from other regions of the brain towards the blood-brain barrier will reduce the water content of the other cells of the brain. This will reduce the size of the brain cells via dehydration and thus reduce the swelling that the brain is suffering from, therefore intracranial pressure will be reduced by mannitol-induced osmosis (Shawkat, Westwood, and Mortimer, 2012).

Secondly, mannitol could help to alleviate brain swelling indirectly by reducing the size of the cells of the blood-brain barrier and their ability to form a cohesive barrier against substances in the blood. Once this has occurred, more substances will be able to enter the brain by diffusion. This allows effective drugs to be administered to the patient and travel to their brain. If the blood-brain barrier were functioning normally then these drugs would not reach their site of intended action.

It is highly likely that both of the above mechanisms are in play to a large extent and thus are both responsible for the reduction of brain swelling.

Figure 1.4 shows the blood-brain barrier. In this diagram the small shaded circles are molecules of mannitol. This figure shows the mechanism of action of mannitol where it is taken up by cells of the blood-brain barrier. This then encourages water retention by these cells from various sources, but most importantly, from the other brain cells. This is one of the ways in which intracranial pressure may be reduced. Since the blood-stream surrounding the blood-brain barrier is hypertonic and containing much mannitol, if the cells of the blood-brain barrier fill with mannitol and then water via osmosis, some of this water may diffuse into the blood-stream, setting up a relatively constant diffusion gradient that allows water to move passively from the cells of the brain, into the blood-brain barrier cells and then into the surrounding blood.

Therefore, in returning to the original question, mannitol is commonly used to treat patients who have suffered serious head trauma because it does not cross the brain-blood barrier and thus provides a way of intervening against brain swelling, without contributing to it via osmosis. If mannitol crossed the blood-brain barrier then its administration would worsen brain oedema. However, because it doesn’t cross the blood-brain barrier, it is able to either draw water away from the rest of the brain, or to simply open up this barrier and allow a greater diffusion and utilisation of drugs into and within the brain.

However, there is controversy over the effectiveness of mannitol as a standard treatment for brain oedema in general (Kaufmann, and Cardoso, 1992) and when compared to hypertonic saline solution (Cochrane, 2013). Kaufman and Cardoso even claim that mannitol can increase intracranial pressure (however, this is to be taken with a grain of salt as the study was conducted on cats, not humans) and is consequently harmful to the brain in cases of head injuries. If this is the case for humans as well then the administration of mannitol could very well be dangerous. If mannitol produced this effect then it may simply be a question of both dosage and frequency of administration. It is possible that if multiple doses of mannitol are given that their effect could stack upon each other. This would happen if mannitol caused a shrinkage that lasted for a long time after the mannitol concentration had been reduced in the blood stream. This would allow some osmotically active substances to diffuse into the brain, potentially even mannitol itself. Then, if mannitol were again administered to the patient, before the blood-brain barrier had a time to undo its shrinkage, this could further induce decrease in size of the cells. Even more substances could diffuse freely into the brain and some of these substances could cause water retention to neural tissue. Thus, mannitol could reduce intracranial pressure in the short-term, by reducing water content in the brain and allowing pressure-reducing drugs the opportunity to diffuse into the brain cells, but excessive use could trigger the opposite effect by letting too many osmotically active substances enter the brain where they could then encourage water retention and subsequent swelling. This would certainly exacerbate the condition of brain oedema and potentially lead to coma and/or death.

Question 1 References:

Coan, M., 2008. Osmoles, osmolality and osmotic pressure: Clarifying the puzzle of solution concentration. [online] Available at: <https://www.researchgate.net/publication/23308894_Osmoles_osmolality_and_osmotic_pressure_Clarifying_the_puzzle_of_solution_concentration> [Accessed 6 April 2015].

Cochrane, 2013.Mannitol for acute traumatic brain injury, [online] Available at: <http://www.cochrane.org/CD001049/INJ_mannitol-for-acute-traumatic-brain-injury> [6 April 2015 2015].

Evans, H.E., ed. 2008. Osmotic and Ionic Regulation: Cells and Animals. Boca Raton, FL: CRC Press.

Kaufmann, A.M., and Cardoso, E.R., 1992. Aggravation of vasogenic cerebral edema by multiple-dose mannitol. Journal of Neurosurgery, 77(4), pp. 584-8.

Pack, P.E., 2001.  Anatomy and Physiology, Hoboken, NJ: Wiley Publishing, Inc.

Seeley, R.R, VanPutte, C.L., Regan, J. and Russo, A.F., 2011. Seeley’s Anatomy & Physiology, 9^th ed. New York, NY: McGraw-Hill.

Shawkat, H., Westwood, M., and Mortimer, A., 2012. Mannitol: a review of its clinical uses. Continuing Education in Anaesthesia, Critical Care & Pain. [online] Available at: <http://www.ceaccp.oxfordjournals.org/content/early/2012/01/12/bjaceaccp.mkr063.full>

Vries, H.E., Kuiper, J., de Boer, A.G., Van Berkel, T.J.C., and Breimer, D.D., 1997 The Blood-Brain Barrier in Neuroinflammatory Diseases. Pharmacological Reviews, 49 (2): pp. 143-156.