The physiology of the bends

Gases and physiology go hand in hand. We breathe oxygen and exhale carbon dioxide. Several molecules that we usually come across in gas form act as signal molecules, for example carbon monoxide and nitric oxide. But sometimes, things go wrong when gases meet physiology. In this blog post, I will talk about one such situation: decompression sickness, also called the bends.

diving_free_stock

What, exactly, is the bends? The bends happens when a person has been under pressure and then that pressure is removed quickly, causing the gases that are normally dissolved in the body fluids to form bubbles. A typical situation is where someone has been diving deep underwater and then resurface too quickly*.

What causes it? The first thing we need to know is which gases are problematic, namely inert gases. There are inert gases in the body. An inert gas is a gas that, under the given conditions, does nothing – i.e. it doesn’t undergo chemical reactions. The inert gases in the body mostly sits there and does not take part in metabolism. Oxygen is not an inert gas in the body – it contributes to several physiological processes including respiration. Nitrogen, however, is an inert gas in the body – it does not contribute. It is the main inert gas in the air, and the most common culprit of the bends.

Nitrogen and other inert gases are typically dissolved in the bodily fluids. This process requires pressure. Henry’s Law describes the relationship between pressure and a gas dissolved in liquid. This law states that when pressure on a liquid is decreased, the amount of gas dissolved in the liquid will decrease to the same extent, and when pressure is increased, the amount of gas dissolved will increase. This means that if the human body is put under high pressure (e.g. when diving), the inert gases will be dissolved in the bodily fluids. This is not a problem. The gases are inert, doing nothing, so we can usually handle the extra amount. The problem occurs when the pressure is reduced, and it is reduced fast. If a diver is moving slowly from the high-pressure underwater environment to the surface, the gases being released may escape bit by bit as the person breathes out, which is fine. But if it happens fast, the lungs may not be able to keep up, and that causes trouble. The inert gases become un-dissolved in places where they really should not be: we get bubbles trapped in the body.

Bubbles are bad news. They disrupt blood flow and can cause damage. Bubbles can form in (or be transported to) any part of the body (although some places such as large joints are more at risk). This means that we can get a great range of symptoms, depending on where the bubbles form. The worst outcome is death, which can happen if the bubbles for example interrupt spinal cord function. Less severe outcomes, although still severely unpleasant, are seizures, paralysis, dizziness, pain, visual disturbances, breathlessness, nausea, incontinence and more. In short, we do not want bubbles of inert gas (usually nitrogen) interfering with our organ function.

The deeper and longer the dive, the more dangerous it can be. We know that pressure increases as you descend. At 10 meters, the pressure has increased from 1 atm (sea level) to 2 atm, and the same level of pressure will therefore be needed for the diving suit to remain inflated. For reference, one atmosphere is the pressure that the air exerts at sea level, and the pressure tends to increase with 1 atm for each 10 m increase in depth. In other words, each 10 m below the surface adds to the body of water pressing down on the diver. The further down, the more weight, and this weight is in addition to the weight of the air (1 atm at sea level). 

At 1 atm (sea level), the oxygen content is approximately 21%, and nitrogen content is approximately 78%. The oxygen pressure is therefore 0.21 * 1 atm =0.21 atm, and the nitrogen pressure is 0.78 * 1 atm = 0.78 atm. There are other pressure units that this can be measured in, such as mmHg or Torr (1 atm = 760 mmHg = 760 Torr) and kPa (1 atm = 101.325 kPa), but we will stick to atm for now.

If a diver is given normal air (with the percentage composition described above), at 10 meters the oxygen pressure will be 0.21 * 2 atm = 0.42 atm, and nitrogen pressure will be 0.78 * 2 atm = 1.56 atm. At this pressure, the blood will start to dissolve the nitrogen. This means that the fluids in the body will contain increasing amounts of nitrogen until an equilibrium is reached. How much nitrogen ends up where in the body depends on the tissue composition. Fat, for example, can dissolve a lot of nitrogen, and will take a bulk share.

Dissolving nitrogen is a slow process, as is releasing dissolved nitrogen. The longer the dive, the more nitrogen is dissolved, and the longer it takes to release it afterwards.

So why nitrogen? Why not oxygen, or carbon dioxide? As a rule of thumb, for the bends to happen, the diver has to experience the gas in question at a pressure of at least 2 atm. This excludes both carbon dioxide and oxygen. Even at depth, carbon dioxide pressure simply does not increase much above its normal level (about 40 mmHg, or around 0.05 atm), and oxygen is used up by the tissues so it does not rise as much either*. Unless the diver uses a gas mixture with a different inert gas (e.g. helium), nitrogen will be the villain of the piece.

There are advantages to using other gases. Helium, for example, is less soluble than nitrogen. This means it dissolves faster into the body fluids, but it is also removed faster, reducing the risk of developing the bends. For long dives, this is good, but for short ones (where nitrogen simply would not have time to dissolve much), it’s not so good. One may ask why use an inert gas at all, if they are so problematic, and the simple answer is that pure oxygen under pressure is toxic**.

What about diving animals? Plenty of animals have an aquatic mode of life, and the list includes a variety of species from the odd rodent, ungulate and marsupial to penguins, seals and whales. Many of these dive to incredible depth, and they ascend and descend quickly.

diving depths
Diving depths of marine mammals, not including the deepest diving whale (Cuvier’s Beaked Whale, 2992 m) or birds (deepest diving is Emperor Penguin at 565 m, and deepest diving flying bird is Brünnich’s guillemot at 210 m). The personal record of this particular mammal is 3 m, which was a proud, yet unpleasant, moment. *Record by Soviet submarine K-278 in 1984, data for current submarines classified. Image not to scale: the submarine was 117 meters long, making it around 4 times the size of a blue whale.

At depth, the lungs of diving animals are almost entirely collapsed and blood flow to the lungs limited. Some diving animals even have collapsible rib cages that forces the air out when they dive. This means that no nitrogen can enter the blood during the dive. Many diving animals have quite small lungs, possibly because of this (and because large lungs work as airbags, making diving difficult). The diving animals do not store oxygen in their lungs for their dives, but rather in their blood, and they are very particular in how they use what little oxygen they have.

Oxygen storage. Diving animals usually have much more blood relative to their body size than non-diving animals, and the blood itself has a greater oxygen capacity (almost 40 ml oxygen per 100 ml blood, twice that of humans). The increased oxygen carrying capacity is due to a greater amount of red blood cells (which contain haemoglobin, the molecule that binds oxygen), and a much higher level of haemoglobin in the muscles (called myoglobin). The sperm whale, for example, has a myoglobin content almost 10 times that of humans, allowing them to store a vast amount of oxygen. Theoretically, the high level should cause the myoglobin to clump together, causing disease, but diving animals’ myoglobin is positively charged and stays nicely separated even when the muscles are packed with it.

Oxygen use. This impressive store of oxygen, however, is not enough. In addition, most diving animals have a lowered heart rate during dives, and blood flow to most of their internal organs is lowered (the exception being the brain). Even the heart gets less blood (but then again, the heart rate is lowered, so less blood is needed). Muscles also get reduced blood flow despite being needed to swim – instead, they rely on anaerobic (non-oxygen) metabolism after the myoglobin oxygen storage is depleted. Many diving animals also reduce muscle use. For example, Weddell and Elephant seals, bottlenose dolphins and blue whales have all been shown not to use their muscles when descending: sinking rather than swimming. And diving animals also reduce their metabolic rate, meaning that they use less energy. This is made easier by many diving animals being large, because oxygen consumption relative to body size is smaller the larger the body size. In other words: a mouse uses much more oxygen per gram of its body (1.65 litre oxygen per kilo per hour) than does an elephant (0.07 litre oxygen per kilo per hour).

In short: diving animals avoid the bends through minimizing the amount of nitrogen entering their blood when they dive***. To stay submerged, they are very good at storing oxygen in their blood (through higher blood volume and greater amounts of red blood cells) and muscles (through greater myoglobin levels), and use less oxygen (through shutting off blood flow to non-important organ systems, reducing their heart rate and metabolic rate, letting their muscles work anaerobically or not work at all).

*Interesting fact: The bends can also happen when ascending rapidly in for example an aircraft, with the danger arising as the pressure is quickly reduced to 0.5 atm. The underlying principles remain the same. A pressurised cabin will prevent this entirely (as with deep-diving submarines).

**Oxygen at pressures above 1 atm is toxic. When diving deeper than 60 meters, the pressure is so high that the gas mix needs to contain less oxygen than at sea level (hypoxic mixture) to avoid oxygen poisoning. There are multiple studies on oxygen toxicity, but the wikipedia article on the topic is quite extensive and accessible: https://en.wikipedia.org/wiki/Oxygen_toxicity

***This does not mean that they cannot get the bends. For example, stranded whales have shown signs of gas-bubble tissue damage (Gas-bubble lesions in stranded cetaceans: Nature 425, 575–576 (2003) https://www.nature.com/articles/425575a). It is possible that this has to do with the animals being stressed (and therefore either diving for too long or ascending or descending too quickly), or having too high blood flow (again due to stress or possibly also cold). While we do not know the exact reason why and how this happens, we do know that the use of sonar has been linked to whales getting the bends.

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Reblog: The Firefly Scanner is Featured on the BBC

Below is a blog post I wrote for our group after the Firefly neonatal scanner was highlighted on BBC. It’s a great, little scanner and I’m thrilled to be involved with this research. (Link to the post on our group’s blog.)

Building on previous work, Professor Martyn Paley has developed the concept of a bespoke MRI scanner for newborn babies (neonates) along with Professor Paul Griffiths. The result is a unique, full-strength neonate scanner, built by GE Healthcare and installed in the Neonatal Intensive Care Unit in the Jessop Wing. Named ‘Firefly’, the scanner is one of only two such prototype scanners in the world, and uniquely marries diagnostic imaging with easy access to our neonatal unit.

Featured last week on the BBC, the Firefly scanner has gained deserved attention. The BBC’s video of the scanner in action shows how important it is for the healthcare of newborn babies to have powerful scanning facilities within quick and easy reach. Tiny compared to adult scanners (which can easily weigh several tons), the Firefly would be able to fit in many small Neonatal Intensive Care Units. This is a major advantage over the more commonly used ultrasound imaging in providing ready access to high quality brain imaging.

Babies can be difficult to image as they rarely stay still. Our group have also recently published one of the first research papers with data collected using the Firefly scanner, in which we discuss a potential new way of correcting motion during MRI in babies. The paper is titled “Wireless Accelerometer for Neonatal MRI Motion Artifact Correction” and freely available.

We are delighted to have the Firefly scanner in Sheffield. It is an important clinical development and opens up exciting new possibilities for linking research on reproduction and development with the health of newborn babies.

technologies-05-00006-g001The Firefly 3-Tesla neonate MRI scanner in the Jessop Wing. Image from Paley et al. 2017. Technologies, 5(1); 6. (CC BY 4.0)

Breathlessness and opioids

We’ve recently published a paper on how opioids can modulate breathlessness. (The whole manuscript is open access here). Low-dose opioids can be used for treating chronic breathlessness, but we don’t know exactly how they work.

Opioid receptors exist across the brain. These are part of the internal opioid system (endogenous opioid system) for natural pain relief. When opioids are used in the clinical setting to treat negative stimuli, such as pain, they influence the unpleasantness and the intensity of the stimulus in different ways(1). In terms of breathing, opioids lower breathing by influencing brainstem respiratory centres(2), causing breathing to stop completely in high doses, and they can also affect higher brain centres(3).

Opioids also have behavioural effects, and may, amongst other things, influence associative learning. Associative learning is when an association between two stimuli is learnt by pairing these together. For example, in chronic breathlessness this could be previously neutral stimuli (e.g. a flight of stairs) and breathlessness. This could create an anticipatory threat response, which means that simply seeing a flight of stairs is enough to bring about breathlessness or the fear associated with breathlessness. This can worsen the breathlessness for the patient in the long run.

In this study, we hypothesized that opioids improve breathlessness in part through changing the anticipatory response to breathlessness. We focused on two brain regions in particular: the amygdala and hippocampus. Both are strongly involved in associative learning(4) and are rich in opioid receptors(5,6).

What did we do? First, we asked healthy volunteers to do a breathing test where we paired three different degrees of breathlessness and three symbols. The symbols were presented immediately before the volunteer was made breathless and were matched to the level of breathlessness that they signified. We invited those volunteers who learnt to associate each different symbol and its corresponding breathlessness to undertake two MRI scans in random order. Before one scan they received a low-level opioid (remifentanil) infusion, and before the other they received a control (saline) infusion. During the scans, the volunteers repeated the breathlessness/symbol task. Below is a schematic of the breathing circuit used for the study, showing how different levels of breathlessness was induced.

breathingsystemFigure 1. Breathing circuit for inducing breathlessness

What did we find? We were able to show that breathlessness anticipation in the control condition was processed in the right anterior insula and operculum, and that the breathlessness itself was processed in the insula, operculum, dorsolateral prefrontal cortex, anterior cingulate cortex, primary sensory cortices and motor cortices. These regions have been identified in other studies on breathlessness (as discussed in a previous blogpost).

However, in the opioid condition, we saw the following:
1. Opioids reduced breathlessness unpleasantness (Figure 2)
2. This reduction correlated with reduced activity in the amygdala and hippocampus during anticipation of breathlessness (Figure 3)
3. This reduction also correlated with increased activity in the anterior cingulate cortex and nucleus accumbens during the actual breathlessness (Figure 3)
4. During the actual breathlessness, the opioid infusion directly reduced activity in the anterior insula, anterior cingulate cortex and sensory motor cortices (Figure 4).

opioid_res1Figure 2. Ratings of breathlessness and intensity. Abbreviation: Remi=remifentanil

Reduction in unpleasantness. The reduction in unpleasantness with opioids was expected – the different effect of opioids on intensity and unpleasantness has been shown in many other negative conditions, including pain(1). Interestingly, we could confirm that this lowered unpleasantness correlated with reduced activity in brain regions linked with associative learning and memory (amygdala and hippocampus) before the breathlessness began (Figure 3, bottom). This reduced activation in the amygdala and hippocampus, regions that are needed for formation of unpleasant memories, may explain how low-dose opioids gradually become more efficient as a therapy over the first week of administration. The reduction in amygdala/hippocampus activation may mean that fewer new negative memories and reaction patterns are formed.

opioid_res2.jpgFigure 3. Brain activity linked to lowered unpleasantness, relating to breathlessness (top) and anticipation of breathlessness (bottom). NA=nucleus accumbens, paraCC=paracingulate cortex, ACC=anterior cingulate cortex, PC=precuneus, ant hipp=anterior hippocampus, amyg=amygdala. 

We also see that the reduced unpleasantness correlates with activation during the actual breathlessness in the anterior cingulate cortex and nucleus accumbens (Figure 3, top). These are parts of the endogenous opioid system which reduces the perception of negative stimuli. This means that the reduced unpleasantness our volunteers felt is linked with these regions being more active. In other words, they may act to further dampen the negative sensation. Less unpleasantness during the breathlessness means that even less negative memories are likely to be formed.

Reduction in breathlessness activation. Finally, opioids directly reduced activity in the anterior insula, anterior cingulate cortex, sensory motor cortices and brainstem (Figure 4). The activation during control (saline) in the figure below is typical of breathlessness, and has been found in several other studies using breathing challenges.

opioid_res3.jpg

Figure 4. Brain activity during breathlessness in the control (saline) condition (top) and where it is reduced by the opioid (remifentanil, bottom). Increased activation is shown in red-yellow, and decreased in blue. M1/S1=primary motor & sensory cortices, OP=operculum, dlPFC=dorsolateral prefrontal cortex, Thal=thalamus, ACC=anterior cingulate cortex, vmPFC=ventromedial prefrontal cortex, PAG=periaqueductal grey, SMG=supramarginal gyrus.

The areas that are reduced in activation with opioids are commonly activated in breathlessness and central to respiratory sensation. For example, the anterior insula, the most commonly activated brain region during breathlessness, is believed to assess the quality of the stimulus and help control interpretation. The anterior cingulate cortex, which is also commonly activated in breathlessness, is similarly involved in control of negative emotions. These regions may be part of an interpretation process that is shaped by expectation and learning(7) similar to other control systems in the body (e.g.(8)).

Summary: Opioids manipulate brain regions associated with learning, negative memory formation and negative stimulus control. We have shown that the opioid remifentanil may alter breathlessness perception and the brain regions associated both with anticipation of and actual breathlessness. This suggests that opioids work to reduce breathlessness in part through direct effects on respiratory control mechanisms in the brainstem, insula and anterior cingulate cortex, and in part through changes in how breathlessness is anticipated, by changing associative learning processes in the amygdala/hippocampus.

References:
1. Pain, 22 (1985), pp. 261–269
2. Br J Anaesth, 100 (2008), pp. 747–758
3. J Neurosci, 29 (2009), pp. 8177–8186
4. Curr Opin Neurobiol, 14 (2004), pp. 198–202
5. Life Sci, 83 (2008), pp. 644–650
6. Pain, 96 (2002), pp. 153–162
7. Nat Rev Neurosci, 16 (2015), pp. 419-429
8. Exp Physiol, 92 (2007), pp. 695-704

DOI: http://dx.doi.org/10.1016/j.neuroimage.2017.01.005
Link: https://www.ncbi.nlm.nih.gov/pubmed?term=10.1016%2Fj.neuroimage.2017.01.005
All images presented with permission, creative commons licence.

Predicting trouble: EEG, NO and stroke

We’ve recently published a paper titled “Electroencephalographic Response to Sodium Nitrite May Predict Delayed Cerebral Ischemia After Severe Subarachnoid Hemorrhage” on how electroencephalography (EEG for short) can be used to figure out which patients with a certain type of stroke (subarachnoid hemorrhage) will develop a complication after the initial brain bleed and which will not.

Subarachnoid hemorrhage is a type of stroke that can happen at any age. It is a bleed on the surface of the brain, in a space between the arachnoid membrane and the pia mater surrounding the brain (see figure below).

Meninges-en

The bleed is usually caused by an aneurysm (a bulging, weak section of a blood vessel) bursting, causing blood to escape into the subarachnoid space. Here, it puts pressure on the brain tissue (causing tissue damage) which can also reduce blood flow to other parts of the brain (causing lack of oxygen and cell death). The released blood may be toxic to the brain tissue and could cause inflammation. At worst, a subarachnoid hemorrhage results in death or severe brain damage.

Some of the damage is caused directly by the bleed (e.g. the pressure on the brain), and some is caused by how the bleed disrupts the normal control of blood flow in the brain. In particular, it is bad when the nitric oxide pathway stops functioning properly. Nitric oxide helps preserve the circulation in small blood vessels in the brain, partly through enlarging the blood vessels (vasodilation) and lowering the gathering of blood-clot forming platelets. After a bleed, nitric oxide levels in the brain are often reduced. This could be because hemoglobin in the blood stops enzymes responsible for producing nitric oxide from working properly. Another contributing factor is that the nitric oxide that is already present in the brain reacts with superoxide (part of the immune response) which leads to the levels of nitric oxide being even further lowered.

Nitric oxide disruption appears to be involved in delayed cerebral ischemia, which is the most common complication after a subarachnoid hemorrhage. For example, people with genetically lower activity in an enzyme that produces nitric oxide have a higher risk of this complication. Delayed cerebral ischemia is the unpredictable lack of oxygen to the brain leading to severe, even fatal, brain damage. This typically happens 3-14 days after the initial subarachnoid hemorrhage.

Which brings us to our question:

Patients who have the same clinical severity, no measurable genetic differences in nitric oxide production, and who seem exactly the same can go on to show very different outcomes. One can develop ischemia and suffer devastating new brain injuries, and the other return to normal without complications. How can we tell who will get it and who will not?

EEG measures neuronal (electrical) activity using electrodes placed on your scalp. It picks up fluctuations in voltage from the electric currents in the neurons, and in the clinical setting it is used to measure the spontaneous electrical activity in a brain over time. Different parts of the brain generate different signals. Some parts show signal with low frequencies (long waves, delta and theta frequencies) and some with short frequencies (rapid waves, alpha frequencies). The signal is linked to blood flow. In subarachnoid hemorrhage, it can be used to see cerebral ischemia develop naturally at an early stage, because as the blood flow is reduced, short frequencies begin to fade and long frequencies steadily increase. In short, the ratio of alpha to delta (for example), will be reduced in the ischemic patients. However, it can take several days of recording to get a result with this method. It is possible, but not practical.

However, we know that nitric oxide disruption seems to be critically involved in delayed cerebral ischemia. This means that we can give patients a nitric oxide donor (sodium nitrite) to stimulate the nitric oxide pathway, and use EEG to measure how well they respond. By doing this, we can speed the process up. We can see if the patient responds well to the sodium nitrite, or not. This means, in short, that we can see which patients show nitric oxide pathway disruption.

Using this method, we showed in our paper that patients who later went on to develop delayed cerebral ischemia showed no change (or a decrease) in the EEG signal whilst infused with sodium nitrite. Those that did not develop delayed cerebral ischemia did the exact opposite, and showed a strong increase in EEG signal.

temp3EEG spectrograms from a patient that did not develop delayed cerebral ischemia, and one who did, plus a scatter plot with the group differences in spectrogram ratio (alpha delta ratio (ADR))

So we have not only shown how the nitric oxide pathway is important in the development of delayed cerebral ischemia, but this also means we now quite possibly may be able to relatively quickly determine who is at risk for this life-threatening complication, and who is not.

Reference: Garry, P.S., Rowland, M.J., Ezra, M., Herigstad, M., Hayen, A., Sleigh, J.W., Westbrook. J., Warnaby, C.E. and Pattinson, K.T.S. (2016) Electroencephalographic Response to Sodium Nitrite May Predict Delayed Cerebral Ischemia After Severe Subarachnoid Hemorrhage. doi: 10.1097/CCM.0000000000001950

Breathing on the brain

As easy as breathing? It may seem like it, but breathing is actually no easy process. It involves the precise integration of several systems, including, of course, the pulmonary and cardiovascular systems (gas exchange and transport, respectively). However, it also requires the direct involvement of the central nervous system: the brain.

The drive to breathe is generated in the brainstem. In a study published almost 100 years ago, the crucial role of the different parts of the brainstem was demonstrated in a somewhat gruesome way by sectioning the brainstem of an anaesthetised cat (Lumsden, 1923). The main areas of interest are in the medulla and pons. Cut above this, and the breathing continue. Cut below this, and it will cease.

brainstem_small2

The brainstem is the part of the brain that connects to the spinal cord. The figure above shows an MRI image of a human brainstem, with the pons (A) and medulla (B) labelled.

The Medulla

On each side of the medulla we find neurons that are grossly organised in vertical columns. These can be divided into dorsal and ventral columns – that means columns situated towards the back of the medulla, and columns that are towards the front.

On the dorsal side of the medulla (towards the back), we find neurons that primarily govern inspiration (as opposed to expiration). These neurons fire when you breathe in. One of the most important clusters of neurons in this area is the nucleus tractus solitarius (NTS), which is an area that receives sensory input from the chest. It is thought that the NTS integrates sensory information from the body and relays this to other regions in the brainstem. In short, it could be viewed as a communication hub. The NTS is involved in a range of important functions as well as respiratory control, including for example the control of blood pressure.

On the ventral side of the medulla (towards the front), we find both inspiratory and expiratory neurons. We also find motor neurons that connect with muscles in the throat and chest. The respiratory column on the ventral side can be grossly divided into three regions: rostral (top/front), intermediate (middle) and caudal (bottom//back). The rostral part holds expiratory neurons. The intermediate part contains mostly inspiratory neurons plus the assumed ‘pacemaker’ of breathing: the pre-Bötzinger complex*. This complex contains pacemaker neurons capable of setting a breathing rhythm, and it can generate different rhythms depending on the level of oxygenation. We don’t yet know if the pre-Bötzinger complex sets the pace on its own, or if uses input from other regions as well. Typically, if not influenced by other factors, this region is ‘on’ for two seconds (inspiration) and ‘off’ for three seconds (expiration). Finally, the caudal regions hold expiratory neurons (that connect with motor neurons controlling expiration).

372px-2327_Respiratory_Centers_of_the_Brain

So to summarise, it is thought that sensory  input relevant to respiration is received in the dorsal medulla, specifically the NTS, and relayed to the ventral side, where we find both expiratory and inspiratory neurons, a ‘pacemaker’ and motor neuron connections to the body. So far so good.

At normal, ‘relaxed’ breathing, we see heightened activity in inspiratory motor neurons during inspiration, but the expiratory phase is silent. This is because we normally exhale by passive recoil (i.e. by simply letting the lung return to its non-stretched state after breathing in). During heavy breathing, such as during exercise, expiration is active. This means that the expiratory muscle neurons will fire whenever we breathe out, forcing a faster and stronger expiration than normal (puffing). Which leads us to one crucial point: the activity in the medulla can be modulated, and even overridden.

The Pons

Further up in brainstem, we find clusters of respiratory neurons in the pons. If the brainstem is cut between the medulla and the pons, the breathing will (most likely) continue, but it will be irregular. The part of the pons involved in the control of breathing is the pneumotaxic centre (or, as it is currently often named: the pontine respiratory group). This is a selection of clusters that modulates breathing through connections to the medulla (i.e. through causing the medulla to change its activity). It controls the timing of breaths through switching the duration of the respiratory phases. For example, stimulation of the pontine respiratory groups can cause your breathing to switch more quickly from inspiration to expiration. It also receives input from receptors in the lung. This input acts to dampen its activity, so that the lungs aren’t made to inflate too much (or too little). In short, the pontine respiratory groups fine-tune how quick and deep you breathe.

A second region of the pons is also often discussed in terms of respiratory control, but this centre – the apneustic centre – has not been found yet. All we know is that cutting the pons in the general region where we believe the apneustic centre is located causes excessively long inspirations with only the occasional expiration (apneustic breathing). We therefore guess that this area is important for stopping inspirations (generating the inspiratory cut-off). Again, it is thought that this region acts by sending signals to the medulla.

So to summarise, the pons is necessary for the finer regulation of our breathing patterns to fit the needs of the body, say, for example, if we need to breathe faster or take deeper breaths. Without the pons, the medulla generates a rhythmic, but slightly gasping, type of breathing.

The higher brain regions

In addition, we have higher brain regions that can modulate breathing. Speaking, holding your breath, eating, being in pain or startled, emotional states – these can all change your breathing, either voluntarily or involuntarily. Generally speaking, voluntary breathing alterations (e.g. speaking, eating, holding your breath) bypass brainstem centres and act directly on spinal motor neurons, while involuntary breathing alterations (e.g. pain, startle) can act both on the spinal motor neurons and through the brainstem nuclei, thus changing the respiratory pattern generation directly.

640px-14082012_-_Apné_(8859961212)

The higher brain regions involved in breathing modulation are not fully described, and it is likely that each of the situations mentioned above may recruit different higher brain regions. For example, being out of breath can induce activation in sensorimotor brain regions and/or emotional regions (like the limbic system) and/or areas associated with cognition (prefrontal regions), each to varying degrees, depending on the level and origin of the breathlessness (Herigstad et al. 2011).

 

In conclusion, we are still some way away from fully characterizing how the brain governs breathing, and it’s not likely to be a simple process. As with many of the important bodily functions, there are several layers of control, both to fine-tune our responses and make it possible for us to adapt to different environments and situations, but also to provide redundancies (‘backup systems’) to ensure that the body keeps on functioning even if one system is not working precisely the way it should. It’s never as easy as breathing.

 

Caveat: This is meant as a brief summary of breathing in the brain. It doesn’t include all the details, mechanisms or exceptions by a long stretch. Squeezing that kind of detail into a single blog post is not really feasible.

* The pre-Bötzinger complex is a projection of the Bötzinger complex, which was named after a bottle of reasonably-priced white wine that happened to be served at the scientific workshop when its discovery was discussed.

References: Lumsden, T. J Physiol (1923), 57: 153-160; Pattinson, K.T.S. Br J Anaesth (2008), 100(6): 747-758; Herigstad, M. et al. Respir Med (2011), 105(6): 809-18.

MATLAB for physiological analysis

I use MATLAB for my number crunching. While data acquisition and analysis programs such as Spike and LabChart have analysis functions that are good, they are not always appropriate for the type of analysis required. Also, I like to know all the calculations made on my raw data, and so my analysis tends to gravitate towards custom-made code. Dealing with time series for the most part, I’ve found MATLAB very useful, but it’s also handy for other types of data.

I therefore thought I’d compile a small list of things that has worked for me for the MATLAB novice looking to start using the program.

Learning MATLAB:

  1. Start small. Create a small matrix (e.g.20×20). Write code for processing your small matrix and make sure that it does what you expect it to do. Learn the basics on this matrix before touching real data.
  2. Break it. Error messages are great. Each message will lead to a better understanding of how to use MATLAB, so make sure you understand what they mean.
  3. Use the forum. MATLAB central will have many (if not all) the answers you can wish for (including: “what does my error message mean”). It’s a great resource! Link: http://uk.mathworks.com/matlabcentral/

Using MATLAB:

  1. Remember that MATLAB is only as good as its user. In many cases, you’ll get a number which might appear reasonable, but errors in your code can still be present. Getting an output does not mean that your code works the way you want.
  2. Learn to plot your data. Plot raw data and superimpose your calculations along the way. Mistakes should be easy to visualise, at least when working with time series. This is an easy quality check and has spared me some embarrassing mistakes in the past. Also, the figures you get can be made quite attractive for publication purposes. Certainly prettier than MS Excel, at least.
  3. Run the code on a known data set first. Make sure that the code produces the results you would expect. Again, this is a simple thing that can spare you a lot of grief. Compare with in-built programs if possible (such as spike detection in LabChart versus custom-written spike detection in MATLAB).
  4. Understand which computations are better done manually. Small, one-off computations could require more work coding up than just processing manually. The point of the exercise is to make things easier, not harder.
  5. Learn how to run a series of analyses with one click. Sometimes you’ll need to run many subjects in quick succession. Writing code that reads a list of subject numbers (one click) and automatically loops through the analysis process for each line on the list is one way of doing this that has saved me some time in the past.

Automated analysis can be very useful as it is less time consuming in the long run and you reduce the risk of silly, manual mistakes (typos etc.). With a few clicks, you could run all your number crunching in the background whilst you have your morning coffee and try to wake up your brain. Sounds good, eh? It also helps you understand the data better. For example, if you want to calculate a waveform mean, you have to know the mathematical formula for doing so. In an in-built program, this would not be needed. In short, it can be very helpful, when done right.

A few other things:

  1. Text files are good. Text files (.txt or .dat) are quite easy to work with in MATLAB. Save raw data in one of these simple formats as well as the custom format.
  2. Company is better. If there is someone else around that knows or wants to learn, join forces. It’s much more fun that way.
  3. Annotation is the best. Annotate and annotate well. Your future self thanks you! Trust me on this. A few simple %this-is-what-this-line-does will make your life infinitely easier.

If you’re not certain about using MATLAB, there’s a 30-day trial version available that you can play with to figure out if it is for you. It’ll take a little while to get to grips with if you’re not used to this type of program, but it’s worth the time.

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Hemoglobin: a gamechanger

The supply of oxygen (O2) is crucial for life and there are two ways O2 can be carried in blood: dissolved or combined with hemoglobin.

Dissolved O2 can only supply a fraction of what the human body needs. One mL blood holds only about 0.003 mL O2 at a partial pressure of oxygen (PO2) of 100 mmHg, and this can only be changed if we increase the PO2 (not easily done) or the solubility of O2 in blood (also not easy). And 0.003 mL O2/ mL blood is not enough.

To put it in perspective, a human with an O2 consumption of 250 mL and only a dissolved supply of O2 would need a cardiac output (blood pumped by the heart) of 83 L/min if the O2 extraction was perfect (hint: it is not). The heart normally pumps 5-6 L/min, and even top Olympic athletes don’t go much above 40 L/min. With the heart ejecting a maximum of 200 mL blood per beat (again, in athletes), that means we’d need more than 400 beats per minute to get to a cardiac output of 83. If we take into consideration that O2 extraction fraction is 25-30%, these figures increase to a cardiac output of 333 L/min and a heart rate of 1665 beats per minute, almost three times that of your average mouse heart rate.

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Hemoglobin (Hb), fortunately, is a gamechanger.

It is a molecule in red blood cells that has a central protein portion (globin), with four protoporphyrin (heme) groups attached, each with a ferrous (Fe2+) iron atom. This Fe2+ atom has a free binding side, which is where O2 binds. In effect, one Hb can bind four O2 molecules. And it does so reversibly. This means that O2 bound in the lungs can be easily released in the tissues.

The capacity of Hb to carry O2 is huge. It allows us to move away from the 83 L/min scenario above, to a much more reasonable required cardiac output. To illustrate how much of a gamechanger Hb truly is:

If a person has 15 g Hb per dL blood, and the O2 carrying capacity of Hb is 1.31 mL/g (normal conditions – the ideal capacity is 1.34 mL/g), it follows that this person’s Hb can carry:
15 g/100mL x 1.31 mL/g = 19.65 mL(O2)/100mL(Blood)

Add this to the dissolved O2 and take O2 exchange fraction into account:
(19.65 mL/100mL + 0.3 mL/100mL) * 0.25 extraction = 4.99 mL(O2)/100mL(Blood)

Calculate cardiac output:
250 mLO2/min / (4.99 mlO2/100mL) = 5012 mL/min = 5.01 L/min

This, of course, means that O2 transport in humans is heavily Hb dependent. The more Hb, the better the O2-carrying capacity. It is this principle that is at the core of blood doping and the use of illegal products like EPO to stimulate red blood cell production in competitive sports. Max physical performance depends on max O2 uptake, which is determined by cardiac output, O2 extraction (how well O2 enters the tissues) and Hb concentration. Of the three, Hb concentration is the only factor that can be artificially manipulated with relative ease to the increased performance of the athlete cheater.

But Hb contributes in other ways than simply by blunt concentration, and it does so by changing its affinity for O2.*

The affinity of Hb for O2 is simply the ease with which O2 is bound and released by Hb. Low affinity means that Hb binds less O2 for any level of PO2 (i.e. O2 is released easily, but not bound easily), and high affinity means that Hb binds more O2 for any level of PO2 (i.e. O2 is not released easily, but bound easily). This is usually described in terms of shifts on the oxyhemoglobin dissociation curve (below).

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The relationship between PO2 and O2 bound to Hb is S-shaped, since the heme groups in Hb bind or release O2 one by one rather than independently of each other. This means that it doesn’t matter much for the actual O2 content of the blood if the PO2 is 70 mmHg or 100 mmHg – Hb is still quite saturated, thank you very much. PO2 in venous blood can fall to 40% under normal circumstances, and we only see a drop in Hb saturation to 75%.

There are mechanisms that can shift this curve, however, including: pH, partial pressure of CO2 (PCO2), 2,3-BPG (produced by red blood cells) and temperature. High acidity, high PCO2, high 2,3-BPG and high temperature lower the affinity (right-shift), and vice versa (left-shift). The shifting of this curve has some interesting physiological consequences.

As blood moves from the lungs into the tissues and gets more O2-poor (deoxygenised), PCO2 increases due to tissue metabolism. This causes a right-shift of the curve. PCO2 can also cause a further right-shift by reacting with water to form H+ and bicarbonate (HCO3-), thus lowering the pH in the blood. We can measure this acidity in venous blood (pH: 7.32 – 7.42, compared to arterial blood pH: 7.35 – 7.45). This together means that as the blood gets deoxygenised, Hb loses some of its affinity for O2, releases O2 more easily, and the O2 delivery can therefore be maintained until the blood returns to the lungs. There, CO2 is offloaded and pH normalised, returning the affinity to normal.

Of course, whilst Hb is essential, it is no guarantee. There are diseases caused by Hb malfunction or damage (sickle cell anaemia, for example), and at lower levels of O2, even Hb fails. For PO2 levels below 60 mmHg, there is a rapid decline in Hb saturation, and therefore also in O2 delivery to the tissues. We’re entering the realm of hypoxia. Down this road lies headaches, dizziness, increased pulmonary blood pressure, increased production of red blood cells, breathlessness, confusion, rapid heart rate and lowered blood pressure (except for in the lungs). This eventually leads to some pretty nasty consequences: cyanosis, right heart ventricle enlargement and failure, and death. The severity of the symptoms depends on how rapid and severe the O2 deprivation is.

Some of these symptoms, however, may be lessened by increasing Hb concentration. And one way to do this, is through iron. The Fe2+ component of Hb is a limiting factor in its production. The body has a store of iron to draw upon, called ferriting, but under long-term hypoxic conditions, such as travelling at altitude, the body will increase its uptake of iron from food, facillitating the generation of more Hb. We can also add iron intravenously. This can limit some of the body’s harmful responses to hypoxia, such as high pulmonary blood pressure, to almost nothing.

Adding iron to your diet is risky in the Marvel Universe. magneto

 

* O2 concentration in the blood can also be affected by PO2 and the gas exchange in the lungs. Neither are easily changed. PO2 is dependent on atmospheric pressure, and gas exchange is optimised in healthy individuals so that ventilation (V) matches blood perfusion in the lung (Q) perfectly – a V/Q ratio of 1.

How do eggs breathe?

Bird’s eggs contain everything that the chick needs, except oxygen. So how exactly does the egg ‘breathe’?

First, the shell needs to be permeable to gases. The shell consists of a calcium carbonate outer layer, with two shell membranes beneath. A typical outer shell of a hen’s egg holds about 10,000 pores, each less than 0.02mm (approximately 0.017mm) in diameter, about 2.3mm2 pore area in total. Through these pores oxygen is taken in and carbon dioxide (CO2) released. However, the pores also allow water vapour to escape. Over the 21-day incubation period, a hen’s egg typically takes in 6 litres of oxygen, releases 4.5 litres of CO2 and loses 11 litres of water vapour. The rate of water loss depends on the porosity of the eggshell and the water vapour pressure gradient between the nest and egg.

Water vapour pressure is the pressure at which water evaporates at the same rate that it condenses. We can change this equilibrium point by changing temperature. At lower temperatures, the vapour pressure is less (fewer molecules have the kinetic energy to evaporate), and at higher temperatures the vapour pressure is higher (more molecules have the kinetic energy to evaporate). A liquid with a higher vapour pressure will evaporate more readily, and it will boil when its vapour pressure reaches the external, atmospheric pressure. For example, water has, at 100°C, a saturated vapour pressure equal to the atmospheric pressure at sea level (760 Torrs). If the atmospheric pressure decreases below 760 Torrs, the saturated vapour pressure of water and the atmospheric pressure would match at lower temperatures. For example, on Mount Everest, the low pressure associated with altitude means water will boil at about 70°C.

To avoid the egg losing too much water, whilst not jeopardising oxygen uptake, birds employ a number of strategies. The water vapour pressure gradient between nest and eggs is regulated by nest construction (insulation, humidity) and incubation patterns. The pore area of the egg is kept within certain biological limits: large enough for the egg to get enough oxygen, and small enough for the egg not to dry out. Species that lay their eggs at lower atmospheric pressures (higher altitude) tend to have lower pore area as the diffusion coefficient for gases is increased at altitude. This means that while more oxygen is delivered (good), more water vapour escapes (bad), and the lowered pore area at altitude is therefore necessary to prevent the egg from drying out. Some pollutants (such as aluminium) can increase shell porosity, causing water loss that is too great for the embryo to survive.

A pocket of air can be found between the inner and outer shell membrane on the blunt side of the egg, and this increases in size during the incubation as water is lost to the environment. This is believed to be the principal site of gas exchange, and there tends to be more and larger pores in this area. In the final day before hatching, the chick breaks through the barrier to this air space, and begins breathing with its lungs (pulmonary breathing).

Before pulmonary breathing is possible, gas exchange occurs via a membrane called the corioallantoic membrane. This is inside the inner membrane of the shell. Air diffuses through the shell and outer membrane into the area between membranes, where the capillary-rich corioallantoic membrane allows diffusion of oxygen into the blood and CO2 out of the blood. The blood is transported from the embryo to this membrane through corioallantoic arteries to the corioallantoic membrane, and oxygenated blood returns from the membrane to the embryo through allantoic veins.

The resistance to diffusion is mostly in the hard shell. The membranes have low permeability at first, but they increase in permeability quickly within the first few days after the egg is laid. After this, the egg doesn’t change its diffusion characteristics, and so the concentration of gases depends on the air composition outside of the egg and the metabolism (oxygen uptake, carbon dioxide production) of the embryo inside.

During the incubation period, the embryo grows and its metabolic demands increase. That means it uses more oxygen and produces more CO2 as it grows. As the partial pressures of gases outside of the egg don’t change, this means the gradients of gases between the inside and outside of the shell begins to increase. In short, oxygen concentration goes down and CO2 concentration goes up inside the egg over time. This change in pressure gradients of the gases across the shell and enables more oxygen to flow in (and more CO2 to flow out) as the embryo’s metabolism increases. Despite this, oxygen is reduced and CO2 increased inside the egg in a gradual change that continues almost until hatching. In this way, the ability of gas to diffuse through the shell places an upper limit on the metabolism of the growing embryo.

(Reptile eggs are a different kettle of …err fish, and tends to be more variable than bird’s eggs. Typically, reptiles use underground nests which have reduced oxygen availability, increased CO2 availability and increased humidity compared to the open nests seen in most birds. As discussed above, gas exchange depends in part on atmospheric conditions, and so reptile eggs’ respiration often depends greatly on the properties of the nest (and its interaction with the environment) as well as the properties of the egg itself. )

Taking a walk on the wild side

One of the things I want to do with this blog is to present a few basic animal physiology topics, in random order. The official reason is that I believe it is important as a scientist to look outside one’s niche every once in a while. The unofficial reason is that taking a step out of the cozy world of chronic lung disease every so often is quite nice. So instead of letting all my undergraduate work and excitement go to waste, I’ll selfishly take the opportunity to occasionally wax poetically about general physiology here on my blog.

(Lou Reed, ladies and gentlemen. Probably infinitely cooler than your average physiologist.)