Dr. Charles J. Marcuccilli examines pathophysiological events in traumatic brain injury, post-traumatic epilepsy in children and electrophysiological techniques.
[MUSIC PLAYING] JAMES J MARCUCCILLI: So as you all know, traumatic brain injury in children is a significant health care concern, with a significant morbidity and mortality. Each year, childhood traumatic brain injury results in an estimated 3,000 deaths, 29,000 hospitalizations, and 400,000 emergency room visits. TBI is six times more likely to cause death than HIV infection, and 20 times more likely to cause death than asthma. Repeated mild TBI, as typically occurs in sports-related settings, results in cognitive impairment. And there's been a wealth of recent data that suggests that these children don't have to lose consciousness to acquire these cognitive deficits. Even mild hits. which apparently are asymptomatic, can lead to these impairments. Each year in the United States, approximately 30,000 pediatric patients incur permanent disability as a result of traumatic brain injury. And the sequelae include a host of things, headaches, epilepsy, motor disturbances, learning disabilities, cognitive impairment, and behavioral problems. So what are the biomechanics of a traumatic brain injury? Well, forces to the brain resulting in injury can be described as either linear or rotational. Linear forces refer to the acceleration or deceleration forces occurring in a straight line. As the brain is floating in the cerebral spinal fluid-- try to use this here-- these forces may result in a compression of the brain at the site of impact, the so-called coup injury, which is shown up here in the left upper panel. As this occurs, a relative area of low pressure develops in the opposite direction to the direct impact. And as a result, the brain can spring backward, and injury can occur again in the opposite direction in what is termed the contrecoup mechanism here. The injuries can include contusion, hemorrhage, and in the right setting, even a skull fracture. And in some cases, the contrecoup injury is actually more deleterious than the direct coup, impact coup injury. In addition to the linear forces, there are also rotational forces. And that's shown in these neurons that are directly in front of the face. Rotational forces occur typically in sports-related accidents and motor vehicle accidents. And this is a situation where you can have a stretch injury to the axon that results in axonal injury, with the development of microhemorrhages, which you see in this image here. Children are at particular risk, or much more sensitive to traumatic brain injury, compared to adults, due to their relatively large head size compared to the rest of their body, their weak neck muscles, and greater flexibility. So these forces can be much greater in a child than an adult. What I've described for you are the primary mechanisms of injury. But there are also secondary mechanisms of injury, which I'll go into in the next slide. I want to distinguish this secondary injury from the second impact syndrome, which is this panel kind of down here in your lower right. This second impact syndrome was, or is used to refer to an individual who sustains mild traumatic brain injury. And then upon sustaining a second traumatic brain injury while recovering from the first, develops massive cerebral edema, which can then result in disruption in cerebral blood flow, and in some cases, death. This is somewhat of a controversial topic, but is something we use clinically, warning kids not to play while they're recovering from even a mild traumatic brain injury. Secondary injury in traumatic brain injury would include things like additional pathological insults that are independent of the original injury. So this could include hypotension due to systemic hemorrhage, hyperthermia do to concomitant infection, airway occlusion, systemic hypo- or hyperglycemia. In addition, the secondary injury may be directly or indirectly related to the original brain injury, such as cerebral edema, intracranial hemorrhage, early posttraumatic seizures, respiratory compromise, or posttraumatic hydrocephalus. And then finally, there as I'll show you in the next couple slides, secondary injuries that are due to ongoing molecular or metabolic processes, which to the pediatric epileptologist, are incredibly important. So results from several animal and human studies have demonstrated the breakdown of the blood-brain barrier following traumatic brain injury. Try saying that 50 times fast, blood brain barrier breakdown. And this has been divided into two responses, the acute response, and the delayed response. In the acute response, the traumatic brain injury leads to direct tissue damage. And so this is the primary insult. And this is believed to occur, due to mechanisms including excitotoxicity, which I'll go into in more detail. But that is essentially the indiscriminate release of excitatory neurotransmitters, such as glutamate, which then leads to cell death and subsequent metabolic imbalance. The same shear forces that can cause diffuse axonal injury in the rotational injury can also cause endothelial damage, which can impair the regulation of cerebral blood flow. And that compromises the blood vessel, and can obviously lead to blood-brain barrier breakdown. As the endothelium are damaged, you get a protein-rich exudate into the extracellular space, which causes vasogenic edema within a few hours of the injury, inflammation, which as you'll see, is caused by macrophages and leukocytes that kind of pass through the damaged endothelium, can further contribute to blood-brain barrier breakdown. And again, this mechanism of hyperexcitability or excitotoxicity can further damage it. Now note the bi-directional nature of these arrows. So the blood-brain barrier breakdown can lead to edema, inflation, inflammation, and hyperexcitability. But likewise, these can cause further damage to the blood-brain barrier. And in fact, the damage to the blood-brain barrier can persist for weeks or months, and in some reports, people have described years. The edema, inflammation, and hyperexcitability can then lead into the delayed response, as shown again by the arrows. This results from neovascularization, with further impaired regulation of cerebral blood flow, dysfunction of astrocytes and microglia, which can contribute to inflammation, and changes to neural network plasticity, and degeneration if the neuron doesn't survive. And then all of these things combined lead to some of the consequences we've seen following trauma, headaches, which I think Dr. Thornton will talk about, cognitive and emotional disabilities, which I think Dr. Hirsch will talk about, and I will discuss seizures and epilepsy. I think coma and death were relatively self-explanatory. No celebration of the 10th anniversary of Comer would be complete without acknowledging the individuals who came before us. And the individual to the left is not only a mentor of mine, but many of my colleagues who are still here at the University of Chicago. That's Peter Huttenlocher. And he demonstrated that the development of synaptic brain connections, as shown in that upper right panel, increased dramatically in early childhood, and persists until about 10 years of age. And synaptic connections are the connections that the brain cells form with one another. And when you look at this active period of development, what you see is an increase in glucose metabolism. And these mechanisms are mediated by excitatory amino acids, such as glutamate, meaning you need excitatory neurotransmitters to provide that development. However, if you have too much glutamate, that can result in excitotoxic damage. And that's shown in this kind of panel or schematic to the left, taken from an article by Giza and Hovda. Essentially, following a traumatic brain injury, what happens is, is you get this indiscriminate release of glutamate, which causes depolarization of brain cells or neurons, which then causes further neurotransmitter release. The neurotransmitter glutamate then binds to multiple glutamate receptors. And this results in a massive influx of sodium and calcium. As a result, the neuron tries to maintain electrical neutrality by effluxing potassium ions. And that results in a high concentration of extracellular potassium ions, which further leads to hyperexcitability. The neuron, overwhelmed by the sodium and the calcium, tries to extrude the sodium ions through the ATP-dependent sodium potassium ATPase. Unfortunately, there's so much sodium that kind of rushes in that the pump can't keep up. So hyperglycolysis ensues. And that is the transport of glucose into the neuron itself. The calcium is a big problem, because calcium can activate certain enzymatic processes, such as proteases, such as calpain, which can break down the microtubules. Or it could start a cascade, known as apoptosis, and further lead to cell death. And so the mitochondria act as a calcium sink. They sequester the calcium, much to their detriment. And when that happens, they're no longer able to produce ATP. And energy failure ensues. And that leads to further death, because then the cell just get swollen to the point of being incapacitated. Downstream in the axon kind of to the right there, what one sees is the effects of calcium on microtubule disassembling. And the microtubules are incredibly important for the transport of messenger RNA from the soma of the neuron down to the terminal boutons. And so if you disrupt that, you have further impairment of neural function. So this is the summary slide of kind of what I've said. So following a traumatic brain injury, you get this massive release of glutamate, which then binds to the NMDA receptors and causes an influx of sodium and calcium. And then the mechanisms I just showed you in the previous slide leads to excitotoxic death. The panel on the right demonstrates the kind of time course for this process. So glutamate is released within the first two minutes. And as you can see by those upper curves, the green and the red curves, you're looking at calcium and potassium levels 400 to 500% normal. The astrocytes try to help by converting the glutamate to glutamine, which is a substrate that could be used for energy by the neuron, but is also overwhelmed. Sheer injury to the endothelium leads to, not only edema and increased ICP, but also macrophage and leukocyte inflammation. In addition, activated astrocytes as well as microglia combined with the leukocytes, produce a number of cytokines, and important to this discussion, neurotrophins, which can cause some of the network reorganization that can lead to epilepsy. OK, so let's go with that foundation. Let's start with posttraumatic seizures in children. This is not epilepsy, right? This is an acute symptomatic seizure. So this is a consequence, or these are seizures provoked by the injury itself. And they have been divided into early and late onset. Early posttraumatic seizures occur within the first week after injury, and are generally directly related to acute or subacute insult. Most occur within the first 24 hours. Late posttraumatic seizures are defined as they occur after the first seven days. These too may be reactive to some subacute process, such as the subsequent development of hydrocephalus or delayed infection due to skull fracture, et cetera. The incidence of posttraumatic seizures is higher in children than in adults following a severe pediatric TBI, with rates of 20 to 39%. And again, as I said, they're much more susceptible, given the biomechanics to injury. There have been several studies suggesting that prophylactic anticonvulsants decrease the risk of early posttraumatic seizures and may improve survival rates. Given the-- sorry-- given this effectiveness and the potential risk of complications from early posttraumatic seizures, as well as the difficulty in identifying a clinical seizure in infants, prophylaxis with antiepileptic medication should be considered for the first week. However, as you'll see, not so much in posttraumatic epilepsy. Now posttraumatic epilepsy is a diagnosis reserved for when a patient has experienced greater than two unprovoked seizures following a traumatic brain injury. Posttraumatic epilepsy is a common cause of morbidity in children. And again, occurs in about 10 to 20% following severe traumatic brain injury. It's greater than 20% following a penetrating injury, such as a bullet. If you look at soldiers returning home from Iraq, the risk of posttraumatic epilepsy is at least as high as 50%. There are multiple proposed mechanisms underlying posttraumatic epilepsy. We've talked about neuronal death. I'm going to talk a little bit about the development of recurrent excitatory neural pathways, this process known as kindling. But in addition, loss of inhibitory GABAergic neurons, interneurons, has been described. Now anticonvulsant prophylaxis does not seem to prevent the subsequent development of posttraumatic epilepsy. So it may prevent a seizure from occurring, but it's not going to prevent the child from developing epilepsy. And in fact, there's animal data that the use of antiexcitatory pharmacological agents like anticonvulsants negatively impact recovery, as excitatory activity is necessary for survival of developing neurons and experience-dependent plasticity. Hence, long term anticonvulsants for prophylaxis are not recommended. So as I mentioned in earlier slide, the graphic to the right is a book published by Dr. Huttenlocher before he retired on neuroplasticity. And again, these mechanisms involve glutamate. So whereas some glutamate is important for development, too much is not so good, as it can lead to excitatory death. But on the other hand, if you block it too vigorously in the recovery period, you may not have enough glutamate to help with redevelopment or reorganization of the synapses. Recently, Park and Chugani published a relatively large cohort looking at posttraumatic epilepsy in children with TBI in Detroit. Epilepsy was diagnosed in about 15%, consistent with previous data. And most of the patients were male, also consistent with previous data. Interestingly, as you see in this lower panel here, they did not observe seizures following moderate traumatic brain injury. But I think this was a relatively small cohort. Looking at the MR neuroimaging in the upper left hand panel here, seven out of eight patients with mild traumatic brain injury had CT findings which were described as normal. The eighth individual had no imaging. That's in comparison to the children with severe TBI, none of which had normal neuroimaging. It should be noted that 54% of the patients in this study with severe TBI had non-accidental trauma. I found the bottom table a little more interesting. About 28% of seizures, including both the mild and severe TBI cases, occurred within the first 24 hours. But 40% occurred after eight days. And to me, that indicates that there's a lot of reorganization of these brain cells following injury. Oh. sorry. So this wouldn't be the University of Chicago if we didn't employee basic science mechanisms to understand disease. Now remember in that summary slide, I mentioned that neurotrophins are released from a variety of cells. And Dr. Andrew Tryba, who is a researcher here at the University of Chicago, and I decided we wanted to understand the mechanisms that lead to epilepsy in these posttraumatic patients a little better. So we looked at a bunch of neuromodulators and decided to study this particular neurotrophin, a brain-derived neurotrophic factor. And the reason we did so is it had already been described in the literature as playing an important role in the recovery from injury. Brain-derived neurotrophic factor, or BDNF, is involved in neurodevelopment, plasticity, and learning and memory. It is a protein which binds-- I'm sorry you can't see this-- to the tropomyosin-related kinase receptor, or the TrkB receptor, to activate a bunch of secondary messenger systems, which can then, as you'll see in subsequent slides, cause some problems perhaps. The idea here is following a traumatic brain injury, BDNF expression and translation is increased, as you see in the panel to the right. And this ultimately leads to increased neurotransmitter release, and perhaps new synapse formation, and overall, an increase in excitation, which is kind of what you want after an injury if, overall, you have a reduction in the general excitation of the nervous system. The problem is, we wondered if too much BDNF could be a bad thing. There is some literature, this is a study by McNamara and colleagues, that looked at conditional deletions of the TrkB receptor. Remember, the BDNF binds to the TrkB receptor. And what they did in this study is they looked at the wild type mice. These are the black squares here. And they used an electrical stimulation model to see how long it would take, or how many stimulations it would take, to develop class four or class five seizures, which are pretty significant seizures. And in the wild type animals, those animals with normal TrkB receptors, it only took about eight stimulations before they got up to a class four seizure. In the heterozygous animals, those animals in which one to allele was normal and one allele had the conditional deletion, it took up to 20 stimulations before they saw seizure activity. And in the animals that were homozygous, meaning both genes were conditionally deleted, even after 50 electrical stimulations, they could not see seizure activity. So several other studies have demonstrated that a decrease in BDNF TrkB signaling results in decreased seizure susceptibility, while over-expression of BDNF TrkB signaling leads to increased seizure susceptibility. However, the role in humans is not clearly documented. So a lot of the studies I just showed you, in fact all of the studies I just showed you, were done in animals. My lab, along with the van Drongelen lab and Dr. Tryba's lab, are one of the few labs in the world that can actually do research on human brain tissue. And so we obtain this tissue from patients undergoing surgery for epilepsy. We identify the seizure focus by placing, actually Dr. Frim does the placing, of intracranial electrodes where we think the seizures are coming from. We identify the seizure focus as that point where the seizures are initiated. We then take a block of that tissue, cut it up, and put it into a dish. We then use electrophysiological techniques to study the electrical properties of that tissue. So in this case, we take a glass pipette with the relatively large bore. We place it over the surface of this brain slice, and then measure the electrical activity of a population of neurons. And so what you're looking at here is just a bunch of neurons that are synchronously firing together. We can then identify a single cell. And using a fine bore pipette, we can then impale that neuron and study the electrical properties of that activity individually. And we can use certain pharmacological agents to isolate that neuron from the rest of the population. This is a paper we published in 2011 that was well-received. Because it was the first description or isolation of the intrinsic bursting neuron. This is a brain cell that many people believe plays a role as a pacemaker in the development of epileptiform activity. Unfortunately, from a histological perspective, you can't tell this neuron, or the pathologist can't tell this neuron, from any other neurons, with the exception of inhibitory neurons. These cells can only be identified functionally, or electrically, after we inject the cell with a current. And what you're seeing here is when we inject in the intrinsic burster with the current, we get this kind of cluster of action potential firing. And we can distinguish that from these neurons, regular spiking neurons, the kind of run of the mill brains cell, by that firing pattern. And it's very obvious. So we wondered whether or not BDNF somehow affected the activity of these neurons, which are believed to play a role in epilepsy. And so we did used simultaneous mouse and human tissue. And interestingly, brain slices of mice are active even at rest, whereas brain slices of human tissue is pretty silent. When we added the BDNF, what we saw is an increase in bursting of the activity in mice quite dramatically. And what we saw in the humans was the start of bursting activity, or the beginning of a seizure, perhaps. We then went on to demonstrate that-- or we were curious as to how the BDNF was doing this. And so we applied the BDNF to the tissue. And what we saw is that after about two to four minutes, the inward current, the amount of sodium that was rushing into the cell, increased dramatically. And that panel to the right shows you that this was true of only the intrinsic bursting neurons. The regular spiking neurons, which constitutes the larger percentage of neurons, were unaffected by the BDNF. And that made sense to us, because these bursting neurons play a role, we believe, in epilepsy. This time course of two minutes is incredibly important. Because as you see in the panel on the right, these green channels here, we were able to block the effects of the BDNF with a compound called SKF. And this compound blocks a particular type of channel, called the TRPC channel. And it takes about two minutes to four minutes for this channel to go from intracellular stores to be incorporated into the extracellular membrane. I'm not going to go in a lot of detail. But these TRP, or T-R-P channels, for transient receptor potential proteins, were first isolated in Drosophila. They were looking at visual signal transduction. And these channels are believed to play a role as chemical and environmental sensors. So let's summarize what I've just told you. And then I just have one more slide after this. Following trauma, we get a massive release of glutamate. But in addition, we also get a release of BDNF. The cell has an underlying bursting pattern, as you can see to the right. But following the release of the BDNF, it binds to the TrkB receptors, which then activate secondary messenger systems, resulting in the incorporation of these TRPC channels into the membrane, which then results in increased bursting. And the idea here is the central nervous system likes to exist in a state where excitation, which is mediated by glutamate, is in balance with inhibition, which is mediated by GABA. And so it likes to kind of stay in this blue range here. And so what we believe is that following traumatic brain injury, there may be injury to some of the excitatory neurons. Or there may be some damage, such that excitation is decreased. The brain doesn't like to be too inhibited. So via some of the mechanisms I showed you, the release of BDNF is going to try to get that state back to homeostasis. But unfortunately, on occasion, it exceeds that. And seizures occur. What I basically have just showed you is we've identified a potential target for what we like to call the after morning pill, right? So if you're out in the field, a soldier, for example, and you sustain traumatic brain injury, the idea is you could inject yourself with the compound similar to SKF. You'd have two to four minutes to do so. And although you would have the consequences of the traumatic brain injury, which will be discussed later, we may be able to prevent epilepsy from occurring. And I think this is a nice example of how these cellular and molecular changes can affect the brain in such a way that it predisposes the individual to cognitive impairment, or behavioral and other psychiatric problems. For example, maybe you don't get all the way up here in the excitatory range. Maybe you're in a state where your inhibition exceeds excitation. And cognitive impairment predominates. And I'd just like to thank Dr. van Drongelen, Dr. Tryba, who's here, and a variety of others who help me do this work.
