Neural circuit mechanisms of post–traumatic epilepsy

1Post-traumatic epilepsy (PTE) is a common long-term consequence of traumatic brain injury (TBI), for which there are few effective therapies. More than one million people are presented for medical care each year in the United States after sustaining a head injury. While the incidence of epilepsy in the general population is about 1–2%, the overall incidence of epilepsy is 7–39% after severe closed-head injury and as high as 57% after penetrating injury, and approximately 20% of all acquired epilepsies are the result of TBI. Injury severity is generally considered the major determining factor for developing a seizure disorder after brain trauma. Focal brain damage and cortical contusions to the frontal, temporal, or parietal lobes are also important risk factor for human PTE. Several other risk factors have been identified, many of which are associated with focal lesions: dural penetration, depressed skull fracture, intraparenchymal hemorrhage, epidural and/or subdural hematomas, reduced brain volume, prolonged impaired consciousness, presence of injury-induced seizures, age (young children and the elderly), and gender (male;;;;; ). PTE occurs as temporal lobe epilepsy (TLE) in 35–62% of trauma patients.

2Here, we review experimental animal models used in PTE research, including the neurophysiological and structural abnormalities believed to underlie the increased propensity of the injured brain to generate spontaneous seizures. Emphasis is placed on modifications of synaptic networks in the injured dentate gyrus that are associated with post-traumatic epileptogenesis. The hippocampus has long been recognized as an important structure in epilepsy. The dentate gyrus is particularly susceptible to injury, often undergoes time-dependent structural reorganization, is a widely used model system for altered synaptic circuitry of cortical structures in epilepsy, and is one of the brain regions that has been best characterized in terms of changes in structure–function relationships after TBI.

3Most information about cellular mechanisms of epileptogenesis has been derived from animal models. Although there is ongoing debate over which experimental paradigms make for appropriate models of PTE, the presence of spontaneous burst discharges, preferably in the form of spontaneous seizures in vivo, that develop following a sudden, mechanical injury (i.e., TBI) seems to be necessary minimal criteria for a “post-traumatic epilepsy” model. The presence of relatively high numbers of animals in which the behavioral, anatomical, and physiological characteristics of epileptic pathology in humans are reproduced, including short but distinct latencies between initial injury and spontaneous seizure onset, also has obvious advantages. Ideally, a model will effectively reproduce the range of tissue deformation and damage observed in mild, moderate, and severe TBI in a controllable way so that the dual pathology of cellular and molecular factors associated with injury dynamics and epileptogenesis might be dissociated. Using these selection criteria, many traditional experimental epilepsy models, such as pharmacologically induced status epilepticus, do not qualify as models of PTE, because they do not induce spontaneous seizures by a mechanical insult. This is not to negate these models, because they have provided valuable information about cellular mechanisms of epileptogenesis. Rather, these criteria provide a more restrictive and specific basis for differentiating between epileptogenesis mediated by a mechanically induced lesion versus brain insults by other causes (e.g., pharmacological agents, electrical stimulation, or models of neurodegenerative diseases). It is important to make this distinction, because there are currently few effective treatments for PTE (; discussed below). Therefore, it is important to understand how the epileptogenic cellular processes in experimental neurotrauma compare with human PTE and other experimental epilepsy models (e.g., TLE induced by status epilepticus).

4The fluid percussion injury (FPI) model is one of the best characterized in terms of epilepsy development after closed-head TBI. In this model, injury is delivered through a craniotomy by rapid fluid injection, accomplished by a hammer swung on a pendulum. The fluid pulse first strikes the intact dura and then moves into the epidural space. The height from which the pendulum hammer is dropped determines the pressure of the fluid pulse transmitted through a fluid-filled cylinder. This pressure can be used to estimate injury severity. Injury severities that have been investigated with regard to long-term changes in excitability can be categorized into two groups: severe injuries, with fluid pressure typically >3 atm; and moderate injuries, with impact forces of 2–2.2 atm. Craniotomies can be applied to the midline to produce more diffuse injuries or laterally to produce mixed focal and diffuse injury.

5Several studies have reported electrographic seizures in rats and more recently in mice after severe FPI. Susceptibility to pharmacologically induced seizures is also increased after more moderate injuries, but clear evidence for spontaneous seizures after moderate FPI has not been demonstrated. After severe lateral FPI, spontaneous seizures are present in up to 50% of rats and 3% of mice by 12 months post-injury. Depth-electrode recordings have inferred that spontaneous electrographic seizures involve hippocampal structures, and these electrographic events are accompanied by obvious behavioral abnormalities defined by a widely used Racine rating scale for rodent seizures. However, seizure frequencies are typically quite low and time to first seizure is long in these animals, making it somewhat difficult to definitively attribute cellular mechanisms that occur following an injury to epileptogenesis.

6In addition to lateral FPI, rats injured by rostral parasagittal FPI develop seizure-like epileptiform activity and seizures that are accompanied by more subtle changes in behavior (e.g., behavioral arrest), but do not generally develop tonic–clonic convulsive seizures. Similar findings were also reported for injuries to parietal and occipital cortices, but fewer animals developed spontaneous epileptiform activity. Originally, behavioral seizures in this model were examined based on the traditionally used Racine rating scale, but later studies developed a new seizure classification scale to describe the subtle behavioral abnormalities associated with electrographic activity in this model. The authors reasoned that post-traumatic seizures after rostral parasagittal FPI did not fit well with the Racine scale and proposed that electrographic abnormalities with ≥2-s duration represent ictal events. These “epileptiform” events were also observed in nearly 40% of sham-control rats by 21 weeks post-injury. It is unclear why control rats also sometimes have epileptiform activity, but this may be due to how a seizure is defined. Thus, it appears that the majority of electrographic abnormalities in rats injured by rostral parasagittal FPI represent relatively brief events that are associated with behavioral inactivity or crouching, compared to robust convulsive seizures observed after lateral FPI, but they may not have been considered “seizure” activity in other PTE studies. Future studies that combine long-term EEG monitoring with electromyogram (EMG) and electro-oculogram (EOG) may be useful to better distinguish ictal activity from interictal events or benign variants of normal electrographic patterns that can morphologically reflect epileptiform activity but are not epileptic.

7The weight drop model, also referred to as an impact-acceleration injury, has been examined as a model of closed-head post-traumatic hyper-excitability. Trauma is delivered to the neocortex by dropping a large blunt weight through a tube to impact the skull. Injury severity is managed by adjusting the height at which the weight is dropped. This injury produces large and extensive damage to cortical and subcortical structures, including the dentate gyrus and hippocampus in rats. Seizures have not been reported in this model. However, increased seizure susceptibility to pentylenetetrazol (PTZ) is observed 15 weeks after injury. The lack of demonstrated spontaneous seizures after weight drop is an obvious limiting factor in using this injury to model PTE. Impact is delivered to the intact skull, not directly to the brain via craniotomy as in FPI. This is sometimes considered a limitation of the model, due to increased risk for skull fracture; and injury dynamics after weight drop can depend somewhat on skull thickness. On the other hand, this characteristic might better reflect the range of variability expected in human closed-head TBI, which is unlikely to occur by craniotomy. However, by using gravitational forces to produce head injury, there can also be a risk for secondary “rebound” injury. Weight drop injury can also be difficult to perform on mice, limiting the use of transgenic animals and genetic manipulation with this model.

8Controlled cortical impact (CCI) injury is a widely used experimental model of closed-head injury that was recently identified as a model of injury-induced epilepsy. First developed by, this model often utilizes an electronically controlled pneumatic impactor to apply a focal contusion injury to the brain surface through a craniotomy. Injury severity is primarily managed by adjusting the depth of tissue compression, but other external injury parameters can also be controlled with precision (i.e., impact depth and velocity, impactor shape and size, and number of craniotomies). This model has unique advantages over weight drop and FPI, because it allows for good control over biomechanical parameters. This allows for a relatively consistent and reproducible focal injury with minimized risk for inaccuracy or secondary “rebound” injury. In addition, the CCI device can be scaled for use in rodents or even large animals, such as sheep or non-human primates.

9Several laboratories have independently demonstrated the presence of spontaneous seizures in mice or rats after CCI. Early seizures, within 24 h of injury, have been reported in rats and mice. Within weeks after CCI, spontaneous convulsive seizures are present in up to 40% of mice after severe injuries and 9–20% of mice after more moderate injuries. CCI injury has also been administered to immature rats, and a percentage of these animals develop spontaneous electrographic seizures. Seizures after CCI are similar to spontaneous behavioral and electrographic seizures that have been described in rats after lateral FPI and in models of TLE. Although seizure frequency after CCI injury appears considerably lower than in widely utilized chemoconvulsant TLE models, spontaneous seizures after CCI injury occur with roughly similar onset latency as pharmacologically induced TLE, which appears to be considerably shorter than the seizure onset latency after severe lateral FPI in rats. For example, previous studies have suggested that limbic involvement typically does not evolve in rats until several months after FPI, but many mice after CCI injury develop seizures and hippocampal pathology by 8 weeks post-injury.

10This model of penetrating TBI involves partial isolation of neocortical circuits by surgical undercut to reproduce the deafferentation and white matter damage caused by neocortical TBI. In vivo electrographic epileptiform activity develops in intact regions of the neocortex within hours after injury in cats, but clear evidence for spontaneous electrographic seizures has not been demonstrated.

11Recently, a new model of penetrating, ballistic-like TBI in rats was developed to more closely recapitulate aspects of war-time TBI. Injury is delivered to frontal cortex using a computer-controlled hydraulic pressure generator to rapidly inflate and deflate an elastic water balloon on a probe, which creates a cortical cavity in the brain. Injury severity can be managed by amount of inflation. In this model, up to 70% of animals develop seizures 72 h after injury, and the frequency and the duration of epileptic events can be scaled according to injury severity. However, it is unknown whether rats injured by ballistic-like TBI develop long-term spontaneous seizures.

12The term “epileptogenesis” refers to a transformation process by which the normal brain develops an increased propensity for generating spontaneous seizures. This process typically involves structural alterations in neural circuitry – due to progressive neuronal damage and “self-repair” mechanisms – which develop through a latent period of variable time and culminate with the emergence of spontaneous, recurrent, seizures. That this process includes a latent period of variable time suggests a progressive series of cellular changes may be involved. As such, human post-traumatic seizures are often classified according to the time of their presentation after injury: immediate or impact-associated (<24 h after injury), early (<1 week after injury), and late (>1 week after injury;;; ). This classification scheme is thought to represent different pathophysiological processes. Understanding the epileptogenic process after TBI should help to elucidate the importance of these cellular mechanisms in PTE and promote new therapeutic targets. Trauma sets into motion a multidimensional cascade of cellular and molecular events that involve three temporally overlapping responses in the brain: primary and secondary injuries and “self-repair” mechanisms (;;; Figure 1). An important goal of studies which investigate post-traumatic epileptogenesis is to dissociate injury-induced cellular alterations that promote seizure generation from compensatory and “self-repair” responses.

13Primary injury refers to the immediate tissue deformation and compression that occurs within seconds to minutes after mechanical brain insult. There is an immediate release of neurotransmitters, glutamate in particular, which is followed by ion channel activation and calcium influx. This can lead to excitotoxic injury, reflected by mitochondrial damage and energy depletion, neuronal and glial swelling, and cell death. Vascular damage and blood–brain barrier (BBB) disruption can also occur in primary injury. Cortical structures such as the hippocampus are especially vulnerable to neuronal damage after moderate and severe TBI. Moreover, the presence of immediate or early seizures can further exacerbate initial damage and complicate injury management. These seizures are considered to be injury-induced (i.e., provoked) and are not epileptic, because they occur as a direct result of neurologic and systemic abnormalities of the acute trauma. Strategies to protect against primary injury are typically aimed at taking preventive rather than therapeutic measures (e.g., wearing protective headgear).

14Secondary injury involves a myriad of cellular and physiological factors associated with progressive tissue damage. Over time, brain injury triggers inflammatory cascades, growth factor responses, edema, mitochondrial dysfunction, oxidative stress due to the build-up of free radicals and reactive oxygen species, delayed cell death, perturbation of cellular calcium homeostasis, and hypoxia and ischemia. The brain also initiates “self-repair” mechanisms over the course of days to months concurrent with the development of secondary tissue damage. This period is characterized by synaptic circuit remodeling, axon sprouting, synaptic plasticity, gliosis, neurogenesis, and angiogenesis. The time-dependant reorganization of synaptic circuitry can promote increased synchronous neuronal activity that contributes to functional recovery, but may also contribute to spontaneous seizure generation. During this period, seizures that occur spontaneously (i.e., late seizures) are considered to represent PTE because they reflect permanent changes in neuronal structure and function. The primary goal of many neuroprotective measures is to prevent or reduce secondary brain damage and to enhance beneficial “self-repair” mechanisms. However, repair mechanisms may not be universally beneficial, as some of these same mechanisms (e.g., axon sprouting and synaptic reorganization) are also correlated with the development of spontaneous seizures.

15Genetics, gender, age, acute medical treatments, and the agent of injury all likely influence the epileptogenic processes, but the contribution of these personal traits and injury dynamics in epilepsy have not been well established. A better understanding of the importance of these factors in post-traumatic epileptogenesis will likely elucidate why some individuals develop PTE after TBI while others do not.

16The central dogma in epilepsy research has long been that seizures occur due to some type of imbalance between excitatory and inhibitory neurotransmission. In humans and animal models of injury-induced epilepsy, the presence of spontaneous seizures is strongly associated with axon sprouting and reorganization of neural circuitry. Within this framework, most experimental injury models show evidence that excitatory connectivity is enhanced and inhibitory influences are decreased. These changes often involve the presence of recurrent excitatory circuits, which form when principal cells are sufficiently interconnected and have long been proposed as a cellular basis for pathologically synchronous neural activity and seizures.

17Several laboratories have independently demonstrated reactive plasticity of glutamatergic axons and formation of new, recurrent excitatory circuits in TBI models. The dentate gyrus has acted as a model system to study excitatory axon sprouting associated with PTE because it is particularly susceptible to injury and subsequently undergoes structural reorganization (Figure 2). Dentate granule cells, which are not normally connected to each other, sprout axon collaterals into the inner molecular layer to form functional recurrent excitatory connections with nearby granule cells during epileptogenesis, a process termed “mossy fiber sprouting.” These dramatic changes in local mossy fiber circuits are relatively easy to detect by Timm’s histochemistry and are consistently reproduced in human tissue and experimental models of TLE. Mossy fiber sprouting into the inner molecular layer of the dentate gyrus is also a common feature of the epileptic dentate gyrus in TLE patients with a history of head injury, and it has been consistently reported in animal models of TBI. Post-traumatic mossy fiber sprouting is generally more robust after severe versus mild TBI and might be related to whether cortical injury directly impinges upon the hippocampus. However, the degree of mossy fiber sprouting after experimental TBI is qualitatively less than the robust, bilateral sprouting observed after experimental status epilepticus, and it is often more regionally localized to areas near the injury.

18These anatomical changes in mossy fiber circuits are associated with a functional alteration in neuronal circuitry of dentate granule cells that may contribute to abnormal network synchronization after TBI. Similar to rodent models of TLE, new functional recurrent excitatory circuits emerge in the presence of mossy fiber sprouting after TBI that are not detected in the normal dentate gyrus. When surgically isolated from afferent input (i.e., entorhinal cortex), spontaneous and evoked reverberating burst discharges, indicative of synchronous network activation, have been observed by single-cell and extracellular field potential recordings from granule cells in slices with mossy fiber sprouting after TBI. Studies using localized glutamate stimulation have suggested monosynaptic granule cell-to-granule cell connections in slices with mossy fiber sprouting, which are absent in the normal dentate gyrus. These new excitatory connections among granule cells are only present in brain slices with mossy fiber sprouting and are not detected in slices without sprouting, even in the same injured animal. Although recurrent excitatory circuits are normally masked by recurrent inhibitory circuits and can only be revealed by experimentally altering the extracellular environment to increase excitation, these new excitatory circuits have been proposed to form the basis from which synchronous network activity can periodically arise in the dentate gyrus, particularly if inhibition periodically fails. Therefore, mossy fiber sprouting may provide a means for regional granule cell network synchronization after TBI that may be “unmasked” if inhibitory control is impaired.

19Other brain regions that are susceptible to injury, such as hippocampus and neocortex also undergo synaptic reorganization after TBI. For example, an increase in spontaneous and evoked burst discharges has been detected in neocortex of rats within 2 weeks after severe CCI injury. Although it is not known whether these changes are due to the formation of new excitatory circuits in neocortex or enhanced intrinsic excitability of existing circuits after injury, similar findings have been observed at similar time points after neocortical undercut (recently reviewed by ), which appear to be associated with an increase in excitatory synapses onto pyramidal neurons. In CA1 of hippocampus, pyramidal neurons show immediate deafferentation following CCI injury, possibly due to loss of CA3 neurons, which is followed by a time-dependent increase in number of synaptic contacts and fiber excitability. Taken together, these findings suggest that changes in functional excitability of other brain regions might also contribute to seizure generation after TBI, independent of mossy fiber sprouting.

20GABAergic interneurons form robust local synaptic connections with principal cells and play an important role in controlling cortical activity. Two primary modes of GABAA receptor-mediated inhibition have been identified: phasic (synaptic) and tonic (extrasynaptic) inhibition (;; Figure 3). Phasic inhibition refers to the transient activation of postsynaptic GABAA receptors at the synaptic junction following exposure to high concentrations of GABA released from presynaptic vesicles. This form of synaptic communication allows for rapid transmission of information from the presynaptic terminal to the postsynaptic membrane in a spatially and temporally restricted manner. In whole-cell voltage-clamp recordings, synaptic events can be easily identified as inhibitory postsynaptic currents (IPSCs). Small amounts of GABA escape the synaptic cleft and activate high-affinity extrasynaptic GABAA receptors on the same or adjacent neurons, an event termed “spillover.” Tonic inhibition refers to the persistent activation of these extrasynaptic GABAA receptors by low concentrations of ambient GABA. Phasic and tonic currents can be isolated by high concentrations of GABAA receptor antagonists. In whole-cell voltage-clamp recordings the tonic current is reflected by a shift in “holding” current.

21In the dentate gyrus, the loss of certain populations of hilar interneurons is a common histopathological feature in human PTE, and several labs have reported a loss in the number of inhibitory interneurons after experimental TBI. Although it is unclear whether subtypes of hilar neurons are preferentially lost after TBI, as is often observed in TLE, inhibitory hilar neuron loss ipsilateral to the site of injury is typically associated with a reduction in synaptic inhibition of granule cells. In contrast, whether there are changes in tonic inhibition after TBI remain unresolved. One study reported an increase in tonic current amplitude in dentate granule cells contralateral to severe CCI injury, whereas other studies reported no change in tonic current amplitude to granule cells or a marked decrease in tonic inhibition in semilunar granule cells ipsilateral to moderate FPI. Recent evidence has also shown that there are changes in the expression of GABAA receptor subunit levels after CCI injury, which might also affect inhibitory influences after TBI.

22Beyond the presence of substantial interneuron loss and a subsequent reduction in synaptic inhibition of granule cells after TBI, relatively little is known about how individual interneuron subpopulations are affected by mechanical trauma. In a recent study, we reported an increase in excitatory input to hilar somatostatin interneurons arising from granule cells and CA3 pyramidal neurons after CCI injury, suggesting that reactive plasticity of glutamatergic axons is not limited to connections among principal neurons. These changes were accompanied by an increase in the decay time constant of spontaneous inhibitory synaptic events in granule cells, which is consistent with increased activity or axon sprouting of this dendritically projecting GABA neuron population after injury. Similar changes have also been reported in a model of status epilepticus. However, whether this phenomenon represents a compensatory response to boost feedback inhibition in the dentate gyrus, which is compromised by injury, or a pathological response that contributes to seizures (e.g., by a periodic failure of already fragile inhibitory circuits or increased risk for GABA-mediated depolarization in distal dendrites) is unknown. It is also not known how other populations of inhibitory neurons are affected by TBI. Given the relatively large number of ways in which inhibitory circuits can be modified by head injury and the important role of inhibitory neurons in restraining excessive cortical excitability, future studies aimed at understanding how inhibition is modified after closed-head TBI, including in brain regions outside of the dentate gyrus, should remain an active area of investigation.

23While the immune system provides crucial protection against infected or damaged tissue, accumulating evidence points to an involvement of inflammation in the pathophysiology of epilepsy. Heightened or abnormal innate immune responses are noted in individuals with epilepsy, in animal models of this disease, as well as with pro-epileptic insults such as infection, trauma, ischemia/hypoxia, fever, and recurrent seizures. Importantly, many innate immune processes play significant roles in cell excitability and survival during inflammation, which have the potential to promote network hyper-excitability. Experimental models of epilepsy that do not involve head injury (non-PTE models) have described several inflammatory pathways that contribute to seizure susceptibility. Despite these indications, surprisingly very few basic studies have studied the role of the immune system in experimental PTE even though inflammation and BBB breakdown are hallmark features of head injury. The causal role of inflammation in PTE development can, at least in part, be assessed by systematic experimental manipulation of individual components of inflammation in animal models. An important consideration in these studies is to discriminate between acute effects of seizure threshold (i.e., anti-convulsant effects) and long-term development of spontaneous seizures (i.e., epileptogenesis), because these processes may have different mechanisms and inflammation may contribute to them in different ways. This section attempts to highlight the need to examine the role of inflammation in epileptogenesis as presently there a very few studies that do so using PTE or non-PTE models.

24Work in brain trauma has extensively characterized patterns of immune responses following TBI (detailed review in; ). Head trauma initially triggers inflammation by a variety of factors including direct damage to the BBB as well as an accumulation of foreign particles, extravasated blood proteins, cellular debris, complement fragments, prostaglandins, and both reactive oxygen and nitrogen species within the brain. The resulting inflammatory cascade involves the coordinated activity of resident central nervous system (CNS) immune and non-immune cells, circulating immune cells, and transport mechanisms across the BBB. Neurons, astrocytes, and particularly microglia located proximal to the site of injury rapidly respond to trauma with pro-inflammatory signals including chemokines, cytokines, and tissue adhesion molecules. The release of these inflammatory mediators supports the further recruitment, migration, and infiltration of leukocytes into the brain parenchyma. The invasion of leukocytes (chiefly neutrophils, monocytes, and lymphocytes) results in further disruption of homeostasis and secretion of pro-inflammatory signals within acutely spared brain regions resulting in a potent inflammatory response to these sites. Additional increases in BBB permeability magnify levels of inflammatory units and blood proteins entering brain tissue.

25In the context of epilepsy, an important feature of inflammation is its contribution to ongoing cell death after trauma. In addition to cell loss by excitotoxicity, ischemia, and disruptions in fluid and metabolic homeostasis, inflammatory cytokines enhance signaling components of apoptosis. Proteases and free radicals released during inflammation support lipid and protein peroxidation, mitochondrial damage, DNA damage, as well as further induction of apoptotic mechanisms. Conversely, many immune components provide a neuroprotective effect following trauma. The mechanisms underlying these neuroprotective effects are not fully characterized, however inflammatory components can provide neurotrophic support by increasing growth factors, reducing oxidative stress, or via anti-inflammatory signaling. Understanding the role that individual inflammatory components serve in post-traumatic cell loss will likely help determine how they affect PTE development. Components of inflammation also affect cell excitability within the CNS in addition to their effects on cell survival. For example, inflammatory cytokines modulate neurotransmitter levels, communication between neurons and glia, GABAA receptor-mediated responses, and calcium currents. Chemokines also affect neuronal excitability by modulation of voltage-dependent channels (sodium, potassium, and calcium), activation of an inward rectifying potassium conductance and increasing the release of neurotransmitters including GABA, glutamate, and dopamine. The effects of inflammation on cell loss and cell excitability may serve a role in the development of network hyper-excitability and epileptogenesis following TBI.

26There are several challenges to understanding the overall contribution of inflammatory pathways to PTE. Individual components of inflammation typically affect many processes; they are pleiotropic, and are integrative such that one process can affect many others via forward and backward regulatory pathways. For example, treatment with antibodies against interleukin (IL)-6 results in elevated serum levels of tumor necrosis factor alpha (TNF-α) whereas treatment with antibodies against TNF-α results in decreased serum levels of IL-6. Individual inflammatory components also have different temporal expression patterns following head injury (for review, see; ). There is also a complex spatial patterning of post-traumatic inflammation where expression may be specific to cell-type or brain region and may be different between peripheral and central tissues. For example, transgenic mice that over-express IL-6 under a glial fibrillary acidic protein (GFAP) promoter (i.e., to reflect astrocytic production of IL-6) exhibit spontaneous seizures, neuronal loss, neural dysfunction, and breakdown of the BBB. In contrast, mice that over-express this same cytokine under a neuron-specific enolase (NSE) promoter display no neuronal damage or seizures. All of these characteristics depend on the type of immune component, unique biomechanics of the TBI, as well as the specific traits of the injured individual. As studies continue to document these pathways in TBI it is becoming increasing important to ascertain more specific profiles of individual inflammatory components that include their spatial and temporal patterning as well as their net effect on cell loss and cell excitability.

27The PTE field can be guided by reports that characterize or manipulate immune processes in non-PTE models and models of trauma. To date, many links between innate immunity and seizure expression in non-PTE models have been made studying cytokine signaling. Cytokines are a diverse family of glycoproteins that are secreted by glia, neurons, leukocytes, endothelial and epithelial cells in response to stress, immune challenge, and injury. Cytokine signaling occurs at receptors found on a variety of cells throughout the body via transcription-dependent and transcription-independent mechanisms. Cytokines are often characterized as either pro-inflammatory (e.g., IL-1β, IL-6, and TNF-α) or anti-inflammatory, with the latter exerting their effects by reducing expression of pro-inflammatory cytokines (e.g., IL-4, IL-10, and IL-13; ). While basal cytokine expression is low, these signals are highly elevated in the acute phase of TBI. Transgenic glial over-expression of IL-6 or TNF-α results in spontaneous seizures and neurodegeneration including a loss of GABAergic cells in cortical and hippocampal structures. IL-6 delivered to naïve animals intranasally increases their seizure susceptibility to the convulsant PTZ. Brain administration of IL-1β increases the duration of seizures induced acutely by kainic acid, whereas administration of the IL-1 receptor antagonist (IL-1ra) provides anti-convulsant effects. Mice with transgenic glial over-expression of IL-1ra or IL-1 receptor type 1 knock-out mice exhibit reduced susceptibility to convulsant-induced seizures. The prostaglandin receptor EP2 has recently been targeted using novel inhibitors because of its role in diverse cytokine signaling and neurotoxicity. Inhibition of prostaglandin receptor EP2 reduces cytokine expression, markers of astrogliosis and microgliosis, mortality, hippocampal neurodegeneration, and BBB leakage in pilocarpine-treated mice in the absence of acute anticonvulsant effects. These results, particularly the long-term development of spontaneous seizures observed with over-expression of IL-6 and TNF-α indicate a role for cytokines in epileptogenesis that needs to be assessed in models of PTE.

28Increases in BBB permeability and tissue adhesion molecules may also reduce seizure threshold or have pro-epileptogenic effects (;; reviewed in; ). The BBB’s constituent components: endothelial cells, pericytes, and astrocytic end-feet work in concert to homeostatically regulate the flow of molecules and cells between the vasculature and brain parenchyma. The resulting “neurovascular unit” that arises from these structures in combination with local neurons is drastically altered by primary and secondary damage following TBI. While a positive correlation between BBB permeability and recurrent seizures has been observed following electrically induced status epilepticus, the relationship between seizures and BBB permeability remains complex. Increased BBB permeability allows entry of inflammatory cells/molecules into the brain, while in parallel, inflammatory signaling from sites within the brain can also promote BBB permeability. Thus, secondary phases of increased BBB permeability and inflammation can occur in response to immune signaling and these secondary phases may play a role in post-traumatic epileptogenesis.

29Emerging work using animal models has further characterized how BBB permeability is altered in pathological conditions and how individual infiltrates within the extracellular space affect local cell survival and excitability. Repeated administrations of a granulocyte-specific antibody to reduce populations of neutrophils produce anti-convulsant and anti-epileptogenic effects in pilocarpine mice. Brain slices from animals given experimental disruption of the BBB using bile salts exhibit epileptiform activity. These bile salts result in an extravasation of serum albumin into the brain and this epileptiform activity is similarly observed in slices from animals given direct cortical application of serum albumin. The mechanistic link between extravasation of serum albumin into the brain and epileptiform activity is suggested to be a transforming growth factor beta (TGF-β)-dependent down-regulation of an inwardly rectifying K+ current in astrocytes that reduces their ability to buffer extracellular K+. Administration of rapamycin or an antagonist of the prostaglandin receptor EP2 are both associated with reduced BBB leakage. Administration of IL-1β or transgenic over-expression of TNF-α result in increased BBB permeability. There is also growing interest in understanding how adhesion molecules that support transport across the BBB may contribute to, as opposed to result from, epileptic pathologies. Pilocarpine-induced seizures increase levels of vascular cell adhesion molecules (VCAMs) including intercellular adhesion molecule-1 (ICAM-1), VCAM-1 and P-selectin glycoprotein ligand-1 (PSGL-1) and these increases enhance leukocyte adhesion to CNS vessels in vivo. Exogenous delivery of antibodies for either α4 integrin or VCAM-1 delivered 1 h after pilocarpine treatment, and repeated (every other day for 20 days), greatly reduces spontaneous seizure expression. Interestingly, magnetic resonance imaging of individuals with TBI identifies increased BBB permeability in the majority of cases involving PTE and this enhanced permeability is sustained from days to years post-injury in some individuals. These findings suggest that BBB permeability may be a common feature of inflammation-dependent changes in seizure threshold and/or epileptogenesis. The long-term suppression of spontaneous seizures in pilocarpine-treated mice by in vivo treatment with antibodies against α4 integrin, VCAM-1, or granulocytes indicates several important potential targets to treat PTE.

30Chemokines (also known as chemoattractant cytokines) may also contribute to the development of epilepsy. Chemokines are chemotactic proteins that are characterized by the relative location of their cysteine (Cys) residues within the NH2 terminal of each protein. Chemokines are classified as CC, CXC, CX3C, and XC where CC refers to both Cys residues adjacent to one another, CXC refers to both Cys residues separated by one amino acid, CX3C refers to both Cys residues separated by three amino acids and XC has only one Cys residue. There is also a lipopolysaccharide inducible CC chemokine receptor termed L-CCR. The central role of chemokines is to direct cell migration including migration of leukocytes during brain inflammation. Importantly, individual (or classes) of chemokines are known to affect specific types of leukocytes. Many chemokines are increased after head trauma including CCL3, CCL9, CCL12, CCL10, and CCL2 (also known as monocyte chemoattractant protein MCP-1; ). Unfortunately few, if any, studies have selectively targeted chemokines to characterize their role in PTE. CCL2 is up-regulated following pilocarpine or kainate induced SE. CCL3 and CCL4 are increased in amygdala stimulation induced SE. Administration of the small molecule Minozac results in reductions in CCL2, IL-1β, IL-6, and TNF-α; behavioral improvement; and attenuates the increased susceptibility to electroconvulsive shock-induced seizures after experimental closed-head injury. These results indicate a potential neuroprotective or neurorestorative role for suppressors of chemokines, cytokines, or other pro-inflammatory responses. Targeted approaches are needed to assess the contribution of individual chemokines to epileptogenesis in models of PTE. Identification of a role for individual chemokines can provide clues as to what types of infiltrating leukocytes are pro-epileptogenic based on established relationships between individual chemokines and their target immune cells.

31Collectively, a number of studies suggest that inflammation may contribute to the development of PTE. Inflammation or other immune changes are shared features of many seizure disorders and these changes often precede the manifestation of behavioral seizures (i.e., during the latent period;; ). An emerging concept is that seizures themselves can result in brain inflammation, thereby raising the possibility of a reciprocal relationship between inflammation and seizures that may support or maintain epilepsy. Inflammation affects cell excitability and cell survival and there is some evidence that these effects can promote network hyper-excitability. There is also a growing need to distinguish immune effects on seizure mechanisms from immune effects on recovery and compensation as there is at least some evidence that individual inflammatory components affect both processes. For example, in addition to TNF-α’s pro-excitatory and pro-epileptic effects, this inflammatory cytokine serves a neuroprotective role, since mice lacking TNF-α show enhanced lesion size and BBB breakdown following CCI. The pleiotropic and integrative nature of inflammatory components will offer some difficulties in this type of pathway-specific targeting. Nonetheless, this pathway-specific approach needs to be further extended in models of PTE and other forms of epilepsy to characterize how individual components of inflammation affect epileptogenesis. While there are clear challenges in identifying the role of inflammatory processes in the pathophysiology of PTE, the possibility to reduce or block the development of epilepsy after TBI provides great encouragement for work in this area.

32The development of medically intractable epilepsy is one of the most common long-term health problems associated with head injury in humans. Recent work using animal models of TBI has shown substantial evidence for the formation of new, excessive recurrent excitatory synaptic connectivity and alterations to inhibitory interneurons in this disorder, although these studies have primarily focused on the dentate gyrus and other regions of the injured brain should be investigated in more detail. However, there are a number of important aspects of post-traumatic epileptogenesis that are not fully understood. For example, there is a need for a more complete profile of how individual components of inflammation and BBB permeability contribute to post-traumatic epileptogenesis. Thus, much progress has been made in modeling epilepsy in rodents after mechanical injury and basic mechanisms of neural circuit reorganization associated with epilepsy have been identified in these models, but new developments in this field should continue to identify precise cellular or molecular mechanisms that control the development of epilepsy after head injury.

33The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.