Modeling Post-Traumatic Epilepsy for Therapy Development

Giulia Curia; Clifford L Eastman; John W Miller; Raimondo D’Ambrosio

Chapter 10Modeling Post-Traumatic Epilepsy for Therapy Development

Curia G, Eastman CL, Miller JW, et al.

INTRODUCTION

Epilepsy is the most prevalent serious neurological disorder, afflicting almost 1% of the population worldwide.¹^,² It is a heterogeneous disorder, comprising numerous syndromes with a wide range of etiologies,³ that is defined by the manifestation of chronic spontaneous recurrent seizures (CSRSs). An epileptic seizure, in turn, is defined by the International League Against Epilepsy (ILAE) as “transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain.”⁴^,⁵

Post-traumatic epilepsy (PTE) arises after mechanical damage to the brain and is diagnosed when spontaneous seizures are observed at least a week after brain injury. PTE is the most prevalent acquired epilepsy in young adults and accounts for 5% of epilepsies overall.⁶^,⁷ There are currently no cures for PTE and no means to prevent the disorder in those at risk.⁸ Available treatments of PTE are symptomatic, and at least 40% of patients have seizures that cannot be controlled with any of the available drugs.⁹^,¹⁰ This dire situation requires rethinking the development and use of animal models for the development of therapies for PTE.¹¹^,¹² In this chapter we will introduce the problem and discuss several topics crucial for modeling PTE for therapy development.

What to Model

Human PTE results from a traumatic brain injury (TBI), which may result from traffic accident (~47%), fall (~30%), recreation or sport accident (14%), and assault (7%).¹³ The complexity and diversity of PTE parallels that of TBI, which includes both focal and diffuse components due to acceleration/deceleration injuries, blunt force closed head injuries with or without skull fracture, contusions, and penetrating brain injuries. Acceleration/deceleration injuries induce diffuse damage, while predominantly focal injuries induce surface contusion and laceration that can be accompanied by skull fracture or hematoma. Thus, TBI may affect different epileptogenic areas of the human brain.¹⁴ Although subcortical structures may be involved when the impact is severe, surface focal injuries are often restricted to the vicinity of the mechanical impact and preferentially affect the frontal and temporal lobes, possibly due to the particular shape of the human skull.¹⁵ Thus, in view of the well documented high epileptogenicity of brain contusions in both humans and experimental animals,¹⁶^–¹⁹ it is not surprising that human PTE presents most often as frontal and temporal lobe epilepsies, with or without secondary generalization.²⁰^–²⁸ In addition, the frontal and temporal lobes are intrinsically highly epileptogenic brain regions, and experiments with frontal, parietal, and occipital FPI demonstrate the much higher epileptogenicity of the rat frontal neocortex.¹⁵

The probability that patients will develop PTE varies greatly depending on the type and severity of TBI, and on such risk factors as intracerebral hemorrhage, subdural hematoma, dural penetration, depressed skull fracture, multilobar injury, retained metal fragments, early (<1 week) post-traumatic seizures, and loss of consciousness or post-traumatic amnesia >24 hours.⁷^,¹³^,²⁹^–³²

The rate of epileptogenesis after head injury is also very variable.³³^–³⁶ Patients may become epileptic within days after the injury or many years later.³⁰^,³¹^,³⁷^–³⁹ The reason for this large variability in epileptogenesis is not well understood. While experimental work clearly shows that the severity of the injury directly affects the speed of epileptogenesis, other physiological and genetic factors must also contribute.¹⁵

The frequency of seizure in human PTE also varies widely and has not been well characterized, because head injury patients do not undergo prospective electroencephalography (EEG) monitoring and PTE patients do not routinely undergo invasive electrocorticographic (ECoG) monitoring. Also, PTE patients may present with localized or spreading focal seizures, termed simple partial seizures (SPSs), complex partial seizures (CPSs), or secondarily generalized seizures (SGSs) in the older ILAE classification.⁴⁰ Focal seizures can have very diverse ictal behaviors, ranging from undetectable to tonic clonic convulsions, depending on the location of the epileptic focus and the pattern and extent of the seizures’ spread.¹⁴^,²⁴^,³³^,³⁵^,⁴¹^–⁴⁶ When these seizures have subtle or no detectable (subclinical) behavioral correlates, PTE may be missed or misdiagnosed.²⁸^,⁴⁷^–⁵³ Human SPSs, especially those producing subjective symptoms without motor output, are often unreported or misdiagnosed.⁵⁴^,⁵⁵ CPSs, which can manifest with a wide variety of convulsive or nonconvulsive behaviors and subjective experiences,⁵⁶^–⁵⁸ can easily be misdiagnosed or confused with psychiatric disorders.⁴⁸^–⁵⁰ In addition, seizures with limited electrical spread that are clearly evident in ECoG often elude detection by standard scalp EEG.⁴⁹^,⁵⁰^,⁵⁵^,⁵⁹^–⁶¹ When patients are invasively monitored, the differences between electrographically detected seizures and seizures detected by other means can differ greatly.⁶² Thus, epidemiological data based on self-reported seizures underestimate nonconvulsive SPSs and CPSs,⁵² and seizures that are not severe enough to lead the patient to seek the advice of a physician are never counted.

In summary, there is no canonical PTE syndrome to target, and efforts to model PTE should focus on mimicking epileptogenic head injuries that closely correspond to head injuries commonly resulting in PTE in humans and adapting them to specific experimental aims.

Animal Models of Post-Traumatic Epilepsy

Existing animal models of PTE include: (1) models developed to investigate particular mechanisms that have been postulated to contribute to PTE, (2) models based on chemoconvulsant or electrical stimulation-induced status epilepticus (SE), and (3) etiologically realistic models in which epilepsy is induced after an experimental head injury. The first class includes the cortical undercut model⁶³ and acute intraparenchymal injection of FeCl₃.⁶⁴ The cortical undercut reproduces trauma-induced hemorrhage and partial deafferentiation,⁶³ while the FeCl₃ model mimics features of intracerebral hemorrhage. However, while several aspects of TBI, such as intraparenchymal hemorrhage, ischemia, and hypoxia, can induce epilepsy in experimental models,⁶⁵^–⁶⁹ neither clinical nor experimental studies have determined which, if any, of theses factors is actually responsible for genesis and progression of human PTE. Thus, until the roles of the hypothesized mechanisms are better understood, mechanism-based models may not be prudent choices for therapy development. Since human PTE often involves the temporal lobes, existing SE-based models of temporal lobe epilepsy have been suggested for the study of PTE. In these widely used models, SE is induced by systemic or intracerebral administration of pilocarpine or kainate, or electrical stimulation of the amygdala or hippocampus. Although convenient to implement and extensively studied, SE-based models have limitations for PTE translational research: (1) the epileptogenic mechanisms involved are not well known and may not necessarily be clinically relevant for human PTE,⁷⁰^–⁷² and (2) it is generally difficult to determine whether experimental preventive treatments for epilepsy modify the disease or merely the initiating insult.⁷³^,⁷⁴

Etiologically realistic PTE models reproduce a mechanical injury that induces TBI in humans. While animal models have been developed to reproduce both the focal and the diffuse injuries found in human TBI, predominantly diffuse injuries have not yet been studied with a focus on epilepsy and will not be discussed further. There are three main experimental models that reproduce predominantly focal contusive brain injuries. The weight-drop model,⁷⁵^,⁷⁶ which has not yet been demonstrated to induce epilepsy, the controlled cortical impact (CCI) model, and fluid percussion injury (FPI) model. In the CCI model, a rigid impactor delivers a calibrated injury to the intact dura.⁷⁷^–⁷⁹ Several laboratories have now reported the CCI-induced development of CSRSs in rodents with behavioral correlates that range from behavioral arrest to tonic-clonic convulsions.⁸⁰^–⁸³ However, the reported incidence (20%–50%) and frequency (0.2–0.3 seizures/day) of CSRSs is low, the latency to seizure onset is long (>75 days),⁸³ and the progression of PTE has yet to be well characterized. In the FPI model, a calibrated fluid pulse is delivered to the intact dura through an open craniotomy. FPI reproduces most of the pathophysiological features of human contusive closed head injury,⁸⁴ and it has become one of the best-studied rodent PTE models.⁸⁵^–⁸⁹ However, FPI is a complex injury, and the character of FPI-induced PTE varies greatly depending on the details of the injury protocol.¹⁵ A recent study⁹⁰ in which FPI was administered with a picospritzer, rather than a conventional FPI apparatus,⁸⁴ failed to induce PTE despite injury parameters (pulse duration and pressure) claimed to be nominally similar to those used routinely to induce PTE with conventional FPI. Fortunately, this sensitivity of the FPI model to the details of implementation also provides opportunities for optimization, and we have found that FPI can be effectively optimized for therapy development.

MODEL OPTIMIZATION FOR THERAPY DEVELOPMENT

An ideal model for therapy development would provide accurate prediction of the effect of investigational interventions in the modeled human population with high throughput and minimal cost (Table 10.1). Given our poor understanding of the mechanisms mediating human post-traumatic epileptogenesis, it is expected that the best prediction would be obtained with models, such as the FPI and CCI models, in which the epileptogenic stimulus is very similar (i.e., realistic) to the corresponding one in humans and, thus, likely to engage the same mechanisms. High throughput requires rapid and reliable epileptogenesis and syndrome assessment, and costs are determined by the number of subjects required to reach statistically valid conclusions and the length of time required for epileptogenesis, treatment, and the collection and analysis of video/ECoG data. We have invested considerable effort in optimizing the throughput and minimizing the number of subjects required for translational studies using the FPI model. Since human data clearly show that the incidence and severity of PTE vary with the character and severity of the initiating injury, the parameters of experimental injury can be manipulated to increase the incidence and frequency of CSRSs without threat to model validity.

TABLE 10.1

Desirable Features of a PTE Animal Model for Therapy Development

The well-studied lateral FPI model induced using the classic cylinder-piston device was adapted by McIntosh for TBI/neuroprotection studies in the rat.⁸⁴ It induces PTE, but epileptogenesis is reported to be slow (several months), seizure frequencies are low (<1/day), and fewer than 50% of injured animals are reported to develop epilepsy by 7–12 months postinjury.⁸⁷^,⁸⁸ In such a model, experiments to assess antiseizure or antiepileptogenic interventions would require 6–9 months with large numbers of animals recorded for long periods of time and would be prohibitively expensive (Figure 10.1). However, we have found that the rate of epileptogenesis and the incidence and frequency of seizures all vary depending upon the location of injury and the amplitude and duration of the pressure pulse.¹⁵ The overall incidence and frequency of seizures was lower after milder (2.0 atm) than more severe (3.4 atm) trauma, and a rostral injury delivered over the frontal neocortex resulted in more rapid and reliable epileptogenesis than injury delivered at more caudal sites, as in the classic FPI model. The parameters of FPI are implementation-dependent and should be interpreted very cautiously. While a 3.4-atm-pressure wave delivered through a rostral parasagittal 3 mm craniotomy with our methods reliably induces PTE, similar pressure and pulse durations are most likely not suitable for a different device with different mechanical properties or different methods. For example, it is known that the mere presence of an air bubble between the dura of the animal and the pressure transducer can dramatically affect the severity of the injury. Effective parameters must be separately identified for any novel implementation and validated. In addition, the use of volatile anesthetics and mechanical ventilation permits better control of the model: (1) it controls post-traumatic apnea, which induces epileptogenic hypoxia; and (2) it reduces post-traumatic mortality (~10%), relative to the classic lateral FPI (30%–35%), which permits severely injured animals to enter the pool of PTE subjects. The FPI model we have developed (Figure 10.2) results in over 90% incidence of CSRSs with a mean frequency of about 2 seizures/hour, by 4–5 weeks postinjury. This permits completion of seizure-control and seizure-prevention studies in less than 6 weeks, and manageable numbers of animals recorded for less than 72 hours per experimental condition provide adequate statistical power to detect robust and clinically interesting treatment effects.⁹²^–⁹⁴

FIGURE 10.1

The statistical power to detect the prevention of epileptic seizures after head injury critically depends on the animal model’s seizure incidence. This Monte Carlo simulation of antiepileptogenesis studies is based on 10 simulated experiments (more...)

FIGURE 10.2

Optimization of the FPI model to achieve the high seizure frequency and incidence needed for therapy development. In the classic FPI model developed for neuroprotection studies, a long fluid pulse is delivered through a large craniotomy centered on the (more...)

This FPI model, which is adapted for epilepsy therapy development (etdFPI), offers additional advantages that could facilitate advances in target identification and therapy development. First, the focus that reliably develops in the perilesional cortex is readily accessible for experimental manipulation. This feature was critical for the demonstration that mild focal cooling of the epileptic focus prevents the development of post-traumatic epileptic seizures.⁹⁴ Also, the PTE syndrome that develops after etdFPI prominently includes nonconvulsive neocortical seizures,⁹⁴^,⁹⁵ which have been largely overlooked in most preclinical studies and which have proven difficult to treat with available antiseizure drugs (ASDs).⁹²^,⁹³ Like human PTE, FPI-induced PTE includes frontal lobe seizures that are associated with behavioral arrest, with or without mild automatisms, and which are electrically and behaviorally consistent with those seen in humans.⁴⁷^,⁴⁸^,⁵⁰^,⁵⁷^,⁹⁸^–¹⁰⁸ The rare parieto-occipital neocortical seizures induced by etdFPI were never associated with detectable behavioral change but might have been associated with subjective visual phenomena that cannot be assessed in animals.¹⁵ Frontal and limbic seizures with loss of body posture became progressively more common in rats over time⁸⁶^,⁹⁵ and had semiology reminiscent of human CPSs. Many of these seizures consist of stereotyped crouching, sometimes with facial or body automatisms.

Although the latency of PTE after etdFPI is shorter, and the incidence of PTE and the frequency of post-traumatic epileptic seizures is much higher than the averages reported for humans, the differences are more apparent than real. First, human data show a very large variability in both speed of epileptogenesis and seizure frequency. Many head injury patients develop epilepsy within 1–2 weeks, just as most animals receiving etdFPI. Second, all estimates of the incidence of epilepsy after head injury are based on aggregated outcomes of diverse injuries that may differ widely in epileptogenicity depending on the character and severity of the initiating injury. Even when stratified by a measure of severity, head injury patients differ widely in the type, number, and location(s) of their injuries, and in the presence of risk factors for PTE. Patients are also typically heterogeneous in age, gender, genetic background, and comorbid conditions that may affect their susceptibility to PTE.¹⁰⁴^,¹⁰⁵ In contrast, the etdFPI protocol is standardized and empirically designed to induce rapid and robust epileptogenesis, and it is administered to rats of identical age and gender that vary much less in genetic background and antecedent comorbidities. In addition, the experimental animals are much more closely and comprehensively monitored than PTE patient cohorts. They are continuously monitored, prospectively, using sensitive video/ECoG, while human head injury patients are not typically monitored prospectively and seizures are most often monitored by self-report, which underestimates the incidence and frequency of post-traumatic CSRSs (see Section “What to Model”). Thus, the model cannot be compared to average epidemiological findings, and its features and performance can be optimized so long as the selected injury protocol induces epilepsy and realistically reproduces the pathophysiology of a clinically important human epileptogenic injury.

ASSESSING EPILEPSY TREATMENTS WITH FLUID PERCUSSION INJURY-INDUCED PTE

The etdFPI-PTE model has been deployed in studies of both antiseizure and antiepileptogenic treatments.⁹²^–⁹⁴ Results from these studies suggest that (1) the pharmacological sensitivity of etdFPI-induced epilepsy differs from other models of evoked and spontaneous seizures, (2) etdFPI-induced PTE may have value as a model of pharmacoresistant epilepsy, and (3) the etdFPI-PTE model is capable of identifying antiepileptogenic interventions.

The limited data available on the pharmacological properties of the SE- and etdFPI-based epilepsy models suggest important differences. Several conventional ASDs (carbamazepine, phenobarbital, phenytoin, and topiramate) have been demonstrated to be effective against convulsive seizures in the chronic phase after SE,¹⁰⁶^–¹⁰⁸ as has the investigational ASD, carisbamate (CRS).¹⁰⁹ In contrast, etdFPI-induced CSRSs were poorly controlled by carbamazepine, CRS, and valproate (in a majority of animals), but well controlled by halothane and, in a subset of animals, by valproate.⁹²’⁹³ The effect of valproate on CSRSs in responders developed progressively over a week of exposure and outlasted the exposure period, suggesting a mechanism distinct from the one mediating its antiseizure action in acute models.⁹² Thus, etdFPI-induced PTE appears less responsive to conventionally developed ASDs than older models. Indeed, the etdFPI-PTE model incorporates most known risk factors for pharmacoresistant epilepsy (Table 10.1), and its poor response to conventional ASDs suggests it may have value as a model of pharmacoresistant epilepsy. This suggestion is supported by studies of CRS—a promising investigational ASD that was withdrawn from the regulatory process for epilepsy treatment after disappointing performance as an adjunctive treatment in pharmacoresistant patients. In stark to its poor performance against etdFPI-induced PTE,⁹³ CRS has demonstrated broad efficacy in evoked seizure models and was reported to perform better than topiramate against convulsive seizures in a SE-based epilepsy model.¹⁰⁹^,¹¹⁰ However, these disparate results may both be consistent with the clinical trial experience. Halford et al.¹¹¹ reported that CRS, at the highest dose tested, resulted in 17%, 12%, and 29% reductions (over placebo levels) in the frequency of SPSs, CPSs, and SGSs, respectively. Thus, while the very modest effects of CRS on partial seizures without generalization are consistent with its effect on nonconvulsive seizures in the etdFPI- PTE model, it may have had a larger effect on SGSs, which were relatively rare in the study population. Together, these data support efforts to determine the scope and mechanisms of treatment resistance in etdFPI rats.

The etdFPI-PTE model has also demonstrated the capacity to distinguish effective and ineffective antiepileptogenic interventions. In a blind, randomized and adequately powered study, a 2-week treatment with CRS begun 15 min after injury, had no significant effect on the development of CSRSs.⁹³ Although the study was not powered to detect less than an 85% reduction in seizure frequency compared to controls, the nearly identical seizure frequencies in control and treated rats at both 4 and 12 weeks after injury suggests a much weaker effect. In contrast, 5 weeks of mild focal cooling of the perilesional neocortex demonstrated prevention of about 99% of CSRSs by the end of treatment in a similar blind and randomized study.⁹⁴ This effect persisted for the duration of the study, which extended over 10 weeks after treatment termination.

TECHNICAL AND METHODOLOGICAL ISSUES IN PTE THERAPY DEVELOPMENT

Definition of Epilepsy and of Seizures

In addition to optimizing an experimental injury to induce a high incidence and frequency of epileptic seizures, the experimentally induced seizures must also be reliably and sensitively detected for an epilepsy model to be practical and useful. Overlooking seizures poses the same threat to a cost-efficient study as low seizure frequency and incidence. Seizure detection is straightforward when attention is restricted to Racine scale 4–5 convulsive seizures that roughly correspond to secondarily generalized convulsive seizures in humans. However, detection of the nonconvulsive seizures that predominate in etiologically realistic epilepsy models, and roughly correspond to human SPSs and CPSs without secondary generalization, is more challenging. Both electrographic and video monitoring are essential for the characterization of these seizures, which may have subtle behavioral correlates, and criteria must be established for their reliable and reproducible identification. To ensure that the same phenomena are being studied in animals and humans, these criteria should closely resemble those that guide seizure identification in human video/EEG monitoring. Currently, there is no consensus on how to identify and classify seizures in animals, and definitions of experimental seizures are varied, arbitrary, and often far more restrictive than those used for humans. The definition of human clinical epileptic seizures accepted by the ILAE⁴^,⁵ is equally applicable to seizures in other species, and its use in preclinical studies should help align clinical and preclinical study endpoints and improve bench to bedside translation.⁹⁶^,⁹⁷

As in human PTE, the epileptic seizures observed in etiologically realistic PTE models come in a wide range of durations and clinical manifestations, and there is no scientific reason to systematically exclude any class of events from preclinical investigation. However, many investigators have required that seizures have minimum durations or specific types of behavioral correlates, which may vary widely among laboratories. Such criteria were introduced because they (1) simplify the distinction between ictal and interictal events, (2) simplify exclusion of both the typically short age-dependent idiopathic seizures that are common in many rodent strains, and brief focal seizures that may be induced by cortical damage inflicted unintentionally during electrode implantation, and (3) reduce false positives in both visual and algorithm-based automated seizure detection. However, application of such arbitrary criteria can exclude large numbers of seizures, introducing large errors in seizure frequency and epilepsy incidence. The duration of clinical seizures in etdFPI rats ranges widely from 1 second to several minutes (See figure 4I in Ref. 94; Figure 10.2), and shows a clear progression over time postinjury regardless of the location of injury.¹⁵ We have previously shown how altering the definition of epileptic seizures can change estimates of seizure incidence and frequency, as well as the latent period (figure 9 in Ref. 95). In addition, an investigational ASD that increased the frequency of seizure but shortened their duration or prevented generalization could be misidentified as an effective antiepileptogenic or antiseizure agent. More important, such criteria can result in exclusion of specific classes of seizure from the study. For example, an investigation that systematically overlooked nonconvulsive seizures could seriously overestimate the clinical effectiveness of a treatment for patients who, like those enrolled in most phase III clinical trials, suffer predominantly from SPSs and CPSs with infrequent or absent secondary convulsions.

Detection of Epileptic Seizures

The clinical manifestations of a seizure depend on its site of origin and the area and extent of its spread. Thus, both animals and humans with PTE may experience seizures with electrographic changes on EEG that lack detectable behavioral accompaniment. Human SPSs and CPSs can be associated not only with convulsive events but also with nonmotor symptoms, including somatosensory, special sensory, autonomic, or affective components.⁵⁴ We have observed that brief focal seizures with very limited spread in human motor cortex areas were associated with a mild stereotyped ictal behavior (eye blinking or muscle twitching), while similar events located outside of the motor cortex were not associated with visible behavioral output.⁹⁵ Similarly, in FPI rat, we observed that frontal cortex focal seizures are associated to behavioral change (e.g., motor arrest with or without facial automatisms), while focal seizures in the occipital cortex are never associated with obvious behavioral changes.¹⁵ Therefore, seizures are most accurately diagnosed on the basis of both behavioral and electrographic data.

Simultaneous epidural ECoG and scalp EEG recordings in etdFPI-injured rats have shown that nonspreading focal cortical seizures were not detected by scalp EEG, which also failed to detect ~62% of the spreading seizures that were detected by the epidural electrodes. These data suggest that scalp EEG only picked up the activity when it had spread to a sufficiently large volume of cortex.⁹⁵ Similar results have been obtained in humans: brief epileptic events that were detected by invasive ECoG were missed by scalp EEG,⁵⁹^,⁶⁰^,⁹⁵ and invasive monitoring often shows many times the frequency of seizures that regular scalp EEG detects.⁵³^,⁶² Thus, assessment of ictal activity is better performed with ECoG.

In animals, as in humans, multiple ECoG electrodes are required to better appreciate focal seizures with limited spread. When the location of an injury-induced epileptic focus is predictable (as it is after etdFPI), strategic montage and analysis can be used to maximize seizure detection using a reasonable number of electrodes. In the rat, we routinely monitor invasively with a five-electrode montage that directly samples the perilesional cortex that reliably generates an epileptic focus after FPI. This guarantees the detection of most seizures. Because many of the focal neocortical seizures that develop after etdFPI are brief and fail to spread, a montage including a perilesional electrode is critical for accurate assessment of FPI-induced PTE⁹⁵ and also to test treatments.⁹⁴

Long-term continuous recording is often considered important to prove experimental subjects to be seizure-free. However, clinical seizures can be both focal and local, and ictal behaviors can be mild or internal. Thus, because it is not possible to comprehensively map the electrical activity of a whole brain with an indefinitely large numbers of electrodes, no subject can ever be proven to be seizure free, even with continuous observation using the most sensitive methods. Indeed, seizure freedom can only be proven for generalized convulsive seizures whose diffuse spread ensures both motor output and electrical detection. In general, the experimenter can only report the number of seizures detected in a specified interval using a particular recording montage, and the effects of interventions must be assessed in terms of treatment-related changes in the numbers and/or duration of seizures. While prolonged recording may be needed for models that exhibit low seizure frequency, treatment effects on frequent absence seizures in WAG/Rij or GAERS rats are routinely assessed on the basis of a few hours of recording (e.g., Refs. 112 and 113). Nonparametric power analyses show that clinically interesting reductions of 50% or more in seizure frequency after etdFPI can be detected (80% power; α = 0.05) in groups of eight rats recorded for just 48–72 hours before and during treatment.⁹²^,⁹³ Thus, the use of sensitive ECoG recording in an optimized acquired epilepsy model permits assessment of antiseizure effects in small groups of subjects recorded for manageable periods of time.

Avoiding Experimental Bias

Experimental outcomes can be biased by comparisons of nonequivalent experimental groups or by biases in investigator judgment. Individual experimental animals may differ in age, gender, weight, genetics, fine details of experimental treatment, responses to injury and treatment, and in innumerable other factors that may or may not affect experimental outcomes. Many factors that are known to affect outcomes (e.g., strain, age, and gender) can be explicitly controlled. However, outcomes may also be affected by factors that are unknown, not measured or not controllable. For example, despite the carefully controlled injury parameters, the duration of posttraumatic apnea, and the age and gender of rats (Figure 10.2), seizure frequency and rate of progression of epileptogenesis still vary greatly among animals.¹⁵ The possibility that experimental outcomes could be biased by unbalanced allocation of subjects to experimental groups can be minimized by formal randomization procedures. Such procedures also protect against another form of bias. Unconstrained and seemingly random subject selection could easily introduce bias if the investigator selected the easiest animal to grab (low activity level) or the first in a cohort to exhibit seizure. Formal randomization measures have recently been recommended for improving the translation of preclinical data in both the epilepsy and TBI fields.¹¹⁴^,¹¹⁵

Experimenter bias in data interpretation can be minimized either by blind manual assessment of experimental data or by using an exclusively algorithmic approach. A recent study determined that when human video EEG monitoring data were analyzed by clinicians blinded to all other clinical data, they might arrive at different conclusions about whether seizures are epileptic in origin.¹¹⁶ For epilepsy studies, blinding entails manual review of video and/or ECoG data by expert personnel who are unaware of the identity of the data. In our previous preclinical studies on carisbamate⁹³ and cooling⁹⁴ all ECoG data were analyzed manually by trained investigators blinded to the treatment group and collection date of data files. Such blinding is not difficult to do and only requires a third person, not involved in the analysis, to rename the data files with a coding software.

Unbiased analysis of ECoG data from etiologically realistic models of PTE could be greatly expedited by improvements in automated ECoG analysis. Currently, expert manual analysis is the only approach to detect all seizures in ECoG, including the focal nonconvulsive ones, with high sensitivity and specificity. This approach, while reliable, requires extensive training and takes qualified investigators about 1.5 hours to analyze 24 hours of continuous rat ECoG. An ECoG-based antiepileptogenesis study to examine a single compound with 20 animals recorded for a week would require one person about 6 weeks just for the primary analysis of the data. Systems capable of detecting both convulsive and nonconvulsive seizures with acceptable sensitivity and specificity to fully automate the process do not yet exist. Significant effort should be put into their development to improve therapy development.

Using the Right Control Groups

Sham Injury and Electrode Damage

Studies of FPI-induced PTE often compare data obtained from injured animals to data obtained from sham-injured animals given a craniotomy but no FPI. While such shams provide a useful comparison group, they may not be regarded as normal. Cole and colleagues have demonstrated that the sham surgery itself caused brain injury distinct from the impact injury.¹¹⁷ In their study, rats that received anesthesia only were compared to rats with craniotomies carefully performed either by manual trephine or by electric drill. Despite their care to avoid damage to the dura, both drill and trephine caused morphological, behavioral, and biochemical changes consistent with TBI. The demonstration that the craniotomy itself, without the fluid percussion, can cause significant proinflammatory, morphological, and behavioral damage, suggests that the routine use of this type of sham controls may lead to confusion in interpretation of conventional experimental brain injury models. Therefore, great care should be taken in selecting the appropriate models and controls for comparing posttraumatic changes in the brain and for evaluating the efficacy of potential treatments.

We routinely take several precautions to ensure that epileptiform ECoG events we examine are induced by FPI and not by compression or frictional heating of the cortex during drilling.⁹⁴^,⁹⁵ We cool the skull and drill bit with room-temperature sterile saline during drilling and take care to not deform the skull or the dura with the drill bit. The depth of epidural electrodes is carefully adjusted to avoid brain compression. In addition, glial fibrillary acidic protein (GFAP) immunostaining can be performed in FPI and sham-injured animals to assess astroglial reactivity beneath the epidural electrodes, and animals exhibiting foci of GFAP immunoreactivity (see figure 1 in Ref. 95) can be excluded from study. While the relationship between the subtle neuropathology induced by suboptimal electrode implantation and the generation of epileptic discharges has not yet been fully characterized, we have never observed focal discharges in control animals that were not associated with foci of glial reactivity beneath the electrode at which they were detected. Similar concerns apply even more urgently to the use of depth electrodes, which penetrate the meninges and brain parenchyma, and locally disrupt the blood-brain barrier. Chronic depthelectrode implantation likely induces inflammation¹¹⁸ and prolonged implantation of depth electrodes has been shown to facilitate kindling, worsen evoked seizures, and increase the severity of electrically induced SE.¹¹⁹^–¹²¹ Thus, depth recordings may have the potential to significantly affect the outcome of acquired epilepsy studies.

Impact of Rat Strain, Age, and Gender on Idiopathic Seizures

In studies of acquired epilepsies, it is desirable that control animals be seizure-free and that seizures in injured animals be solely attributable to the experimental injury. Idiopathic epilepsy is common in rats, however, with incidence and onset depending on strain, age, and gender.¹²²^–¹²⁵ Since acquired epilepsy studies frequently involve recording from animals well beyond 6 months of age, many investigators have reported brief epileptiform events in control animals. These potentially confounding events can be minimized by using younger animals of a strain with low incidence and/or late onset of idiopathic seizures. Willoughby and MacKenzie¹²² reported Sprague Dawley rats to have the lowest rate of idiopathic discharges among eight strains examined. More recently, Pearce et al.¹²⁵ reported the appearance of idiopathic seizures in female Sprague Dawley rats and in males older than 6 months, but not in younger male Sprague Dawley rats. This is consistent with our experience. Most of our studies utilized male Sprague Dawley rats younger than 6 months of age, and seizures were not observed in undamaged controls.⁸⁶ Idiopathic seizures were observed in undamaged control rats in one study in which recordings were obtained from rats older than 6 months. These events, typically 2–10 seconds long and bilateral at onset, were characterized by a sharp-wave pattern and were readily distinguished from PTE because they were significantly larger in amplitude in the parietal-occipital cortex (see figure 1D in Ref. 86). This posterior dominance was similar to that previously reported in idiopathic seizures recorded from Wistar rats using a similar bilateral fronto-occipital multielectrode montage.¹²²^,¹²⁶ We observed these events in about 33% of the rats that were recorded through 7–8 months of age and they represented just 3.6% of the cortical discharges recorded from FPI rats at 27–28 weeks postinjury. Thus, young male Sprague Dawley rats can provide seizure-free controls for acquired epilepsy studies. In acquired epilepsy studies that must extend past the age at which idiopathic seizures appear, such seizures can be identified and excluded from analysis if a montage is employed that permits spatial characterization of the cortical discharge.⁸⁶ A recent study⁹⁰ claimed to detect SWDs in two-thirds of young male Sprague Dawley rats from Harlan Laboratories, but it is unclear whether this colony of rats is genetically more prone to SWDs because the detection was based on an unvalidated automated algorithm with unknown specificity. Since behaviorally salient genetic differences have been documented in Sprague Dawley rats from different colonies,¹²⁷ it may be prudent to use animals from colonies known to have a low incidence of idiopathic epilepsy. Pearce et al.¹²⁵ used rats from Charles River in Wilmington, Massachusetts, and our rats have predominantly been shipped from Charles River in Hollister, California.

CONCLUSIONS

Limitations and diminishing returns of conventional methods of epilepsy drug development and the absence of treatments to prevent acquired epileptogenesis have spurred interest in epilepsy models featuring etiologically realistic epilepsy syndromes. An ideal model would engage mechanisms that contribute to human epilepsy, exhibit pathology consistent with human epilepsy, produce CSRSs, and neurobehavioral impairment consistent with human epilepsy, and yet be practical to use in translational studies. Models for translational research must deliver adequate statistical power to detect treatment effects, and throughput issues are an additional concern. The etdFPI-PTE model has benefited from optimizations that make it suitable for therapy development. Indeed, it has already contributed to novel insights on investigational treatments. This demonstrates the feasibility and value of adapting etiologically realistic models to the development of novel treatments for the corresponding human epileptic syndromes.

ACKNOWLEDGMENTS

This work was supported by the Italian Ministry of Education, University and Research (“Rientro Cervelli” 17DZE8RZEA to GC), by the National Institutes of Health (NS076570 to RD), and by Citizens United for Research in Epilepsy (Prevention of Acquired Epilepsies Award to RD).

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

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Publication Details

Author Information and Affiliations

Authors

Giulia Curia, Clifford L Eastman, John W Miller, and Raimondo D’Ambrosio.

Copyright

Publisher

CRC Press/Taylor and Francis Group, Boca Raton (FL)

NLM Citation

Curia G, Eastman CL, Miller JW, et al. Modeling Post-Traumatic Epilepsy for Therapy Development. In: Laskowitz D, Grant G, editors. Translational Research in Traumatic Brain Injury. Boca Raton (FL): CRC Press/Taylor and Francis Group; 2016. Chapter 10.