Kevin Graber

Epilepsy following cortical injury: cellular and molecular mechanisms as targets for potential prophylaxis.

The sequelae of traumatic brain injury, including posttraumatic epilepsy, represent a major societal problem. Significant resources are required to develop a better understanding of the underlying pathophysiologic mechanisms as targets for potential prophylactic therapies. Posttraumatic epilepsy undoubtedly involves numerous pathogenic factors that develop more or less in parallel. We have highlighted two potential “prime movers”: disinhibition and development of new functional excitatory connectivity, which occur in a number of animal models and some forms of epilepsy in humans. Previous experiments have shown that tetrodotoxin (TTX) applied to injured cortex during a critical period early after lesion placement can prevent epileptogenesis in the partial cortical (“undercut”) model of posttraumatic epilepsy. Here we show that such treatment markedly attenuates histologic indices of axonal and terminal sprouting and presumably associated aberrant excitatory connectivity. A second finding in the undercut model is a decrease in spontaneous inhibitory events. Current experiments show that this is accompanied by regressive alterations in fast‐spiking γ‐aminobutyric acid (GABA)ergic interneurons, including shrinkage of dendrites, marked decreases in axonal length, structural changes in inhibitory boutons, and loss of inhibitory synapses on pyramidal cells. Other data support the hypothesis that these anatomic abnormalities may result from loss of trophic support normally provided to interneurons by brain‐derived neurotrophic factor (BDNF).

Homeostatic Plasticity Studied Using In Vivo Hippocampal Activity-Blockade: Synaptic Scaling, Intrinsic Plasticity and Age-Dependence

Homeostatic plasticity is thought to be important in preventing neuronal circuits from becoming hyper- or hypoactive. However, there is little information concerning homeostatic mechanisms following in vivo manipulations of activity levels. We investigated synaptic scaling and intrinsic plasticity in CA1 pyramidal cells following 2 days of activity-blockade in vivo in adult (postnatal day 30; P30) and juvenile (P15) rats. Chronic activity-blockade in vivo was achieved using the sustained release of the sodium channel blocker tetrodotoxin (TTX) from the plastic polymer Elvax 40W implanted directly above the hippocampus, followed by electrophysiological assessment in slices in vitro. Three sets of results were in general agreement with previous studies on homeostatic responses to in vitro manipulations of activity. First, Schaffer collateral stimulation-evoked field responses were enhanced after 2 days of in vivo TTX application. Second, miniature excitatory postsynaptic current (mEPSC) amplitudes were potentiated. However, the increase in mEPSC amplitudes occurred only in juveniles, and not in adults, indicating age-dependent effects. Third, intrinsic neuronal excitability increased. In contrast, three sets of results sharply differed from previous reports on homeostatic responses to in vitro manipulations of activity. First, miniature inhibitory postsynaptic current (mIPSC) amplitudes were invariably enhanced. Second, multiplicative scaling of mEPSC and mIPSC amplitudes was absent. Third, the frequencies of adult and juvenile mEPSCs and adult mIPSCs were increased, indicating presynaptic alterations. These results provide new insights into in vivo homeostatic plasticity mechanisms with relevance to memory storage, activity-dependent development and neurological diseases.

Tetrodotoxin prevents posttraumatic epileptogenesis in rats.

Severe cortical trauma frequently causes epilepsy that develops after a long latency. We hypothesized that plastic changes in excitability during this latent period might be initiated or sustained by the level of neuronal activity in the injured cortex. We therefore studied effects of action potential blockade by application of tetrodotoxin (TTX) to areas of cortical injury in a model of chronic epileptogenesis. Partially isolated islands of sensorimotor cortex were made in 28‐ to 30‐day‐old male Sprague–Dawley rats and thin sheets of Elvax polymer containing TTX or control vehicle were implanted over lesions. Ten to 15 days later neocortical slices were obtained through isolates for electrophysiological studies. Slices from all animals (n = 12) with lesions contacted by control‐Elvax (58% of 36 slices) exhibited evoked epileptiform field potentials, and those from 4 rats had spontaneous epileptiform events. Only 2 of 11 lesioned animals and 6% of slices from cortex exposed to TTX in vivo exhibited evoked epileptiform potentials, and no spontaneous epileptiform events were observed. There was no evidence of residual TTX during recordings. TTX‐Elvax was ineffective in reversing epileptogenesis when implanted 11 days after cortical injury. These data suggest that development of antiepileptogenic drugs for humans may be possible. Ann Neurol 1999;46:234–242

Postlesional Epilepsy: The Ultimate Brain Plasticity

Summary: Lesions that occur either during fetal development or after postnatal brain trauma often result in seizures that are difficult to treat. We used two animal models to examine epileptogenic mechanisms associated with lesions that occur either during cortical development or in young adults. Results from these experiments suggest that there are three general ways that injury may induce hyperexcitability. Direct injury to cortical pyramidal neurons causes changes in membrane ion channels that make these cells more responsive to excitatory inputs, including increases in input resistance and a reduction in calcium‐activated potassium conductances that regulate the rate of action potential discharge. The connectivity of cortical circuits is also altered after injury, as shown by axonal sprouting within pyramidal cell intracortical arbors. Enhanced excitatory connections may increase recurrent excitatory loops within the epileptogenic zone. Hyperinnervation attributable to reorganization of thalamocortical, callosal, and intracortical circuitry, and failure to prune immature connections, may be prominent when lesions affect the developing neocortex. Finally, focal injury can produce widespread changes in γ‐aminobutyric acid and glutamate receptors, particularly in the developing brain. All of these factors may contribute to epileptogenesis.

A critical period for prevention of posttraumatic neocortical hyperexcitability in rats.

Penetrating cortical trauma frequently results in delayed development of epilepsy. In the rat undercut model of neocortical posttraumatic hyperexcitability, suppression of neuronal activity by exposing the injured cortex to tetrodotoxin (TTX) in vivo for approximately 2 weeks prevents the expression of abnormal hypersynchronous discharges in neocortical slices. We examined the relationship between neuronal activity during the latent period after trauma and subsequent expression of hyperexcitability by varying the timing of TTX treatment. Partially isolated islands of rat sensorimotor cortex were treated with Elvax polymer containing TTX to suppress cortical activity and slices obtained for in vitro experiments 10 to 15 days later. TTX treatment was either started immediately after injury and discontinued after a variable number of days or delayed for a variable time after the lesion was placed. Immediate treatment lasting only 2 to 3 days and treatment delayed up to 3 days prevented hyperexcitability. Thus, there is a critical period for development of hyperexcitability in this model that depends on cortical activity. We propose that the hyperexcitability caused by partial cortical isolation may represent an early stage of posttraumatic epileptogenesis. A hypothetical cascade of events leading to subsequent pathophysiological activity is likely initiated at the time of injury but remains plastic during this critical period. Ann Neurol 2004;55:860–870

The microtubule interacting drug candidate NAP protects against kainic acid toxicity in a rat model of epilepsy.

NAP (NAPVSIPQ, generic name, davunetide), a neuroprotective peptide in clinical development for neuroprotection against Alzheimer’s disease and other neurodegenerative indications, has been recently shown to provide protection against kainic acid excitotoxicity in hippocampal neuronal cultures. In vivo, kainic acid toxicity models status epilepticus that is associated with hippocampal cell death. Kainic acid toxicity has been previously suggested to involve the microtubule cytoskeleton and NAP is a microtubule‐interacting drug candidate. In the current study, kainic acid‐treated rats showed epileptic seizures and neuronal death. Injection of NAP into the dentate gyrus partially protected against kainic acid‐induced CA3 neuron death. Microarray analysis (composed of > 31 000 probe sets, analyzing over 30 000 transcripts and variants from over 25 000 well‐substantiated rat genes) in the kainic acid‐injured rat brain revealed multiple changes in gene expression, which were prevented, in part, by NAP treatment. Selected transcripts were further verified by reverse transcription coupled with quantitative real‐time polymerase chain reaction. Importantly, among the transcripts regulated by NAP were key genes associated with proconvulsant properties and with long‐lasting changes that underlie the epileptic state, including activin A receptor (associated with apoptosis), neurotensin (associated with proper neurotransmission) and the Wolfram syndrome 1 homolog (human, associated with neurodegeneration). These data suggest that NAP may provide neuroprotection in one of the most serious neurological conditions, epilepsy.

Targets for preventing epilepsy following cortical injury

Prophylaxis of posttraumatic epilepsy will require a detailed knowledge of the epileptogenic pathophysiological processes that follow brain injury. Results from studies of experimental models and human epilepsy highlight alterations in GABAergic interneurons and formation of excessive new excitatory synaptic connectivity as prominent targets for prophylactic therapies. Promising laboratory results suggest that it will be possible to experimentally modify these aberrant processes and interfere with epileptogenesis. However, a number of key issues must be addressed before these results can be used to frame clinical antiepileptogenic therapy.

Direct electrical stimulation for epilepsy: The laboratory evidence

Studies of deep brain stimulation in laboratory models of seizures and epilepsy are invaluable for understanding of mechanisms of stimulation and for identifying best targets and parameters of stimulation. Recent successes of clinical trials may give more impetus to laboratory studies, which in turn will further our clinical practice. For an expanded treatment of this topic see Jasper’s basic mechanisms of the epilepsies. 4th ed. (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado‐Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at http://www.ncbi.nlm.nih.gov/books).

Neocortical posttraumatic epileptogenesis

Development of new excitatory connectivity and decreases in γ‐aminobutyric acid (GABA)ergic inhibition are mechanisms underlying posttraumatic epileptogenesis in animal models. Experimental strategies that interfere with these processes, applied between the trauma and seizure onset, are antiepileptogenic in the laboratory, and have promise for prophylaxis of epileptogenesis after cortical injury in humans. For an expanded treatment of this topic see Jasper’s Basic Mechanisms of the Epilepsies, Fourth Edition (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado‐Escueta AV, eds) National Library of Medicine Bookshelf [NCBI] at http://www.ncbi.nlm.nih.gov/books).