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Purpose: Infantile spasms is one of the most severe epileptic syndromes of infancy and early childhood. Progress toward understanding the pathophysiology of this disorder and the development of effective therapies has been hindered by the lack of a relevant animal model. We report here the creation of such a model.
Methods: The sodium channel blocker, tetrodotoxin (TTX), was chronically infused into the developing neocortex or hippocampus of infant rats by way of an osmotic minipump starting on postnatal day 10–12.
Results: After a minimum of 10 days of infusion, approximately one-third of these rats began to display very brief (1–2 s) spasms, which consisted of symmetric or asymmetric flexion or extension of the trunk and sometimes involvement of one or both forelimbs. The typical ictal EEG pattern associated with the behavioral spasms consisted of an initial generalized, high amplitude, slow wave followed by an electrodecrement with superimposed fast activity. The interictal EEG revealed multifocal spikes and sharp waves, and in most animals that had spasms a hypsarrhythmic pattern was seen, at least intermittently, during NREM sleep. Like in humans, the spasms in the rat often occurred in clusters especially during sleep–wake transitions. Comparison of the ictal and interictal EEGs recorded in this model and those from humans with infantile spasms revealed that the patterns and the frequency components of both the ictal events and hypsarrhythmia were very similar.
Discussion: The TTX model of infantile spasms should be of value in furthering an understanding of the pathophysiology of this seizure disorder.
Infantile spasms (IS) is a disorder of infancy and early childhood characterized by mental retardation and epileptic spasms (Nordli, 2002; Hrachovy & Frost, 2003; Frost & Hrachovy, 2003). The spasms usually consist of sudden and brief flexions or extensions of the body and extremities which often occur in clusters during sleep–wake transitions. Another hallmark of IS is the interictal EEG pattern called hypsarrhythmia, which consists of high amplitude slow waves intermixed with multifocal spikes. The fundamental cause of this disorder is not known and as many as 200 associated conditions have been identified (Lacy & Penry, 1976; Frost & Hrachovy, 2003). These range from brain insults such as hypoxia/ischemia, and intraventricular hemorrhage to congenital malformations such as tuberous sclerosis and lissencephaly. The lack of an animal model has severely hampered an understanding of the biological basis for this disorder as well as a means to develop therapies (Stafstrom & Holmes, 2002; Stafstrom et al., 2006). Several attempts have been made to develop animal models but none have been able to completely recapitulate the human condition (Brunson et al., 2001a; Velisek et al., 2007).
Previously, we described an animal model of early-onset epilepsy where recurring seizures can be induced by chronic infusion of the sodium channel blocker, tetrodotoxin (TTX) into the hippocampus beginning on postnatal days 10–12 (Galvan et al., 2000; Galvan et al., 2003). Within two weeks after initiating the infusion, behavioral seizures were observed that consisted of brief flexions of the head and upper trunk. EEG recordings revealed a concurrent brief run of high frequency activity. During the course of the initial studies of this model we were aware that the behavioral and electrographic features of the seizures had features reminiscent of IS. However, since a very limited recording montage was used, we did not feel that we had enough evidence to suggest that this model was a potential model of IS.
We recently undertook a more detailed neurophysiological characterization of the animal model. In many animals TTX was infused into the developing neocortex beginning on postnatal days 10–12. In other animals our original protocol was used in which the hippocampus was infused. Moreover, in these experiments recordings were made at many sites across the cortex in order to determine if the EEG abnormalities seen in human infants were reproducible in rats. Here we report that the EEG and behavioral features seen in these animals are very similar to those seen in IS patients. Preliminary results of this work have previously been presented in abstract form (Lee et al., 2006).
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The results of this study demonstrate that infusion of TTX into the neocortex or hippocampus of rats at an early stage of postnatal development can induce an epileptic process with neurophysiological and behavioral characteristics essentially identical to those associated with human IS. As summarized in Table 1, the major similarities include (1) an interictal EEG pattern characterized by multifocal spikes and sharp waves, which in most animals resembles hypsarrhythmia, (2) ictal EEG complexes identical to those seen in human IS, (3) brief clinical extensor or flexor spasms that vary in intensity, (4) spasms may occur as isolated seizures or in clusters, (5) spasms typically occur during wakefulness, or upon arousal from sleep, and (6) ictal complexes may occur without accompanying clinical seizures. Also, it should be noted that in this model, just as in the human condition, the spasms and EEG changes do not occur acutely following a brain perturbation. Instead, there is a latent period between the infusion of TTX and the development of the behavioral and EEG abnormalities. The fact that only a third of animals infused with TTX developed spasms is also consistent with the human condition since not all infants experiencing the same apparent brain insult develop IS (Frost & Hrachovy, 2003). In addition, TTX-infused animals often develop other types of seizures later in life, just as in the human condition. In rats these consisted primarily of prolonged limbic motor seizures (Galvan et al., 2000).
Table 1. Comparison of human and animal model
|Interictal EEG-multifocal spikes/hyps||+||+|
|Ictal EEG complexes—SW, ED, FA||+||+|
|Seizures—single or clusters||+||+|
|Seizures may occur awake or on arousal||+||+|
|Ictal complexes occur without clinical seizures||+||+|
Certain features of the human condition still need to be confirmed in this model. For example, it has been suggested that response to ACTH or other therapies, as well as the presence of cognitive impairment, are essential criteria for a comprehensive model of this disorder (Stafstrom & Holmes, 2002; Stafstrom et al., 2006). To evaluate response to therapy, more prolonged EEG/video monitoring sessions are required to accurately quantify spasm frequency. Experiments are currently being designed to address these issues. However, it should be noted that the diagnosis of IS in the human does not require these additional criteria.
In the current rat model, spasms begin during the four-week TTX infusion period. Thus far, the earliest we have observed spasms is P21 but long-term video EEG monitoring may reveal they begin earlier. Furthermore, it is also known that the TTX model is developmentally dependent, with chronic epileptogenesis resulting when TTX infusion is initiated on P10–12 but not later (Galvan et al., 2000). It is conceivable that if treatment was initiated during the first week of life, seizures might take place even earlier in development. Consequently, this model shares certain developmental features that are consistent with the human condition: In humans, IS typically begins within the first year of life, with a peak age of onset at 4–6 months (Hrachovy & Frost, 1989), although it can occur as early as the first week of life, or, rarely, as late as 3 years of age or longer. Stafstrom & Holmes (2002) have suggested that P13–20 in the rat represents the time window corresponding to the period when humans are susceptible to developing IS. However, it has been and continues to be very difficult to directly compare maturational processes in the human and rat brain. In any given species, different brain regions and systems exhibit different maturational rates, and these system-specific rates may vary considerably between species (Clancy et al., 2001; Avishai-Eliner et al., 2002). Commonly, P5 to P7 in the rat has been considered comparable to birth in the human and the first postnatal year of human life approximately equivalent to postnatal days 7–14 in the rat (e.g., Gottlieb et al., 1977; Avishai-Eliner et al., 2002), but recently even these interspecies comparisons have been called into question (Clancy et al., 2001).
Nonetheless, it is important to note that our model of IS is not the first in which seizures or behavioral abnormalities that occur in human infants or young children are observed in what are commonly considered more mature rodents. Indeed, in genetic animal models of neurodevelopmental disorders such as Rett, Angelmann, and Fragile X syndromes, clinically relevant behavioral abnormalities are observed in adulthood (Watase & Zoghbi, 2003). For instance, in Rett mouse models, animals appear normal until six weeks of age after which symptoms similar to those of very young girls (6–18 months of age) begin to appear (Chen et al., 2001; Guy et al., 2001). In terms of epilepsy, spike-and-wave discharges begin to be recorded in GAERS and WAG/Rij rats at one month and two to three months of age, respectively and in both strains discharging increases in intensity with age (Vergnes et al., 2003; Schridde & van Luijtelaar, 2005)—this despite the fact that human absence seizures commonly occur in childhood. Thus, comparisons between animal and human brain maturation will likely to continue to be debated but with ever-increasing intensity since this issue could impact interpretation of the underlying neuropathological mechanisms discovered in a variety animal models of neurodevelopmental disorders.
The pathophysiological basis of IS is not known, although there has been much speculation (Chugani et al., 1992; Brunson et al., 2001a; Lado & Moshe, 2002; Frost & Hrachovy, 2005; Dulac et al., 1999). Recently, we have suggested that desynchronization of two or more developmental processes could be the basis for this disorder (Frost & Hrachovy, 2005). The entire process of normal development is dependent on a complex series of interactions that is finely tuned temporally and spatially. Unanticipated disruptions in any of these processes could potentially lead to pathologic consequences. It is well known that neuronal activity plays an important role in normal neuronal development (Katz & Shatz, 1996). Numerous studies have shown that when the activity of neurons is experimentally altered early in life, the maturation of neurons and their patterns of connectivity can be drastically modified. Perhaps the visual system is the best studied in this regard where blockade of neuronal activity in the retina by infusion of TTX disrupts the formation of ocular dominance columns in visual cortex (Stryker & Harris, 1986; Antonini & Stryker, 1993). However, activity can also regulate the growth of dendrites (Wong & Ghosh, 2002), formation of synapses (Waites et al., 2005), and expression of neurotransmitter receptors (Dumas, 2005). Thus the TTX-induced blockade of neuronal activity undertaken in the present study could be expected to desynchronize the developmental processes that normally take place in neurons at the infusion site and their synaptic partners at more distant sites. The occurrence of an epileptic condition in rats that closely resembles human IS lends further credence to the developmental desynchronization hypothesis.
Our results and the desynchronization model are also consistent with the model of Chugani and colleagues (Chugani et al., 1992) who reported hypometabolic regions in neocortex in the majority of human IS patients. This observation has been confirmed independently by others (Itomi et al., 2002; Metsahonkala et al., 2002). It would also be expected, and indeed has been shown, that infusion of TTX into the hippocampus results in focal hypometabolism in the region of infusion (Galvan et al., 2000). Since focal cortical hypometabolism has been reported in the majority of patients with IS and since TTX induces a focal hypometabolism in rats that develop an epilepsy closely resembling IS, it is possible that this epilepsy results from regional neuronal inactivity early in life.
In the past, a number of attempts have been made to develop an animal model of IS. One is based on the “CRH hypothesis,” which suggests that IS results from an excessive release of CRH from limbic and brainstem regions (Brunson et al., 2001a). The hypothesis was developed based on the ability of ACTH to control IS (Hrachovy et al., 1983; Mackay et al., 2004) and the demonstration that ACTH suppresses the production of CRH in the brain (Brunson et al., 2001b). CRH has also been shown to produce seizures in infant rats (Baram et al., 1992). However, the behavioral and electrographic features of these events in rats do not resemble the human condition. A second model relies on an intraperitoneal injection of NMDA in infant mice to induce acute, flexion-like spasms that last for tens of seconds (Kabova et al., 1999; Velisek et al., 2007). EEG recordings during these spasms resemble the electrodecremental response that characterizes the ictal discharge in humans. However, these animals do not display the interictal EEG pattern (hypsarrhythmia) and the condition does not persist beyond the acute injection period. A third model was recently described by Snead et al. (2007) in which GABA receptor agonists were administered in a mouse model of Down's syndrome with resulting extensor spasms and an accompanying electrodecremental response in the EEG. However, these spasms occurred only acutely (10–25 min) following IP injection of the agent. These animals also exhibited altered behavioral patterns compared to controls. The seizures in this model were blocked or reduced by several anticonvulsants, including ACTH (Cortez et al., 2007). As in the case of the NMDA model, the condition apparently does not persist beyond the acute injection period. The crucial aspect of the TTX model of IS described here is that it recapitulates many of the features of the clinical syndrome, including spontaneous seizures that persist for weeks. Thus, it should be an especially important resource for exploring the neurobiological mechanisms that are responsible for this epilepsy of infancy.
Disclosure of conflicts of interest
We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The authors do not have conflicts of interest to report. This work was supported by NIH Grants: NS 37171 and NS18309.