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Summary: Purpose: To use animal models of variable seizure induction in rats at different developmental stages to determine contributing factors for spontaneous seizures resulting from status epilepticus (SE) early in life.
Methods: Two models of SE with distinct modes of seizure induction, lithium–pilocarpine (LiPC) and perforant path stimulation (PPS), were used at different ages. Multiple methods of determining neurodegeneration during an acute period and plastic changes in those monitored during the chronic phase were used.
Results: Different modes of seizure induction lead to varying types and extents of damage, dependent on the age of the animals at the time of insult. LiPC resulted in injury to animals as young as 2 weeks and became widespread in animals 3 weeks old, whereas widespread damage after PPS was not seen until P35. Rats at an age with widespread damage in response to seizures also showed extensive immediate-early gene activation and often developed spontaneous seizures and features of hippocampal plasticity seen in the epileptic brain.
Conclusions: SE early in life results in multiple consequences to the developing brain. These changes, coexisting in the nonepileptic brain, can overlap in a maladaptive combination to result in the diseased state of epilepsy. The consequence of early seizures in immature animals is a function of both the developmental stage and the method of seizure induction.
The consequences of seizures in the developing brain continue to be a dynamic topic of discussion (1) and debate (2,3). This issue is not readily answered by human studies. Historically, febrile convulsions have been cited as the classic example of the benign nature of seizures in children with no previously demonstrated brain pathology (4,5). However, some studies report adverse sequelae after febrile status epilepticus (SE) (6,7). Additionally, recent studies suggest that prolonged seizures in children, including complex febrile seizure, may result in brain injury that becomes discernible with new, noninvasive imaging techniques (8–11).
Over the years, numerous investigators have attempted to provide further insight into this controversy using the inherent advantage of animal research to control factors that are problematic in the clinical situation. Nevertheless, the general concept of age-specific seizure-induced vulnerability has been further complicated by the multiple ages examined in diverse studies, and the method used to produce seizures or SE. For example, opinions of the resistance of the developing brain to seizures were bolstered by early work showing lack of histologic damage or mossy fiber sprouting (MFS) in response to kainic acid (KA)–induced SE in 2-week-old animals (12–15). Conversely, corticotropin-releasing hormone (CRH) is a potent convulsant that induces limbic seizures at picomolar doses in infant rats (P10–P13) (8). CRH-induced SE causes neuronal damage in the CA3b subfield of the hippocampus and mossy fiber reorganization in these rats (17,18). These effects are not demonstrable in mature rats. Investigations in our laboratory and others with the lithium–pilocarpine (LiPC) model have shown that young animals can undergo region-specific brain damage after SE (19,20) and that those patterns can vary with age (21).
Further, SE-induced damage may have been underestimated in the past. Many studies evaluated only cresyl violet sections to count remaining neurons in an animal with a chronic disorder, rather than label acutely damaged neurons with high sensitivity. In addition, markers of apoptosis were not customarily examined as they are today. DNA fragmentation in response to SE has been shown in subpopulations of damaged hippocampal (21) and extrahippocampal neurons (22). Meanwhile, some damaged hippocampal neurons may have been replaced by increased neurogenesis reported in the mature as well as in developing brain (23–27).
Even in the absence of discernible injury, many changes have been described in recent years as a consequence of early-life seizure. For instance, in a model of hyperthermic seizures in rat pups, postnatal age 10–12 (P10–P12) designed to mimic febrile convulsions in children, even seizures lasting ∼20 min resulted in lasting changes in presynaptic, γ-aminobutyric acid (GABA)ergic inhibitory transmission (28). Moreover, such relatively brief hyperthermia-induced seizures have been shown to reduce thresholds to chemical convulsants in vivo and electrical stimulation in vitro, indicating persistent enhancement of limbic excitability that may facilitate the development of epilepsy (29). Seizures induced by kainic acid in P15 pups enhanced the damage sustained by these animals when rechallenged by KA at P45 (30). Holmes et al. (31) examined the effect of recurrent, brief, flurothyl-induced seizures in neonatal rats. These rats demonstrated impaired learning and decreased activity levels. They also displayed MFS in area CA3 and the supragranular region. When studied as adults, these animals had a decreased threshold to pentylenetetrazol (PTZ)-induced seizures. The authors concluded that recurrent brief seizures during the neonatal period have long-term detrimental effects on behavior, seizure susceptibility, and brain development. In another article, these authors reported MFS in the CA3 region after brief PTZ-induced seizures in P1 and P10 rats (32). Not surprisingly, in a review in Neuron, the authors concluded that seizures were not so benign to the immature brain after all (33). More recently these authors showed that when neonatal rats are subjected to flurothyl seizures, and the hippocampal slices are studied subsequently, there are differences in the intrinsic membrane properties of CA1 pyramidal cells (34). CA1 pyramidal cells from rats treated with flurothyl in the neonatal period displayed a marked reduction in the spike frequency adaptation and after hyperpolarizing potential after a spike train compared with controls.
We present some of our findings of SE-induced changes in the immature brain and attempt to delineate multiple factors that together may ultimately result in the chronic epileptic state in these animals.
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Our initial evaluation of SE-induced damage in developing rats involved determination of s-NSE. We were interested in this enzyme because of the published human data on adults after SE (35,36). Furthermore, recognizing the inherent difficultly in quantifying the total amount of neuronal injury in the brain, we used s-NSE as a biochemical correlate that takes into account differing regional vulnerabilities in the entire CNS, ignores the type of the injury itself (necrosis or apoptosis), and represents elevations corresponding to the appearance of neuronal injury.
The age-related damage seen in the rats with elevated s-NSE appeared to show particularly interesting patterns in the hippocampus. A detailed examination of the injury and also a study of substance P (SP) expression as a consequence of SE (37) revealed region-specific vulnerabilities that were age dependent and also paralleled changes in SP expression. By using multiple techniques sensitive to distinctive mechanisms of neuronal damage, we were able to demonstrate that some of these cells undergo apoptosis. With further maturation, apoptosis seemed quite rare, as indeed has been confirmed by Fujikawa et al. (38).
Some rats that had undergone LiPC SE at age 3 weeks began to display spontaneous seizures. These rats had demonstrated significant hippocampal hilar damage as well as damage to extrahippocampal structures such as the amygdala, thalamus, entorhinal cortex, and cortex in response to LiPC SE. In a separate set of studies using the PPS model of SE, we observed that similarly aged rats (P20) responded very differently, with even 8 h of stimulation rarely resulting in self-sustained SE (SSSE) (39). However, a significant number of P35 animals did so. The very fact that some of the P35 animals could tolerate 8 h of PPS without developing SSSE confirmed that even by this age (prepubescent to pubescent), synaptic maturation has not reached an adult level.
Some of the rats that underwent PPS at P35 did develop spontaneous seizures, and they had also shown more widespread damage than those that underwent PPS at P20. We hypothesized that the difference in the epileptogenic potential between LiPC SE and 8 h of PPS in the P21 rat may be attributable to differences in the recruitment of extrahippocampal circuits by these two methods. To demonstrate this difference, we undertook an immunohistochemical study for c-Jun, a transcription factor product of an immediate-early gene, c-jun, that may reflect early recruitment of epileptogenic areas preceding the onset of neuronal injury. We confirmed that PPS on a P21 pup resulted in activation restricted to the hippocampus, and extrahippocampal spread could be demonstrated well in this method only on the P35 or adult animals (40). In contrast, LiPC SE caused extensive hippocampal and extrahippocampal expression of c-Jun, consistent with widespread activation of circuits. Bertram (41,42) investigated the role of such extrahippocampal involvement and found the involvement of midline thalamic structures to be crucial in the development of limbic epilepsy.
We also wanted to examine for changes in plasticity in the epileptic state and chose both an anatomic marker, MFS, and a physiologic method, dentate inhibition as measured by population response to short interstimulus interval (40 ms) PPS of the perforant path. The most established anatomic method is one that involves the Timm stain for zinc, which is abundant in the mossy fiber terminals and synapses. Sprouting and neosynapse formation by mossy fibers is readily discerned by this technique. PPI, meanwhile, is an electrophysiologic correlate of the strength of recurrent inhibition in the dentate gyrus (43). Our studies demonstrated that the two phenomena, anatomic evidence of sprouting and enhanced inhibition to paired-pulse stimuli, correlated well with the evolution of an epileptic state.
In summary, our data suggest that several factors influence the development of an epileptic state. We propose a schematic with the central feature to be the condition of chronic epilepsy (CE) in Fig. 3. A key concept expressed by this diagram is the possibility that areas of overlap can vary with the relative contributions of each component, which in turn modify the area representing the epileptic state. No single component is likely responsible for CE by itself. For example, neurodegeneration alone cannot result in spontaneous seizures due to the presence of the silent period after limbic SE. Plastic changes (neurogenesis), meanwhile, have been seen in naïve mice placed in an enriched environment (44). Although numerous studies show an overlap of these two areas, a current study using older animals that undergo both these changes reported the incidence of CE to be almost certain, approximating 90% (see Mazarati et al. same issue). The stage of development of the brain is an important factor as well, but it appears that some provocations are more epileptogenic with advancing maturation (KA, LiPC, PPS), whereas others (hypoxia in P10–12 pups, CRH in P10–12 pups) may be more likely to cause injury and/or lead to plasticity, resulting in epileptogenicity in younger animals.