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Summary: Purpose: In temporal lobe epilepsy, it remains to be clarified whether hippocampal sclerosis is the cause or the consequence of epilepsy. We studied the temporal evolution of the lesions in the lithium–pilocarpine model of epilepsy in the rat with magnetic resonance imaging (MRI) to determine the progressive morphologic changes occurring before the appearance of chronic epilepsy.
Methods: MRI was performed on an MR scanner operating at 4.7 T. We followed the evolution of lesions using T2- and T1-weighted sequences before and after the injection of gadolinium from 2 h to 9 weeks.
Results: At 2 h after status epilepticus (SE), a blood–brain barrier breakdown could be observed only in the thalamus; it had disappeared by 6 h. At 24 h after SE, edema was present in the amygdala and the piriform and entorhinal cortices together with extensive neuronal loss; it disappeared progressively over a 5-day period. During the chronic phase, a cortical signal reappeared in all animals; this signal corresponded to gliosis, which appeared on glial fibrillary acidic protein (GFAP) immunohistochemically stained sections as hypertrophic astrocytes with thickened processes. In the hippocampus, the correlation between histopathology and T2-weighted signal underscored the progressive constitution of atrophy and sclerosis, starting 2 days after SE.
Conclusions: These data show the reactivity of the cortex that characterizes the initial step leading to the development of epilepsy and the late gliosis that could result from the spontaneous seizures. Moreover, it appears that hippocampal sclerosis progressively worsened and could be both the cause and the consequence of epileptic activity.
The pilocarpine model, whether or not associated with lithium, is a well-studied model of temporal lobe epilepsy that reproduces most clinical and neuropathologic features of human temporal lobe epilepsy (1,2). In adult rats, pilocarpine leads to status epilepticus (SE), starting usually within 50–60 min after the injection, lasting for up to 8–12 h and followed by recurrent seizure episodes for up to 24–48 h. This acute seizure phase is followed by a “silent” seizure-free phase of a mean duration of 14–25 days. Thereafter, all animals exhibit spontaneous recurrent seizures (SRSs). This epilepsy lasts for the whole life of the animals (2,3).
In this model, neuronal damage is located mainly in the hippocampal formation, piriform and entorhinal cortices, septum, thalamus, amygdala, and neocortex (2,4). The extent of neuronal damage in the hippocampal formation correlates directly with the duration of the initial SE (5). The lithium–pilocarpine-induced neuronal injury occurs as a phenomenon of necrosis rather than apoptosis (6). Lesions in the hippocampus (CA1, CA3, and the hilus), amygdala, and piriform and entorhinal cortices begin to appear as soon as the first hour of SE and worsen or extend thereafter (7). During SE, brain areas where most damage occurs are those with highest expression of Fos (marker of cellular hyperactivation), acid fuchsin staining (marker of dying neurons), and largest metabolic activation (8–10).
An anatomic correlation has been found between large hypermetabolic levels measured during the second hour of seizures and neuronal damage assessed at 6 days after SE (10). At 14 days after SE (i.e. during the silent phase), cerebral metabolic rates for glucose (CMRglc) decrease in damaged forebrain areas that are involved in the circuitry of spontaneous seizures (cortical regions, hippocampal CA1 and CA3 areas, anterior and lateral thalamic nuclei) and increase in nondamaged brainstem areas involved in the remote control of epilepsy (substantia nigra reticulata, superior colliculus, red nucleus) (11). Later, the interictal period of the chronic phase of the epilepsy is characterized by normometabolic levels in the intact brainstem areas and hypometabolic levels in the damaged forebrain regions (12).
However, all the studies described above give only information about the nature of the regions injured or involved in the circuitry of the epilepsy. They do not provide information about the nature of the regions responsible for the induction of this epilepsy and about the changes that are the consequence of recurrent seizures. Moreover, all autoradiographic and histologic methods used in previous studies need to kill the animals, and therefore they represent either the SE, the silent phase, or the chronic epilepsy, but they do not allow any progressive study of the lesions and related phenomena occurring in diverse brain regions. For this reason, we chose to use magnetic resonance imaging (MRI), which allows a follow-up of the animals, thus giving a better insight into the lesional and plastic mechanisms underlying the establishment of temporal lobe epilepsy.
In the present study, we explored the temporal evolution of the MRI signal in adult rats from 2 h after the onset of lithium–pilocarpine-induced SE up to the occurrence of the SRSs. This procedure allowed a detailed follow-up of the progressive morphologic and lesional changes occurring during the acute, silent, and chronic phases that allow the establishment of the chronic epilepsy.
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It has been well established that MRI is the most sensitive in vivo technique permitting the diagnosis of structural abnormalities that underlie seizure disorders. This great sensitivity is because the signal amplitude is related to the water environment of tissues (i.e., the concentration and mobility of water), but MRI fails to provide detailed information about the histopathologic nature of lesions. In almost all structural changes, including edema, gliosis, and neuronal loss, the concentration of free water is increased in the tissue. In the present study, in agreement with previously published data (2,6,12,18), the nature of lesions at different stages of the lithium–pilocarpine model of temporal lobe epilepsy has been clearly established in the 15 rats that underwent histopathologic examination (Fig. 3). In this context, MRI is a powerful technique allowing assessment of the temporal evolution of the different lesions.
Role of the BBB breakdown in the genesis of the lesions
Many observations in epilepsy patients (19–24), confirmed by animal studies (25–28), demonstrated that SE provokes transient BBB breakdown and vasogenic edema in the area of the seizure focus. It is commonly proposed that local (relative hypoxia secondary to increased ictal metabolism with an increase in local Pco2 and lactate) and systemic (increase in blood pressure and decrease in blood pH) physiologic changes induce a vasodilatation, resulting in increased regional blood flow and BBB breakdown (25,26,29).
In the present study, we observed a BBB breakdown during SE only at the level of the thalamus. The role of this phenomenon in the genesis of the lesions is not clear. However, some hypotheses can be advanced. First, the known anatomic connections of the thalamus to the limbic system and its excitatory input to these structures indicate its central role in limbic seizures. Indeed, thalamic stimulation yields short-latency excitatory responses in the entorhinal cortex and hippocampus (30–32). Moreover, in a number of studies using different models for limbic SE, the 2-deoxyglucose mapping of metabolic activity has underscored clearly the involvement of the midline thalamic nuclei (33–36). Second, we did not analyze BBB breakdown before 2 h or between 2 and 6 h after the onset of SE. The thalamus may be the structure most sensitive to SE and the only one with a BBB breakdown, but it is also possible that other structures may present a BBB breakdown but at a time that we did not explore. Third, MRI may be not sensitive enough or the contrast medium, gadolinium, too large to detect a small increase in permeability of the BBB.
Despite the presence of marked edema in the amygdala and piriform and entorhinal cortices at 24 and 48 h, we did not see any obvious BBB breakdown in those regions. These MRI observations were associated with early selective neuronal loss in the amygdala and the cerebral cortex. Of the various theories for the pathophysiology of the neuronal damage in SE, one suggests that excessive presynaptic neuronal activity causes postsynaptic neuronal damage (37,38). The excitotoxic phenomenon responsible for the excessive presynaptic activity is presumed to be the release of normal endogenous neurotransmitters (e.g., glutamate and aspartate) in excessive, neurotoxic amounts (37,39). This concept is supported by studies with kainate (an excitotoxic analogue of glutamate), which causes excessive neuronal excitation at distant postsynaptic sites, leading to neuronal damage (38).
Key role of the cerebral cortex in epileptogenesis
We saw a hypersignal during the acute and the beginning of the silent phase (i.e., at 24 h to 5 days after the SE) in the piriform and entorhinal cortices and in the amygdala. This signal disappeared by 5 days and reappeared at the onset of the chronic phase. We associated this hypersignal with a edema that is present at the time at which marked loss of neurons is found on thionine-stained sections in the same regions. In fact, most neuronal loss in those regions, mainly in the cortex, was complete as soon as 24 h after SE. As neuronal injury, in this model, has been shown to be a phenomenon of necrosis (6), the neurons of the piriform and entorhinal cortices undergo swelling during the first 24 h after SE, which increased the amount of free water. Because the signal disappeared thereafter, it means that the T2-weighted signal reflected edema rather than only the neuronal loss. In the future, it will be interesting to couple the MRI scans with magnetic resonance spectroscopy to distinguish neuronal damage and edema (40,41). In previous studies on kainate-induced SE, diffusion-weighted MRI allowed detection of early and minimal brain damage, whereas no such changes could be seen on T2-weighted images (42,43). The present data confirm this observation. Indeed, an early T2-weighted signal was detected in regions like the cerebral cortices and amygdala in which neuronal damage is rapid and massive. Conversely, in regions like the hippocampus, where the damage is more limited and progressive, the signal intensity does not change over the first 24 h, and in the latter case, diffusion-weighted MRI would probably be more appropriate to detect early changes.
With the onset of spontaneous recurrent seizures, the T2-weighted signal reappeared in the cortex. This can be correlated with the results obtained by GFAP immunohistochemistry, which shows swollen, reactive astrocytes, and underlines the glial nature of the late MRI signal in the cortex. The common reaction of astrocytes to injury in central nervous system is the striking hypertrophy and occasional proliferation of these cells (44–47). The hypertrophy involves an increase in the size of the soma as well as an increase in the length and width of astrocytic stellae, with a hallmark accumulation of intermediate filaments such as GFAP and an increase of free water. Studies on animals model of epilepsy show that an apparent gliosis is almost always present in brain regions that exhibit epileptiform activity because of neuronal injury as well as seizure activity per se (48–53). In kindled seizures (a nonlesional model), it has been shown that astrocytes present a prominent hypertrophy that is accompanied by a reorganization of astrocytic cytoskeleton. The changes in their morphology appear to be seizure-intensity dependent, occur early in the kindling process, and persist for weeks after the last seizure (51–53).
In the group of rats that received a number of DZP injections related to the behavioral state, we noticed that the intensity of the cortical edema seen at 24 h after the injection of pilocarpine was inversely proportional to the number of injections of DZP a given rat received. Moreover, it appeared that the rat that presented the most prominent edema at 24 h after SE had received fewer injections of DZP and entered the chronic phase, and thus presented SRSs earlier than rats that received a higher number of DZP injections. However, the more or less marked severity of the edema at 24 h after SE did not influence the volume of lesions in the hippocampus, neither in the group of rats with a fixed schedule of DZP injections nor in the group receiving DZP injections according to their behavioral state. In both cases, the piriform and entorhinal cortices were always the first structures affected, and the hippocampus reacted always after some delay. The early MRI signal and lesional process of the cortex compared with the later and prolonged neuropathology of the hippocampus also was reported recently in adult rats subjected to pilocarpine SE (54). Thus the present results suggest the predictive character of the early involvement of the cortex as well as the key role of the piriform and entorhinal cortices in epileptogenesis induced by the administration of lithium and pilocarpine. Conversely, the secondary involvement of the hippocampus seemed to be rather independent of the intensity of the cortical signal.
These results are in agreement with previous studies from our group showing that the rapid destruction of the entorhinal cortex induced by a pretreatment consisting of a series of maximal electroshocks before lithium–pilocarpine SE prevents the occurrence of SRSs (17,55), whereas the mechanical lesion of the same structure 4 days after SE does not prevent the occurrence of SRSs in adult rats (56). The prominent role of the entorhinal cortex in the primary step of the process of epileptogenesis appears also in our metabolic studies. Indeed, during SE, the entorhinal cortex is the structure the most largely activated area in adult rats, whereas in P10 rats, which do not become epileptic after lithium–pilocarpine SE, it is the least activated within the forebrain (10). Our data also are in accordance with a study performed in the rat hippocampal–entorhinal slice preparation in which an epileptiform activity can be induced by pilocarpine (57). This study indicates that epileptiform discharges originate in the entorhinal cortex, from where they project to the hippocampus proper via the perforant path.
Progressive setup of the hippocampal sclerosis
At the level of the hippocampus, the T2-weighed signal appeared ∼2 days after SE and gradually intensified until the end of the study, fully correlating with the progressive loss of neurons seen in the thionine-stained sections. Indeed, although neuronal loss was apparent in the hilus as soon as 24 h after SE, it became significant only at 14 days in CA1 and CA3a. CA3b was significantly injured only later, at 9 weeks after SE. Thus the T2-weighted signal observed on the MRI scan corresponds to the progressive constitution of atrophy and gliosis in the hippocampus.
Finally, our data confirm the results of a previous study (58) using the kainate model of epilepsy in mice, which leads to hippocampal sclerosis in two steps. The first period is characterized by an edema provoked by the neurotoxin underlying a period of limited histologic damage. The second phase is characterized by the extension of the damage, leading to a progressive and complete degeneration of the hippocampal CA1 area, partly spreading to CA3. This late phase is accompanied by the onset of the occurrence of SRSs and gliosis. Therefore, it appears that in these two models of human temporal lobe epilepsy, the development of hippocampal sclerosis is the consequence of the primary excitotoxic event, the SE, and the ongoing seizure activity during the chronic phase of SRSs.