Epilepsia, 46(9):1371–1379, 2005 Blackwell Publishing, Inc. C 2005 International League Against Epilepsy
Increased Expression of Ferritin, an Iron-storage Protein, in Specific Regions of the Parahippocampal Cortex of Epileptic Rats ∗ †Jan A. Gorter, ∗ Ana R.M. Mesquita, ∗ †Erwin A. van Vliet, ∗ Fernando H. Lopes da Silva, and ‡Eleonora Aronica
∗ Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam; †Stichting Epilepsie Instellingen Nederland, Heemstede; and ‡Department of (Neuro)Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Summary: Purpose: Iron accumulation in the brain has been associated with neurodegenerative disorders, including epilepsy. In our previous SAGE study, we showed that ferritin, an ironstorage protein, was one of the genes (Ferritin-H) that showed overexpression before the chronic epileptic phase. In this study we used ferritin as indicator for disturbed iron homeostasis to acquire insight into whether this could play a role in the pathogenesis of temporal lobe epilepsy. Methods: With immunocytochemistry, we studied the regional and cellular distribution of ferritin protein in an animal model for temporal lobe epilepsy in which spontaneous seizures develop a few weeks after electrically induced status epilepticus (SE). Results: Increased ferritin expression was observed in regions known to be vulnerable to cell death, mainly in reactive microglial cells of epileptic rats. Ferritin expression after SE was
initially high, especially throughout the hippocampus, but decreased over time. In the chronic epileptic phase, it was still upregulated in regions where extensive cell loss occurs during the early acute and latent period. Within the parahippocampal region, the most persistent ferritin overexpression was present in microglial cells in layer III of the medial entorhinal area. The upregulation was most extensive in rats that had developed a progressive form of epilepsy with frequent seizures (approximately five to 10 seizures per day). Conclusions: The fact that ferritin upregulation is still present in specific limbic regions in chronic epileptic rats, when neuronal loss is absent or minimal, suggests a role of iron in the pathogenesis and progression of epilepsy. Key Words: Iron—Hippocampus—Entorhinal cortex—Microglia—Immunostaining—Epileptogenesis—Progression.
Disruption in the metabolism of iron has been postulated to have a role in the pathogenesis of various neurologic diseases, such as Alzheimer’s and Parkinson’s diseases (2–6). Although iron is essential to life, it can be toxic. In the presence of O2 , free iron is potentially harmful, as it can lead to the production of toxic reactive oxygen species. For this reason, cells have evolved highly regulated mechanisms for controlling intracellular iron levels so that under physiologic conditions, very little free iron is present in biologic systems. Normally it is combined with functional proteins such as the cytochromes, or it is bound in protein-storage complexes such as ferritin or to irontransport proteins such as transferrin. Ferritin is the main player in the sequestration of iron. Inadequate storage of iron by ferritin creates an environment of oxidative stress, leading to free radical generation and consequently cell damage. This is illustrated by the finding that mutant mice
heterozygous for ferritin-H have increased indices for oxidative stress, whereas their homozygous littermates die in utero (7,8). Moreover, overexpression of either ferritin-H or ferritin-L reduced the accumulation of reactive oxygen species in response to oxidant challenge (9). Some evidence indicates that iron can play a role in epileptogenesis, especially as the result of lesion/trauma of the brain. Several studies have shown that injection of ferric cations can cause gliosis and neuronal loss and may eventually lead to recurrent spontaneous seizures (10–12). Moreover, an interesting association has been reported between a haptoglobin phenotype (Hp2-2) and the presence of recurrent seizures in patients with idiopathic seizures. Hp 2-2 is the least effective of three major Hpphenotypes in binding free hemoglobin, suggesting less efficient removal of iron in these patients and involvement of hemoglobin in the etiology of seizures (13). Interestingly, in a previous study in which we performed a serial analysis of gene expression (SAGE), we found an upregulation of ferritin within the hippocampus 8 days after status epilepticus (SE) induced by electrical
Accepted May 1, 2005. Address correspondence and reprint requests to Dr. J.A. Gorter at Kruislaan 320, 1098 SM, Amsterdam, The Netherlands. E-mail:
[email protected]
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stimulation (1). The upregulation was observed during the latent period, just before the occurrence of the chronic epileptic phase. This led us to investigate ferritin protein expression in the parahippocampal region during epileptogenesis in more detail. Because the SE in this model is followed by either a progressive or nonprogressive form of epilepsy, we focused especially on the relation between ferritin expression and the progression of the disease in this post-SE model for temporal lobe epilepsy. MATERIALS AND METHODS Experimental animals Adult male Sprague–Dawley rats (Harlan CPB laboratories, Zeist, The Netherlands) weighing 350–550 g were used in this study, approved by the University Animal Welfare committee. The rats were housed individually in a controlled environment (21 ± 1◦ C; humidity, 60%; lights on 08:00 AM to 8:00 PM; food and water available ad libitum). The EEG was measured 24 h/day, until the animals were killed. Electrode implantation, seizure induction, and EEG monitoring Rats were anesthetized with an intramuscular injection of ketamine (57 mg/kg; Alfasan, Woerden, The Netherlands) and xylazine (9 mg/kg; Bayer AG, Leverkussen, Germany) and placed in a stereotactic apparatus. To record hippocampal EEG, a pair of insulated stainless steel electrodes (70-µm wire diameter; tips were 80 µm apart) were implanted into the left dentate gyrus under electrophysiologic control as previously described (14). A pair of stimulation electrodes was implanted in the angular bundle. Two weeks after recovery from the operation, each rat was transferred to a recording cage (40 × 40 × 80 cm) and connected to a recording and stimulation system (NeuroData Digital Stimulator; Cygnus Technology Inc., Delaware Water Gap, PA, U.S.A.) with a shielded multistrand cable and electrical swivel (Air Precision, Le Plessis Robinson, France). A week after habituation to the new condition, rats underwent tetanic stimulation (50 Hz) of the hippocampus in the form of a succession of trains of pulses every 13 s. Each train had a duration of 10 s and consisted of biphasic pulses (pulse duration, 0.5 ms; maximum intensity, 500 µA). Stimulation was stopped when the rats displayed sustained forelimb clonus and salivation for minutes, which usually occurred within 1 h. However, stimulation never lasted >90 min. Behavior was observed during electrical stimulation and several hours thereafter. Immediately after termination of the stimulation, periodic epileptiform discharges (PEDs) occurred at a frequency of 1–2 Hz and were accompanied by behavioral and EEG seizures (SE). Four hours after termination of the tetanic stimuli, rats were injected intraperitoneally with pentobarbital (PTB; 60 mg/kg; Nembutal, Sanofi Sant´e, Maassluis, The Netherlands) to avoid lethal SE. Epilepsia, Vol. 46, No. 9, 2005
Most of the animals were monitored continuously until they were killed (1 day, 1 week, and 3 to 5 months after SE) to determine whether, when, and how frequently spontaneous seizures occurred. In a subset of rats, stimulation did not lead to SE, although stage V seizures during stimulation were observed; in these rats, PEDs disappeared within several minutes after stimulation or were absent altogether (non-SE; n = 3). Sham-operated control rats (n = 5) were handled and recorded identically, but did not receive electrical stimulation. Differential EEG signals were amplified (×10) via an FET transistor that connected the headset to a differential amplifier (×20; CyberAmp; Axon Instruments, Burlingame, CA, U.S.A.), filtered (1– 60 Hz), and digitized by a computer. A seizure-detection program (Harmonie; Stellate Systems, Montreal, Quebec, Canada) sampled the incoming signal at a frequency of 200 Hz per channel. All EEG recordings were visually monitored and screened for seizure activity on a daily basis until the animals were killed. Differential EEG recordings assured that EEG artifacts were minimized so that seizures could be easily determined. EEG seizures also were validated by combined EEG-video monitoring in each rat during 1 day in the chronic epileptic phase. Tissue preparation for immunocytochemistry Thirty-six rats were used in the immunocytochemical study. Rats were continuously EEG monitored and killed at different time points after SE for subsequent immunocytochemical and histologic analysis. Rats were killed at 1 day (n = 2), 1 week (n = 4), 4 weeks (n = 2), 6 weeks (n = 3), and between 3 and 5 months after SE (n = 17). The last group was subdivided into rats with a progressive seizure evolution (n = 10; approximately five to 10 seizures per day; van Vliet et al., 2004) and rats with a nonprogressive form of seizure evolution (n = 7; on average, approximately one to two seizures per week; van Vliet et al., 2004); rats that were stimulated but that did not develop SE (non-SE, n = 3) and control rats (n = 5) also were included. Rats were disconnected from the recording apparatus and deeply anesthetized with PTB (Nembutal; intraperitoneally, 60 mg/kg). The animals were perfused through the ascending aorta with 300 ml of 0.37% Na2 S solution and 300 ml 4% paraformaldehyde and 0.2 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were postfixed in situ overnight at 4◦ C, dissected, and cryoprotected in 30% phosphate-buffered sucrose solution, pH 7.4. After overnight incubation at 4◦ C, the brains were frozen in isopentane (−25◦ C) and stored at −80◦ C until sectioning. Horizontal sections (40 µm) were cut on a sliding microtome and collected in 0.1 M phosphate buffer for immunocytochemistry. In a subset of rats (n = 4), sagittal sections (40 µm) were cut. Horizontal sections between 5,100 and 5,600 µm below cortex surface (midlevel) of control and post-SE rats were stained with different
FERRITIN EXPRESSION IN EPILEPTIC RATS immunocytochemical markers. For each animal, two sections were analyzed per level. Sections were washed in 0.05 M phosphate-buffered saline (PBS), pH 7.4, and incubated for 30 min in 0.3% hydrogen peroxide in PBS to inactivate endogenous peroxidase. Sections were then washed (2 × 10 min) in 0.05 M PBS, followed by washing (1 × 60 min) in PBS + 0.4% bovine serum albumin (BSA). Sections were incubated with anti–horse spleen ferritin (ferritin; polyclonal rabbit; 1:2,000, no F6136; SigmaAldrich, Zwijndrecht, The Netherlands) or monoclonal anti-rat CD11b/c (OX42; monoclonal mouse; Pharmingen, San Diego, CA, U.S.A.; 1:100, as a marker for microglia) in PBS + 0.1% Triton X-100 + 0.4% BSA at 4◦ C. Twenty-four hours after the incubation with the primary antibody, the sections were washed in PBS (3 × 10 min) and then incubated for 1.5 h in biotinylated sheep antirabbit/mouse immunoglobulin (Ig) (Amersham Pharmacia Biotech, Roosendaal, The Netherlands), diluted 1:200 in PBS. Sections were washed in PBS (3 × 10 min) and incubated for 30 min with AB-mix (Vectastain ABC kit, Peroxidase Standard pk-4000; Vector Laboratories, Burlingame, CA, U.S.A.). After washing (3 × 10 min) in 0.05 M Tris-HCl, pH 7.9, the sections were stained with 3,3 -diaminobenzidine tetrahydrochloride (30 mg DAB; Sigma-Aldrich), 750 µl 1% hydrogen peroxide in a 100ml solution of Tris-HCl. The staining reaction was monitored under the microscope and stopped by washing the sections in Tris-HCl. After mounting on gelatine-coated slides, the sections were air dried, dehydrated in alcohol and xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany). Omission of the primary antibody eliminated all specific immunoreactivity. The intensity of ferritin immunoreactive cells was estimated semiquantitatively in the hippocampus, parahippocampus, and piriform cortex. The immunoreactivity was classified as follows: (+), moderate; +, strong; and ++, very strong. The frequency of such cells was classified as 1, sparse; 2, high; 3, very high; and 4, clusters formation. Sections were photographed by using bright-field illumination on an Olympus-Vanox microscope, equipped with a digital camera, and imported into Adobe Photoshop (version 7.0). This program was used to optimize contrast and brightness, but not to enhance or change the image content in any way. Fluorescent immunocytochemistry For double labeling of ferritin with the markers OX-42 for microglia [mouse anti-rat CD11b/c (OX42), 1:100, Pharmingen, San Diego, CA, U.S.A.], anti– neuron-specific antigen; monoclonal mouse anti–neuronspecific nuclear protein (NeuN), 1:1,000, Chemicon International, Harrow, U.K.), glial fibrillary acidic protein (GFAP) for glial cells (mouse anti-GFAP, 1:500, Boehringer Mannheim, Germany), vimentin for reactive astrocytes (mouse anti-V9, 1:25, DAKO, Denmark), and ED1 (mouse anti-ED1, MAB 1435, 1:100, Chemicon,
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Hampshire, UK) for macrophages, a subset of freefloating sections was incubated with primary antibodies, followed by washing. Hereafter the sections were incubated with Alexa Fluor 568, goat anti-rabbit IgG, and Alexa Fluor 488, goat anti-mouse IgG Alexa (Molecular Probes; dilution, 1:200). After three additional washes in PBS, sections were mounted on slides and coverslipped with Vectashield. Images were acquired by using a confocal-laser scanning microscope (Zeiss LSM510) and processed by using LSM software (Zeiss) and Adobe Photoshop. RESULTS Ferritin immunoreactivity in control tissue Hippocampus Ferritin staining in control tissue showed that neuropil staining is present in all hippocampal regions but with different staining intensities [dentate gyrus (DG) > subiculum/CA1 > CA3). Within the dentate molecular layers, the intensity was moderate in the middle molecular layer and slightly lighter within the inner molecular layer (< outer molecular layer). Ferritin staining was weak in the principal neurons, especially in the cell soma cytoplasm (granule cells >CA neurons). Moderate ferritin staining intensity was found in the stratum lucidum and hilus, corresponding to the presence of ferritin in the mossy fiber terminals (arrows in Fig. 1A). The strongest ferritin immunoreactivity was observed in individual cells with two types of morphology. The predominant cell type expressing ferritin has a small and irregular cell body with branched processes and is morphologically similar to microglia cells. The immunoreactivity was visible throughout the cell. Cell densities were higher in the hilus and stratum lucidum and to a lesser extent in the subiculum than in the rest of the hippocampal formation. Another ferritin-positive cell type had a small and round soma with no processes. These cells were less abundant than microglia cells and were probably oligodendrocytes (15). Entorhinal cortex Within the entorhinal cortex (EC), ferritin staining was stronger in the superficial layers (I–III) than in the deep layers (V–VI). Layer IV had the lightest appearance (Fig. 1A). Individual cells within these layers had strong ferritin staining. Cell densities were somewhat higher in the superficial than in deeper layers. Parenchymal ferritin staining was moderate in the parasubiculum and somewhat lighter in the presubiculum. Most of the immunopositive cells in the parahippocampal region had irregular soma and small processes, resembling resting microglia cells. This is further described later. Ferritin immunoreactivity after SE The changes of cellular ferritin expression after SE are summarized in Table 1. At 1 day after SE, ferritin staining Epilepsia, Vol. 46, No. 9, 2005
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FIG. 1. Horizontal section of the hippocampal region showing ferritin immunoreactivity in a control (A); during the latent phase [1 week after status epilepticus (SE)] (B); and in a chronic epileptic rat with daily seizures (p-SE; 5 months after SE) (C). Increased ferritin expression in post-SE versus control (D) tissue is especially evident in CA regions, as shown in more detail for the CA1 region at 1 week (E) and 5 months (chronic epileptic state) after SE (F). G–I: The OX-42 staining (a marker for complement receptor type 3 that is present in microglia) in CA1 area of the same control (G), 1 week (H), and chronic epileptic rat (I). Insets (G–I) show corresponding hippocampal OX-42 expression. Calibration bar for A, 800 µm; for B and C, 600 µm; for D–I, 100 µm.
had increased at several hippocampal regions, mainly at clusters near the CA pyramidal cell layers and within the hilar region. One week after SE, when the rats were still in the latent period, ferritin protein expression was increased throughout the hippocampal formation. The most significant differences occurred in the CA1 and CA3 (stratum oriens) (Fig. 1B) and in the superficial layers of the EC (mainly in layer III). At this time point, not only an increase in the number of ferritin-positive cells is present, but also a change in their morphology. In the pyramidal cell layer, cells with large rodlike processes were observed along the dendrites of CA1 and CA3 neurons and intensely stained somata (Fig. 1E). Ferritin-immunoreactive cells with a bushy appearance were observed in the medial as well as in the lateral EC. All these cells had the morphologic characteristics of reactive microglia. An upregulation of the protein also was found in other limbic regions, such as dorsal thalamic nucleus, amygdala, septum, perirhinal cortex, and piriform cortex. A semiquantitative analysis showed also a dramatic upregulation of Epilepsia, Vol. 46, No. 9, 2005
ferritin expression in layers II/III of the piriform cortex (Table 1). After the end of the latent period and in the beginning of the chronic epileptic phase (until 6 weeks after SE), ferritin immunoreactivity progressively decreased throughout the hippocampus along with a decrease in OX-42 intensity (see later). Nevertheless, strong ferritin staining was still observed in cells resembling reactive microglia. These cells had generally smaller processes than at 1 week after SE. In the EC, strongly stained ferritin immunoreactive cells were observed mainly in layers I, III, and V. The expression of ferritin also was still increased in other limbic structures, especially piriform cortex (along the entire anteroposterior axis). Chronic epileptic post-SE rats (3–5 months after SE) could be divided into rats that had progressive seizure frequency over time (progressive SE rats; p-SE) and post-SE rats that had only an occasional seizure (“nonprogressive” SE rats; np-SE) (14,16). On average, p-SE rats exhibited six to 10 seizures/day during the week before death. In
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TABLE 1. Cellular ferritin protein expression in the hippocampal formation and piriform cortex after status epilepticus Control DG CA3 CA1 Sub PrS PaS EC II/III EC V/VI PC II/III PC V/VI
(+) (+) (+) (+) (+) (+) (+) (+) (+) (+)
1 day 2 2 2 2 2 2 2 2 2 2
++ ++ ++ + (+) ++ ++ + + +
1 wk 3–4 3–4 3–4 2–3 2 3 3 2 2 2
++ ++ ++ + (+) ++ ++ + + +
4–6 wk 3–4 3–4 3–4 2–3 2 3 3 2 3 3
+ ++ ++ + + ++ ++ + ++ ++
2 2–3 3 2 2 2 2 2 3 3
np-SE (+) + + (+) (+) (+) (+) (+) ++a (+)
p-SE 2 2 2 2 2 2 2 2 3 2
+ + + (+) (+) (+) ++ + ++ +
Non-SE 2 2 2 2 2 2 2 2 3 2
(+) (+) (+) (+) (+) (+) (+) (+) (+) (+)
2 2 2 2 2 2 2 2 2 2
Immunoreactivity was classified as follows: (+), moderate; +, strong; ++, very strong. Frequency was classified as 1, moderate; 2, high; 3, very high; 4, clusters formation. DG, dentate gyrus (hilus); Sub, subiculum; PrS, presubiculum; PaS, parasubiculum; EC, entorhinal cortex; PC, piriform cortex. a If upregulated: only at stimulated site.
np-SE rats, seizure frequency was low (one seizure every 5 days) and did not increase over time. In both groups of rats, strong ferritin immunoreactivity could still be observed in CA1-3 regions, even 5 months after the SE (Fig. 1C and F). This was associated with the cell loss observed in those regions that is extensive in CA1-3 in both p-SE and np-SE rats (17). In the hilar region, ferritin immunoreactivity was higher in p-SE than in np-SE rats, corresponding to the milder cell loss in this region in np-SE rats (17). Outside the hippocampus, differences between the two groups were evident, especially in EC layer III. In this layer, large ferritin-positive cells were observed in chronic epileptic rats that underwent a progressive evolution of seizures (p-SE rats; Fig. 2B) and not in rats that did not exhibit this progressive evolution (np-SE rats). The large ferritin-positive cells also were observed in deeper EC layers of p-SE rats. Ferritin expression in extrahippocampal regions of np-SE rats was less pronounced than in rats with progressive seizure evolution, although it could still be high in specific regions, especially at the (electrically) stimulated site. Ferritin staining in brains of stimulated non-SE was similar to staining patterns in control rats (Table 1). Because ferritin-positive cells appeared to be morphologically similar to microglial cells, a diaminobenzidine (DAB) staining with OX-42 (a marker for complement receptor type 3 that is present in microglia) was performed. In control rats, resting microglia was expressed throughout the hippocampal formation, with somewhat higher density in the hilar region (Fig. 1G, inset). The cells have an irregular soma and branched processes. The microglial reaction after electrically induced SE was very similar to that described after kainite-induced SE (18). Microglia staining with reactive morphology could be observed at the first day after SE, especially in nestlike clusters near the cell layers within the hippocampus. At 1 week after SE, a dramatic increase of OX-42 immunoreactivity was observed in all hippocampal subfields, indicating further activation of microglial cells (Fig. 1H, inset). Middle and outer molecu-
lar layer of the DG appeared to be relatively unaffected. Cells in the CA1 had a characteristic reactive bushy appearance and distended clubfoot-like endings (Fig. 1H), whereas in the CA3, they had a stellate morphology with larger cell bodies and coarse processes. In the hilar region of the DG, the microglial cells tended to form clusters. Clustering also was observed in the inner molecular layer, probably linked to degeneration of hilar neuron projections that target this layer. Within the parahippocampal cortex, the entorhinal layer III showed the highest reactivity; the presubiculum appeared to be unaffected. Other structures such as the limbic cortex, the perirhinal cortex, and the piriform cortex, as well as the dorsal thalamic nucleus and amygdala, also showed activation of microglial cells (data not shown). Between 4 and 6 weeks after SE, the OX-42–positive cells started more and more to look like resting microglial cells, although clusters of reactive microglia were still observed in some regions, including CA1, CA3, and layer III and V of the EC. In chronic epileptic rats, OX-42 staining appeared to stain cells that resembled microglial cells of the resting type. Closer examination, however, still showed reactive microglial cells with abnormal morphology. This was especially true in the CA1-3 region, which was often markedly shrunken. In contrast to the obviously increased ferritin staining in the soma of the cells, the microglial staining appeared normal in the hilar region and in layer III of the EC. However, closer examination showed several microglial cells with stronger OX-42 staining (Fig. 2C). This difference in appearance may be related to the fact that the ferritin stain is a cytoplasmic stain, and OX-42 only stains the complement factor (at the outside of the cell). The increased OX-42 staining in some cells of EC layer III was mainly observed in p-SE rats. Np-SE rats appeared more similar to those with control staining, especially at the nonstimulated site. In contrast to the expression pattern during the early chronic epileptic phase (4–6 weeks), we did not observe clusters of microglial cells in the hippocampal formation during the late Epilepsia, Vol. 46, No. 9, 2005
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FIG. 2. Horizontal sections of layers II and III of the superficial entorhinal cortex (EC) region showing ferritin immunoreactivity in an (A) control and (B) chronic epileptic rat with seizure progression (p-SE); arrows, increased expression in cells with microglia morphology. C: OX-42 stain in same EC layers of the same rat. OX-42 staining shows that expression in EC layer III in p-SE tissue is sometimes slightly darker (arrows) than that in layer II. Increased ferritin staining is found mainly in the superficial layer III. Calibration bar, 100 µm.
chronic epileptic phase (3–5 months). OX-42 staining in the parahippocampal region of non-SE rats had a very similar distribution and morphology to that of control rats. Cellular distribution A double-labeling was performed to identify the cellular localization of ferritin. Because ferritin-positive cells were morphologically similar to microglia, we performed a double-labeling with OX-42. Figure 3A–C shows ferritin labeling in an OX-42–positive cell. This was by far the predominant type of ferritin-positive cell. Nevertheless, ferritin appeared also to be present in OX-42–negative cells with a round cell body and without processes, probably representing oligodendrocytes (15). Interestingly, ferritinpositive cells (in red) in tissue of chronic epileptic rats were much larger (Fig. 3D, K, L) than ferritin-positive cells in control tissue (Fig. 3G and J). Ferritin also colocalized with ED1, which labels macrophages (not shown). Double-labeling with vimentin did not reveal the presence of ferritin in reactive astrocytes (Fig. 3I). To investigate the presence of ferritin in neurons, we performed a double-labeling with NeuN. Although the presence of ferritin in neurons has been reported (15,19), our results are not conclusive about the subcellular localization of the protein in these cells. In regions such as the granule cell layer of the DG, a weak staining was visible just beneath the neuronal membrane (Fig. 3D). We did not observe a change in neuronal ferritin immunoreactivity after SE. DISCUSSION In this study, we investigated the expression pattern of ferritin during epileptogenesis that was initiated by an SE. The hypothesis to be tested was whether a change in iron metabolism in epileptic rats could be associated with the progressive nature of temporal lobe epilepsy. We used ferritin, an iron-storage protein, as an indicator of changed iron metabolism. If changes in this protein are evident, one can assume that iron metabolism is disturbed. The findings of our study are (a) a widespread increase of ferritin Epilepsia, Vol. 46, No. 9, 2005
immunoreactivity was noted in microglial cells within the hippocampal formation (and other limbic regions) during the latent and early chronic epileptic phase; (b) in chronic epileptic rats, ferritin protein expression was persistently increased in more restricted regions such as specific layers of the parahippocampal cortex, compared with control expression; and (c) Increased ferritin expression is more extensive in rats with a progressive seizure evolution versus rats that have only occasional seizures. The data confirmed our previous SAGE results that showed a twofold upregulation of ferritin at 8 days after SE just before the start of the chronic epileptic phase (1). Previous studies also showed increased ferritin expression shortly after kainate-induced SE (20) or after an ischemic insult (21). However, these studies showed the acute effects only at early time points after the insult. A more prolonged (1 month) increased ferritin protein expression also was observed after experimental stroke in the rat (22). Because of the characteristics of the ferritin antibody, we could not discriminate between the L and H isoforms. However, our recent findings in a microarray study indicate an increased expression of both mRNA isoforms in the parahippocampal cortex at 1 day and 1 week after SE. Because the L isoform is more associated with long-term storage of iron, it is most likely that this isoform is most prominent in the chronic epileptic phase. In addition to the dynamic regulation of ferritin, other mRNAs related to the “iron ion homeostasis pathway” also were upregulated, and this was identified as a significantly altered biologic process during the chronic epileptic phase (23). It is reasonable to assume that ferritin is upregulated as a compensatory mechanism to counteract the iron overload that is caused by the release of iron from heme proteins. The most important heme proteins in this respect are (a) cytochrome c and (b) hemoglobin. SE is associated with excitotoxic cell death, mitochondrial disruption, and release of cytochrome c, a heme protein involved in the electron transfer chain (24–26). The fact that ferritin expression is especially strong in limbic regions where
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FIG. 3. Confocal microscope images of a reactive microglial cell in the cortex of an epileptic rat (3 months after status epilepticus) shows intracellular presence of ferritin (A) and (mainly membranous) OX-42 staining (B). C: Merged image. D–F: Detail of dentate gyrus (DG) of chronic epileptic rat (p-SE, 5 months) shows strong ferritin staining in cells with microglial morphology cells and weak ferritin staining in granule cells (D–E). F: Merged image. G, H: Merged images of ferritin (red) and NeuN (green) in DG of control and 1-week post-SE rat (latent period). I: Image of ferritin immunoreactivity (red) and the reactive astrocytes marker vimentin (green) in the hilar region of a 1-week animal. Note that no colocalization appears between ferritin and vimentin. White arrow, Example of a blood vessel labeled with vimentin. J–L: Ferritin staining (red) combined with NeuN (green, J–K) or vimentin (green, L) in control (J) and chronic epileptic rat (K, L: pSE, 5 months). Note that ferritin-positive cells with microglial morphology in layer III of epileptic rats are much larger than ferritin-positive cells in control rat. Also note that only blood vessels are stained by vimentin; vimentin-positive astrocytes are much rarer at this stage. Calibration bar: A–C, 10 µm; D–F, 20 µm; J–L, 40 µm. Epilepsia, Vol. 46, No. 9, 2005
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extensive cell loss generally occurs shortly after SE suggests that iron liberated from cytochrome c plays a major role. Although we did not look in detail to ferritin expression outside the parahippocampal region, the sagittal sections showed that ferritin expression was strongly and persistently upregulated in other limbic regions including the dorsal thalamic nucleus, amygdala, piriform cortex (PC), and olfactory bulb. However, the upregulation (with the exception of the olfactory bulb and PC) was less abundant than in EC layer III. Without the localized upregulation of ferritin, the neuronal damage in these regions would probably be even more extensive. Prolonged seizures such as SE can disrupt the blood–brain barrier and result in extravasation of blood and breakdown of red blood cells and hemoglobin (27). Iron liberated from hemoglobin, and hemoglobin itself, are associated with the generation of reactive oxygen species and subsequent cell death. For instance, in the PC, the blood–brain barrier is disrupted by the SE, suggesting that release of iron from haemoglobin-containing blood cells probably also plays a role in the cellular destruction of this region and the upregulation of ferritin in microglial cells. Studies have shown that injection of iron can induce epileptiform activity when injected in specific brain regions (10,12). Whether this effect is caused by ironinduced lesions or by iron-induced destabilization of nearby neuronal networks is not known. In our study, ferritin protein expression was still high at specific brain areas in chronic epileptic rats at a time when cell loss is negligible (17,24,28). Thus although no contribution occurs from released iron from dying cells, iron homeostasis seems still to be disrupted in these regions. Because ferritin also is capable of releasing iron, it is tempting to speculate that ferritin-containing microglia cells remain a permanent potential source for release of iron that could affect and destabilize the neuronal networks. In this respect, it is noteworthy that ferritin is significantly increased in the superficial layers of the EC of rats with progressive seizure evolution. The superficial layers of the EC in chronic epileptic rats are very prone to oscillatory activity and are therefore considered to be highly epileptogenic (29,30). Nevertheless, the relation between increased ferritin expression and seizure progression does not necessarily imply that progression is caused by a disturbed iron homeostasis. It might be just the result of SE-induced cell death, which positively correlates with the probability to develop a progressive form of epilepsy later (17). However, assuming that it reflects a compromised iron homeostasis, ferritin expression could provide a biologic marker with potential relevance for an insight into the progressive state of the disease. Conclusion The increased ferritin expression in chronic epileptic rats that occurred in specific cells (microglia) at deEpilepsia, Vol. 46, No. 9, 2005
fined cortical layers of the parahippocampal region was observed in rats with a progressive form of epilepsy. Whether this increased expression might alter cellular properties and network excitability must be further investigated. Manipulation of this ferritin protein expression (e.g., via dietary iron restriction in rats with progressive seizure evolution) could provide an answer as to whether increased ferritin levels contribute to the progression of temporal lobe epilepsy or that its increased expression reflects the aftermath of cell death incurred early after SE. Acknowledgment: We thank Dr. M. de Sousa for her valuable comments on the manuscript. Part of this work was supported by a grant of the National Epilepsy Fund “Power of the Small” nr 01-09 and 03-03.
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