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Keywords:

  • Pharmacoresistance;
  • Epilepsy;
  • Antiepileptic drugs;
  • Progression;
  • Status epilepticus;
  • Spontaneous seizures

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Summary: Purpose: Because drug transporters might play a role in the development of multidrug resistance (MDR), we investigated the expression of a vesicular drug transporter, the major vault protein (MVP), in a rat model for temporal lobe epilepsy.

Methods: By using real-time polymerase chain reaction (PCR) analysis and immunocytochemistry, we quantified MVP mRNA and protein from the dentate gyrus (DG) and parahippocampal cortex (PHC) taken from EEG-monitored rats at 1 week after electrically induced status epilepticus (SE) and at 5–9 months after SE, when rats exhibit spontaneous seizures.

Results: Within 1 week after SE, MVP mRNA levels increased in both DG and PHC compared with those in controls. In chronic epileptic rats, MVP mRNA was still significantly upregulated in the PHC, whereas in the DG, the expression returned to control levels. MVP protein increased within 1 day after SE in reactive microglial cells within most limbic regions; the hippocampus showed the highest expression at 1 week after SE. In chronic epileptic rats, MVP protein expression was largely decreased in most brain regions, but it was still high, especially in the piriform cortex. The occurrence of SE was a prerequisite for increased MVP expression, because no increase was found in electrically stimulated rats that did not exhibit SE.

Conclusions: MVP expression is upregulated in chronic epileptic rats and may contribute to the development of pharmacoresistance.

In one third of patients with epilepsy, antiepileptic drugs (AEDs) are ineffective (1). The causes and mechanisms underlying this multidrug resistance are not fully understood but might be due to alterations of drug targets or to a decreased penetration of AEDs into the brain. Overexpression of drug transporters MDR1/P-glycoprotein (P-gp) and MRP1 has been shown in brain tissue from patients with refractory epilepsy (2–5), but also in epileptic mice and rats (6–8). Recent indications were noted that increased expression of multidrug transporters, such as P-gp, may lead to reduced drug concentrations in the brain, shortly after status epilepticus (SE), thereby suggesting a possible mechanism for drug-resistance (8). Other factors that may play a role in the development of pharmacoresistance are intracellular transport processes. A specific cell organelle, the vault, could be involved in this, although this is yet not clear. Vaults are evolutionary highly conserved, large ribonucleoprotein particles [for review see (9)]. The particles represent multimeric RNA–protein complexes with one predominant component, the major vault protein (MVP), identical to lung resistance–related protein (10). Vaults appear to have a transport function by acting as carriers, mediating bidirectional nucleo–cytoplasmic exchange as well as vesicular transport of compounds such as cytostatic drugs (11). In P-gp–negative drug-resistant tumor cells, vaults are frequently overexpressed (12–14). Human leukemia cells, which accumulate MVP during culturing, become multidrug resistant to various cytostatic drugs after successive passages (15). In a recent study, the upregulation of human MVP was shown in the neuronal component of gangliogliomas and a population of tumor glial cells of patients that underwent resection of gangliogliomas for medically intractable epilepsy (16). In patients with hippocampal sclerosis, MVP was found to be upregulated in specific neurons and blood vessels of the dentate gyrus (DG) (16–18). Because studies on human brain material are limited to biopsy or postmortem material, it is not known whether increased expression is due to an initial insult, to recurrent seizures, or both. To investigate the spatial and temporal expression of MVP in relation to the progression of epilepsy, we used a rat model for temporal lobe epilepsy, (TLE) in which spontaneous seizures develop after electrically induced SE. We studied the relation between altered expression of MVP, seizure onset, and severity of the chronic epileptic condition by using permanently EEG-monitored epileptic rats.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Experimental animals

Adult male Sprague–Dawley rats (Harlan CPB laboratories, Zeist, the Netherlands) weighing 350–550 g were used in this study, which was 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 a.m. to 8:00 p.m.; 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 the hippocampal EEGs, a pair of insulated stainless steel electrodes (70-μm wire diameter, tips were 0.8 mm apart) were implanted into the left DG under electrophysiological control, as previously described (19). 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; maximal 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. Immediately after termination of the stimulation, periodic epileptiform discharges (PEDs) occurred at a frequency of 1 to 2 Hz and were accompanied by behavioral seizures (SE). The animals were monitored continuously until they were killed (1 day to 9 months after SE induction) to determine whether, when, and how frequently spontaneous seizures occurred. Sham-operated control rats (n = 12) 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 (20x; 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. EEG recordings were visually monitored and screened for seizure activity on a daily basis until death.

Total mRNA isolation and cDNA synthesis

For quantification of MVP RNA expression, real-time quantitative polymerase chain reaction (PCR) analysis was performed on the DG and parahippocampal cortex (PHC, including entorhinal and part of posterior piriform and perirhinal cortex). Rats were killed at 1 week (n = 5) and 4–9 months (pSE, n = 8; non-SE, n = 5) after the induction of SE; five sham-operated control rats were included (see also Table 1). The brain was rapidly dissected, and the PHC, located at the rear end of the cerebral hemisphere, was removed by incision at the ventrocaudal part underneath the rhinal fissure. The hippocampus was dissected and sliced into smaller parts. The DG was cut out of the slice under a dissection microscope. All material was frozen on dry ice and stored at –80°C until use. Total RNA was isolated by using the TRIzol LS reagent, following the manufacturer's instructions (Invitrogen-Life Technologies, Breda, the Netherlands). The concentration and purity of the RNA were determined spectrophotometrically at 260/280 nm. Five micrograms of total RNA were reverse-transcribed into cDNA by using 125-pmol two-base anchored oligo dT primers [5'(dT)14-d(A/G/C)-d(A/G/C/T); Amersham Biosciences Europe, Roosendaal, the Netherlands]. The reverse transcription was performed in 50-μl reactions. Five nanomoles of oligo dT primers were annealed to 5 μg total RNA in a total volume of 20 μl by incubation at 72°C for 10 min and cooled to 4°C. Reverse transcription was performed by the addition of 25 μl RT-mix, containing 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 20 mM DTT, 0.1 mM dNTPs (Pharmacia, Germany), 60 U RNAse inhibitor (Roche Applied Science, Indianapolis, IN, U.S.A.), and 400 U M-MLV reverse transcriptase (Invitrogen-Life Technologies). This mixture was incubated at 37°C for 60 min, heated to 95°C for 10 min, and cooled to 4°C.

Table 1. Overview of the different groups of rats
GroupNo. of ratsSurvival timeMeasurement
Control54–9 moq-PCR
1 week SE5  1 wkq-PCR
p-SE84–9 moq-PCR
Non-SE54–9 moq-PCR
Control54–9 moImmuno
1 day SE5  1 dayImmuno
1 wk SE5  1 wkImmuno
4–6 wk SE54–6 wkImmuno
np-SE54–9 moImmuno
p-SE74–9 moImmuno
Non-SE64–9 moImmuno
Control2  8 moWestern blot
1 wk SE2  1 wkWestern blot
p-SE2  8 moWestern blot

Quantitative real-time RT-PCR analysis

Real-time monitoring of PCR reactions was performed by using the LightCycler system (Roche Applied Science). Two pairs of primers specific for MVP and β-actin (Sigma-Genosys, Haverhill, U.K.) were designed on intron/exon boundaries (Table 2). For each PCR, a master mixture was prepared on ice, containing per sample: 1 μl of cDNA, 1 μl of FastStart Reaction Mix SYBR Green I (Roche Applied Science), 0.5 μl of 10 μM primers and 1.6 μl of 25 mM MgCl2. The final volume was adjusted with H2O to 10 μl. After the reaction mixture was loaded into a glass capillary tube, the cycling conditions for both MVP and β-actin were carried out as follows: initial denaturation at 95°C for 6 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 5 s, and extension at 72°C for 10 s. The temperature transition rate was set at 20°C per second. Fluorescent product was measured by a single acquisition mode at 72°C after each cycle. Separate calibration (standard) curves for MVP and β-actin (as reference) were constructed by using serial dilutions of total cDNA from a whole brain of a control rat. The standard curve samples were included in each PCR. Standards for both MVP and β-actin RNAs were defined to contain an arbitrary starting concentration, because no primary calibrators exist. Hence all calculated concentrations are relative to the concentration of the standard.

Table 2. Primer characteristics
GeneGenBankForwardaReverseaAmpliconSequence forwardSequence reverse
  1. aCoordinates according to Genbank.

MVPNM_022715501–518648–631148 bp5′ GAC CTG GCA CCT ACA TCC 3′5′ CAC TCC TCA CCT GTC ACG 3′
β-actinNM_031144974–9951,093–1,072120 bp5′ TGA AGA TCA AGA TCA TTG CTC C 3′5′ ACT CAT CGT ACT CCT GCT TGC 3′

For distinguishing specific from nonspecific products and primer dimers, a melting curve was obtained after amplification by holding the temperature at 65°C for 15 s followed by a gradual increase in temperature to 95°C at a rate of 0.1°C per second, with the signal-acquisition mode set at continuous. Product identity was confirmed by electrophoresis on a 12% nondenaturating polyacrylamide gel, stained with ethidium bromide afterward. The product size was verified by using a 10-bp ladder (1 μg/μl; Invitrogen-Life Technologies).

Quantification data were analyzed by using the LightCycler analysis software. Background fluorescence was removed by setting a noise band. The log-linear portion of the standard's amplification curve was identified, and the crossing point is the intersection of the best-fit line through the log-linear region and the noise band. With calibration curves, the concentration of a product was calculated. The amount of MVP was divided by amount of β-actin for each sample and normalized to control values. Statistical analysis between groups was performed by using analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons with SPSS software (SPSS Inc., for Windows, release10). A correlation between two ordinal variables was calculated by using a Spearman rank correlation test (p < 0.05) with SPSS software.

Immunohistochemistry

Rats were disconnected from the recording apparatus and deeply anesthetized with pentobarbital (Nembutal; intraperitoneally, 60 mg/kg). The animals were perfused through the ascending aorta with 300 ml of 0.37% Na2S solution, 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. The brains were cut on a sliding microtome and 40-μm horizontal sections were collected in 0.1 M phosphate buffer for immunocytochemistry. Rats were killed 1 day (n = 5), 1 week (n = 5), 4–6 weeks (n = 5), or 4–9 months (np-SE, n = 5; p-SE, n = 7; non-SE, n = 6) after the stimulation, including five sham-operated control rats (see also Table 1). Horizontal sections between 5,100 and 5,600 μm below cortex surface (midlevel) and 7,600 and 8,100 μm below cortex surface (ventral level) of the brain [according to (20)] of control and post-SE rats were stained with different 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 + 0.5% Triton X-100 + 0.4% bovine serum albumin (BSA). Sections were incubated with rabbit anti-MVP [1:100, a generous gift from Dr. L.H. Rome, described in (21)] 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 anti-rabbit or anti-mouse immunoglobulin (Ig; Amersham Pharmacia Biotech, Roosendaal, the Netherlands), diluted 1:200 in PBS + 0.1% Triton X-100 + 0.4% BSA. Sections were washed in PBS (3 × 10 min) and incubated for 1.5 h in streptavidin–horseradish peroxidase (Zymed Laboratories, San Francisco, CA, U.S.A.), diluted 1:200 in PBS + 0.1% Triton X-100 + 0.4% BSA. After washing in 0.05 M Tris-HCl, pH 7.9, the sections were stained with 3,3'-diaminobenzidin tetrahydrochloride (30 mg DAB; Sigma-Aldrich, Zwijndrecht, the Netherlands) and 750 μl 1% hydrogen peroxide in a 100-ml 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 primary antisera, preincubation with excess of MVP protein, and incubation with normal goat serum eliminated all specific immunoreactivity. The intensity of MVP immunoreactive (IR) cells in microglia was estimated semiquantitatively in the hippocampus, parahippocampus, and piriform cortex. The immunoreactivity was classified as follows: (+), weak; +, moderate; and ++, strong. Frequency 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 MVP with the markers OX-42 for microglia [mouse anti-rat CD11b/c (OX-42), 1:100; PharMingen, SanDiego, CA, U.S.A.), glial fibrillary acidic protein (GFAP) for glial cells (mouse anti-GFAP, 1:500; Boehringer Mannheim, Mannheim, Germany), vimentin for reactive astrocytes (mouse anti-V9, 1:25; DAKO, Glostrup Denmark), and ED1 (mouse anti-ED1, MAB 1435, 1:100; Chemicon, Hampshire, U.K.) for macrophages, a subset of free-floating 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, Breda, the Netherlands; 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.

Western blot analysis

For quantification of MVP protein expression, a Western blot was performed on the parahippocampal cortex. Rats were killed 1 week (n = 2) or 8 months (n = 2) after the induction of SE. Two controls also were included (see also Table 1). The PHC was dissected and stored at –80°C until use. Samples were homogenized in lysis buffer containing, per 20 ml, 200 μl 1 M Tris, pH 8.0; 1 ml 3 M NaCl; 2 ml 10% NP-40; 4 ml 50% glycerol; 800 μl Na-orthovanadate (10 mg/ml); 200 μl 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 8.0; 400 μl protease inhibitors; 200 μl 0.5 M NaFI; and 11 ml H2O. Protein content was determined by using the bicinchoninic acid method (22). Homogenate was diluted to a concentration of 3.3 mg protein/ml in SDS/bromophenol blue loading buffer and boiled for 5 min. After protein isolation, 50-μg samples were separated by gel electrophoresis (10% polyacrylamide mini-gels) and transferred to nitrocellulose by electroblotting. The blots were then processed for immunolabeling with MVP antibody. MVP optical density values were normalized with the amount of β-actin in each sample. Blots were washed in 50 mM Tris-HCl + 154 mM NaCl, pH 7.5, + 0.1% Tween 20 (TBS-T), and then incubated in TBS-T + 5% nonfat dry milk) for 60 min. Hereafter blots were incubated with rabbit anti-MVP (1:250) in TBS-T+ 5% nonfat milk. After overnight incubation, blots were washed in TBS-T (3 × 10 min) and incubated for 1 h in anti rabbit-horseradish peroxidase (1:1,000; Dako). After washing, enhanced chemiluminescence reagents were applied to the blots (ECL; Amersham Pharmacia Biotech), which then were exposed to an x-ray film (Fuji Safelight Super RX, glass no. 84). For quantification of Western blots, the exposed x-ray films were digitized, and the optical density of each sample was measured by using Scion Image (release beta 3b; Scion Corporation, MD, U.S.A.) software. For each sample, the background was subtracted.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

SE was successfully induced in 44 rats; in 11 rats, electrical stimulation did not lead to SE (non-SE rats). In rats that were killed 4 to 9 months after SE, we distinguished two groups of rats based on their registered EEG seizure activity: rats with frequent daily seizures and a progressive seizure evolution after SE (p-SE rats; n = 17) and rats with occasional seizures (one to two per week) without seizure progression (np-SE rats; n = 5). Because SE yields many more p-SE rats than np-SE rats, this latter group was included only in the immunostaining experiment and not in the PCR study described later. Non-SE rats (n = 5 PCR; n = 6 immunostaining) exhibited behavioral and electrographic seizures during stimulation but did not develop SE and had only occasional spontaneous seizures later in life. Histologically, these rats displayed minor cell loss in several parahippocampal regions (16,19).

MVP mRNA expression

To investigate how MVP expression changes after SE, we investigated mRNA levels in two regions that are assumed to play a role in epileptogenesis in TLE: the DG and the PHC. MVP mRNA expression was increased significantly, compared with that in controls, both in DG (2.5X ± 0.6) and PHC (2.7X ± 0.3) 1 week after SE (Fig. 1). MVP mRNA expression was still elevated in the PHC of chronic epileptic rats with frequent seizures (2.4X ± 0.5), but not in non-SE rats that had only occasional seizures (Fig. 1B). In the DG, no significant increase was observed in chronic epileptic rats.

image

Figure 1. Major vault protein (MVP) mRNA (normalized to control values) in the dentate gyrus and parahippocampal cortex of control rats, rats that were killed 1 week after status epilepticus (SE) induction (1 wk); rats that were killed 4–9 months after SE induction with a progressive seizure evolution (p-SE), and rats that were electrically stimulated but that did not exhibit SE (non-SE). In the dentate gyrus (A), a transient increase of MVP RNA expression was observed. In the parahippocampal cortex (B), the increase was persistent only in chronic epileptic rats with progressive seizure activity.

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Seizure activity and changes of MVP expression

To correlate changes of MVP mRNA expression with the induced seizure activity (the initial insult), the duration of the SE was plotted against MVP mRNA (normalized to control values) for rats that were killed at 1 week after SE (Fig. 2A and B). All rats that had exhibited SE (>4 h) showed increased expression, but no linear relation was found between the MVP expression and the duration of SE. Chronic epileptic rats with frequent and daily seizures also showed increased MVP mRNA expression, but only in the PHC and not in the DG. MVP mRNA expression was correlated with the number of lifetime seizures in the PHC (p < 0.01) but not in the DG (Fig. 2C and D).

image

Figure 2. Duration of status epilepticus (SE) versus amount of major vault protein (MVP) mRNA (normalized to control values) in the dentate gyrus (A) and parahippocampal cortex (B) 1 week after SE. For both regions, MVP expression is not related to the duration of SE. C, D: The lifetime seizures versus MVP mRNA expression in the dentate gyrus and parahippocampal cortex, respectively, of rats that were killed 4 to 9 months after SE induction. MVP expression is related to the number of lifetime seizures in the parahippocampal cortex but not in the dentate gyrus.

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MVP protein expression

To investigate whether the RNA increase also is translated into increased protein expression, we performed immunocytochemistry on horizontal brain sections. In control rats, we observed a low MVP immunoreactivity (IR) in parenchyma and blood vessels throughout all layers of the hippocampus (Fig. 3A and Table 3) and parahippocampal region (including presubiculum, parasubiculum, and entorhinal cortex). Whereas staining in glial cells was highly reproducible, staining in blood vessels was less consistent. In agreement with a previous study, control rats had low-moderate MVP IR in cytoplasm of microglial cells (21). Remarkably, we also observed MVP IR in layer II neurons of the piriform cortex (Figs. 4A and 5A).

image

Figure 3. Increase of major vault protein (MVP) in cells with microglial morphology in the hippocampus, 1 week after status epilepticus (SE) (C), compared with control (A). In chronic epileptic rats, MVP expression returned to control levels (E). Insets: Higher magnification of MVP immunoreactive cells. OX-42 immunostaining, a microglia marker, shows a pattern similar to that of MVP. Controls have weak OX-42 immunoreactivity (B); a strong increase can be observed at 1 week after SE (D). At this time, the morphology of microglia also changed (inset in D). OX42 expression returned to control levels in the hippocampus of most chronic epileptic rats (F). Scale bar, 500 μm.

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image

Figure 5. Confocal images of major vault protein (MVP) expression. In controls, MVP is expressed in piriform cortex layer II neurons (A–C). After status epilepticus (SE), MVP also was present in macrophages (1 week after SE; D–F) and in OX-42–positive microglia, up to 9 months after SE (G–I). Scale bar in A, D, and E, 20 μm.

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Table 3. MVP IR in the hippocampal formation and piriform cortex after status epilepticus
 Control1 day1 wk4–6 wknp-SEp-SENon-SE
  1. Immunoreactivity was classified as follows: (+), weak; +, moderate; ++, strong. Frequency was classified as 1, sparse; 2, high; 3, very high; 4, clusters formation.

  2. DG, dentate gyrus; Sub, subiculum; PrS, presubiculum; PaS, parasubiculum; EC, entorhinal cortex; PC, piriform cortex.

DG(+)2++3–4++3–4  +2(+)2(+)2(+)2
CA3(+)2++3–4++3–4++2+2+2(+)2
CA1(+)2++3–4++3–4++2(+)2(+)2(+)2
Sub(+)2  +2–3  +2–3  +2(+)2(+)2(+)2
PrS(+)2(+)  2(+)  2  +2(+)2(+)2(+)2
PaS(+)2++  3++  3++2(+)2(+)2(+)2
EC II/III(+)2++  3++  3++2(+)2(+)2(+)2
EC V/VI(+)2  +  2  +  2  +2(+)2(+)2(+)2
PC II/III(+)2  +  2  +  3++3++3++3(+)2
PC V/VI(+)2  +  2  +  3++3(+)2(+)2(+)2
image

Figure 4. Major vault protein (MVP) in layer II neurons of the piriform cortex in control rats (A). B: Neu-N staining of the piriform cortex of a control rat; box indicates the part of the piriform cortex where the detailed images (A, C, and D) are taken. In chronic epileptic rats that were killed 8 months after status epilepticus (SE) induction, (a part of) layer II neurons were lost; increased MVP immunoreactivity was present in microglia in layer III of both np-SE (stimulated side) and p-SE rats (both sides) (arrows in C and D). Scale bar, 130 μm.

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Acute period

Within 1 day after SE, an increase of MVP IR was observed in all rats (n = 5) in microglia at different regions of the hippocampal formation, including the molecular layers of the DG, the hilus, stratum radiatium, pyramidale and oriens of CA3 and CA1, part of the subiculum, the parasubiculum, and entorhinal cortex layers II and III (Table 3). The distal part of the subiculum, the presubiculum, and deep layers of the entorhinal cortex showed no or only a slight increase of IR. We also observed increased IR in microglia in other regions outside the hippocampal formation (e.g., perirhinal cortex, amygdala, septum, piriform cortex, and thalamus).

Latent period

One week after SE, the intensity of MVP IR and the number of MVP IR cells was increased in the parenchyma of the hippocampus in all rats (n = 5; Fig. 3C, Table 3) compared with controls. The cellular staining changed and clearly resembled the morphology and the pattern of reactive microglial cells (Fig. 3D shows an OX-42 immunostaining, specific for microglial cells). Indeed, double-label immunostaining showed that MVP was present in cells that also were IR for OX-42 and ED1 (Fig. 5G–I). All OX-42 cells were also positive for MVP. No OX42-positive/MVP-negative cells were present. No colocalization was observed with GFAP or vimentin (data not shown).

Early chronic epileptic phase

Four to six weeks after SE, intense MVP IR could still be observed in individual cells at specific and various locations of the entorhinal cortex (mainly layers II/III), hilus, CA3 (n = 5, Table 3), and CA1 (n = 5). Outside the hippocampal formation, the piriform cortex was the most conspicuous region with high MVP expression in cells that were colocalized with OX-42. This region was always extensively damaged after SE and therefore fewer layer II IR neurons were observed compared with controls.

Late chronic epileptic phase

To relate protein expression to seizure frequency in chronic epileptic rats, we investigated MVP staining in rats with frequent daily seizures (p-SE rats) and rats with occasional seizures (np-SE rats). Furthermore, we studied expression in non-SE rats. Within the hippocampus, MVP IR was still increased (mainly CA3) compared with controls, in three of seven p-SE rats and only in one of five np-SE rat. In these rats, the damage in that specific region was more extensive than that in the other chronic epileptic rats. Other regions were similar to those in controls (e.g., DG; Fig. 3E, Table 3). In the piriform cortex, the number of MVP IR neurons in layer II had decreased because of extensive cell loss in both np-SE and p-SE rats (Fig. 4C and D). In contrast, we observed increased expression in deeper layers, both in p-SE and np-SE rats, although to a greater extent in the former group (arrows in Fig. 4C and D). Double labeling showed that MVP was expressed in microglial cells that were OX-42 positive (Fig. 5G-I). Microglia activation in chronic epileptic rats was observed mainly in the hippocampal formation and piriform cortex. In p-SE rats, increased MVP IR was present bilaterally in the piriform cortex, whereas this was unilateral in np-SE rats. MVP expression in rats that had not experienced SE (non-SE rats) was comparable to that in control rats.

Western blot analysis

Additional quantification of MVP protein in the PHC was performed by using Western blot. Figure 6 shows the optical density of MVP levels as percentage of control values; an immunoblot for each experimental condition is shown. MVP levels are low in control rats and strongly upregulated 1 week after SE (∼×3 compared with controls). MVP upregulation is still present in the chronic epileptic phase (∼×2.5).

image

Figure 6. Western blot of major vault protein (MVP) levels in the parahippocampal cortex in controls, 1 week after status epilepticus, and in chronic epileptic rats [8 months after status epilepticus (SE)]. MVP levels are expressed as percentage of control values. MVP levels are low in control rats and strongly upregulated 1 week after SE. MVP upregulation is still present in the chronic epileptic phase.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

We investigated the spatial and temporal expression pattern of MVP during epileptogenesis in a rat model for TLE in which seizures evolve after a latent period after an electrically induced SE. The main findings of our study are (a) MVP RNA and protein levels increased within 1 day and peaked at 1 week after SE; (b) in chronic epileptic rats, MVP mRNA was still increased in PHC, especially in the piriform cortex; (c) MVP mRNA upregulation was related to the occurrence of SE and to the frequency of spontaneous seizures; (d) MVP protein was abundantly present in both ramified microglia (OX-42 positive) and ameboid microglia (ED1 positive) throughout the hippocampal formation early after SE. In chronic epileptic rats, MVP expression decreased but was still present in microglial cells within the piriform cortex.

In the rat model, we found upregulation of MVP mRNA shortly after SE, and this persisted in epileptic rats. The functional role of overexpression of vaults is still speculative, but a role in drug resistance has been proposed. Several studies showed that MVP upregulation is an excellent marker and predictor of resistance in both cancer cell lines and a broad variety of clinical tumors (13,14,23–25). Moreover, it has been shown that cultured human leukemia cells that upregulate the MVP mRNA level 1.5-fold within one cell passage can accumulate vault particles and become multidrug resistant by passage 25 and thereafter, independent of contributions from other drug transporters like MDR1, MRP1, MRP2, or BCRP (15). Treatment of a carcinoma cell line with sodium butyrate induced vault expression and resulted in cells resistant to various cytostatic drugs. Expression of MVP-specific ribozymes, which prevent MVP synthesis, led to the reversal of this drug resistance (11,26). Nevertheless, the way vaults contribute to pharmacoresistance is not completely clear, because a direct involvement in the MVP mouse-knockout model could not be demonstrated (27). Likewise, transfection of MVP in a carcinoma cell line leads to increased numbers of vault particles but fails to confer drug resistance to various cytostatic drugs (25). Thus from these studies, it was concluded that upregulation of vaults can contribute but is not sufficient for multidrug resistance. However, whether MVP also acts as carrier of AEDs remains to be established. In addition, other factors, including multidrug transporters that are carriers for AEDS, will be important. Strikingly, P-gp and mdr1b RNA follow a pattern similar to that of MVP mRNA in the PHC. In chronic epileptic rats, mdr1 increases 5.5-fold in this region (8).

Because MVP is highly expressed in tissues that are exposed over the long term to elevated levels of xenobiotics, metabolically active tissue, and macrophages (24), MVP also has been implicated in the protection of cells after stress responses (23). Sequestering of xenobiotics by vaults could protect cells against toxic concentrations of those substances. Our results show that upregulation of MVP was related to the occurrence of SE, which leads to cell death in vulnerable brain regions (28). The increased MVP expression in the PHC during the chronic epileptic phase suggests that MVP expression is not only related to the extent of cellular death that occurs in the aftermath of the SE (28,29), but that it also is regulated by spontaneous seizure activity and accompanying cellular stress. In our previous studies (8), we also observed regulation of two different mdr1 isoforms by seizure activity.

In drug-refractory seizures, an upregulation of various multidrug transporters has been observed (18). To understand whether MVP and other drug transporters can play a role in pharmacoresistance, one should know whether upregulation also occurs in TLE patients who respond well to AED treatment. Our data suggest that MDR proteins may contribute to the development of pharmacoresistance, but other etiologic factors are certainly not excluded. One of these factors might be astrocytic loss of the tumor-suppressor gene p53. In the epileptic brain, loss of p53 was associated with cell types that expressed abnormal levels of MDR proteins (30). Loss of p53 in glial cells may protect them from apoptosis and force them into a permanently diseased state.

In the present study, we showed activation of MVP protein mainly in reactive microglial cells. These cells are abundantly present in both hippocampal and parahippocampal regions shortly after SE (31). In chronic epileptic rats with frequent seizures, overexpression of MVP-positive microglia was restricted mainly to the piriform cortex. In a previous study, we observed that in the piriform cortex of chronic epileptic rats, cells were positive for Fluoro-Jade, a degeneration marker. No staining was present in the hippocampus at this time (28). The ongoing degeneration or the presence of debris may activate glial cells in the piriform cortex, which in turn express increased levels of MDR proteins. This may explain the persistent overexpression of MVP in glial cells within the piriform cortex versus the transient increase in the hippocampus. Because the piriform cortex is considered to be highly epileptogenic (32,33), this is particularly interesting.

Because vaults might act as carriers of different drugs (11), the upregulation of MVP in microglia might change the distribution volume of drugs entering the brain (34). When drugs are sequestered by microglia, the extracellular concentration of drugs will be reduced, at least locally, which could contribute to pharmacoresistance, if strategically placed brain areas are involved. In TLE patients, MVP is overexpressed in astrocytes (30) and in specific neurons (16–18). In rats, however, we could not demonstrate upregulation of MVP in neurons after SE. Interestingly, MVP expression was present in layer II neurons of the normal piriform cortex of control rats, but many of these cells appeared not to be protected after SE by the MVP presence.

In conclusion, this study demonstrates that the expression pattern of MVP is regulated by SE and later spontaneous seizure activity. Upregulation of MVP is most evident shortly after SE in reactive microglial cells in regions that are involved in limbic epilepsy, suggesting that MVP also plays a role in cellular protection during a strong stress response. Whether MVP contributes to pharmacoresistance still must be established.

Acknowledgments

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Acknowledgment:  We thank Drs. L.H. Rome and S. Raval-Fernandes for providing the MVP antibody and MVP protein (David Geffen School of Medicine at UCLA, Department of Biological Chemistry, CHS 33-131). We also thank Prof. F.H. Lopes da Silva for his valuable comments on the manuscript. This work was supported by Stichting Epilepsie Instellingen Nederland (Lopes da Silva fellowship).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES