SEARCH

SEARCH BY CITATION

Keywords:

  • Epilepsy;
  • Pharmacoresistance;
  • Breast cancer resistance protein;
  • Multidrug resistance;
  • associated protein;
  • Astrocytes;
  • Blood;
  • brain barrier;
  • Probenecid

Abstract

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

Summary: Purpose: Overexpression of multidrug transporters may play a role in the development of pharmacoresistance by decreasing extracellular drug levels in the brain. However, it is not known whether overexpression is due to an initial insult or evolves more gradually because of recurrent spontaneous seizures. In the present study, we investigated the expression of different multidrug transporters during epileptogenesis in the rat. In addition, we determined whether these transporters affected phenytoin (PHT) distribution in the brain.

Methods: Expression of multidrug resistance–associated proteins MRP1 and MRP2 and breast cancer–resistance protein (BCRP) was examined after electrically induced status epilepticus (SE) by immunocytochemistry and Western blot analysis. Brain/blood PHT levels were determined by high-performance liquid chromatography (HPLC) analysis in the presence and absence of the MRP inhibitor probenecid.

Results: Shortly after SE, MRP1, MRP2, and BCRP were upregulated in astrocytes within several limbic structures, including hippocampus. In chronic epileptic rats, these proteins were overexpressed in the parahippocampal cortex, specifically in blood vessels and astrocytes surrounding these vessels. Overexpression was related to the occurrence of SE and was present mainly in rats with a high seizure frequency. Brain PHT levels were significantly lower in epileptic rats compared with control rats, but pharmacologic inhibition of MRPs increased the PHT levels.

Conclusions: Overexpression of MRP and BCRP was induced by SE as well as recurrent seizures. Moreover, overexpression was associated with lower PHT levels in the brain, which was reversed through inhibition of MRPs. These data suggest that administration of antiepileptic drugs in combination with specific inhibitors for multidrug transporters may be a promising therapeutic strategy in pharmacoresistant patients.

Poor seizure control by antiepileptic drug (AED) treatment (pharmacoresistance) is a critical problem in human epilepsy. One postulated mechanism of pharmacoresistance includes involvement of drug efflux transporters of the adenosine triphosphate (ATP)-binding cassette (ABC) family. The drug transporters P-glycoprotein (P-gp or ABCB1), multidrug resistance–associated proteins MRP1 (ABCC1) and MRP2 (ABCC2), and breast cancer–resistance protein (BCRP, or ABCG2) are present in brain endothelial cells, mainly at the luminal side of blood vessels (1–5). Multidrug transporters might have a toxicologic protective role because of their luminal localization in endothelial cells (1–5). Studies using knockout mice or mutant rats demonstrate that drug transporters can prevent the entry of drugs into the brain and play a role in drug transport from the brain to the blood (1). MRP1 knockout mice have increased sensitivity for various anticancer drugs (6–8), whereas MRP2-deficient rats have higher brain levels of phenytoin (PHT), compared with wild-type rats after kindling (9). BCRP has been shown to be functional at the mouse blood–brain barrier, limiting the permeability of the brain to drugs (10). MRPs transport carbamazepine (11) and valproate (12). BCRP has some substrates in common with P-glycoprotein, although it is not known whether it transports AEDs (5,13,14). Overexpression of P-gp, MRP1, and MRP2 has been reported in human epileptogenic brain tissue (15–20) as well as in experimental models (9,21–23). Elevated expression of BCRP has been detected in a variety of human tumors (24,25) and drug-resistant tumor cell lines (26,27). Thus the overexpression of drug transporters, together with their luminal localization and the ability to transport a variety of AEDs, might be responsible for an increased efflux of drugs from the brain to the blood, leading to lower extracellular concentrations of AEDs and ineffective treatment.

To understand whether increased multidrug transporter expression is due to an initial insult or to the subsequent epileptogenesis, we examined the cellular distribution and expression of MRP1, MRP2, and BCRP in relation to the progression of temporal lobe epilepsy that evolves after electrically evoked status epilepticus (SE) in the rat. The expression of MRP1, MRP2, and BCRP was investigated at different phases during epileptogenesis: in the acute, latent, and chronic epileptic phase. In addition, we determined whether changes in protein expression and inhibition of MRPs affected the distribution of the AED PHT in chronic epileptic rats.

METHODS

  1. Top of page
  2. Abstract
  3. 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 am to 8:00 pm; food and water available ad libitum).

Electrode implantation

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 0.8 mm apart) were implanted into the left dentate gyrus under electrophysiological control, as previously described (28). A pair of stimulation electrodes was implanted in the angular bundle.

Status epilepticus induction

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, NJ 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 continuously monitored during electrical stimulation and for several hours thereafter. Immediately after termination of the stimulation, periodic epileptiform discharges (PEDs) occurred at a frequency of 1–2 Hz in most rats and were accompanied by behavioral generalized seizures and EEG seizures [status epilepticus (SE)]. Rats were injected intraperitoneally with pentobarbital (PTB; 60 mg/kg; Nembutal; Sanofi Santé, Maassluis, The Netherlands) 4 h after termination of the tetanic stimuli to avoid lethal SE. After injection, PEDs disappeared quickly but often reappeared later during the night. The total PEDs duration was considered the total SE duration. Electrical stimulation did not lead to SE in some rats (non-SE rats), although they experienced generalized seizures during stimulation. Sham-operated control rats (n = 15) were handled and recorded identically, but did not receive electrical stimulation.

Video-EEG monitoring

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 rats were monitored continuously from the SE induction onward until the time of death (≤9 months after SE induction) to determine whether, when, and how frequently spontaneous seizures occurred. EEG recordings were visually monitored and screened for seizure activity on a daily basis until death. Differential EEG recordings assured that EEG artifacts are minimized, so that seizures can be easily determined [see (29)]. EEG seizures also were validated by combined video-EEG monitoring in the chronic epileptic phase. The time span between the last spontaneous seizure and death ranged from a few days in rats with infrequent seizures to a few hours in rats with frequent seizures.

Tissue preparation for immunohistochemistry

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% Na2S 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 by using a sliding microtome. Sections were collected in 0.1 M phosphate buffer and processed for immunocytochemistry.

Immunocytochemistry

Rats were killed for immunocytochemistry in the acute period (1 day after SE; n = 3), latent period (1 week after SE; n = 5), or in the chronic period (4–9 months after SE; nonprogressive, n = 5; progressive, n = 5; non-SE, n = 5). Horizontal sections of control (n = 5) and post-SE rats were stained with different immunocytochemical markers. 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 monoclonal mouse anti-MRP1 (1:25, clone QCRL-2, IgG2b subtype; Kamiya Biomedical Company, Seattle, WA, U.S.A.), anti-MRP2 (1:25, m2III-6, IgG2a subtype), or anti-BCRP (1:50, bxp-21, IgG2a subtype) in PBS + 0.4% BSA. MRP2 and BCRP antibodies were a generous gift of Dr. Scheffer. Specifications of these antibodies were published previously (24,30). 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-mouse Ig (Amersham Pharmacia Biotech, Roosendaal, The Netherlands), diluted 1:200 in PBS. Sections were washed in PBS (3 × 10 min) and incubated for 60 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, Zwijndrecht, The Netherlands) and 2.5 μl 30% hydrogen peroxide in a 10-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 gelatin-coated slides, the sections were air-dried, dehydrated in alcohol and xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany). Omission of primary or secondary antisera and incubation with a specific serum eliminated all specific immunoreactivity. The intensity and the number of MRP1-, MRP2-, and BCRP-immunoreactive cells was estimated semiquantitatively. The intensity of immunoreactivity was classified as (+), weak; +, moderate; or ++, strong. The frequency of immunoreactive cells was classified as 1, sparse; 2, moderate; 3, high; or 4, very high. Sections were photographed by using bright-field illumination on an Olympus microscope, equipped with a digital camera (DP11; Olympus, Paes Nederland, Zoeterwoude, The Netherlands), 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

To detect whether MRP1, MRP2, and BCRP were expressed in astrocytes, a double labeling was performed with the astrocytic marker anti–glial fibrillary acidic protein (rabbit anti-GFAP, 1:1,000; DAKO, Glostrup, Denmark). To show labeling of blood vessels, an endothelial cell marker, anti–von Willebrand factor, was used (rabbit anti-VWF, 1:100; DAKO). A subset of free-floating sections was incubated with primary antibodies, as described earlier, followed by washing. Hereafter, the sections were incubated with Alexa Fluor 568 [goat anti-rabbit immunoglobulin (Ig)G, 1:200, Molecular Probes] and Alexa Fluor 488 (goat anti-mouse IgG Alexa, 1:200, Molecular Probes). 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 Zeiss software (Zeiss LSM Image browser) and Adobe Photoshop.

Western blot

To quantify MRP1, MRP2, and BCRP protein levels, Western blots were made by using tissue of the parahippocampal cortex. Rats were killed in the latent period (1 week after SE; n = 5), or in the chronic period (6–8 months after SE; progressive, n = 5). Five controls were also included. The parahippocampal cortex (which included the entorhinal cortex and parts of the perirhinal and posterior piriform cortex) was removed by incision at the ventrocaudal part underneath the rhinal fissure until ∼5 mm posterior to bregma, and stored at –80°C until use. Brain 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 EDTA, pH 8.0; 400 μl protease inhibitors; 200 μl 0.5 M NaF; and 11 ml H2O. Protein content was determined by using bicinchoninic acid method (31). Homogenate was diluted to a concentration of 6.7 μg protein/μl in sodium dodecylsulfate (SDS)/bromophenol blue loading buffer, and incubated for 5 min at 100°C. Proteins (100 μg total protein per lane) were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by semi-dry electroblotting (Transblot SD; Bio-Rad, Hercules, CA, U.S.A.). 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 primary antibodies (MRP1 and MRP2, 1:200; BCRP, 1:500; ß-actin, 1:10,000) in TBS-T + 5% nonfat milk. After overnight incubation, blots were washed in TBS-T (3 × 10 min) and incubated for 1 h in secondary antibody labeled with horseradish peroxidase (1:1,000, Dako). After washing, enhanced chemiluminescence reagents were applied to the blots (ECL, Amersham Pharmacia Biotech, Roosendaal, the Netherlands), which then were exposed to 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 (Scion Corporation, release beta 3b, Frederick, MD, U.S.A.) software. For each sample, the background was subtracted, and optical density values were normalized with the amount of ß-actin in each sample. Statistical analysis between groups was performed by using analysis of variance (ANOVA), followed by the one-tailed Student's t test. Differences with p < 0.05 were considered significant.

Drugs

Probenecid (Sigma-Aldrich, Zwijndrecht, The Netherlands), a MRP inhibitor, was dissolved in distilled water alkalinized with NaOH and administered 15 min before PHT at a dose that does not exert a significant effect on seizure threshold (50 mg/kg, i.p.; 3 ml/kg) (9). PHT sodium (5,5-diphenylhydantoin sodium salt; Katwijk Chemie, Katwijk, The Netherlands) was dissolved in distilled water alkalinized with NaOH (50 mg/kg, i.p.; 3 ml/kg).

Phenytoin analysis

To determine PHT brain-to-plasma ratios, control (n = 5) and chronic epileptic rats (PHT treatment, n = 5; PHT+probenecid treatment, n = 4) were decapitated 1 h after PHT injection. Blood was collected from the trunk and centrifuged at 3,000 rpm for 10 min to obtain plasma. The brains were removed, and the parahippocampal cortex was dissected as described earlier.

Phenytoin detection in plasma

Plasma (50 μl) was diluted with 50 μl blank plasma and 400 μl of 2.5 μg/ml ethyl-tolylbarbituric acid (ETB, internal standard) in 40 mM phosphate buffer (pH 2.1). Of this mixture, 200 μl was added on SPE extraction columns (Bakerbond C18, 50 mg), and then columns were washed with 1 ml of 10 mM K2HPO4 and 0.5 ml of 10 mM NaH2PO4. Elution was done with 100 μl acetonitrile; the eluate was diluted with 1,200 μl of 10 mM Na H2PO4, and 100 μl of this mixture was injected into the chromatograph. The HPLC system consisted of a P1000 solvent-delivery system (Thermo Electron), an ASPEC XL automatic sample injector (Gilson), and a FOCUS scanning UV detector (Thermo Electron) at a fixed wavelength of 205 nm. Data processing was performed with Spectrasystem PC1000 software (Thermo Separation Products). Separation was performed on a homemade reversed phase analytic column (15 × 0.46 cm) packed with Spherisorb 3ODS2 (Waters) kept at room temperature. The mobile phase consisted of a mixture of acetonitrile (16 vol%), methanol (23 vol%), and 10 mM Na H2PO4, pH 4.5 (61%), and was delivered isocratically at a flow rate of 1.2 ml/min. ETB and PHT retention times were 15.7 and 18.5 min, respectively. The extraction recoveries were >95%. Within-day precision for PHT was 2.5% for a 7.44-μg/ml control sample (n = 10) Limit of detection was 0.38 μg/ml (VC, <10%), and the assay was linear in the range to 48 μg/ml.

Phenytoin detection in brain

Brain samples were homogenized in methanol/water (60/40 vol/vol;10 mg tissue/100 μl) and centrifuged for 6 min at 3,000 rpm; 50 μl of the supernatant was diluted with 100 μl of an internal standard (1.4 μg/ml ETB in water), and 75 μl of the mixture was injected into the chromatograph. The HPLC system consisted of a P1000 solvent delivery system (Thermo Electron), a MIDAS automatic sample injector (Spark), and an UV 6000LP diode-array detector (Thermo Electron) at a wavelength of 205 nm. Data processing was performed with Spectrasystem PC1000 software (Thermo Separation Products). Separation was performed on a homemade reversed-phase analytic column (15 × 0.46 cm) packed with Alltima 3C18 (Alltech/Applied Science, Breda, the Netherlands) kept at a constant temperature of 35°C. The mobile phase consisted of a mixture of acetonitrile (14 vol%), methanol (29 vol%), and 40 mM phosphate buffer pH 7.0 (57%) and was delivered isocratically at a flow rate of 1.0 ml/min. ETB and PHT retention times were 11.2 and 13.8 min, respectively. The extraction recoveries were >95%. Within-day precision for PHT was 3.1%, and the assay was linear in the range to ≥25 μg/ml.

Statistical analysis between groups was performed by using ANOVA, followed by a post hoc Student's t test. Differences with p < 0.05 were considered significant.

RESULTS

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

Spontaneous seizure activity in chronic epileptic rats

After induction of SE, EEG recordings were analyzed to identify the evolution of spontaneous seizures. As described previously (28), chronic epileptic rats exhibited either a low frequency of spontaneous seizures (approximately one seizure every 5 days) without progression of seizure activity (nonprogressive rats; n = 5), or had frequent seizures (∼10 seizures/day during the week before death) and initial seizure progression, during the first 9–10 weeks after the first recorded spontaneous seizure (progressive rats; n = 10). Rats that were stimulated but that did not exhibit SE (non-SE rats) had occasional spontaneous seizures (less than one per month).

Immunocytochemistry

Control rats

Weak MRP1, MRP2, and BCRP expression was detected, mainly in large-diameter blood vessels that were stained throughout the whole brain (Tables 1–3). A colocalization study showed that MRP1, MRP2, and BCRP were expressed in endothelial cells, which were von Willebrand factor positive (Fig. 4A, D, and G). No expression was found in GFAP-positive cells (Fig. 4B, E, and H).

Table 1. Changes of MRP1 immunoreactivity in the (para)hippocampal cortex during epileptogenesis (acute, latent, and chronic periods)
 ControlAcuteLatentChronic nonprogChronic progressiveChronic non-SE
  1. Immunoreactivity was classified as (+), weak; +, moderate; ++, strong.

  2. Frequency was classified as 1, sparse; 2, moderate; 3, high; 4, very high.

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

DG(+)2++3–4++3–4(+)2(+)2(+)2
CA3(+)2++3–4++3–4(+)2(+)2(+)2
CA1(+)2++3–4++3–4(+)2(+)2(+)2
Sub(+)2+2–3+2–3(+)2(+)2(+)2
PrS(+)2(+)2(+)2(+)2(+)2(+)2
PaS(+)2++3–4++3(+)2(+)2(+)2
EC II/III(+)2++3–4++3–4+2–3+3(+)2
EC V/VI(+)2+2+2(+)2(+)2(+)2
PR II/III(+)2++3–4++3(+)2++4(+)2
PR V/VI(+)2++3–4++3(+)2++4(+)2
PC II/III(+)2+2+3++3++4(+)2
PC V/VI(+)2+2+3(+)2(+)2(+)2
Table 2. Changes of MRP2 immunoreactivity in the (para)hippocampal cortex during epileptogenesis (acute, latent, and chronic periods)
 ControlAcuteLatentChronic nonprogChronic progressiveChronic non-SE
  1. Immunoreactivity was classified as (+), weak; +, moderate; ++, strong.

  2. Frequency was classified as 1, sparse; 2, moderate; 3, high; 4, very high.

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

DG(+)2++3–4++3–4(+)2(+)2(+)2
CA3(+)2++3–4++3–4(+)2(+)2(+)2
CA1(+)2++3–4++3–4(+)2(+)2(+)2
Sub(+)2+2–3+2–3(+)2(+)2(+)2
PrS(+)2(+)2(+)2(+)2(+)2(+)2
PaS(+)2++3++3(+)2(+)2(+)2
EC II/III(+)2++3++3+2–3+3(+)2
EC V/VI(+)2+2+2(+)2(+)2(+)2
PR II/III(+)2++3–4++3(+)2++4(+)2
PR V/VI(+)2++3–4++3(+)2++4(+)2
PC II/III(+)2+2+3++3++4(+)2
PC V/VI(+)2+2+3(+)2(+)2(+)2
Table 3. Changes of BCRP immunoreactivity in the (para)hippocampal cortex during epileptogenesis (acute, latent, and chronic periods)
 ControlAcuteLatentChronic nonprogChronic progressiveChronic non-SE
  1. Immunoreactivity was classified as (+), weak; +, moderate; ++, strong.

  2. Frequency was classified as 1, sparse; 2, moderate; 3, high; 4, very high.

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

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

Figure 4. Confocal images of MRP1 (A–C), MRP2 (D–F), and BCRP (G–I), showing the presence of drug transporters in blood vessels (green), in which they colocalize with von Willebrand factor–positive endothelial cells (red; A, D, G). In controls, MRP1, MRP2, and BCRP (green) were not present in glial fibrillary acidic protein (GFAP)-positive astrocytes (red; B, E, H), whereas in chronic epileptic rats, overexpression of these transporters was observed in astrocytes (C, F, I) surrounding blood vessels. Scale bar, 20 μm.

Download figure to PowerPoint

Acute and latent period

Increased expression of MRP1, MRP2, and BCRP was observed in GFAP-positive astrocytes in different regions of the (para)hippocampal cortex during the acute and latent periods (1 day and 1 week after SE). Overexpression was evident in the dentate gyrus, particularly in astrocytes within the hilar region (Figs. 1B and F, 2B and F, 3B and F), but also in CA3, CA1, and subiculum (Tables 1–3). These astrocytes were often located around blood vessels that also had increased immunoreactivity (Figs. 1F and 2F). In parahippocampal areas, increased glial immunoreactivity was restricted to entorhinal cortex layers II and III. In addition, the piriform and perirhinal cortex had increased glial immunoreactivity. In contrast, no or just a few immunoreactive glial cells were observed in the presubiculum and deep layers of the entorhinal cortex (Tables 1–3).

image

Figure 1. MRP1 expression in a control rat (A, E), in a rat that was killed in the latent period (B, F) and in chronic epileptic rats; rat with progressive seizure evolution (C, G), nonprogressive rat (D, H). In control rats, no detectable IR was observed in glia or neurons of the hippocampus (A, E). In the latent period, MRP1 overexpression was observed in astrocytes within the hippocampus (arrows in B), which surrounded blood vessels (arrow in F). The inset in B shows a high-power magnification of reactive astrocytes that were present in the hilus. A detail of a hippocampal astrocyte surrounding a blood vessel is shown in F. In chronic epileptic rats, a persistent upregulation was present in astrocytes within the perirhinal cortex (arrows in C). This was evident in rats with a progressive type of epilepsy (C) but not in nonprogressive rats (D). Overexpression was found mainly in astrocytes that surrounded blood vessels. A detail of an astrocyte surrounding a blood vessel in a progressive rat is shown in G. MRP expression in the hippocampi of both progressive and nonprogressive rats (H) was similar to that in control rats. gcl, granule cell layer; Scale bar A, B, 300 μm; E, G, 100 μm; C, D, 1,200 μm; F, 20 μm; H, 1,400 μm.

Download figure to PowerPoint

image

Figure 2. MRP2 expression in a control rat (A, E), in a rat killed in the latent period (B, F), and in chronic epileptic rats; progressive rat (C, G), nonprogressive rat (D, H). In control rats, no detectable IR was observed in glia or neurons of the hippocampus (A, E). During the latent period, MRP2 was overexpressed in astrocytes within the hippocampus, mainly in the dentate gyrus (B) and CA3 (F). The inset in B shows a high-power magnification of reactive astrocytes that were present in the hilus. A detail of the CA3 region with many reactive astrocytes is shown in F. Some of them were localized next to blood vessels (arrowhead in F). In chronic epileptic rats, a persistent upregulation of MRP2 was present in astrocytes within the perirhinal cortex of progressive rats, especially around blood vessels (arrow in G). In the piriform cortex, (C) bilateral MRP2 overexpression was observed mainly in cortex layer III. Nonprogressive rats also had increased IR of MRP2 in the piriform cortex (D), but only at the stimulated side of the brain. The hippocampus of both progressive and nonprogressive rats (H) had similar MRP2 expression as controls. gcl, Granule cell layer, piri, piriform cortex. Scale bar A, B, 300 μm; E, G, 100 μm; C, D, 1,200 μm; F, 20 μm; H, 1,400 μm.

Download figure to PowerPoint

image

Figure 3. BCRP expression in a control rat (A, E), in a rat killed in the latent period (B, F) and in chronic epileptic rats; progressive rat (C, G), and nonprogressive rat (D, H). In control rats, no detectable immunoreactivity was observed in glia or neurons of the hippocampus (A, E). In the latent period, increased BCRP expression was present in astrocytes within the dentate gyrus (arrows in B and F; a higher-power magnification of the dentate gyrus is shown in F). Some of the reactive astrocytes were localized next to blood vessels (arrowhead in F). In chronic epileptic rats, a persistent BCRP upregulation was present in astrocytes, especially around blood vessels within the perirhinal cortex of rat with progressive seizure evolution (G). In the piriform cortex (C), BCRP overexpression was observed mainly in cortex layer III. In the piriform cortex of nonprogressive rats, an upregulation of BCRP was observed only at the stimulated side of the brain (D). In contrast, no overexpression was observed in the hippocampus of both progressive and nonprogressive rats (H). gcl, Granule cell layer; piri, piriform cortex. Scale bar A, B, 300 μm; E, G, 100 μm; C, D, 1,200 μm; F, 20 μm; H, 1,400 μm.

Download figure to PowerPoint

Chronic period

MRP1, MRP2, and BCRP were each persistently increased to a similar extent in specific brain regions of chronic epileptic rats (≤9 months after SE). The overexpression was associated with the progressive nature of epilepsy, because most changes were not found in brains of rats without progressive seizure evolution. Increased expression was observed bilaterally in GFAP-positive astrocytes (Fig. 4C, F, and I) within the piriform cortex (Figs. 2C and 3C), perirhinal cortex (Fig. 1C), and entorhinal cortex (Tables 1–3). In these regions, immunoreactive astrocytes surrounded blood vessels, which were also immunoreactive (Figs. 1G, 2G, and 3G). MRP1, MRP2, as well as BCRP colocalized with the von Willebrand factor in these vessels.

Less drastic changes were observed in nonprogressive rats: in most brain regions, the staining pattern was comparable to that in control rats (Figs. 1D, 1H, 2H, 3H, and Tables 1–3). However, an increased immunoreactivity was observed in superficial layers of the piriform cortex (2D and 3D) and entorhinal cortex, but only at the stimulated side of the brain.

We did not observe increased staining in the hippocampus of chronic epileptic rats, either progressive or nonprogressive rats (Figs. 1H, 2H, 3H, and Tables 1–3).

Rats that did not exhibit SE during or after stimulation (non-SE rats) were comparable to control rats (Tables 1–3).

Western blot

Western blots for MRP1, MRP2, and BCRP were performed by using tissue of the parahippocampal cortex (which includes the entorhinal cortex and parts of the perirhinal and posterior piriform cortex). Figure 5 shows the optical density of protein levels as a percentage of control values; a representative immunoblot for each experimental condition also is shown. MRP1, MRP2, and BCRP levels were low in control rats and significantly increased in the latent period (MRP1, ×2.7; MRP2, ×1.7; BCRP, ×1.6 increase compared with controls). Increased expression was still present in the chronic epileptic phase (MRP1, ×2.1; MRP2, ×1.5; BCRP, ×1.4 increase compared with controls). BCRP was most abundant in both control and epileptic rats, followed by MRP1 and MRP2, which were nearly equally abundant.

image

Figure 5. Western blot of MRP1 (A), MRP2 (B), and BCRP (C) protein levels in the parahippocampal cortex of control rats (n = 5), rats killed in the latent period (n = 5), and chronic epileptic rats (killed 6–8 months after SE; n = 5). Protein levels are expressed as percentage of control values (average ± SEM). MRP1, MRP2, and BCRP levels were low in control rats and significantly increased in the latent period (MRP1, ×2.7; MRP2, ×1.7; BCRP, ×1.6 increase compared with controls). Significantly increased expression was still present in the chronic epileptic phase (MRP1, ×2.1; MRP2, ×1.5; BCRP, ×1.4 increase compared with controls). D: Phenytoin brain-to-plasma ratio (average ± SEM) in the parahippocampal cortex of control rats and chronic epileptic rats treated with either phenytoin (PHT) or phenytoin and probenecid (PHT+PB). The brain-to-plasma ratio in chronic epileptic rats was significantly decreased compared with control rats (20 ± 6%; p < 0.05). When the MRP inhibitor probenecid was coadministered with PHT, the PHT brain-to-plasma ratio significantly increased (20 ± 7%; p < 0.05), compared with rats that were treated with PHT only, and was similar to control values. *Significant difference compared with control values (Student's t test, p < 0.05).

Download figure to PowerPoint

Phenytoin levels in chronic epileptic rats

To determine whether overexpression of multidrug transporters can influence penetration of AEDs into the brain (parahippocampal cortex), PHT was i.p. administered to chronic epileptic rats and control rats. One hour after injection, the brain-to-plasma ratio of PHT was measured (Fig. 5D). The brain-to-plasma ratio was significantly decreased in chronic epileptic rats compared with control rats (Fig. 5D; 20 ± 6%; p < 0.05). When the MRP inhibitor probenecid was coadministered, the PHT brain-to-plasma ratio significantly increased (20 ± 7%; p < 0.05) compared with rats that were treated with PHT only. The levels were comparable to PHT levels in control rats (Fig. 5D).

DISCUSSION

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

Investigation of multidrug transporter expression MRP1, MRP2, and BCRP during epileptogenesis indicated the following:

  • 1
    MRP1, MRP2, and BCRP protein were upregulated in astrocytes and blood vessels during epileptogenesis within several limbic structures, including hippocampus.
  • 2
    In chronic epileptic rats, MRP1, MRP2, and BCRP protein expression was restricted to the parahippocampal cortex (including piriform, perirhinal, and entorhinal cortex) and specifically increased in astrocytes that were in close apposition to strongly stained blood vessels.
  • 3
    In chronic epileptic rats, upregulation of MRP1, MRP2, and BCRP protein was present only in rats that experienced SE and was most evident in rats that had developed a progressive form of epilepsy.
  • 4
    The phenytoin brain-to-plasma ratio decreased significantly in chronic epileptic rats compared with control rats. Administration of the MRP inhibitor probenecid reversed the PHT brain-to-plasma ratio to control levels.

In our study, overexpression of MRP1, MRP2, and BCRP protein was evident in both astrocytes and blood vessels within 1 day after SE. This was associated with the occurrence of SE, because rats that did not exhibit SE had expression levels comparable with those in controls. Similarly, in epileptic human brain, MRP1 and MRP2 are overexpressed in blood vessels and glial cells shortly after SE (32), whereas in normal human brain, MRP1, MRP2 (2,15–18), and BCRP (2,25,33,34) are exclusively expressed in blood vessels. The fact that MRPs are upregulated shortly after SE, when seizure activity provokes brain inflammation, cell death, and gliosis (35,36), suggests that induction of these proteins is at least partly related to cellular stress that occurs during SE and shortly thereafter. MRPs as well as BCRP transporter proteins were primarily upregulated in reactive glial cells, which also suggests that the increased MDR production is related to the inflammatory response (e.g., production of cytokines and inflammatory proteins) that occurs after SE (36,37). A protective role of MRPs has been proposed in oxidative stress, because enhanced levels of oxidized glutathione can be controlled by MRPs (38). Moreover, MRPs might play a role in the control of inflammatory responses, because MRP knockout mice have an impaired response to inflammatory stimuli (7,39–42).

Overexpression of MRP1, MRP2, and BCRP did not only appear to depend on the extent of cellular death that occurs after SE (35,43). Spontaneous seizure activity also was associated with a persistent increased expression level, (although more restricted, compared with the acute and latent period) in astrocytes and blood vessels within the parahippocampus of chronic epileptic rats, possibly through seizure-induced cellular stress. Similar to our studies in which we characterized P-gp expression (mdr1a/b) in epileptic rats (21,44), this was evident in rats with seizure progression and not in rats that had only occasional seizures. Clinical data also show overexpression of P-gp, MRP1, and MRP2 in astrocytes and blood vessels of pharmacoresistant patients that had had epilepsy for years (15–20,45,46). In contrast to hippocampal expression in human, MRP1, MRP2, and BCRP in epileptic rats was more restricted to the parahippocampal region. BCRP was not found to be overexpressed in the human hippocampus of MTLE patients (46,47).

In the present study, we show that an inhibitor of MRPs can increase the PHT brain-to-plasma ratio in chronic epileptic rats, indicating that MRPs can alter drug distribution in the brain. Increased drug penetration into the brain has been reported in MRP2-deficient rats and kindled rats after treatment with the MRP inhibitor probenecid, together with PHT (9) or carbamazepine (11). Because no specific inhibitors are available for BCRP, we could not provide functional data concerning this transporter. Evidence for a functional role for BCRP as drug transporter was presented previously in a study in which BCRP limits the drug permeability into the mouse brain (10). Because BCRP is more abundant than MRP1, MRP2, or P-gp (2,46), it is not unlikely that BCRP upregulation in a specific brain region can have effects on drug distribution in the brain. This will be further investigated when more specific BCRP inhibitors are available.

In conclusion, this study demonstrates that the expression pattern of MRP1, MRP2, and BCRP protein in chronic epileptic rats is associated with the occurrence of SE, as well as spontaneous seizure activity, and is most evident in rats with frequent daily seizures. PHT brain-to-plasma ratio is significantly decreased in chronic epileptic rats compared with control rats, indicating compromised drug access in the epileptic brain. Because inhibition of MRPs can reverse these effects on drug access, MRP inhibitors might be promising drugs in the pharmacologic treatment of refractory epilepsy. Unraveling the role of multidrug transporters and other related proteins in the development of pharmacoresistance may ultimately lead to a better therapy for refractory epilepsy. This may include either application of AEDs together with specific multidrug-transporter inhibitors, or alternatively, the development of AEDs that are not substrates for these transporters.

Acknowledgments

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

Acknowledgment:  We are thankful to Dr. G.L. Scheffer for providing MRP2 and BCRP antibodies (Department of Pathology, Free University Hospital, Amsterdam, The Netherlands) and A. Tierlier for editorial assistance (Epilepsy Institute of the Netherlands, Heemstede, The Netherlands). We also thank M.C. de Rijke and W.E. Dieters (Epilepsy Institute of the Netherlands, Heemstede, The Netherlands) for technical assistance on phenytoin analysis in plasma and brain samples.

This work was supported by the Epilepsy Institute of the Netherlands (SEIN-Lopes da Silva fellowship).

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES
  • 1
    Miller DS, Nobmann SN, Gutmann H, et al. Xenobiotic transport across isolated brain microvessels studied by confocal microscopy. Mol Pharmacol 2000;58: 135767.
  • 2
    Cooray HC, Blackmore CG, Maskell L, et al. Localisation of breast cancer resistance protein in microvessel endothelium of human brain. Neuroreport 2002;13: 205963.
  • 3
    Zhang Y, Schuetz JD, Elmquist WF, et al. Plasma membrane localization of multidrug resistance-associated protein (MRP) homologues in brain capillary endothelial cells. J Pharmacol Exp Ther 2004;311: 44955.
  • 4
    Huai-Yun H, Secrest DT, Mark KS, et al. Expression of multidrug resistance-associated protein (MRP) in brain microvessel endothelial cells. Biochem Biophys Res Commun 1998;243: 81620.
  • 5
    Allen JD, Schinkel AH. Multidrug resistance and pharmacological protection mediated by the breast cancer resistance protein (BCRP/ABCG2). Mol Cancer Ther 2002;1: 42734.
  • 6
    Allen JD, Brinkhuis RF, Van Deemter L, et al. Extensive contribution of the multidrug transporters P-glycoprotein and Mrp1 to basal drug resistance. Cancer Res 2000;60: 57616.
  • 7
    Wijnholds J, Evers R, Van Leusden MR, et al. Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein. Nat Med 1997;3: 12759.
  • 8
    Lorico A, Rappa GA, Finch D, et al. Disruption of the murine MRP (multidrug resistance protein) gene leads to increased sensitivity to etoposide (VP-16) and increased levels of glutathione. Cancer Res 1997;57: 523842.
  • 9
    Potschka H, Fedrowitz M, Loscher W. Multidrug resistance protein MRP2 contributes to blood-brain barrier function and restricts antiepileptic drug activity. J Pharmacol Exp Ther 2003;306: 12431.
  • 10
    Cisternino S, Mercier C, Bourasset F, et al. Expression, up-regulation, and transport activity of the multidrug-resistance protein Abcg2 at the mouse blood-brain barrier. Cancer Res 2004;64: 3296301.
  • 11
    Potschka H, Fedrowitz M, Loscher W. P-glycoprotein and multidrug resistance-associated protein are involved in the regulation of extracellular levels of the major antiepileptic drug carbamazepine in the brain. Neuroreport 2001;12: 355760.
  • 12
    Gibbs JP, Adeyeye MC, Yang Z, et al. Valproic acid uptake by bovine brain microvessel endothelial cells: role of active efflux transport. Epilepsy Res 2004;58: 5366.
  • 13
    Litman T, Brangi M, Hudson E, et al. The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J Cell Sci 2000;113: 201121.
  • 14
    Schinkel AH, Jonker JW. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev 2003;55: 329.
  • 15
    Sisodiya SM, Heffernan JH, Squier MV. Over-expression of P-glycoprotein in malformations of cortical development. Neuroreport 1999;10: 343741.
  • 16
    Sisodiya SM, Lin WR, Squier MV, et al. Multidrug-resistance protein 1 in focal cortical dysplasia. Lancet 2001;357: 423.
  • 17
    Aronica E, Gorter JA, Jansen GH, et al. Expression and cellular distribution of multidrug transporter proteins in two major causes of medically intractable epilepsy: focal cortical dysplasia and glioneuronal tumors. Neuroscience 2003;118: 41729.
  • 18
    Aronica E, Gorter JA, Ramkema M, et al. Expression and cellular distribution of multidrug resistance-related proteins in the hippocampus of patients with mesial temporal lobe epilepsy. Epilepsia 2004;45: 44151.
  • 19
    Tishler DM, Weinberg KI, Hinton DR, et al. MDR1 gene expression in brain of patients with medically intractable epilepsy. Epilepsia 1995;36: 16.
  • 20
    Marchi N, Hallene KL, Kight KM, et al. Significance of MDR1 and multiple drug resistance in refractory human epileptic brain. BMC Med 2004;2: 37.
  • 21
    Van Vliet EA, Aronica E, Redeker S, et al. Selective and persistent upregulation of mdr1b mRNA and P-glycoprotein in the parahippocampal cortex of chronic epileptic rats. Epilepsy Res 2004;60: 20313.
  • 22
    Volk HA, Burkhardt K, Potschka H, et al. Neuronal expression of the drug efflux transporter P-glycoprotein in the rat hippocampus after limbic seizures. Neuroscience 2004;123: 7519.
  • 23
    Volk HA, Potschka H, Loscher W. Increased expression of the multidrug transporter P-glycoprotein in limbic brain regions after amygdala-kindled seizures in rats. Epilepsy Res 2004;58: 6779.
  • 24
    Diestra JE, Scheffer GL, Catala I, et al. Frequent expression of the multi-drug resistance-associated protein BCRP/MXR/ABCP/ABCG2 in human tumours detected by the BXP-21 monoclonal antibody in paraffin-embedded material. J Pathol 2002;198: 2139.
  • 25
    Zhang W, Mojsilovic-Petrovic J, Andrade MF, et al. The expression and functional characterization of ABCG2 in brain endothelial cells and vessels. FASEB J 2003;17: 20857.
  • 26
    Scheffer GL, Maliepaard M, Pijnenborg AC, et al. Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines. Cancer Res 2000;60: 258993.
  • 27
    Han B, Zhang JT. Multidrug resistance in cancer chemotherapy and xenobiotic protection mediated by the half ATP-binding cassette transporter ABCG2. Curr Med Chem Anticancer Agents 2004;4: 3142.
  • 28
    Gorter JA, Van Vliet EA, Aronica E, et al. Progression of spontaneous seizures after status epilepticus is associated with mossy fibre sprouting and extensive bilateral loss of hilar parvalbumin and somatostatin-immunoreactive neurons. Eur J Neurosci 2001;13: 65769.
  • 29
    Gorter JA, Van Vliet EA, Proper EA, et al. Glutamate transporters alterations in the reorganizing dentate gyrus are associated with progressive seizure activity in chronic epileptic rats. J Comp Neurol 2002;442: 36577.
  • 30
    Scheffer GL, Kool M, Heijn H, et al. Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies. Cancer Res 2000;60: 526977.
  • 31
    Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150: 7685.
  • 32
    Sisodiya SM, Thom M. Widespread upregulation of drug-resistance proteins in fatal human status epilepticus. Epilepsia 2003;44: 2614.
  • 33
    Maliepaard M, Scheffer GL, Faneyte IF, et al. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res 2001;61: 345864.
  • 34
    Eisenblatter T, Huwel S, Galla HJ. Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood-brain barrier. Brain Res 2003;971: 22131.
  • 35
    Gorter JA, Goncalves Pereira PM, Van Vliet EA, et al. Neuronal cell death in a rat model for mesial temporal lobe epilepsy is induced by the initial status epilepticus and not by later repeated spontaneous seizures. Epilepsia 2003;44: 64758.
  • 36
    Aronica E, Van Vliet EA, Mayboroda O, et al. Upregulation of metabotropic glutamate receptor subtype mGluR3 and mGluR5 in reactive astrocytes in a rat model of mesial temporal lobe epilepsy. Eur J Neurosci 2000;12: 233345.
  • 37
    De Simoni MG, Perego C, Ravizza T, et al. Inflammatory cytokines and related genes are induced in the rat hippocampus by limbic status epilepticus. Eur J Neurosci 2000;12: 262333.
  • 38
    Konig J, Nies AT, Cui Y, Leier I, Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta 1999;1461: 37794.
  • 39
    Cole SP, Deeley RG. Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP. Bioessays 1998;20: 93140.
  • 40
    Ballerini P, Di Iorio P, Ciccarelli R, et al. Glial cells express multiple ATP binding cassette proteins which are involved in ATP release. Neuroreport 2002;13: 178992.
  • 41
    Renes J, De Vries EE, Hooiveld GJ, et al. Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4-hydroxynonenal. Biochem J 2000;350: 55561.
  • 42
    Leslie EM, Deeley RG, Cole SP. Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters. Toxicology 2001;167: 323.
  • 43
    Pitkanen A, Nissinen J, Nairismagi J, et al. Progression of neuronal damage after status epilepticus and during spontaneous seizures in a rat model of temporal lobe epilepsy. Prog Brain Res 2002;135: 6783.
  • 44
    Rizzi M, Caccia S, Guiso G, et al. Limbic seizures induce P-glycoprotein in rodent brain: functional implications for pharmacoresistance. J Neurosci 2002;22: 58339.
  • 45
    Dombrowski SM, Desai SY, Marroni M, et al. Overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsy. Epilepsia 2001;42: 15016.
  • 46
    Aronica E, Gorter JA, Redeker S, et al. Localization of breast cancer resistance protein (BCRP) in micro-vessel endothelium of human control and epileptic brain. Epilepsia 2005;46: 84957.
  • 47
    Sisodiya SM, Martinian L, Scheffer GL, et al. Major vault protein, a marker of drug resistance, is upregulated in refractory epilepsy. Epilepsia 2003;44: 138896.
  • 48
    Vogelgesang S, Kunert-Keil C, Cascorbi I, et al. Expression of multidrug transporters in dysembryoplastic neuroepithelial tumors causing intractable epilepsy. Clin Neuropathol 2004;23: 22331.