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Keywords:

  • Epilepsy;
  • Hippocampal sclerosis;
  • Major vault protein;
  • Multidrug resistance–associated protein;
  • P-glycoprotein

Abstract

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

Summary: Purpose: This study investigated the cellular distribution of different multidrug resistance (MDR)-related proteins such as P-glycoprotein (P-gp), the multidrug resistance–associated proteins (MRP) 1 and 2, and the major vault protein (MVP) in normal and sclerotic hippocampus of patients with medically refractory mesial temporal lobe epilepsy (MTLE).

Methods: Single- and double-label immunocytochemistry was used on brain sections of control hippocampus and of hippocampus of refractory MTLE patients.

Results: In TLE cases with hippocampal sclerosis (HS), all four MDR proteins examined that had low or no expression in control tissue were upregulated, albeit with different cellular distribution patterns. P-gp immunoreactivity (IR) was observed in astrocytes in regions with diffuse reactive gliosis. In 75% of HS cases, strong P-gp IR was detected in blood vessels, with prominent endothelial labeling. Reactive astrocytes displayed low MRP1 IR. However, glial MRP1 expression was noted in glial endfoot processes around blood vessels. Neuronal MRP1 expression was observed in hypertrophic hilar neurons and in a few residual neurons of the CA1 region. Hippocampal MRP2 expression was observed in the large majority of HS cases in blood vessels. Hypertrophic hilar neurons and blood vessels within the sclerotic hippocampus expressed major vault protein (MVP).

Conclusions: These findings indicate that MDR proteins are upregulated in concert in the hippocampus of patients with refractory MTLE, supporting their role in the mechanisms underlying drug resistance. The specific cell-distribution patterns within the sclerotic hippocampus suggest different cellular functions, not necessarily linked only to clinical drug resistance.

In human epilepsy, the failure to respond to anticonvulsant drug (AED) treatment is a crucial clinical problem, because in ∼30% of epilepsy patients, seizures persist despite appropriate polytherapy at maximal tolerated doses (1,2). The basis of this multidrug resistance is still elusive but is likely to be multifactorial. Resistance to pharmacologic treatment with a broad range of AEDs with different mechanisms of action, but common physical characteristics, supports the involvement of nonspecific mechanisms responsible for different types of clinical drug resistance (for example, as seen with drug resistance to cytostatic drugs in cancer treatment) (3,4). One possible general mechanism to account for this medical intractability is the inadequate drug concentration in the epileptogenic areas. In recent years, attention has been focused on multidrug transporters such as P-glycoprotein (P-gp) and the family of multidrug resistance–associated proteins (MRPs) (reviewed in 3,5–7). Subcellular particles called vaults, possibly involved in sequestration and compartmentalization of drugs away from the intracellular target, also may play a role in clinical drug resistance (8,9). Overexpression of multidrug resistant (MDR)-related proteins has been recently shown in several causes of human refractory epilepsy (10–15).

Hippocampal sclerosis (HS) constitutes the most frequent neuropathologic finding in adult patients with medically intractable temporal lobe epilepsy (TLE) (2). Previous studies analyzed HS together with other lesions associated with intractable epilepsy, focusing mainly on the expression of P-gp and MRP1 in a limited number of specimens (10,13). Another study analyzed the expression MDR genes and P-gp only at the level of the endothelial cells (12).

In the present histologic study, we analyzed the expression of four major MDR proteins (P-gp, MRP1, MRP2, and major vault protein) in a surgical series of hippocampal specimens from adults with temporal lobe intractable epilepsy and the classic pattern of HS (grades 3 and 4). Our major aim was to provide data that may help to define the specific cellular distribution of different MDR proteins in normal and sclerotic hippocampus and may provide better insights into the functions of these proteins, including their potential role in the MDR in patients with HS.

MATERIALS AND METHODS

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

Subjects

The TLE cases included in this study were obtained from the files of the departments of neuropathology of the Academic Medical Center (University of Amsterdam), the VU University Medical Center in Amsterdam and the Department of Medical Biology & DNA/Cell Bank (Hacettepe University, Turkey). Patients underwent resection of the hippocampus for medically intractable TLE. Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. Two neuropathologists reviewed all cases independently. In 12 cases, a pathologic diagnosis of HS (without extrahippocampal pathology) was made. Four non-HS cases in which a focal lesion [ganglioglioma (GG)], not involving the hippocampus proper, was identified also were included to provide a comparison group to HS cases. Comparison with tissue from patients without pharmacoresistant epilepsy was not possible. This tissue was not available (patients responsive to AEDs do not normally undergo surgical resection of the hippocampus). Control hippocampal tissue was obtained at autopsy from eight patients without history of seizures or other neurologic diseases. All autopsies were performed within 12 h after death. Autopsy specimens of human brain tissue from patients with nonneoplastic brain pathologies associated with reactive gliosis (three cases with multiple sclerosis and three cases of Alzheimer's disease) and no history of seizures also were included in the study to assess whether changes in glial expression of MDR proteins (such as P-gp) are a feature of astrocyte activation alone.

Table 1 summarizes the clinical features of epilepsy patients, with particular attention to the characteristics of seizures (type and frequency of seizures, age at seizure onset). In all patients, the lesion was localized by brain magnetic resonance imaging (MRI) and computed tomography (CT) scan. Video monitoring and intraoperative electrocorticography also were part of the presurgical evaluation. The predominant type of seizure pattern was that of complex partial seizures, which were resistant to maximal doses of AEDs. Pharmacoresistant epilepsy was defined by the presence of repetitive and disabling seizures, despite treatment with maximal doses of AEDs. Adequate treatment with two or more conventional AEDs such as carbamazepine (CBZ), phenytoin (PHT), and phenobarbital (PB) was attempted before presurgical evaluation in both HS and non-HS cases. The postoperative seizure outcome was classified according to Engel (16). Class I was defined as seizure free and included patients who are free of habitual preoperative seizures; class II are almost seizure free or have rare or nocturnal seizures only; class III have worthwhile improvement; and class IV, no worthwhile improvement. Follow-up period ranged from 1 to 6 years.

Table 1. Summary of clinical findings of epilepsy patients and controls
 Total or mean (Range or percentage)
TLE (n = 16)Control (n = 8)
  1. CPS, complex partial seizure; SGS, secondarily generalized seizure; SE, status epilepticus; HS, hippocampal sclerosis.

Male/female9/75/3
Mean age (yr)28 (15–55)49 (16–78)
Seizure typeCPS (100%);
  SGS (44%); SE (6%) 
CPS/mo10 (1–35)
Mean age at seizure9 (1–19)
 onset (yr)
Duration of epilepsy (yr)19 (7–36)
HS: present/absent12/4
Postoperative epilepsy:IA (75%); ID (12.5%);
 Engel class IIA (6.25%); 
  IIIA (6.25%) 

Tissue preparation

Tissue had been fixed in 10% buffered formalin (autopsy tissue, for 2 weeks; surgical specimens, for 12–24 h). Formalin-fixed, paraffin-embedded tissue was sectioned at 6 μm and mounted on organosilane-coated slides (Sigma, St. Louis, MO, U.S.A.). Tissue from three autopsy (mean age, 26.3 ± 7 years) and three HS specimens (mean age, 27.6 ± 5 years) was snap frozen in liquid nitrogen and stored at –80°C. Cryosections (5 μm thick) were cut, air-dried overnight, fixed for 10 min in 100% acetone before use. Representative sections of all specimens were processed for hematoxylin and eosin, Nissl and Bielschowsky silver, and Hoechst 33258 stains, as well as for immunocytochemical reactions, as described later.

Antibody characterization

Glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Denmark; 1:2,000), vimentin (mouse clone V9, DAKO; 1:400), synaptophysin (polyclonal rabbit, DAKO; 1:200), and neuronal nuclear protein (NeuN; mouse clone MAB377; Chemicon, Temecula, CA; 1:1,000) neurofilaments [monoclonal antibody (mAb) clone 2F11, DAKO, 1:500; mAb clone RT97 immunoglobulin G (IgG1), 1:300, Chemicon; mAb SMI-32, IgG1, 1:300, Sternberger Monoclonals, Inc., MD, U.S.A.), and caspase-3 (polyclonal; 1:100 dilution; Cell Signaling technology, Beverly, MA, U.S.A.) were used in the routine immunocytochemical analysis of TLE specimens.

For the detection of MDR proteins, well-characterized mAbs, directed against different separate MDR protein epitopes, were used. MDR1 P-gp detection was with JSB-1 and C219 mAbs. MRP1 detection was with MRPr1 and MRPm6. MRP2 detection was with M2I-4 and M2III-6. MVP detection was with LRP-56 and MVP-37. Except for the C219 (purchased from Alexis, San Diego, CA, U.S.A.; 1:100), all the other Abs were produced at the Department of Pathology (Academic Hospital Vrije Universiteit, Amsterdam) and used at the final concentration of 10 μg/ml. All mAbs are murine (JSB-1, MRPm6, and M2I-4 (IgG1); M2-IIII-6, C219 (IgG2a); MVP-37 and LRP-56 (IgG2b), except for MRPr1, which is a rat Ab (IgG2a). Extensive characterization of mAbs used was previously reported in human tissue (15,17–21).

Immunocytochemistry

Sections of routinely processed formalin-fixed, paraffin-embedded tissues were deparaffinated in xylene and, after rinses in ethanol (100% and 95%), were incubated with 1% H2O2 diluted in methanol for 20 min. Slides were then washed with phosphate-buffered saline (PBS; 10 mM, pH 7.4). The slides were placed into sodium citrate buffer (0.01 M, pH 6.0) and heated in a microwave oven (650 W for 10 min, except for GFAP). The slides were allowed to cool for 20 min in the same solution at room temperature and then washed in PBS. They were incubated with a mixture of 10% normal goat serum (NGS), for 1 h before the incubation with the primary Ab (30 min at room temperature and at 4°C overnight). Sections were then washed thoroughly with PBS and incubated at room temperature for 1 h with the appropriate biotinylated secondary Ab diluted in PBS (1:400 goat-antirabbit immunoglobulin (Ig) or 1:200 goat-antimouse or goat-antirat Ig; Dako). Single-label immunocytochemistry was carried out by using the avidin-biotin peroxidase method (Vector Laboratories, Burlingame, CA, U.S.A.) and 3,3′-diaminobenzidine as chromogen. Sections were counterstained with hematoxylin, dehydrated in alcohol and xylene, and coverslipped. Sections incubated without the primary Ab or with preimmune sera were essentially blank. As positive controls for immunocytochemical staining, paraffin-embedded human specimens of normal human cortex (the capillary endothelium is labeled by anti–P-gp mAbs), human choroid plexus epithelium (labeled by anti–P-gp and MRP1 mAbs), normal human liver (labeled by anti-MRP2 mAbs), and normal human lung tissue (for MVP) were used. Because JSB-1 may crossreact with pyruvate carboxylase, a ubiquitous mitochondrial enzyme (22), human brain tissue from patients with nonepileptic brain pathologies associated with reactive gliosis also was included. As previously reported, no detectable JSB-1 (MRP1, 2 or MVP) labeling was observed in the large majority of reactive astrocytes from autopsy specimens of patients with nonneoplastic brain pathologies associated with reactive gliosis (and no history of seizures) [data not shown (14)]. Cryosections (5 μm thick) were air-dried overnight and fixed for 10 min in acetone at room temperature before immunostaining with anti-MDR Abs. Two representative frozen sections per case (stained with each Ab) were assessed by two investigators independently. The immunoreactivity (IR) pattern observed in frozen sections was similar to that reported in paraffin-embedded material.

For double labeling, sections (after incubation with primary Abs; GFAP and JSB-1; GFAP, NeuN, or CD31, and MRPr1, M2-IIII-6, or MVP-37) were incubated for 2 days at 4°C with fluorochrome [fluorescein isothiocyanate (FITC) or Cy3]-conjugated anti-mouse or anti-rabbit Ig subclass-specific antibodies (1:200; purchased from either Southern Biotechnology Associates, Inc., Birmingham, AL, U.S.A., or Jackson ImmunoResearch, West Grove, PA, U.S.A.). To block autofluorescence due to the presence of lipofuscin pigment in the tissue, sections were stained with Sudan Black B (Merck) for 10 min, as previously described (23). Sections were then analyzed by means of a laser scanning confocal microscope (Bio-Rad, Hercules, CA, U.S.A.; MRC1024) equipped with an argon-ion laser. As both JSB-1 and CD31 mAbs are anti-mouse IgG1 type, this combination could not be directly examined with immunofluorescent procedures.

Evaluation of immunostaining

Labeled tissue sections from both types of lesions were examined by two observers with respect to the presence or absence of various histopathological parameters and specific IR for the different markers. Two representative paraffin sections per case were stained and assessed with each Ab. P-gp (JSB-1), MRP1 (MRPr1), MRP2 (M2-IIII-6), and MVP (MVP-37) immunoreactive staining were evaluated by using a 3-point scale (−, no; +, moderate; ++, strong staining; Table 2). Hypertrophic hilar neurons were detected on the basis of morphology, the hypertrophy of cell body (>50 μm), and by staining of serial sections for neurofilaments, as previously described (24–26). These hypertrophic neurons showed cytoplasmic accumulation of neurofilaments, but they did not display apoptotic changes in nuclear morphology by Hoechst-33258 or hematoxylin counterstaining, nor did they express apoptotic markers, such as caspase-3 (data not shown).

Table 2. Cell-type distribution of MDR protein immunoreactivity in TLE cases with hippocampal sclerosis
 HS (n = 12)
EndotheliumAstrocytesNeuronsa
  1. Values expressed as percentage of cases with immunoreactive cells.

  2. P-gp (JSB-1), MRP1 (Mrpr1), MRP2 (M2III-6), MVP (MVP-37) staining; –, no; +, moderate; ++, strong staining.

  3. aHypertrophic hilar neurons.

P-gp++++++
 0336702575100
MRP1++++++
 10058420502525
MRP2++++++
 8672567258100
MVP+++++++++
 33254283170502525

RESULTS

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

Case material and histologic features

A total of 16 surgical specimens from patients with TLE were examined. The clinical features of the cases included in this study are summarized in Table 1. All patients had a history of chronic pharmacoresistant epilepsy. Postoperatively, 12 (75%) TLE patients were completely seizure free (Engel IA). The classic pattern of HS was observed in 11 cases with neuronal loss predominantly in CA1 and hilar regions and with sparing of the CA2 region. In one case, severe neuronal cell loss was observed in all hippocampal subfields. Severe dispersion of the granule cells (GCs) of the dentate gyrus and cytoskeletal abnormalities in residual hilar neurons (including hypertrophy, abnormal dendritic ramification, and accumulation of neurofilaments (24–26) were observed in 50% of HS cases. GFAP and vimentin immunostaining demonstrated the presence of prominent astrogliosis in CA1 and hilar regions in all HS cases.

P-gp, MRP1, MRP2, and MVP expression in normal human hippocampus

Immunocytochemistry with different mAbs directed against P-gp, MRP1, MRP2, and MVP showed no neuronal or glial labeling in control normal hippocampus (Figs. 1–4A–D). Of the four MDR proteins examined, only the P-gp IR was consistently detectable with JSB-1 mAb in capillary endothelial cells of control hippocampus (Fig. 1B–D). In agreement with previous reports, in paraffin-embedded human tissue, the sensitivity of mAb C219 was low, and the endothelium labeling was too weak and variable to be evaluated (13,14,27,28). Histologically, nonsclerotic hippocampus did not show changes in MDR protein expression compared with autopsy specimens of normal hippocampus from cases with no history of epilepsy.

image

Figure 1. Distribution of P-glycoprotein (P-gp) immunoreactivity in the hippocampus of control and temporal lobe epilepsy patients with hippocampal sclerosis. Representative photomicrographs of immunohistochemical staining with the JSB-1 monoclonal antibody (mAb). A–E: Normal hippocampus. A: Weak immunoreactivity (IR) is detected throughout the hippocampus (DG, dentate gyrus). B–E: No neuronal or glial labeling is observed in the different hippocampal fields (CA1, B; dentate gyrus granule cells (gcl), C; hilus, D) and subiculum (Sub, E). P-gp IR is detected only in blood vessels (arrows, B–E). F–K: Hippocampal sclerosis (HS). F: Increased P-gp staining is observed within the sclerotic hippocampus. G: CA1 sector showing P-gp immunopositivity in reactive astrocytes (arrows). H: Residual neurons in the CA1 are not expressing P-gp (arrowheads), but are surrounded by P-gp–positive glial cells. Insert in H: Merged image, showing colocalization (yellow) of P-gp (red) with the astroglial marker glial fibrillary acidic protein (GFAP; green). I: Dentate gyrus granule cell layer showing strong P-gp labeling in blood vessels (arrows and insert). J: Hilar region showing P-gp–positive reactive astrocytes. K: Subiculum showing P-gp IR in astrocytes (arrowheads) and blood vessels (arrow). Sections are counterstained with hematoxylin. Scale bar: A and F, bar in A, 1 mm; B–E, G, I–K, bar in B, 100 μm; H, 50 μm.

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image

Figure 2. Distribution of multidrug resistance–associated protein 1 (MRP1) immunoreactivity in the hippocampus of control and temporal lobe epilepsy patients with hippocampal sclerosis. Representative photomicrographs of immunohistochemical staining with the MRP1 monoclonal antibody (mAb). A–D: Normal hippocampus without detectable expression of MRP1 (DG, dentate gyrus). B–D: No neuronal or glial labeling is observed in the different hippocampal fields (CA1, B; DG granule cells (gcl), C; hilus, D). E–J: Hippocampal sclerosis (HS). E: CA1 sector with prominent gliosis, but modest MRP1 labeling. G: Residual neurons in the CA1 are occasionally expressing MRP1 (arrow). H: DG granule cell layer without detectable expression of MRP1. I: Hilar region showing MRP1 immunostaining in a hypertrophic hilar neuron (arrow). Insert in I: Merged image, showing colocalization (yellow) of MRP1 (red) with NeuN (green). J: MRP1 immunoreactivity was detected in glial endfoot processes around blood vessels (arrows). Insert in J: Merged image, showing colocalization (yellow) of MRP1 (red) with glial fibrillary acidic protein (GFAP; green) around a blood vessel. Sections are counterstained with hematoxylin. Scale bar: A and E, bar in A, 1 mm; B–D, F, H–J, bar in B, 100 μm; G, 50 μm.

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image

Figure 3. Distribution of multidrug resistance–associated protein 2 (MRP2) immunoreactivity in the hippocampus of control and temporal lobe epilepsy patients with hippocampal sclerosis. Representative photomicrographs of immunohistochemical staining with the M2III-6 monoclonal antibody (mAb). A–D: Normal hippocampus (DG, dentate gyrus). B–D: No significant neuronal or glial labeling is observed in the different hippocampal fields (CA1, B; DG granule cells (gcl), C; hilus, D). E–I: Hippocampal sclerosis (HS). F: CA1 sector with prominent gliosis and MRP2 labeling (arrows). Insert in F: Merged image, showing colocalization (yellow) of MRP2 (red) with the astroglial marker glial fibrillary acidic protein (GFAP; green). G and H: MRP1 immunoreactivity in blood vessels (arrows) in the DG granule cell layer (G) and in the hilar region (H). Insert in H: Merged image, showing colocalization (yellow) of MRP2 (red) with the endothelial marker CD31 (green). I: MRP2 immunoreactivity was detected in glial processes around blood vessels (arrows) and in endothelial cells (arrowheads). Sections are counterstained with hematoxylin. Scale bar: A and E, bar in A, 1 mm; B–D, E, G, H, bar in B, 100 μm; F, 100 μm; I, 50 μm.

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image

Figure 4. Distribution of major vault protein (MVP) immunoreactivity in the hippocampus of control and temporal lobe epilepsy patients with hippocampal sclerosis. Representative photomicrographs of immunohistochemical staining with the MVP-37 monoclonal antibody (mAb). A–D: Normal hippocampus without detectable expression of MVP (DG, dentate gyrus). B–D: No neuronal or glial labeling is observed in the different hippocampal fields (B, CA1; C, DG granule cells (gcl), C; hilus, D). E–I: Hippocampal sclerosis (HS). F: CA1 sector showing clear MVP immunoreactivity in blood vessels (arrows). Reactive glial cells displayed moderate MVP labeling (arrowheads). G: DG cell layer with detectable expression of MVP in blood vessels. H and I: MVP immunostaining within the hilar region in a hypertrophic hilar neuron (arrow in H) and in blood vessels (arrow in I). Sections are counterstained with hematoxylin. Scale bar: A and E, bar in A, 1 mm; B–D and G–I, bar in B, 100 μm; F, 100 μm.

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P-gp, MRP1, MRP2, and MVP expression in hippocampal sclerosis

In TLE cases with HS (n = 12), all four MDR proteins examined were upregulated, albeit with different distribution patterns (Figs. 1–4).

Moderate to strong P-gp IR with the JSB-1 mAb was localized to cells with the morphology of reactive astrocytes in the CA1 and hilar regions in all HS cases (Fig. 1G, H, and J;Table 2). Double labeling confirmed P-gp expression in GFAP-positive reactive astrocytes. (Fig. 1H). In eight HS cases, strong P-gp IR was detected in blood vessels, with prominent endothelial labeling (Fig. 1I; Table 2). Astroglial and endothelial P-gp labeling also was observed in the subiculum in three cases (Fig. 1K) and the C219 mAb-labeled astrocytes only in six cases; in the remaining cases, both astrocytes and capillary endothelium were negative (data not shown).

Positive MRP1 staining was observed in six HS cases (Table 2). Both anti-MRP1 mAbs (MRPr1 and MRPm6) showed a similar pattern of IR, but the most intense labeling was detected with the MRPr1 mAb (Fig. 2E–J). Reactive astrocytes displayed moderate MRP1 IR in five cases (Table 2; Fig. 2F). MRP1 IR was detected in glial foot processes around blood vessels, where it colocalized with GFAP (Fig. 2J). No detectable MRP1 labeling was observed in the capillary endothelium with both mAbs used (Fig. 2; Table 2). In six cases, moderate to strong MRP1 expression was observed in hypertrophic hilar neurons (Table 2; Fig. 2I). In three cases, MRPr1 IR also was detected in residual neurons of the CA1 region (Fig. 2G).

Hippocampal MRP2 expression was observed in 11 of 12 HS cases by using both M2I-4 and M2III-6 mAbs (Fig. 3). MRP2 IR was detected in cells with astroglial morphology in the CA1 and hilar regions in four cases, with the M2III-6 showing labeling in GFAP-positive reactive astrocytes (Table 2; Fig. 3F). In the majority of cases, both mAbs detected MRP2 labeling in blood vessels within the sclerotic hippocampus (Fig. 3F and G). Double labeling confirmed MRP2 expression in CD31-positive endothelial cells (Table 2; Fig. 3H).

Immunocytochemistry with mAbs (MVP-37and LRP-56) directed against MVP showed increased protein expression in the hippocampus of TLE patients (Table 2; Fig. 4E–I). MVP expression was detected mainly in blood vessels; only in two cases was moderate IR observed in reactive astrocytes (Table 2; Fig. 4F–H). Similar to the MRP1 staining, MVP expression also was observed in residual hypertrophic hilar neurons in six HS cases (Table 2; Fig. 4H).

In all four non-HS cases examined, the expression pattern of all four MDR proteins (P-gp, MRP1, MRP2, and MVP did not differ from that observed in normal control autopsy hippocampal specimens.

DISCUSSION

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

This study investigated the cellular distribution of different MDR proteins in normal and sclerotic hippocampus. Detection of the cell types in which different MDR proteins are expressed represents the first step to understanding their functions and the possible effects of their pharmacologic inhibition within the hippocampus. Compared with expression in normal human hippocampus, specimens of TLE patients with HS showed increased expression of all four proteins. The cell-specific distribution in relation to their possible involvement in MDR is discussed later.

MDR proteins in human hippocampus

In normal hippocampus and in non-HS cases, P-gp, MRP1, MRP2, and MVP proteins were not detectable in glial and neuronal cells. This is in agreement with the results of other immunocytochemical studies, in which P-gp, MRP1, or MVP expression was evaluated in human normal brain tissue (10,13–15,29). To our knowledge, this is the first study in which MRP2 IR was investigated in normal and epileptic human hippocampus.

In normal human hippocampus, we were able to show vascular expression only for P-gp, with IR localized in capillary endothelial cells. The fact that we could not detect MRP protein expression in endothelial cells of normal human hippocampus is in agreement with our previous study in human normal cortex (14). Accordingly, MRP expression is entirely lacking in isolated human brain microvessels (30) and is detected only in endothelial cells grown out from the microvessels and maintained in culture (12,30).

Cell-specific distribution of MDR proteins in hippocampal sclerosis

In the present study, we demonstrated that HS, the most common type of neuropathologic substrate in patients with refractory TLE, is associated with the overexpression of several MDR proteins (P-gp, MRP1, MRP2, and MVP) with different distribution patterns.

P-gp

Immunocytochemical studies of MDR proteins are complicated by the crossreactivity of some anti–P-gp Abs (22,31) and the known variability of staining in fixed human tissue (27,28). For these reasons, we used two different Abs for each MDR protein examined. P-gp was localized in cells with the morphology of reactive astrocytes. Astroglial IR was observed in CA1 and hilar regions in all HS cases by using the JSB-1 mAbs and in 50% of cases using the C219 mAb. However, in cases in which the C219 did not label the glial cells, the vascular endothelial staining also was absent, reflecting the lower sensitivity of mAb C219 in paraffin-embedded human tissue (13,14,27,28). Although the JSB-1 may crossreact with a ubiquitous mitochondrial enzyme (22), no detectable JSB-1 labeling was observed in the large majority of reactive astrocytes from autopsy specimens of patients with nonneoplastic brain pathologies associated with reactive gliosis (and no history of seizures) or in the cortex and nonsclerotic hippocampus adjacent to malformative and neoplastic epileptic lesions [(14); and present results]. Thus the expression of P-gp appears to be a feature of astrocytes within the epileptic region, which is in agreement with previous reports showing overexpression in glial cells of different epileptic human disorders (10,13,14).

Expression of P-gp in astrocytes around blood vessels may critically influence the responsiveness to AEDs, reducing the intraparenchymal concentration and contributing to the barrier function as a “second line” mechanism [reviewed in (5)]. However, P-gp is not exclusively localized in glial cells around vessels. The diffuse astroglial staining observed within the sclerotic hippocampus suggests other functions in glial cells, not necessarily linked to pharmacokinetics. Accordingly, recent evidence suggests a role for P-gp in the regulation of glial cell survival in the epileptic tissue (32,33). No detectable neuronal P-gp IR was observed in HS specimens. Neuronal expression of P-gp has been previously observed in malformations of cortical development (MCDs), but only in a population of dysplastic and abnormal neuronal and glioneuronal cells (14,34).

By using the JSB-1 mAbs, P-gp IR was clearly detected in blood vessels in all HS specimens, with stronger endothelial cell labeling observed in eight of 12 HS specimens, compared with control hippocampus or non-HS cases with focal lesions not involving the hippocampus proper. This observation indicates that the upregulation of P-gp expression in endothelial cells is a major feature of HS in TLE patients and is in agreement with the endothelial overexpression observed in different pathologies associated with refractory epilepsy (10,12,14). No detectable changes in both glial and endothelial P-gp expression were present in histologically normal hippocampus (non-HS) from patients undergoing extensive surgical resection of the mesial structures for the treatment of medically intractable epilepsy associated with brain tumors. As previously shown (14), P-gp overexpression was observed only in the epileptogenic tumor region.

In our study, with fixed material, it is not possible to investigate the spatiotemporal development of this overexpression or the role of epileptic activity in regulating the expression of MDR proteins. For this purpose, the use of experimental models of pharmacoresistant epilepsy is required. In a rat TLE model, an increase in the genes that encode MDR proteins (such as P-gp) is observed in the hippocampus and parahippocampal cortex of rats with chronic epilepsy and is related to the induction of status epilepticus and occurrence of spontaneous seizures (35,36; Gorter, unpublished observations). These observations indicate that the P-gp overexpression in the epileptogenic sclerotic hippocampus can be related to seizure activity. In addition, it has been shown that treatment with therapeutic doses of phenytoin (PHT) or carbamazepine (CBZ) does not change the MDR mRNA expression in the mouse hippocampus (35). However, we cannot exclude the possibility of a constitutive (rather than induced or acquired) expression, as suggested for the overexpression observed in different developmental lesions associated with refractory epilepsy (13,37). Accordingly, an underlying hippocampal maldevelopment has been suggested in mesial temporal sclerosis (MTS) (26).

It is not likely that treatment with AEDs per se is responsible for the observed intralesional overexpression of multidrug transporter proteins. Overexpression in MCDs is already present before the onset of seizures or exposure to AEDs (11). In patients with lesional focal epilepsy (tumors or MCDs), the major changes are observed only in the lesion and not in perilesional tissue that has theoretically been exposed to the same drugs (13,14,36). In addition, it has been shown that treatment with therapeutic doses of PHT or CBZ does not change the MDR mRNA expression in mouse hippocampus (35).

MRP1

MRP1 staining was detected within the sclerotic hippocampus of six of 12 HS cases. The MRP1 IR pattern was similar for both MRP1 mAbs used, but clearly different from P-gp IR. MRP1 displayed modest astroglial labeling compared with the prominent and diffuse P-gp expression in reactive astrocytes. This is in agreement with previous reports in MCDs, showing MRP1 glial staining mainly around blood vessels (13,14). Capillary endothelial cells did not display detectable MRP1 IR in any of the HS specimens examined with both MRP1 mAbs. Accordingly, the expression of the gene encoding for MRP1 also has been shown to be unaffected in endothelial cells isolated from epileptic tissue (12). Whether the different localization of P-gp and MRP1 in brain microvessels reflects different physiologic functions is still unclear and requires further functional investigation.

Intriguing is the detection of MRP1 IR in neurons within the sclerotic hippocampus. Strong MRP1 expression was observed, particularly in hypertrophic cells in the dentate hilus. This cellular enlargement has generally been considered an adaptive cellular phenomenon, resulting from an altered hippocampal circuitry and increased metabolic demands (38). Overexpression of MRP1 (and MVP) could be a protection mechanism that contributes to the survival of these cells under hostile conditions (32).

MRP2

In the present study, we report MRP2 expression in the large majority of HS specimens. MRP2 IR was consistently detected in blood vessels in endothelial cells by using two different mAbs. Expression of MRP2 mRNA has been recently demonstrated in endothelial cells isolated from human tissue, with increased mRNA expression in the endothelium of patients with refractory epilepsy (12). Our observation is the first confirmation of endothelial MRP2 overexpression at the protein level in TLE patients with HS. Although immunogold electron microscopic studies (in non–paraffin-embedded material) will be necessary to define the subcellular localization of MRP2 within the endothelium, previous studies in other cell types clearly indicated the location of this transporter protein in the luminal membrane (i.e., facing the blood), whereas other MRPs are located basolaterally [reviewed in (39)]. The critical role of MRP2 at the blood–brain barrier in restricting penetration and activity of specific AEDs has been recently demonstrated in rats. Both inhibition and lack of MRP2 significantly increase the extracellular brain levels and the anticonvulsant activity of PHT (40). MRP2 is, however, not expressed exclusively in endothelial cells; MRP2 astroglial labeling (with the M2III-6 mAb) has been observed in four HS cases in regions with prominent reactive gliosis (i.e., CA1) and around blood vessels. Although no crossreactivity has been reported for the M2III-6 mAb with other MRPs (18), additional studies are needed to investigate whether this glial IR is based on protein processing and masking of different epitopes. We cannot completely exclude other crossreacting antigens in glial cells. However, no glial MRP2 IR is observed in tissue from patients with nonepileptic brain pathologies associated with reactive gliosis with both anti-MRP2 mAbs.

MVP

The functional role of MVP has increased in significance in view of the finding that MVP/vaults are overexpressed in many human tumors and since a connection has been shown between their expression and MDR (19,41–47). In a recent study, we demonstrated that GGs, the most common tumor entity in young patients with medically intractable epilepsy, express high amounts of MVP. MVP IR also was increased in sclerotic hippocampus from patients with dual pathology compared with non-HS specimens (15). In the present study, we showed that overexpression of MVP also is detected in HS specimens of adult patients with TLE, without associated malformative or neoplastic lesions. This is in line with the persistent increase of MVP mRNA/protein observed in an experimental model of TLE, suggesting a relation to seizure activity (48).

With two different mAbs, MVP IR was consistently detected in blood vessels in endothelial cells. Although vault function remains undetermined, some evidence supports the role of vaults in the vesicular transport of several compounds, mediating a bidirectional nucleocytoplasmic exchange (43,49–52). Intriguing also is the presence of MVP (similarly to MRP1) in residual hypertrophic hilar neurons in HS specimens. Further research is necessary to investigate whether MVP expression could contribute to the survival of these cells in TLE.

CONCLUSIONS

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

The data presented in this study demonstrate that several MDR proteins (P-gp, MRPs, and MVP) are regulated in concert in human TLE specimens. This overexpression is likely to contribute to the drug resistance, with multiple mechanisms operating at different levels within the hippocampus. Expression of drug transporters (P-gp, MRP1, and MRP2) at the level of the blood–brain barrier (endothelial cells) and the astrocytic endfoot processes surrounding brain capillaries may impair the penetration of therapeutic agents, leading to decreased tissue drug concentrations. Several inhibitors of P-gp and of MRPs are available and are currently used in the clinical trial stage in human cancer patients (53–55). Thus evaluation of the possible use of these inhibitors as adjunctive treatment in TLE with HS is certainly worth while. However, activation of expression at the level of residual hypertrophic hilar neurons suggests additional functions, not necessary linked to pharmacoresistance, for some proteins (such as MRP1 and MVP), which deserve further consideration.

Acknowledgments

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

Acknowledgment:  This work was supported by the Stichting AZUA-funds (E. Aronica), National Epilepsy Fund: “Power of the Small,” and Hersenstichting Nederland (NEF 02-10; E. Aronica).

REFERENCES

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