Bilateral, vascular and perivascular glial upregulation of heat shock protein-27 after repeated epileptic seizures

Journal of Chemical Neuroanatomy 30 (2005) 1–16 www.elsevier.com/locate/jchemneu

Bilateral, vascular and perivascular glial upregulation of heat shock protein-27 after repeated epileptic seizures Hans-J. Bidmon a,*, Boris Go¨rg b, Nicola Palomero-Gallagher c, Freimut Schliess b, Ali Gorji d, Erwin-J. Speckmann d, Karl Zilles a,c,e a

C. & O. Vogt Institute for Brain Research, Heinrich-Heine-University, Universita¨tsstr. 1, Bldg. 22.03.05, D-40225 Du¨sseldorf, Germany b Department of Gastroenterology, Heinrich-Heine-University, Moorenstr. 5, D-40225 Du¨sseldorf, Germany c Institute of Medicine, Research Ctr. Ju¨lich, D-52425 Ju¨lich, Germany d Institute of Physiology I, Westfa¨lische-Wilhelms-University, Robert-Koch-Str. 27a, D-48149 Mu¨nster, Germany e Biomedical Research Center, BMFZ, Heinrich-Heine-University, Universita¨tsstr. 1, D-40225 Du¨sseldorf, Germany Received 15 October 2004; received in revised form 19 January 2005; accepted 7 March 2005 Available online 25 May 2005

Abstract Heat shock protein-27 (HSP-27) is an inducible stress response protein. It inhibits apoptotic cell death and is a reliable marker for oxidative stress. We studied the induction of HSP-27 in rat brains on days 1, 4 and 14 after repeated, pentylenetetrazole (PTZ)-induced seizures using immunohistochemisty. Saline treated control rats showed no induction of HSP-27. HSP-27 reactive astrocytes were rarely seen 1 or 4 days after PTZ injection. When present, single astrocytes were located in the cortex and/or the hippocampus. After 14 days PTZ treatment, a bilateral distribution of HSP-27 immunoreactive glia was present in piriform and entorhinal cortices and in the dentate gyrus of most brains. Rats with most intense HSP-27 upregulation showed HSP-27 in amygdala and thalamic nuclei. Astrocytes associated with blood vessels presented strongest HSP-27 staining, but did not show upregulation of gial fibrillary acidic protein and none responded with HSP-47 expression. Additionally, HSP-27 immunoreactivity increased in the endothelial cells of blood vessels in the affected brain regions, although no neuronal induction occurred. Contrastingly, a subconvulsive dose of the glutamine synthetase inhibitor L-methionine sulfoxime, which acts directly on astrocytes, resulted in a rapid, homogeneous astrocyte-specific HSP-27 upregulation within 24 h. Thus, repeated PTZ-induced seizure activity elicits a focal ‘‘heat shock’’ response in endothelial cells and astrocytes of selected cerebral regions indicating that expression of HSP-27 occurred in a seizure-dependent manner within the affected cerebral circuitries. Therefore, this PTZ-model of repeated seizure activity exhibited a cortical pattern of HSP-27 expression which is most comparable to that known from patients with epilepsy. # 2005 Elsevier B.V. All rights reserved. Keywords: Epilepsy; Astrocytes; Endothelial cells; Blood–brain-barrier; Oxidative stress; Pentylentetrazole

1. Introduction Heat shock proteins (HSPs) belong to a highly conserved family of proteins, some of which are inducible by various noxious stimuli and are part of the cellular defence system. HSPs represent chaperones acting as specific carriers and/or participate in protein-folding (Jakob and Buchner, 1994; Kiang and Tsokos, 1998; Hartl and Hayer-Hartl, 2002). HSP-70 and HSP-27 serve as binding proteins for Vitamin D and estradiol, respectively (Gacad and Adams, 1998; Lu * Corresponding author. Tel.: +49 211 81 12766; fax: +49 211 81 12336. E-mail address: [email protected] (H. Bidmon). 0891-0618/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2005.03.009

et al., 2002; Chen et al., 2003). Both HSP-70 and HSP-27 are induced in the CNS during heat shock, ischemia, hypoxia and kainate-induced seizures contributing to the phenomenon of preconditioning (Currie et al., 2000; He and Lemasters, 2003). For HSP-27 and the mouse homolog HSP-25, it is known that they inhibit apoptotic neuronal death (Wagstaff et al., 1999; Akbar et al., 2003) by acting at the permeability transition pore of mitochondria (He and Lemasters, 2003), thus inhibiting FAS/APO-1 signalling and preserving the endogenous antioxidant glutathione (Mehlen et al., 1996a,b). In a more general view, HSPs are also considered to represent a reliable marker for tissues affected by oxidative stress (Kregel, 2002). More recently free

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radicals and oxidative stress were identified to play a major role in epilepsy. They contribute mainly to cell loss (Kovacs et al., 2002; Bidmon et al., 2002; Patel, 2002; Patel and Li, 2003; Chung and Han, 2003; Savaskan et al., 2003; Bashkatova et al., 2003). Several animal models have been developed specifically addressing certain aspects of epileptic activity or pathological consequences found in human patients (White, 2002). Between the kainate model and the pentylenetetrazole (PTZ) models certain differences exist, the most important one which is the fact that PTZ does not cause direct neurotoxicity (Valente et al., 2004). These differences seem to result in model specific heat shock responses, since HSP70 is upregulated after seizures induced by kainate but not after seizures elicited by PTZ, NMDA, or lindane (Planas et al., 1994). In addition, after treatment with kainate most authors reported an induction of HSP-27 (Akbar et al., 2001; Kato et al., 1999; Plumier et al., 1996), which is especially linked to apoptotic cell death in this model and which affects glial cells and neurons (Akbar et al., 2003). In comparison, for the PTZ model only a strong induction of HSP-72 has been established (Nehlig and Pereira de Vasconcelos, 1996; Motte et al., 1997; Arzimanoglou et al., 2002). In the temporal cortex and hippocampus of human patients suffering from intractable epilepsy HSP-27 is homogenously or focally induced and its expression remained confined to astrocytes and blood vessels (Erdamar et al., 2000; Bidmon et al., 2004). Therefore, we were searching for an animal model showing a seizure-related HSP-27 induction comparable to that reported for temporal cortex of patients with epilepsy. Since generalized seizure activity elicited by PTZ i.p. (40 mg/kg) results in a sequence of bioelectrical events which are indistinguishable from those seen in the EEG of human epileptic patients (Caspers and Speckmann, 1972), we used this model to study seizure induced HSP-27 induction.

2. Materials and methods 2.1. Pentylenetetrazole treatment Male Wistar rats (220–250 g bodyweight) were placed in cages mounted onto a recording stage in order to register seizure activity and duration. One group of rats (n = 8) was injected i.p. with a single dose of PTZ at a concentration of 40 mg/kg dissolved in physiological saline (10 mg/0.5 ml) to induce acute seizures. Control rats (n = 4) received saline only. These animals were sacrificed 24 h after injection. Additional groups of rats were injected every 48 h (except on weekends, 72 h), with 40 mg PTZ/kg for a total of 4 days (n = 4) or 14 days (n = 12), respectively. According to previously described experimental protocols (Caspers and Speckmann, 1972; Rauca et al., 2004), the intervals of PTZ applications at the mentioned concentrations of the drug

Fig. 1. Western blot analysis showing examples for the presence of a increased HSP-27 immunoreactive bands in protein extracts prepared from cerebral cortex and hippocampus of rats injected repeatedly with 40 mg PTZ/kg bodyweight for 14 days (c, d) in comparison to saline treated control rats (a, b) and rats treated with PTZ for 4 days. Note that only two consecutive injections with PTZ up to day 4 were not enough to induce HSP-27 expression up to the level of detectable amounts of immunoreactive protein.

guaranteed continuous epileptic activity with lowest side effects. Control rats (n = 8) received saline injections only. Rats were anesthetized with sodium pentobarbital and fixed by cardiac perfusion using ice-cold physiological saline followed by Zamboni’s fixative 24 h after their last injection with PTZ. Some treated and some control rats (Fig. 1) were perfused with saline only, the brains were removed, and cortical as well as hippocampal tissue probes of one hemisphere were used for western blot analysis whereas the other hemisphere was immersion-fixed in Zamboni fixative for immunohistochemistry. Following fixation, all brains were cryoprotected in PBS containing 25% sucrose, frozen and sectioned as described (Bidmon et al., 2001). In order to determine whether PTZ affects astrocytes directly, we compared the induction of HSP-27 seen in PTZtreated animals with rats in which the astrocyte-specific glutamine synthetase had been inhibited with L-methionine sulfoxime (MSO; Sigma, Deisenhofen). For this, eight additional rats were injected i.p. with a subconvulsive dose of MSO 100 mg/kg dissolved in physiological saline (Paulsen et al., 1988). After 8 h (n = 3) and 24 h these rats were anesthetized with sodium pentobarbital and perfusionfixed as described above. 2.2. Western blot analysis Tissue probes were lysed at 4 8C with 10 mmol/L Tris– HCl buffer (pH 7.4) containing 1% Triton X-100, 150 mmol/ L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 20 mmol/L NaF, 0.2 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 0.5% Nonidet P-40 (NP-40). Following homogenization, the lysates were centrifuged at 20,000
g at 4 8C.

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For sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis the supernatant was added to an identical volume of 2
gel loading buffer containing 200 mmol/L dithiothreitol (DTT) (pH 6.8) and heated to 95 8C for 5 min. The probes were subject to gel electrophoresis (10% gels) using 30 mg protein per lane. Thereafter, gels were equilibrated with transfer buffer (39 mmol/L glycine, 48 mmol/L Tris–HCl, 0.03% SDS, 20% methanol). Proteins were transferred to nitrocellulose membranes using a semi-dry transfer apparatus

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(Pharmacia, Freiburg, Germany) Blots were blocked overnight in 5% bovine serum albumin (BSA), solubilized in 20 mmol/L Tris–HCl, pH 7.5, containing 150 mmol/L NaCl and 0.1% Tween 20, and incubated for 2 h with antiserum against HSP-27 (SPA-801, Stressgen, Canada) diluted 1:5000 at 4 8C. After several rinses and incubation with horseradish peroxidase coupled anti-rabbit-IgG (Sigma, Deisenhofen) diluted 1:10,000 at 4 8C for 2 h, blots were washed and developed using enhanced chemiluminescent (ECL) detection (Amersham, Braunschweig, Germany).

Fig. 2. (A–F) Frontal sections through rat brains 14 days after their first PTZ or saline (E) injection, stained for HSP-27 (A–E) or GFAP (F). Sections A–D show the distribution patterns of HSP-27 immunoreactive glial cells in a rostro-caudal direction for strongly responding rats. Note the strong labelling in piriform (p) and entorhinal cortices (e, in D) as well as in the amygdaloid-piriform transition area (ap). Within these animals no comparable focal upregulation of GFAP was detectable (F, compare with B). c, central amygdaloid nucleus; d, dorsal grey; dg, dentate gyrus; rf, rhinal fissure. Bar: 2 mm.

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microglia; Serotec, Biozol, Eiching, 1:100), the rat endothelial cell specific RECA-1 (Serotec, Oxford, UK; 1:20) or the neuronal marker SMI-311 (Sternberger Monoclonals; 1:500). After incubation in blocking serum, sections were incubated for 48 h at 4 8C under gentle shaking in the primary antibodies diluted in PBS containing 0.1% Triton X-100. Following four rinses in PBS, they were incubated with the second antibodies Cy-3 coupled goat anti-rabbit (1:150) and FITC-coupled goat-anti-mouse (1:150, Dianova, Hamburg) for 24 h at 4 8C. Sections were rinsed four times and mounted with flouromount G (Southern Biotechnology Assoc., Burmingham, USA). Flourescence labelled sections were evaluated using a laser-scanning microscope (Leica, Bensheim). In order to check for neuronal degeneration, we used Fluoro-Jade B (Chemicon, Hofheim, Germany) according to manufacturer’s instructions and the experimental protocol described by Schmued et al. (1997). Sections were taken from cryoprotective storage medium, rinsed in PBS and mounted on gelatine-coated slides. Slides were dehydrated with ethanol and rehydrated in distilled water followed by immersion in 0.06% potassium permanganate (KMnO4) 15 min and stained in 0.01% acetic acid containing 0.0004% Fluoro-Jade B for 20 min. Slides were rinsed three times in distilled water, dried, submerged in xylene and coverslipped with DPX (Fluka).

2.3. Immunohistochemistry Sections were stained free floating under constant gentle shaking starting with three rinses in PBS (pH 7.4) followed by inhibition of endogenous peroxidase with 3% H2O2 in PBS for 20 min, and followed by four rinses in PBS. Sections were then incubated for 1 h in PBS containing 0.1% Triton X-100 and 10% normal goat serum (NGS, Dianova, Hamburg). Afterwards, sections were incubated in antiserum against polyclonal HSP-25 (SPA-801, Stressgen Canada) at a final dilution of 1:500 at 4 8C for 48 h. Following four rinses in PBS of 15 min each, sections were incubated with biotinylated second antibody goat anti-rabbit (Dianova, Hamburg) at a final dilution of 1:200 for 24 h at 4 8C. After additional four rinses, sections were incubated at room temperature with AB-complex (Vectastain) for 90 min and antibody binding was visualized with 3,30 -diamino benzidine tetrahydrochloride according to standard protocols (Gladilin et al., 2000). In one animal (which was not included in the study) treated with a single dose of PTZ we observed a unilateral hippocampal HSP-27 and HSP-32 immunoreactive vascular lesion. Therefore, we also stained sections for HSP-32 (heme oxygenase-1, Stressgen, SPA895, 1:500), in order to discard the possibility that our HSP27 labelling was due to ruptured blood vessels. Additionally, we stained sections with a monoclonal antibody directed towards glial fibrillary acidic protein (GFAP, 1:25, Boehringer) or HSP-47 (1:400, SPA-470, Stressgen) using goat-antimouse as the secondary antibody. For colocalization studies using double immunofluorescence, we applied the following antibody combinations as primary antibodies, polyclonal HSP-27 (SPA-801) and monoclonal anti-GFAP, the OX 42 (marker for activated

2.4. Semiquantitative evaluation of the distribution patterns for HSP-27 Since we observed a wide spectrum of HSP-27 immunoreactivity among the individual PTZ treated rats (compare Figs. 2B and 3B), which in addition varied in

Table 1 Topographical localization of HSP-27 induction in response to repeated pentylenetetrazol injections at day 14 Affected brain regions

Individual animals 1

2

3

4

5

6

7

8

9

10

11

12

Motor cortex M1 and M2 Somatosensory cortex S1 and S2 Insular cortex Piriform cortex and amygdaloid-piriform transition area Entorhinalcortex Hippocampus CA1/CA2/CA3

1 2 2 5 5 2

2 1 1 2 3 1

1 1 3 5 5 1

1 2 2 5 5 2

1 / 1 1 1 2

2 1 1 3 3 1

1 2 2 5 4 1

1 1 2 4 4 2

/ 1 1 3 3 1

1 1 2 4 4 2

1 / / 1 1 /

1 1 3 4 4 1

Dendate gyrus Granular layer Molecular layer

2 5

1 3

1 4

2 5

1 4

1 3

1 4

1 4

/ 3

1 4

/ 3

1 4

Amygdala Central Basolateral

4 2

1 /

3 1

1 /

/ /

1 /

2 /

1 1

/ /

3 /

/ /

1 /

Central thalamic nucl. Paraventricular thalamic nucl.

2 3

1 1

4 3

3 3

/ 2

/ 1

2 2

1 2

/ 1

1 1

/ /

2 1

Periaqueductal dorsal grey

2

–

1

–

–

–

1

1

–

–

–

–

/: no HSP-27 above background; –: not found in all brains; 1: single HSP-27 positive cells or a single tiny cluster; 2: 2–3 small clusters per 4
104 mm; 3: larger groups of cells; 4: the whole area covered by immunoreactive cells; 5: whole area tightly covered by intensely stained astrocytes (see entorhinal cortex in Fig. 2C and D). Brain regions defined according to Paxinos and Watson (1986).

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staining intensity in the rostro-caudal direction (e.g. compare the molecular layer of the dentate gyrus in one and the same animal as shown in Fig. 2B–D), we carried out a semiquantiative evaluation, listing the affected cerebral regions and indicating their relative staining intensity (Table 1).

3. Results From the clinical point of view, the application of PTZ lead to typical tonic-clonic seizures with both phases lasting for about 30 s. In the epicortical EEG the tonic phase was associated with generalized fast spikes and the clonic phase by spike-wave complexes. The seizures were terminated by an electrical silent period in which the animals showed a generalized paralysis for 10–20 s. After repeated applications of PTZ, seizures recurred spontaneously for a period of up to 30 min. With repetition semiology of the attacks as well as EEG patterns remained unchanged. In western blots, HSP-27 was clearly upregulated in hippocampi and cerebral cortices dissected from rats which had been repeatedly treated with the GABAA-receptor antagonist PTZ for 14 days (Fig. 1), whereas no HSP-27 upregulation could be detected at day 4, 24 h after their second PTZ injection, or in control rats following repeated injections with saline for 14 days. Rats treated with a single dose of PTZ, however, showed seizures, but no corresponding increase in HSP-27 positive cells was found after 24 h. However, as seen in control rats, an occasional single immunoreactive glial cell was seen in the piriform and entorhinal cortices (Fig. 3A), in addition to the constitutively HSP-27 positive hypothalamic neurons and HSP-27 positive pia-associated glial processes of the tuberculum olfactorium (Fig. 4E). Nearly the same results were found in rats at day 4, i.e. 24 h after the second PTZ injection (Fig. 1). Only one animal showed tiny clusters of HSP-27 immunoreactive astrocytes in the piriform and entorhinal cortices when compared to animals studied at day 1 after their first PTZ injection or to controls (not shown). Animals which had received repeated PTZ injections showed, however, a considerably increased HSP-27 expression in astrocytes at day 14 (Figs. 2, 3B and 4–6). The intensity and the extent of HSP-27 expression differed among the individual animals (Table 1). Two animals of the 14 days-group showed tiny perivascular foci of astrocytic HSP-27 expression throughout the cortex, preferentially in the piriform and entorhinal areas in addition to some HSP-27 immunoreactivity within endothelial cells (Fig. 3B). The ventral portion of the molecular layer of the dentate gyrus was the most intensely labelled region in these animals. In addition tiny foci of HSP-27 immunoreactive glial cells were present around the blood vessels of the stratum lacunosum moleculare or in CA2/CA3 and in the hilus (Fig. 3B0 ). The two animals which reacted with the strongest induction of HSP-27 also showed a considerably increased and

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widespread immunoreactivity in the piriform and entorhinal cortices, amygdaloid-piriform transition area, and in the molecular layer of the dentate gyrus (Fig. 2, Table 1). In addition these two animals showed increased HSP-27 immunoreactivity in the amygdala, thalamic nuclei, and dorsal central grey (Figs. 2A–D and 4, Table 1). In the other functionally characterized areas of the cerebral cortex various degrees of HSP-27 positive astrocytes and blood vessels were present (Fig. 2; Table 1), but no immunoreactive cells could be found in the cingulate or retrosplenial cortices (Figs. 2 and 3). Fluoro-Jade B positive, degenerating neurons were not found in any of the examined brains (not shown). In sections of all animals which were HSP-27 positive, no corresponding increase in GFAP positive astrocytes was present (compare Figs. 2B, F and 5A–A00 ). Furthermore, no increase of GFAP immunoreactivity was observed after 14 days of repeated PTZ treatment when compared to control rats (not shown). In order to exclude the possibility that major vascular lesions may have contributed to strong focal HSP-27 expression, we studied heme oxygenase-1 (HSP-32) immunoreactivity, since HSP-32 is induced by heme and systemic iron leakage (Bergeron et al., 1998), which was neither upregulated in glial cells nor in the constitutively heme oxygenese-1 expressing interneurons of the hippocampus after 2 weeks of repeated PTZ treatment (Fig. 3D). Furthermore, none of the rats displayed an induction of HSP47 in the astrocytes of cerebral regions in which HSP-27 had been induced (Fig. 3C). Compared to the grey matter, HSP-27 immunoreactivity remained weaker and less frequent in white matter. Only the external capsule along the ependymal fusion zones of the lateral ventricles (Fig. 4C) and below the third ventricle (Fig. 4G) showed localized HSP-27 expression. Most HSP27 positive glial cells where associated with the wall of larger blood vessels in the cerebral cortex (Figs. 4A, B and 5A–A00 ). HSP-27 positive cells in the amygdala and the thalamic nuclei were also associated with larger vessels or even small capillaries (Fig. 4D and F). While HSP-27 immunoreactive blood vessels were present in all affected regions, some regions showed only immunoreactive blood vessels, such as the dorsal hypothalamic area (Fig. 4H). Colocalization of the astrocytic GFAP and HSP-27 immunostainings revealed that the HSP-27 positive cells were astrocytes which contacted blood vessels (Fig. 5A and D–F). Also the strongly labelled larger foci of immunoreactive cells in the piriform and entorhinal cortices were represented by astrocytes similar to those in the stratum moleculare of the dentate gyrus (Fig. 5B and E). HSP-27 was homogeneously distributed over the whole cytoplasm of HSP-27 positive astrocytes, whereas the GFAP immunoreactivity was solely confined to cytoskeletal fibrils, which showed a normal intracellular arrangement (Fig. 5A–A00 ). Using the rat endothelial cell specific antigene RECA-1 (Duijvestijn et al., 1992) in combination with HSP-27, we found in most affected regions three patterns of blood vessel associated

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Fig. 3. Frontal sections through PTZ (A–D) and MSO (E) treated rats. One day after a single PTZ treatment some animals exhibited single or tiny clusters of HSP-27 immunoreactivity as indicated by arrowheads (A). (B–B0 ) The distribution of HSP-27 14 days after repeated PTZ induced seizures in a weakly responding rat with tiny blood vessel associated glial HSP-27 immunoreactivity in the cerebral cortex and in the hippocampus (arrowheads in B–B0 ) and larger intensely stained clusters in the ventral part of the molecular layer of the dentate gyrus (arrows, in B0 ). (C) A consecutive section of the same brain shown in B0 stained for HSP-47 showing that repeated PTZ treatment did not cause HSP-47 induction. (D) Heme oxygenase-1 (HSP-32) immunoreactivity is not detectable after repeated PTZ-treatment. (E–E0 ) A single treatment with MSO induced a widespread homogenous upregulation of glial HSP-27 immunoreactivity within 24 h. Bars: 2 mm (A, B, D, E); 500 mm (B0 , C, E0 ).

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Fig. 4. (A–H) Sections through rat brains after 14 days of PTZ treatment (A–D, F–H) and a control (E). In the parietal cortex HSP-27 immunoreactive glial cells surround mainly blood vessels (bv, A, B). Some HSP-27 positive cells are associated with the ependymal fusion zone (ef, C, G). HSP-27 immunoreactive foci were associated with blood vessels in the amygdala (D) and submedial thalamic nuclei and dorsal hypothalamic area (F, H). HSP-27 positive subpial, glial processes were also seen in control animals at the tuberculum olfactorium (E). 3V, third ventricle, CT, central medial—rheuniens thalamic nuclei; S, submedial thalamic nucleus. Bar: 100 mm.

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Fig. 4. (Continued ).

HSP-27 distribution. First of all there were blood vessels or certain regions of blood vessels which contained HSP-27 immunoreactivity within their endothelial cells and which were not surrounded by or in contact with HSP-27 positive glial cells (Fig. 6A–C0 ). In these blood vessels very strong staining was seen in regions were the vessel branched, e.g. as at the ventral edge of the molecular layer of the dentate gyrus (Fig. 6C–C0 ). In addition, there were blood vessels which expressed HSP-27 in their endothelial cells and were surrounded by HSP-27 positive astrocytic processes (Fig. 6D–D0 ). These were often located within larger, strongly HSP-27 positive foci. Especially in the cerebral cortex, this combined endothelial astrocytic staining pattern was found along some vessels, but was often restricted to the upper subpial part (cortical layers I–II) of the blood vessel, as indicated in (Fig. 5A–A00 ). The third type of blood vessels showed no or non-detectable HSP-27 immunoreactivity in their endothelial cells, but they were in close contact with HSP-27 positive astrocytic processes, which usually derived from single well separated astrocytes (Fig. 6E) and sometimes the processes of the same astrocyte were attached to one or more branches originating from the same blood vessel (Fig. 6F–F00 ) in deeper cortical layers as well as in the small glial foci seen in the amygdala. Glutamine synthase is an enzyme specifically confined to astrocytes, were it is involved in glutamate metabolism and the synthesis of glutamine, which is the precursor for both neuronal glutamate and GABA synthesis. Therefore, the treatment with a subconvulsive dose of the glutamine synthase inhibitor MSO was expected to affect astrocytes directly and independently from seizure activity. MSO

treatment resulted in a rapid induction of HSP-27, which was already seen after 8 h post injection. As shown (Fig. 3E–E0 ), MSO induced HSP-27 within 24 h in astrocytes in the same cortical and hippocampal regions as seen after repeated treatment with PTZ. However, as shown in detail for the hippocampus, the population of affected astrocytes was much more homogeneously distributed among the subdivisions and laminae (Fig. 3E0 ). Such a homogenous distribution was also present in the piriform and entorhinal areas.

4. Discussion The present study demonstrates a strong expression of HSP-27 in rats treated chronically with PTZ which remained restricted to astrocytes and endothelial cells in cerebral regions not yet affected by neurodegeneration. Similar to other animal models for the study of epilepsy (White, 2002; Morimoto et al., 2004), repeated PTZ treatment induced seizure activity which preferentially affected the hilus of the hippocampus as well as the entorhinal and piriform cortices, the latter of which is known to be directly connected to the cerebral regions which had responded to seizure activity (Schwabe et al., 2004). As indicated by the results obtained 1 and 4 days after the first PTZ treatment, glial HSP-27 upregulation seems to start in the piriform and/or entorhinal cortices. The piriform- and entorhinal cortices as well as the hippocampal hilus have been described as key structures in various paradigms of experimental epilepsy. In contrast to other allocortical areas,

Fig. 5. Sections through cortex (A–C) and dentate gyrus (D–F) of repeatedly PTZ treated rats stained for astrocytic GFAP (green) and HSP-27 (red). Note the strong blood vessel associated HSP-27 localization in cerebral cortex (A–A00 ) and in the molecular layer of the dentate gyrus (D–D0 , enlargement F–F0 ). In the larger accumulation of HSP-27 positive astrocytes in entorhinal cortex (B–C) and in the molecular layer of the hippocampal formation HSP-27 positive astrocytes were not directly associated with blood vessels. Note that GFAP stained only parts of the astrocytic cytoskeleton whereas HSP-27 filled up the whole cytoplasm (A0 –A00 and C). Bar: 100 mm.

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these brain regions do not respond with the phenomenon of preconditioning before status epilepticus is reached (for review see White, 2002; Morimoto et al., 2004). This is in agreement with the consistent HSP-27 induction in these cerebral regions in our chronically PTZ treated animals. As shown by the present observations, the PTZ-treatment (40 mg/kg) had to be repeatedly applied and the seizures must have occurred frequently before increased amounts of HSP-27 can be demonstrated as immunoreactive protein. Therefore, tonic-clonic seizures seem to affect the involved cerebral circuitries in a less severe manner than a single treatment with kainate, the latter of which results in accompanying neuronal death. Furthermore, not all individuals responded with the same intensity of HSP-27 induction. Such heterogeneous responses are particularly known from several epilepsy models, in which a wide range of individual responses was observed during treatment with anticonvulsive drugs (Brandt et al., 2004a,b). This is a especially well known fact for the PTZ model, because not all rats respond to PTZ injection with the same intensity regarding seizure amplitude or seizure duration (Andre et al., 1998). In addition, a much more widespread and rapid induction of c-fos-like immunoreactivity has been described (Shehab et al., 1992; Del Bel et al., 1998) after treatment with a single dose of 60 mg PTZ/kg. This indicates, that more cerebral regions could be initially affected than those which responded with increasing HSP-27 immunoreactivty in the present study. A comparison of c-fos induction and HSP-27 could be done on a regional level only, because c-fos is induced in neurons only. Therefore, comparing the topographical HSP-27 induction found in our study with that known for c-fos expression in the PTZ-model (Andre et al., 1998; Del Bel et al., 1998) or in the kainate model (Silveira et al., 2002), we found an almost identical regional distribution pattern. The only major differences were obtained for the habenulae (Del Bel et al., 1998), the substantia nigra and the locus coeruleus (Silveira et al., 2002), which expressed c-fos protein, but did not express detectable amounts of HSP-27. In comparison to the c-fos distribution reported by Andre et al. (1998), we have not found HSP-27 induction within the cingulate and retrosplenial cortices, but in contrast had a weak, mostly vascular, HSP-27 induction in the paraventricular hypothalamus which corresponded with the results reported by Del Bel et al. (1998), Shehab et al. (1992) and Silveira et al. (2002). These comparative data indicate that the reported glial and endothelial HSP-27 induction may occur in response to neuronal impairment within the affected regions except for

the hippocampus, since, within the anterior hippocampal formation, clusters of strong HSP-27 positive astrocytes remained restricted to the ventral aspect of the molecular layer of the dendate gyrus, whereas seizure related c-fos expression is reported for the pyramidal layer especially in CA3 (Silveira et al., 2002). Furthermore, the data indicate that the effects elicited in the distinct cerebral regions are dependent on the strength of the stimulus (60 mg PTZ/kg; Del Bel et al., 1998) and the age of the animals (Andre et al., 1998; Silveira et al., 2002). As shown by the lack of FluoroJade B positive neurons during our treatment with PTZ, glial HSP-27 induction occurs as a response to pathologic neuronal activity rather than to acute neurodegeneration. This is especially evident in the hilus of the hippocampus, for which it is known that the granular neurons are resistant to neuronal death (Valente et al., 2004) and which showed adjacent strong glial and endothelial HSP-27 induction. It is, however, noteworthy to mention that PTZ does not seem to affect the glial cells neither directly nor globally, but in a specific focally localized and seizure-dependent manner. This was additionally supported by the comparison with the results obtained for MSO-induced HSP-27 expression in astrocytes. Because glutamine synthetase is restricted to glial cells (Martinez-Hernandez et al., 1977), its inhibitor MSO affects astrocytes directly (Gutierrez and Norenberg, 1977). As shown here, a subconvulsive dose of MSO (Hindfelt and Plum, 1975) caused a homogenous, astrocytic HSP-27 induction within 24 h in all regions known to express the astrocyte-marker GFAP under normal conditions (Zilles et al., 1991). Taken together, repeated PTZ-induced seizures resulted in a much more focal pattern of HSP-27 protein distribution than known from the kainate model (Plumier et al., 1996; Kato et al., 1999), or as it occurs in response to hyperthermia (Krueger-Naug et al., 2000, 2002; Bechtold and Brown, 2003), or MSO injection. Furthermore, the lack of detectable amounts of HSP-27 24 h after a single application indicates that the mild hyperthermia which occurs after PTZ (maximum +1 8C, according to Speckmann, unpublished) or kainate (Ahlenius et al., 2002) injection does not contribute to the focal induction of HSP-27. Therefore, the observed PTZ-elicited HSP-27 induction seems to be related to the repeatedly induced acute seizure activity within certain cerebral circuitries, including especially the piriform-entorhinal cortex, the molecular layer of the dendate gyrus and the central nuclei of the amygdala. Focusing on the cerebral cortex a multifocal, astrocytic and blood vessel-associated pattern of HSP-27 immunor-

Fig. 6. (A–F) Co-localization (yellow) of HSP-27 (red) and the rat endothelial cell-specific marker RECA-1 (green) at day 14 after repeated PTZ-induced seizures in the cerebral cortex (A–A00 ), amygdale (B–B0 , arroweads mark the position of cell nuclei) and in the dentate gyrus (C–C0 ) in blood vessels which were not contacted by HSP-27 positive perivascular astrocytes. (D–D0 ) Image of a large HSP-27 immunoreactive cortical astrocyte which contacts a blood vessel with HSP-27 labelled endothelial cells (inset, arrow; arrowhead, nucleus of the endothelial cell). (E) Large cortical blood vessel which shows no detectable amounts of HSP-27 in its endothelial lining, which is contacted by HSP-27 immunoreactive astrocytic processes. The tiny yellow line (arrow) indicates a pseudocolocalization caused by tightly overlapping astrocytic endfeet. (F–F00 ) Images of a 3D-reconstructed HSP-27 negative, cortical blood vessel which branches in the deeper layers. Shortly below the branching all braches of the vessel were contacted by the endfeet of a strongly HSP-27 immunoreactive astrocyte (F). The endfeet of the astrocyte gave of net-like processes which surrounded the endothelial cells (F0 –F00 ). Bars: 10 mm (B, E); 16 mm (F00 ); 30 mm (A, D); 50 mm (C, F).

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eactivity was present in our repeatedly PTZ-treated rats similar to that described for the temporal cortex obtained after neurosurgery of patients with intractable epilepsy (Erdamar et al., 2000; Bidmon et al., 2004). Furthermore, in humans a similar solely astrocytic and/or endothelial HSP27 induction is reported which corresponds well to that seen after repeated PTZ-induced seizure activity, whereas in the kainate model glial and neuronal HSP-27 upregulation has been reported (Kato et al., 1999). In the specimens obtained from these patients HSP-27 was consistently found, regardless of their seizure frequencies (Bidmon et al., 2004) indicating that when induced, HSP-27 remains increased over longer periods in a manner similar to the situation reported for other neuropathological conditions (Lu et al., 2002). Therefore, it seems to be unlikely that HSP-27 was only transiently induced during the first hours after the first and second PTZ injections, indicating that repeated seizures, which may weaken the brain regions affected by PTZ induced seizures seem to be essential for HSP-27 induction in this model. The latter indicates that this PTZ-model may be the model of choice to study epilepsy related heat shock responses and their consequences.

both endothelial cells and their surrounding astrocytes show HSP-27 immunoreactivity, indicating that the strength of the pathologic stimuli may have affected both cell types. One explanation for these differential, vascular responses may be related to the seizure-associated, focal changes in local cerebral blood flow (LCBF) known to take place in epileptic foci (Hirase et al., 2004) which occur in much more cerebral regions (Andre et al., 2002) than those known to respond with glial or neuronal changes in the expression of heat shock proteins. For the blood vessels which enter the cerebral cortex it could be expected that the upper part, which is in contact with the glia limitans as well as with the subarachnoidal space and the surrounding cerebrospinal fluid (Jancsik and Hajos, 1999), may be affected more severely than their deeper distal branches, because here endothelial and perivascular astrocytic HSP-27 immunoreactivity was much stronger and occurred more frequently. HSP-27 has been shown to stabilize intracellular microfilaments (Lavoie et al., 1993; Hout et al., 1996), particularly in endothelial cells (Hout et al., 1997), which suggests that endothelial HSP-27 induction represents a cytoprotective stress response elicited by focal hyperperfusion. 4.2. HSP-27 expression in astrocytes

4.1. HSP-27 induction in cerebral blood vessels In our animals PTZ was injected i.p. and had to reach the cerebral parenchyma via the cerebral blood vessels. Therefore, it was not surprising that strong HSP-27 induction had occurred in blood vessels and particularly in glial cells associated with them almost symmetrically in both hemispheres. This finding may indicate that HSP-27 induction started around blood vessels. Since the blood vessel associated HSP-27 induction remained focal, it further suggests, that blood vessels and their associated glial cells were not affected globally as would be expected for drugs directly and globally affecting endothelial or glial cells. Our findings indicate that the focal, vessel associated induction of HSP-27 seems to be elicited by specifically localized seizure activity within certain affected cerebral circuits, since a more pharmacological effect of PTZ would have affected all endothelial cells and thus result in a much more generalized vascular and astrocytic response as found, e.g. following treatment with MSO. However, our findings indicate that at least in certain regions of the thalamus and hypothalamus blood vessels may respond first with HSP-27 induction, which may be followed by increasing glial responses. This could be similar to the results reported by Lu et al. (2002), who described an endothelial induction of HSP-27 and HO-1 after cerebral ischemia. At the moment it is still unclear why there were blood vessels which showed HSP-27 immunoreactive endothelial cells without an associated glial HSP-27 induction, whereas others showed a clear association with strongly HSP-27 positive glial cells, but exhibited no or non-detectable levels of HSP-27 in their endothelia. Only in the strongly HSP-27 positive foci did

The PTZ-induced seizures in our model may represent a relatively milder pathologic stimulus compared to other neuropathological conditions and animal models as already discussed above. However, focusing on the astrocyte population which responded with an upregulation of HSP-27, it was obvious that at the time points studied no concomitant induction of GFAP had occurred as is known from many other experimentally induced neuropathological conditions (Schroeter et al., 1995). Furthermore, no immunoreactivity for HSP-47 was detectable after PTZ treatment. During normal conditions, HSP-47 serves as a chaperone for pro-collagen. It is expressed during early steps of CNS formation and differentiation (Walsh et al., 1997), whereas its re-expression in adult brains seems to be pathology related and confined to proliferating and migrating cells. It is known that seizure activity could cause proliferation and/or migration in astrocytes, stem cells and microglia (Morimoto et al., 2004). Such migratory astrocytes and stem cells do not stain for GFAP, but express HSP-47 and HSP-27 together, e.g. after cerebral injury (Acarin et al., 2002; Turner et al., 1999) and in glioma cells (Morino et al., 1997). Also in comparison to the model used by Turner et al. (1999), PTZ treatment did not result in an activation of HSP-47 positive microglia. Therefore, it could be concluded that in our repeatedly PTZ-treated animals, seizure activity had not yet resulted in glial migration within the affected regions indicating that HSP-27 induction seems to represent an early seizure-related glial stress response. This indicates an important role of glial cells in the pathology of epilepsy. The factors which link glial HSP-27 induction to neuronal seizure activity may include glial

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glutamate transport (Watanabe et al., 1999), astrocytic glutamate metabolism (Kondziella et al., 2003; Sonnewald and Kondziella, 2003; Waagepetersen et al., 2003; Eloqayli et al., 2004), as well as glia-synaptic interactions (Haydon, 2001; Beattie et al., 2002; Piet et al., 2004). Since glial cells express a variety of receptors for excitatory neurotransmitters (Aronica et al., 2003; Kirchhoff et al., 2003; Krebs et al., 2003; Park et al., 2003; Seifert et al., 2004), direct excitotoxic stimuli could possibly affect glial cells as well as neurons. Furthermore, the appearance of apoptotic cell death after kainate application has been shown to contribute to HSP-27 induction (Wagstaff et al., 1999; Akbar et al., 2003). In addition, potassium chloride induced spreading depressions (Plumier et al., 1997a,b) and kainic acid induced elevated extracellular potassium concentrations (Kato et al., 1999) cause HSP-27 expression which depends on the activity of heat shock factor 2 (Iwaki et al., 1995; Sadamitsu et al., 2001). Furthermore, the PTZ-induced expression of HSP-27 in endothelial cells and blood vessel contacting astrocytes indicates, that HSP-27 was induced in regions where the blood–brain barrier (BBB) had been affected to some degree by PTZ (Ziylan et al., 1992; Goldman et al., 1992; Sahin et al., 2003) or by PTZ-induced seizure activity influencing neuron-to-astrocyte signalling. The latter aspect is essential for the dynamic control of cerebral microcirculation (Reilly, 2003; Zonta et al., 2003). Since no induction of heme oxygenase-1 was detectable in our rats, the BBB was certainly not injured to the point where systemic heme or iron could have leaked into the cerebral parenchyma as known from other neuropathological, as well as physiological conditions (Bergeron et al., 1998; Maines, 2002). The latter does not rule out BBB disturbances that may allow the leakage of even smaller molecules (Binder et al., 2004), which could affect perivascular astrocytes, eliciting a stress response involving HSP-27 induction. It has been hypothesised that disturbances in blood supply and seizure related cellular hypoxia may contribute to epileptic brain pathology (Caspers and Speckmann, 1972; Hoshi and Tamura, 1993; Zgodzinski et al., 2001). Such hypoxic episodes may cause the formation of reactive oxygen species (ROS), potentiating oxidative stress and inhibiting mitochondrial metabolism and the NO-sensitive soluble guanylyl cyclase causing a loss of functional vasodilation (Francois and Kojda, 2004). In fact, during PTZ-induced seizures the formation of ROS, namely hydroxyl radicals and increasing NOx levels have been reported (Rauca et al., 1999, 2004; Bashkatova et al., 2003; Han et al., 2000; Kaneko et al., 2002) and an oxidative stress induced increase in Mn-SOD has been reported for PTZtreated rats (Rauca et al., 2004), and for temporal cortex of patients with intractable epilepsy (Bidmon et al., 2002). Kato et al. (1999) described the loss of the antioxidant glutathione after kainic acid induced seizures. A recent study showed, that kindling with PTZ down-regulates the endogenous defence mechanisms against oxidative stress, a

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process which has been most potently prevented by the application of taurine (El-Abhar and El Gawad, 2003), whereas HSP-27 stabilizes intracellular glutathione levels (Mehlen et al., 1996a). It is important that the piriform and entorhinal cortices (Bidmon et al., 1997; Oermann et al., 1999) as well as CA3–CA4 (Che et al., 2001), which contain highest densities of constitutively neuronal nitric oxide synthase (nNOS) expressing neurons, become most strongly affected by seizure-related ROS. These findings indicate that NO and superoxide derivatives such as peroxynitrite may affect these cortical areas most severely in the vicinity of blood vessels, because nNOS expressing cortical neurons are mainly located next to branching arteriolae (Regidor et al., 1993). This hypothesis is further supported by the fact that the inhibition of NO prevents the induction of alpha b-crystallin in mice following kainic acid application (Che et al., 2001). In summary, as discussed above, HSP-27 has a very high potential for cytoprotection (He and Lemasters, 2003; Mehlen et al., 1996b; Akbar et al., 2003) and it could become induced in response to a variety of seizure-related stressors. Interestingly, HSP-27 induction in our PTZ-model remained restricted to glial cells and endothelial cells, which highlights their involvement in epileptic pathology beside that of neurons. According to these actions, it may be upregulated as a cellular defence mechanism. At the regional level, HSP-27 expression may be a sensitive marker for the demarcation of cerebral regions affected by seizure activity, which respond by the initiation of a cellular stress response program, as has been demonstrated in animal models, including the PTZ model of epilepsy, as well as in human patients. The linkage, by means of molecular engineering, of pathology related, focal HSP-27 expression to the expression of other anticonvulsant proteins or to an endothelial repression of multidrugtransporters (to cope with pharmacoresistance) may open new avenues for the development of therapeutic strategies, since it would enable their sitespecific action, restricted to the affected brain regions and thus limiting associated negative side effects for the remaining CNS.

Acknowledgements The authors thank L. Igdalova, C. Opfermann-Ru¨ ngeler, E. Boening for excellent assistance and artwork. This study was supported by the Deutsche Forschungsgemeinschaft (DFG).

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