This study utilized the progeny of male Sprague-Dawley (SD) rats (7 weeks old) obtained from Experimental Animal Center, Hallym University (Chuncheon, South Korea). The animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity and lighting conditions (22 ± 2°C, 55 ± 5% and a 12:12 light/dark cycle with lights). Experimental procedures were approved by the Institutional Animal Care and Use Committee of the Hallym university (Chuncheon, South Korea). The number of animals used and their suffering was minimized in all cases. All reagents were obtained from Sigma-Aldrich (USA), unless otherwise noted.

Animals were anesthetized with 1–2% Isoflurane in O₂ and placed in a stereotaxic frame. Animals were then implanted with a brain infusion kit 1 (Alzet, Cupertino, CA, USA) into the right lateral ventricle (1 mm posterior; 1.5 mm lateral; −3.5 mm depth). The infusion kit was sealed with dental cement and connected to an osmotic pump (1007D, Alzet, Cupertino, CA, USA) containing control siRNA or HSP25 siRNA. For knockdown of HSP25, we applied a set of four on-target HSP25 rat siRNAs and a non-targeting control was used in the preliminary study. Of four on-target siRNAs, a siRNA sequence corresponding to coding region (sense 5-GGAACAGUCUGGAGCCAAGUU-3; antisense 5-CUUGGCUCCAGACUGUUCCUU-3) was selected as the best probe (reduction efficiency: 43.2%, Genolution Pharmaceuticals, Inc., South Korea), and used for the final experiments. A siRNA sequence coding region 5-GCAACUAACUUCGUUAGAAUCGUUAUU-3 was used as a non-targeting control siRNA. The pump was placed in a subcutaneous pocket in the dorsal region. Animals received 0.5 μl/h of saline or (100 μM in saline) for 1 week (Kim et al., 2011, 2014b). Some animals were also implanted with monopolar stainless steel electrode (Plastics One, Roanoke, VA, USA) into the left dorsal hippocampus (−3.8 mm posterior; 2.0 mm lateral; −2.6 mm depth). Connecting wire and electrode socket were then inserted in an electrode pedestal (Plastics One, Roanoke, VA, USA), secured to the exposed skull with dental acrylic. Three days after surgery, rats were induced SE by LiCl-pilocarpine.

Animals were given LiCl (3 mEq/kg, i.p) 24 h before the pilocarpine treatment. To reduce the peripheral effects of pilocarpine, atropine methylbromide (5 mg/kg, i.p.) was also received 20 min before the pilocarpine treatment. To induce SE, animals were treated with pilocarpine (30 mg/kg, i.p.). Control animals received saline in place of pilocarpine. For evaluation of the effect of siRNA knockdown on seizure susceptibility in response to LiCl-pilocarpine, animals were recorded EEG signals with a DAM 80 differential amplifier (0.1–3000 Hz bandpass; World Precision Instruments, Sarasota, FL, USA). EEG activity was measured during the 2 h recording session from each animal. The data were digitized (400 Hz) and analyzed using LabChart Pro v7 (AD Instruments, Bella Vista, NSW, Australia). Time of seizure onset was defined as the time point showing paroxysmal depolarizing shift, which lasted more than 3 s and consisted of a rhythmic discharge between 4 and 10 Hz with amplitude of at least two times higher than the baseline EEG (Kim and Kang, 2011). EEG activity was measured during the 2 h recording session from each animal. Spectrograms were also automatically calculated using a Hanning sliding window with 50% overlap. Two hours after SE onset, diazepam (Valium; Roche, France; 10 mg/kg, i.p.) was administered and repeated, as needed.

Animals were perfused transcardially with phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) under urethane anesthesia (1.5 g/kg i.p.). The brains were removed, postfixed in the same fixative for 4 h and rinsed in PB containing 30% sucrose at 4°C for 2 days. Thereafter the tissues were frozen and sectioned with a cryostat at 30 μm and consecutive sections were collected in six-well plates containing PBS. For western blot (WB), animals were decapitated under urethane anesthesia. The hippocampus was rapidly removed and homogenized in lysis buffer. The protein concentration in the supernatant was determined using a Micro BCA Protein Assay Kit (Pierce Chemical, Rockford, IL USA).

Table 1 is a list of the primary antibodies used in the present study. Sections were incubated in a mixture of antisera in PBS containing 0.3% Triton X-100 overnight at room temperature. After washing, sections were incubated in a mixture of FITC- and Cy3-conjugated IgG (or streptavidin, Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA; diluted 1:250) for 2 h at room temperature. Images were captured using an AxioImage M2 microscope. Fluorescent intensity was measured using computer-based image analysis program (AxioVision Rel. 4.8 software, Germany). Fluorescent intensity was then standardized by setting the threshold level (mean background intensity obtained from five image input). Manipulation of the images was restricted to threshold and brightness adjustments to the whole image.

TUNEL staining was performed with the TUNEL apoptosis detection kit (Merck Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Following TUNEL reaction, GFAP immunofluorescence (IF) staining was performed. For nuclei counterstaining, we used Vectashield mounting medium with DAPI (Vector, Torrance, CA, USA).

For quantification of immunohistochemical data, cells in 2–4 regions (1 × 10⁵ μm²) from each section were counted on 20× images. Results are presented as means ± SD of 15–24 regions from seven animals. All immunoreactive cells were counted regardless of the intensity of labeling. Cell counts were performed by two different investigators who were blind to the classification of tissues.

Aliquots containing 20 μg total protein were loaded into a polyacrylamide gel. After electrophoresis, gels were transferred to nitrocellulose transfer membranes (Schleicher and Schuell BioScience Inc., Keene, NH, USA). To reduce background staining, membranes were incubated with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 for 45 min, followed by incubation with the primary antibody (Table 1), and subsequently with an HRP-conjugated secondary antibody. Immunobands were detected with an ECL Western Blotting Detection Kit (Amersham, Piscataway, NJ, USA). Intensity measurements were represented as the mean gray-scale value on a 256 gray-level scale (Kim and Kang, 2011).

All data obtained from the quantitative measurements were analyzed using Mann-Whitney or Kruskal-Wallis test to determine statistical significance. Bonferroni’s test was used for post hoc comparisons. A p-value below 0.05 was considered statistically significant (Kim and Kang, 2011; Kim et al., 2014a).

Figure 1A shows that HSP25 band was detected from 12 h post-SE animals (p < 0.05 vs. non-SE animal). HSP25 protein expression increased until 3 days after SE (p < 0.05 vs. non-SE animal), and gradually decreased 1 and 4 weeks after SE (p < 0.05 vs. 3 days after SE; Figures 1A,B). Immunohistochemical study revealed that HSP25 expression was observed in astrocytes within the all hippocampal region 3 days after SE. One week after SE, HSP25 expression was reduced in the CA3 field and the DG (p < 0.05 vs. 3 days after SE; Figures 1C,D). Thus, HSP25 expression was mainly observed in astrocytes within the stratum radiatum of the CA1 field. Four weeks after SE, HSP25 expression was also declined in the CA1 field (p < 0.05 vs. 3 days after SE; Figures 1C,D). These findings indicate that HSP25 expression showed the distinct spatio-temporal specific pattern in the hippocampus following SE.

In the molecular layer of the DG, 50% of astrocytes showed HSP25 expression 3 days after SE (Figures 2A,B). One week after SE, total number of astrocytes was reduced as compared to 3 days after SE (p < 0.05, respectively; Figures 2A,B). The fraction of HSP25 positive astrocytes in total astrocytes was also declined to 9.3% in this region (p < 0.05, respectively; Figures 2A,B). Four weeks after SE, the fraction of HSP25 positive astrocytes in total astrocytes was decreased to 4.3%, since the total number of astrocytes was increased as compared to 3 days after SE (p < 0.05; Figures 2A,B).

In the CA1 field, about 70% of astrocytes showed HSP25 expression 3 days after SE (Figures 3A,B). One week after SE, the fraction of HSP25 positive astrocytes in total astrocytes was 48% of astrocytes (Figures 3A,B). However, total number of astrocytes was unaltered. Four weeks after SE, total number of astrocytes was reduced in this region, as compared to 3 days after SE (p < 0.05; Figures 3A,B). The fraction of HSP25 positive astrocytes in total astrocytes was also decreased to 19% (p < 0.05 vs. 3 days after SE; Figures 3A,B). Interestingly, HSP25 was accumulated in clasmatodendritic astrocytes, which had round-shaped edematous cell body, short blunt processes, loss of distal processes, vacuoles, GFAP tangles (aggregation) and nuclear dissolution (absence of nucleus or watery pale nuclear staining), more than that in reactive astrocytes (Figure 3A). These findings indicate that the distinct HSP25 induction may be relevant to the different patterns of SE-induced astroglial loss in the hippocampus.

To investigate the role of HSP25 in SE-induced astroglial responses, we applied HSP25 knockdown before SE induction. Control- or HSP25 siRNA-infusion could not induce neurotoxicity (hind-limb paralysis, vocalization, food intake, seizure or neuroanatomical damage), indicating that both siRNA did not affect brain activity under normal condition. After SE induction, the first behavioral seizure occurred 32.1 and 34.3 min in control siRNA- and HSP25 siRNA-infused animals, respectively. In addition, both control siRNA- and HSP25 siRNA-infused animals showed episodes of high-amplitude and high-frequency discharges representing typical SE. An EEG analysis revealed no difference in the latency of seizure on-set and total EEG power during SE between control siRNA- and HSP25 siRNA-infused animals (Figures 4A–C). As shown in Figure 4D, HSP25 siRNA resulted in an approximate 43.2% reduction of HSP25 protein expression level following SE, as compared with control siRNA (p < 0.05). These findings indicated that HSP25 siRNA effectively inhibited SE-induced HSP25 induction without alterations in seizure susceptibility or severity in response to pilocarpine.

Next, we investigated the effect of HSP25 knockdown on SE-induced astroglial apoptosis in the DG using TUNEL staining. Consistent with our previous studies (Kang et al., 2006; Kim et al., 2010b), control siRNA-infused animals showed apoptotic astroglial degeneration in the DG 1 week after SE (Figures 5A–C). HSP25 siRNA exacerbated astroglial loss in this region, and increased the number of TUNEL positive astrocytes (p < 0.05 vs. control siRNA infusion; Figures 5A–C). Thus, it is likely that HSP25 may play an anti-apoptotic role in astrocytes within the DG.

Since up-regulations of LAMP1 and LC3-II represent clasmatodendrosis (Ryu et al., 2011a,b), we applied double immunofluorescent studies for GFAP and LAMP1/LC3-II to evaluate the effect of HSP25 siRNA on autophagic astroglial death in the CA1 region. Four weeks after SE, control siRNA-infused animals showed clasmatodendrosis in the stratum radiatum of the CA1 region (Figure 6A). Consistent with our previous studies (Ryu et al., 2011a,b), clasmatodendritic astrocytes showed LAMP1-positive vacuolization and up-regulated LC3-II expression (Figures 6B,C). HSP25 siRNA effectively abrogated LAMP1-positive vacuolization and LC3-II over-expression. In addition, HSP25 siRNA prevented the decrease in the total number of astrocytes (p < 0.05 vs. control siRNA; Figures 6A–E). These findings indicate that sustained HSP25 expression may contribute to SE-induced autophagic astroglial death in the CA1 region.

Protein kinase RNA (PKR)-like ER kinase (PERK) activation mediates the initial ER stress response by phosphorylating eukaryotic initiation factor 2α (eIF2A). In turn, the activation of the PERK/elF2A leads to ER stress-induced cell death via CCAAT/enhancer-binding protein homologous protein (CHOP)-dependent apoptosis (Moreno et al., 2012; Moreno and Tiffany-Castiglioni, 2015) or CHOP-independent autophagy (Bernales et al., 2006; Ito et al., 2009; Matsumoto et al., 2013). Recently, we have reported that clasmatodendritic astrocytes contained phospho-PERK (pPERK) and phospho-eIF2A (peIF2A) immunoreactivities without CHOP expression, and that up-regulated calnexin (CNX) expression is associated to the activation of autophagy leading to clasmatodendrosis (Ryu et al., 2011a,b; Ko et al., 2015). Therefore, it is likely that sustained HSP25 expression in CA1 astrocytes may be relevant to clasmatodendrosis via ER stress-mediated autophagy. To confirm this hypothesis, we investigated the effect of HSP25 knockdown on astroglial ER stress induced by SE. In non-SE animals, pPERK expression was rarely observed in CA1 astrocytes (Figure 7A). Following SE, clasmatodendritic astrocytes showed pPERK and peIF2A immunoreactivity, which was attenuated by HSP25 siRNA infusion (p < 0.05 vs. control siRNA; Figures 7A,B). In addition, most of clasmatodendritic astrocytes showed strong CNX expression without CHOP, protein-disulfide isomerase (PDI) or glucose-regulated protein 78 (GRP78) expressions (Figures 7C, 8A). HSP25 knockdown effectively decreased CNX expression in astrocytes as well as clasmatodendritic astroglial death (p < 0.05 vs. control siRNA infusion; Figures 8A,B). These findings indicate that accumulation of HSP25 protein may induce autophagic astroglial death via prolonged ER stress.