Lithium chloride, distrene plasticizer xylene (DPX), pilocarpine hydrochloride, potassium permanganate and cresyl violet acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA) and atropine methyl nitrate was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan). Fluoro-Jade B and glial fibrillary acidic protein (GFAP) were purchased from Millipore (Temecula, CA, USA). The CD11b antibody came from Abcam (Cambridge, MA, USA).

The pilocarpine model of SE in mice was previously described [1213]. Briefly, male C57BL/6 mice (7~8 weeks of age) were administered atropine methyl nitrate (1.2 mg/kg, i.p.) 30 min before the injection of pilocarpine hydrochloride (320 mg/kg, i.p.). After pilocarpine administration, the behavior of the mice was closely monitored for approximately 6 h to evaluate the onset time of stage 4 seizure, SE, severity, and mortality. SE was defined as a continuous motor seizure at stage 4 (rearing and falling), stage 5 (loss of balance, continuous rearing and falling) and stage 6 (severe tonic clonic seizures) (Racine, 1972) [14]. In this study, only mice that showed severe tonic-clonic seizures were included. After 15 min of pilocarpine administration, lithium chloride (80 mg/kg, i.p.) (n=29) or vehicle (saline, n=31) was administered. The mice were sacrificed 3 days after SE induction. All procedures were approved by the Institutional Animal Care and Use Committee for Dankook University (DKU-14-034).

The mice were anesthetized with ethyl ether and transcardially perfused with cold saline, followed by 4% paraformaldehyde in phosphate buffered saline (PBS), pH 7.4. Their brains were post-fixed for 4 h, then cryoprotected in 30% sucrose in PBS. Sequential coronal sections (25 µm thick) through the hippocampus were prepared using a cryocut microtome (CM3050S, Leica, Germany).

Live cells were labeled using cresyl violet. The tissues were mounted on gelatin-coated slides for overnight before use. After dehydration in a graded alcohol series, hippocampal sections were stained for 20 min with pre-warmed 0.3% cresyl violet solution at room temperature. After destaining with a solution of 95% ethanol and 0.3% glacial acetic acid, the sections were dehydrated using 100% ethanol, followed by 100% xylene. The sections were then mounted with DPX.

Dead or dying cells were labeled using Fluoro-Jade B. The tissues were mounted on gelatin-coated slides for overnight before use. After dehydration in a graded alcohol series, hippocampal sections were incubated in 0.06% potassium permanganate solution for 10 min. Next, the sections were stained with 0.0004% Fluoro-Jade B solution containing 0.1% glacial acetic acid for 20 min at room temperature. They were then washed with distilled water, dried, and mounted with DPX.

For immunohistochemical labeling, free-floating sections were sufficiently washed with PBS, then blocked with 5% BSA and 0.4% Triton X-100 in PBS. The tissues were then incubated for overnight at 4℃ with mouse anti-glial fibrillary acidic protein antibody (1:500; Millipore, Temecula, CA, USA) or rat anti-CD11b antibody (1:200; Abcam, Cambridge, MA, USA). After washing, the sections were incubated with Alexa Fluor 488 anti-rat IgG (1:500; Life Technologies, Carlsbad, CA, USA) and Alexa Fluor 555 anti-mouse IgG (1:500; Life Technologies, Carlsbad, CA, USA) in PBS for 2 h at room temperature. After washing in PBS, coverslips were inverted onto the slides over a drop of VECTASHIELD Mounting Medium (Vector Laboratories, Inc., Burlingame, CA, USA). Alexa Fluor 488 (excitation, 488 nm; emission, >519 nm) and Alexa Fluor 555 (excitation, 555 nm; emission, >565 nm) -labeled neurons were imaged using confocal microscopy (LSM 700, Carl Zeiss, Germany).

Data are expressed as means±SEM. Student's t test was used for statistical comparisons (GraphPad Prism 5 software v5.01, San Diego, CA, USA). A p value of < 0.05 was considered statistically significant.

To investigate whether lithium could inhibit seizure susceptibility after pilocarpine-induced SE, we employed the pilocarpine model of SE in mice. Eight-week-old, C57BL/6 male mice were injected with atropine (1.2 mg/kg, i.p.), then injected with pilocarpine hydrochloride (320 mg/kg, i.p.) 30 min later. Lithium (80 mg/kg, i.p.) was administered 15 min after the pilocarpine injection. Behavioral changes after the injection with pilocarpine hydrochloride were recorded according to Racine's scale [14] as described in Materials and Methods. Briefly, all mice developed diarrhea, piloerection, and other signs of cholinergic stimulation about 5 min after pilocarpine injection. Mice then, exhibited head bobbing, scratching, chewing, and exploratory behavior during the following 15 to 20 min. Recurrent seizures started around 15 to 20 min after pilocarpine administration. These seizures progressed to SE at about 35 to 40 min after pilocarpine administration [1516]. The mean latency to onset of SE was significantly delayed in the lithium-treated group compared to the vehicle-treated group (vehicle, 28.6±1.6 min; lithium, 36.0±3.9 min) (Table 1). The percentages of seizures and SE were also decreased in the lithium-treated group (vehicle, 96.8%; lithium, 79.3%). Mortality observed over a period of 3 days following pilocarpine hydrochloride injection was reduced in the lithium-treated group (vehicle, 38.7%; lithium, 27.6%). In all of the animals with SE, myoclonus persisted for more than 6 h after pilocarpine injection and no difference in behavioral patterns was observed between the two groups. These data indicate that lithium significantly delayed the onset time of SE and reduced mortality compared to the vehicle-treated group.

We observed reproducible neuronal death in the hippocampus at 3 days after pilocarpine injection, consistent with a previous study [17]. Neuronal cell death was determined using cresyl violet staining (Fig. 1). Many pyknotic nuclei were observed in the hippocampal CA1, CA3 and the hilus subfields of vehicle-treated mice (Fig. 1A(e)~A(h), 1B and 1C). In the sham-treated mice, cresyl violet-positive cells were detected in CA1 at 103.9±7.0 cells/0.2 mm, followed by CA3 at 82.4±1.8 cells/0.2 mm and the hilus at 776.8±25.0 cells/mm² (Fig. 1B and 1C). Compared to the sham-operated mice, cresyl violet-positive cells were dramatically reduced in CA1 at 73.1±1.3 cells/0.2 mm, followed by CA3 at 49.9±1.9 cells/0.2 mm and the hilus at 399.2±13.8 cells/mm² in the vehicle-treated mice (Fig. 1B and 1C). However, cresyl violet-positive cells were significantly increased in CA1 at 80.4±1.5 cells/0.2 mm, CA3 at 55.8±2.1 cells/0.2 mm and the hilus at 537.0±32.1 cells/mm² in the lithium-treated mice compared to the vehicle-treated mice (Fig. 1B and 1C). In parallel with the cresyl violet staining data, we confirmed that obvious neuronal cell death at 3 days post SE was detected in CA1, CA3 and the hilus (Fig. 2) (vehicle, 23.6±1.5, 7.8±3.1, and 644.9±63.1, respectively). Furthermore, neuronal cell death was significantly decreased in all subfields of the hippocampus in the lithium-treated mice (CA1, 16.8±2.9, CA3, 1.5±0.4, hilus, 442.9±74.9). However, Fluoro-Jade B-positive cells were not detected in the hippocampal regions of the sham-treated mice (data not shown). Quantitative analyses of Fluoro-Jade B-positive cells indicate that lithium post-treatment induced robust neuroprotective effects against pilocarpine-induced neuronal cell death in the hippocampus (Fig. 2B and 2C).

Extensive reactive astrogliosis and microglial activation (microgliosis) have been associated with SE-induced neuro-pathological changes [18]. Therefore, we examined the effects of lithium on astrocytic and microglial changes by testing GFAP and CD11b immunoreactivity (astrocyte and microglia markers, respectively). At 3 days after pilocarpine-induced SE, more GFAP-labeled astrocytes were observed in CA1, CA3 and the hilus (Fig. 3B and 3B1). However, there was no prominent difference in GFAP immunoreactivity between the vehicle- and lithium-treated groups. In addition, CD11b-labeled cells were increased in all three hippocampal subfields 3 days following pilocarpine-induced SE, while no distinct CD11b immunoreactivity was observed in any of the hippocampal subfields of the lithium-treated animals (Fig. 4).