Adult Sprague-Dawley male rats (Charles Rivers, Spain) were housed in pairs in standard cages on a ventilated rack (Tecniplast, Buguggiate, Italy). The animal room was under controlled conditions of temperature (22 $\pm$ 2 °C) and was under a light/dark cycle of 12 h (8 AM–8 PM). Rats were left undisturbed for a minimum of 5 d to allow adaptation to the new environment. Animals had free access to food chow and water. Access to food only was restricted for the 12 h before the PET scan procedure to minimize the eventual competition for the glucose transporters between circulating glucose and [${}^{18}$F]FDG. Rats were weighed daily in the morning, and body weights (BW) were recorded. All procedures were carried out in accordance with animal welfare regulations of the European Union (2010/63/UE) and Spain (RD53/2013) and was approved by the Animal Research Ethical Committee of the Universidad Complutense de Madrid. All efforts were made to minimize the suffering of the animals.

The lithium-pilocarpine model was used to trigger the SE as previously reported [28, 29, 30]. Briefly, lithium chloride (3 mEq/kg ($\approx$127 mg/kg i.p.); Sigma–Aldrich, St. Louis, MO, USA) was administered approximately 18 h before the injection of pilocarpine to induce the SE. Methyl-scopolamine (2 mg/kg, i.p.; Sigma–Aldrich, St. Louis, MO, USA) was administered 30 minutes before pilocarpine-induced SE (25 mg/kg, i.p.; Sigma–Aldrich, St. Louis, MO, USA) to minimize the cholinergic peripheral effects on muscarinic receptors. Forty-five minutes after rats reached the SE, pentobarbital (25 mg/kg, i.p.) was administered to stop the SE. When after 30 minutes the convulsant activity persisted, another half-dose of pentobarbital was administered. In this regard, although diazepam is more used to terminate SE, pentobarbital has shown a high efficacy to stop SE as shown by previous studies carried out by our group [28, 29, 30] and others [35, 36, 37, 38, 39]. Rats from the corresponding control group received the same treatments, with the exception that saline was administered instead of pilocarpine.

The LD50 for FLU in rats when administered i.p. is 185 mg/kg (https://cdn.caymanchem.com/cdn/msds/21447m.pdf). In addition, studies in rats have shown that FLU administered i.p at a dose of 66 mg/kg did not affect movement, feeding and drinking behavior, alertness, and sustained attention to the environment, while reaching body fluids concentrations able to block TRPM2 channels [40]. Based on this information, and to evaluate the eventual effects of FLU, a single intermediate dose of this NSAID (100 mg/kg in DMSO, i.p; Sigma–Aldrich, St. Louis, MO, USA) was chosen to be administered 2 h before methyl-scopolamine. The respective control groups were injected with DMSO as vehicle (1 mL/kg). Thus, initially the experiment consisted of 4 experimental groups with 32 rats distributed as follows: (i) vehicle + saline (VEH + SAL, n = 6), FLU + saline (FLU + SAL, n = 6), vehicle + pilocarpine (VEH + PILO, n = 8) and FLU + pilocarpine (FLU + PILO, n = 12) (Fig. 1A). The number of rats included in the groups subjected to SE was higher having in view the risk of mortality associated to the procedure.

Fig. 1.

Descriptive data. (A) Schematic representation of seizure model and drug treatment methodology with the resultant experimental groups. (B) Graphical representation of animals BW change relative to the initial BW. Data is expressed as mean $\pm$ SD, where *p 0.05, **p 0.01 ***p 0.001 when compared to VEH + SAL group and ##p 0.01 ###p 0.001 when compared to their respective basal measure; mixed-measure analysis followed by post-hoc Tukey test. (C) Mortality rates representation per treatment group. (D) Latency time (in min) to SE after pilocarpine injection in pilocarpine injected rats. Data is expressed as mean $\pm$ SD; Student t-test.

Three days after the SE, during the latent period, brain glucose metabolism was evaluated by in vivo [${}^{18}$F]FDG-PET imaging as previously reported [28, 29, 30]. In brief, from the previous evening the rats were fasted (at least 12 h) and then, they were short-term anesthetized with isoflurane (2% isoflurane in 100% oxygen). A blood sample was collected from the tail vein to measure glucose concentrations and immediately after [${}^{18}$F]FDG was injected into the tail vein (12.99 $\pm$ 0.04 MBq in 0.2 ml of 0.9% NaCl; Curium Pharma, Madrid, Spain). Then, a 30 min period, during which animals were awake, was left to ensure the brain uptake of the radiotracer. For the images acquisition, rats were again anesthetized with isoflurane. Studies consisted in a static PET acquisition followed by a high-resolution computed tomography (CT) scan. The equipment used was a dedicated-small animal PET/CT (Albira PET/CT dual scanner, Bruker, Ettlingen, Germany).

After acquisitions, PET images were reconstructed by maximum likelihood expectation maximization algorithm applying decay, random and scatter corrections (Albira Reconstructor, Bruker, Ettlingen, Germany). CT scans were co-registered to a magnetic resonance imaging (MRI) rat brain with pre-defined volumes of interest (VOIs). Each CT mathematical transformation was applied to co-register PET images to the MRI template to obtain tracer uptake values in the different brain areas (kBq/mL; PMOD 4.1, PMOD Technologies Ltd., Zurich, Switzerland). The regional glucose metabolic activity was evaluated in brain areas known to be involved in epileptogenesis by calculating the standard uptake value (SUV), in order to correct the individual weight differences, by applying the following equation: [Tracer uptake (kBq/mL) * BW (g)) / (Tracer dose administered (kBq))].

Animals were sacrificed by decapitation the day after the PET scan. Brains were collected and stored at –80 °C. Thirty-$\mu$m thickness coronal slices at –20 °C were obtained using a cryostat (Leica CM1850, Leica Biosystems, Wetzlar, Germany). Slices were placed onto Superfrost Plus slides (Epredia, Kalamazoo, MI, USA) and stored again at –80 °C until further histochemical analyses.

Nissl staining was performed to obtain a qualitative histological representation of hippocampal integrity. Sections containing the anterior hippocampus were fixed in formaldehyde (4% in phosphate buffer saline, 30 min) and then transferred to a solution of 0.5% of cressyl violet in 0.1% acetic acid for 1 h. Slices were washed in distilled water, dehydrated in ethanol graded solutions (70%, 95% and 100%), cleared in xylene and finally covered with DPX mounting medium (Merck Sigma-Aldrich, Darmstadt, Germany). The images were collected with a digital camera (DFC425 camera, Leica, Wetzlar, Germany) coupled to a microscope (Leitz Laborlux S, Leica, Wetzlar, Germany).

Fluoro-Jade C labeling was used as a marker of hippocampal neurodegeneration [41]. Briefly, brain slices were fixed in 4% formaldehyde, washed in phosphate buffer, and then rinsed in basic alcohol, 100% ethanol and distilled water. Afterwards, slides were rinsed in 0.06% potassium permanganate, 0.1% acetic acid solution containing 0.0001% Fluoro-Jade C (Merck Millipore, Darmstadt, Germany), distilled water and xylene, and mounted with DPX.

GFAP immunofluorescence was evaluated as a marker of reactive astrogliosis in the hippocampus. The sections were fixed in 4% formaldehyde, rinsed in Tris-buffered saline (TBS pH 7.6, 30 minutes) and blocked in 3% bovine serum albumin (BSA), 0.1% triton X-100 in TBS one hour. For GFAP immunofluorescence, slices were incubated with conjugated anti-GFAP-Cy3 antibody (1:500 1% BSA/TBS; Sigma–Aldrich, St. Louis, MO, USA) at 4 °C overnight and cleared in 0.1% Tween 20 and mounted with Mowiol.

NeuN immunofluorescence was evaluated as a biomarker of neuronal integrity. To this end, slides were incubated at 4 °C overnight with a primary antibody (mouse anti-NeuN 1:200, ref. MAB377, Millipore, Darmstadt, Germany), washed in TBS-0.1% tween 20 and, incubated with the anti-mouse secondary antibody (1:140, 1% BSA/TBS, ref. T5393, Sigma–Aldrich, St. Louis, MO, USA) conjugated with tetramethylrhodamine isothiocyanate (TRITC) for 2 h at room temperature. Then, slides were washed again in TBS-0.1% tween 20 and covered with Mowiol.

Images from Fluoro-Jade C, GFAP and NeuN were acquired using a Leitz Laborlux S microscope (Leica Biosystems, Wetzlar, Germany). The filters used were as follows: FITC filter for Fluoro-Jade C and TRITC filter for GFAP and NeuN. In order to carry out the quantification, the different hippocampal subareas (CA1, CA3 and hilus) of each brain slice were manually delimited by an operator blinded to the animal’s identification and the mean fluorescence signal value of those regions were obtained. The average value for each animal was obtained from 4 different brain slices containing dorsal hippocampus. Both delimitation and quantification steps were performed with ImageJ software (NIH, available at the following website: https://imagej.nih.gov/ij/).

Data are shown as mean $\pm$ standard deviation (SD). In the BW analysis, a mixed-measures statistical test was used due to the high mortality rates. For mortality, a z-test for rates and proportions was used. Latency time to SE was evaluated only in the PILO-treated groups, and the results were analyzed by Student t-test. The differences among groups were evaluated using two-way ANOVA with FLU and pilocarpine treatments as the two main factors and, followed when appropriated by post-hoc comparisons with the Tukey’s tests. The analyses were carried out using the SigmaPlot 11.0 (Systat Software Inc., Palo Alto, CA, USA). Statistically significance was considered when p 0.05.

Results were analyzed as cumulative BW changes relative of the initial BW. When groups were compared to their main control (VEH + SAL), VEH + PILO animals only showed significant differences in BW at days 1 and 4 after SE (p = 0.003 and p = 0.01) whether FLU + PILO was different until the sacrifice of animals (p = 0.04, p = 0.01, p = 0.005 and p 0.001). Surprisingly, even FLU + SAL showed a significant difference in BW at the end of the experiment (p = 0.03). Nevertheless, VEH + PILO groups were able to significantly regain BW from day 2 to day 4 (1 day after the weight loss induced by the SE; p = 0.069 and p = 0.66) whereas the FLU + PILO rats kept losing weight (p 0.001; Fig. 1B).

Pilocarpine administration triggered SE after 39.9 $\pm$ 11.6 min in the VEH + PILO group. Pre-treatment with FLU did not significantly affect the latency time (p = 0.302), the SE occurring after 26.9 $\pm$ 5.8 min (Fig. 1D).

Unexpectedly, there was no mortality associated with the SE in the VEH + PILO group. By contrast, FLU pre-treatment arkedly increased mortality (z = 2.513; p = 0.012). Thus, from the 12 initial animals in the FLU + PILO group, 8 rats died (66.6%). The temporal mortality occurrence was as follows: 1 rat died 1h after the SE, 2 rats died 24 h after the SE and 5 rats 48 h after the SE. Surprisingly, 2 rats out of 6 also died in the FLU + SAL group (Fig. 1C). Thus, the following results were obtained from data collected from the surviving rats. The final number in the 4 experimental groups were: (i) VEH + SAL, n = 6; (ii) FLU + SAL, n = 4; (iii) VEH + PILO, n = 8 and (iv) FLU + PILO, n = 4.

Comparing VEH + PILO vs VEH + SAL (Fig. 2), pilocarpine-induced SE resulted in significant brain glucose hypometabolism, measured as percentage of [${}^{18}$F]FDG uptake, in the following areas: hippocampus (–13.94%, p = 0.03), cortex (–18.95%, p = 0.0127), striatum (–17.5%, p = 0.0137), and amygdala (–14.23%, p = 0.02). No effects were found in thalamus (–9.86%, p = 0.0796) or in cerebellum (–8.98%, p = 0.811). Independently of pilocarpine treatment, FLU pre-administration had no effects. Thus, comparing FLU + PILO vs FLU + SAL the percentage of [${}^{18}$F]FDG uptake was as follows: Hippocampus: –19.54%; Cortex: –21.36%; Thalamus: –17.86%; Amygdala: –19.24%; Striatum: –22%; Cerebellum: –8.98%.

Fig. 2.

SE induced by pilocarpine decreased [${}^{\mathrm{18}}$F]FDG-PET uptake, causing an hypometabolic condition. (A) Representative [${}^{18}$F]FDG-PET imaging corrected by SUV. (B) In insulted groups (rats injected with the convulsant pilocarpine), [${}^{18}$F]FDG-PET uptake was lower in hippocampus, cortex, amygdala and striatum, without any protective effect of FLU treatment; data is expressed as mean $\pm$ SD *p 0.05; two-way ANOVAs followed by post-hoc Tukey test.

The Fig. 3 depicts representative images obtained from the Nissl staining in brain sections containing the anterior hippocampus. The images are shown as qualitative data. As can be observed, pilocarpine reduced the intensity of the staining in all the hippocampal subregions, being more marked in the CA1. Regarding FLU, pre-administration of this NSAID had no apparent effects in FLU + SAL or in FLU + PILO in comparison to their vehicle control groups.

Fig. 3.

Neurohistochemical images of cresyl violet (Nissl staining) show a severe degeneration of CA1 and CA3 regions in pilocarpine-insulted groups. As shown, this disruption of the hippocampal integrity was not ameliorated by previous administration of 100 mg/kg FLU.

Results from Fluoro-Jade C fluorescence intensity in the different hippocampal areas were calculated in the VEH + PILO and FLU + PILO as percentage of their respective control groups (refer to section 2.9). Pilocarpine treatment increased hippocampal neurodegeneration. Comparing VEH + PILO vs VEH + SAL, the increase in Fluoro-Jade C fluorescence were as follows: CA1: 179.1%, p 0.006; CA3: 28.7%, p = 0.168 and hilus: 97.4%, p = 0.017. In line with the results from the Nissl staining, FLU had no significant effects either in control or insulted rats. Comparing FLU + PILO vs FLU + SAL, Fluoro-Jade C fluorescence were as follows: CA1: 181.8%, p = 0.003; CA3: 72.8%, p = 0.013 and hilus: 84.8%, p = 0.05 (Fig. 4).

Fig. 4.

Hippocampal neurodegeneration (assessed by Fluoro-Jade C staining) triggered by pilocarpine injection was not reduced by FLU administration. (A) Representative images from Fluoro-Jade C staining. (B) Plot of fluorescence intensity values of Fluoro-Jade C (mean $\pm$ SD), expressed as percentage of the signal obtained in the SAL + VEH. *p 0.05, and **p 0.01; two-way ANOVAs followed by post-hoc Tukey test.

As shown in Fig. 5, pilocarpine treatment, independently from administration of FLU, significantly reduced NeuN immunofluorescence intensity in the hippocampal CA1 subregion (p = 0.013). Neither pilocarpine nor FLU significantly altered NeuN signaling in the hippocampal CA3 and hilus subregions. Nonetheless, there was a trend (p 0.1) for the effects of FLU pre-treatment (p = 0.067) as well as for the interaction between FLU and PILO treatments (p = 0.097) suggesting a potential effect of FLU reducing SE-induced neuronal death (VEH-PILO, –26.1% vs FLU-PILO, –13.6%).

Fig. 5.

FLU pretreatment (100 mg/kg) was ineffective at rescuing CA1 neuronal loss (evaluated by NeuN immunofluorescence) in rats injected with pilocarpine. (A) Representative images of hippocampal subregions showing immunofluorescence NeuN signal. (B) Plot of NeuN fluorescence signal intensity (mean $\pm$ SD), expressed as percentage of the signal obtained in the SAL + VEH. *p 0.05; two-way ANOVAs followed by post-hoc Tukey test.

GFAP immunofluorescence intensity was measured as percentage vs their respective control groups. Pilocarpine treatment resulted in reactive astrogliosis in the 3 hippocampal subregions analyzed, but FLU by itself did not have significant effects. Thus, comparing VEH + PILO vs VEH + SAL, the increments in GFAP intensity were as follows: CA1: 63.6%, p 0.001; CA3: 47.1%, p = 0.003 and hilus: 47.8%, p = 0.001. Comparing FLU + PILO vs FLU + SAL, the increments in GFAP intensity were as follows: CA1: 60.5%, p = 0.015; CA3: 51.4%, p = 0.028 and hilus: 56.0%, p = 0.01 (Fig. 6).

Fig. 6.

SE triggered by pilocarpine injection induced a marked astrocyte activation (evaluated by GFAP immunofluorescence), which was not significantly modified by a previous administration of FLU. (A) Representative images of hippocampal subregions showing immunofluorecent GFAP signal. (B) Plot of GFAP fluorescence signal intensity (mean $\pm$ SD), expressed as percentage of the signal obtained in the SAL + VEH. *p 0.05, **p 0.01 and ***p 0.001; two-way ANOVAs followed by post-hoc Tukey test.