European Journal of Pharmacology 939 (2023) 175453 Available online 11 December 2022 0014-2999/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). The vasodilator naftidrofuryl attenuates short-term brain glucose hypometabolism in the lithium-pilocarpine rat model of status epilepticus without providing neuroprotection Luis García-García a,b,e,*, Francisca Gomez a,b, Mercedes Delgado c, Rubén Fernández de la Rosa b,c, Miguel Ángel Pozo b,d,e a Department of Pharmacology, Pharmacognosy and Botany. Faculty of Pharmacy, Complutense University of Madrid, Madrid, Spain b Brain Mapping Unit, Instituto Pluridisciplinar, Complutense University of Madrid, Madrid, Spain c BIOIMAC, Complutense University of Madrid, Madrid, Spain d Department of Physiology, Faculty of Medicine, Complutense University of Madrid, Madrid, Spain e Health Research Institute, Hospital Clínico San Carlos (IdISSC), Madrid, Spain A R T I C L E I N F O Keywords: Lithium-pilocarpine model [18F]FDG PET Glucose hypometabolism Cerebral blood flow Hippocampal damage Neuroinflammation A B S T R A C T Status epilepticus (SE) triggered by lithium-pilocarpine is a model of epileptogenesis widely used in rats, reproducing many of the pathological features of human temporal lobe epilepsy (TLE). After the SE, a silent period takes place that precedes the occurrence of recurrent spontaneous seizures. This latent stage is charac- terized by brain glucose hypometabolism and intense neuronal damage, especially at the hippocampus. Importantly, interictal hypometabolism in humans is a predictive marker of epileptogenesis, being correlated to the extent and severity of neuronal damage. Among the potential mechanisms underpinning glucose metabolism impairment and the subsequent brain damage, a reduction of cerebral blood flow has been proposed. Accordingly, our goal was to evaluate the potential beneficial effects of naftidrofuryl (25 mg/kg i.p., twice after the insult), a vasodilator drug currently used for circulatory insufficiency-related pathologies. Thus, we measured the effects of naftidrofuryl on the short-term brain hypometabolism and hippocampal damage induced by SE in rats. 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET) neuroimaging along with various neurohistochemical assays aimed to assess brain damage were performed. SE led to both severe glucose hypometabolism in key epilepsy-related areas and hippocampal neuronal damage. Although naftidrofuryl showed no anticonvulsant properties, it ameliorated the short-term brain hypometabolism induced by pilocarpine. Strikingly, the latter was neither accompanied by neuroprotective nor by anti-inflammatory effects. We suggest that naftidrofuryl, by acutely enhancing brain blood flow around the time of SE improves the brain metabolic state but this effect is not enough to protect from the damage induced by SE. 1. Introduction Epilepsy is a chronic neurological disorder characterized by a persistent tendency to generate seizures and by its neurobiological, cognitive, psychological, and social consequences (Berg et al., 2010; Fisher et al., 2014). According to the World Health Organization, around 50 million people worldwide have epilepsy. Furthermore, it is estimated that up to 70% of epilepsy patients could live seizure-free if properly diagnosed and treated. Consequently, therapeutic tools to prevent or slow down the progression of the deleterious consequences of this dis- ease are still needed. Temporal lobe epilepsy (TLE) is the most predominant form of focal epilepsy in adults. It is frequently accompanied by hippocampal sclerosis (Janszky et al., 2005), being highly refractory to the current pharma- cological treatments (Tang et al., 2017; Téllez-Zenteno and Hernán- dez-Ronquillo, 2012). One hallmark of human TLE is the interictal glucose hypometabolism in the epileptogenic focus (Kumar and Chu- gani, 2017a, 2017b) that can be evaluated by 2-deoxy-2-[18F] * Corresponding author. Unidad de Cartografía Cerebral, Instituto Pluridisciplinar, Universidad Complutense de Madrid, Paseo Juan XXIII nº 1, 28040, Madrid, Spain. E-mail address: lgarciag@ucm.es (L. García-García). Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar https://doi.org/10.1016/j.ejphar.2022.175453 Received 20 July 2022; Received in revised form 17 November 2022; Accepted 8 December 2022 mailto:lgarciag@ucm.es www.sciencedirect.com/science/journal/00142999 https://www.elsevier.com/locate/ejphar https://doi.org/10.1016/j.ejphar.2022.175453 https://doi.org/10.1016/j.ejphar.2022.175453 https://doi.org/10.1016/j.ejphar.2022.175453 http://crossmark.crossref.org/dialog/?doi=10.1016/j.ejphar.2022.175453&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ European Journal of Pharmacology 939 (2023) 175453 2 fluoro-D-glucose ([18F]FDG) positron emission tomography (PET) im- aging (Sarikaya, 2015). One of the most used animal models of epilepsy is the lithium- pilocarpine rat model of status epilepticus (SE) (García-García et al., 2017, 2016; Kandratavicius et al., 2014; Leite et al., 2002; Shiha et al., 2015). This model reproduces hippocampal sclerosis as well as many other neuropathologic, electrographic and behavioral features of human TLE (Turski et al., 1983). The SE induced by pilocarpine is followed by a seizure-free period characterized by a generalized brain glucose hypometabolism (Gar- cía-García et al., 2017, 2016; Lee et al., 2012; Shiha et al., 2015). Be- sides, hypometabolism is concurrent with neurodegeneration, neuronal death, neuroinflammation and reactive gliosis (García-García et al., 2017; Rossi et al., 2013; Shapiro et al., 2008; Shiha et al., 2015). Brain hypometabolism has been attributed to neuronal death, diaschisis, reduction in synaptic density (Kumar and Chugani, 2017a, 2017b), and cerebral hypoperfusion (Bascuñana et al., 2021; Kumar and Chugani, 2017b). Regarding the latter, reduced brain hypoperfusion occurs shortly after pilocarpine administration, lasting at least for 12 weeks (Bascuñana et al., 2021). Impaired cerebral blood flow might contribute to cognitive decline in epilepsy and in other neurological diseases (Daulatzai, 2017). Nafti- drofuryl, (known as nafronyl in the US), is a vasodilator drug currently prescribed for the treatment of peripheral vascular disease, intermittent claudication, cerebral vascular disease and Raynoud’s disease. Nafti- drofuryl acts as an antagonist of the 5-hydroxytryptamine 2 (5-HT2) receptors (Maloteaux et al., 1986; Wiernsperger, 1994), leading to vasodilation (Endemann et al., 2002; Oudart, 1990) and reduced platelet aggregation (Jagroop and Mikhailidis, 2000), therefore increasing brain perfusion (Young et al., 1983). Clinical studies indicate that naftidrofuryl seems to have beneficial effects on cognitive impair- ment conditions (Admani, 1978; Cox, 1975; Emeriau et al., 2000; Gerin, 1974; Judge and Urquhart, 1972; Lu et al., 2011). To address whether naftidrofuryl might protect against some of the deleterious consequences of the SE in the rat lithium-pilocarpine model, we evaluated its effects on brain glucose metabolism during the early phase of epileptogenesis by [18F]FDG PET. We also studied behavioral variables related to SE, as well as various histochemical markers asso- ciated with brain damage. 2. Materials and methods 2.1. Animals A total of 40 adult male Sprague-Dawley rats (Charles River Labo- ratories Spain) weighing 293.1 ± 5.8 g at the beginning of the study were used. Rats were housed in standard rat cages (2 rats/cage), on a ventilated rack (Tecniplast, Italy) under controlled temperature (22 ± 2 ◦C) and a 12 h light/dark cycle (8:00 a.m.-8:00 p.m.). During the acclimation period as well as during the experimental procedure, rats had ad libitum access to standard rodent food, except for the 12h before the PET scans. The study was approved by the Animal Research Ethical Committee of the Universidad Complutense de Madrid, and it was car- ried out in accordance with regulations of the European Union (2010/ 63/UE) and Spain (RD53/2013) regarding animal welfare. All efforts were made to minimize, as much as possible, both the number of ani- mals used and their suffering. 2.2. Lithium-pilocarpine model of SE The procedure has been previously reported (García-García et al., 2017; Shiha et al., 2015). Briefly, lithium chloride (127 mg/kg i.p., Sigma–Aldrich) was administered 18–20 h before SE induction by pilocarpine. To minimize the peripheral effects of pilocarpine, methyl-sc opolamine (2 mg/kg, i.p.) was administered 30 min previous to pilo- carpine injection (25 mg/kg, i.p.; Sigma–Aldrich, St. Louis, MO). Regarding pilocarpine, the dose of 25 mg/kg has been used also by other research groups (Rigoulot et al., 2004; Walton and Treiman, 1988; Yang et al., 2014). The convulsive behavior was evaluated according to the Racine scale (Racine, 1972). The onset of SE was considered when the animal reached the stage 4 (rearing with forelimb clonus) and showed contin- uous seizure activity. Rats stayed in seizure period for a maximum of 45 min and then it was ended by injecting pentobarbital (25 mg/kg, i.p.). The control rats that did not undergo SE were exposed to the same administration schedule, but saline solution (SAL) was used instead of drugs. 2.3. Naftidrofuryl administration Naftidrofuryl (Sigma-Aldrich, St. Louis, MO) was i.p. administered at a dose of 25 mg/kg twice, dissolved in saline as vehicle. The first naf- tidrofuryl administration took place 5 min after the pilocarpine injec- tion. The second dose was administered 8 h later. The dose of naftidrofuryl was selected based on previous studies reporting that similar doses had beneficial effects in brain stroke models (Miyake et al., 1993; Taguchi et al., 1994). The final experimental groups were as follows: (1) control rats (SAL + VEH group, n = 7); (2) rats injected with pilocarpine (PILO + VEH group, n = 13); (3) rats injected with naftidrofuryl (SAL + NAFTI group, n = 7) and finally; (4) rats injected with both pilocarpine and nafti- drofuryl (PILO + NAFTI group, n = 13). Three days after pilocarpine (or saline) injection, brain metabolism was evaluated by in vivo PET. One day later, the rats were sacrificed in order to carry out the different neurohistochemical evaluations. A diagram schematizing the timeline of the experimental procedures is depicted in Fig. 1. 2.4. [18F]FDG PET neuroimaging As stated before, to evaluate brain glucose metabolism, [18F]FDG PET scans were carried out 3 days after the SE, during the silent period. Brain glucose hypometabolism can be detected as early as 3 days after SE (García-García et al., 2017; Shiha et al., 2015; Slowing et al., 2022) and it has been widely considered as an early marker of epileptogenesis (Shultz et al., 2013; Zhang et al., 2015). The protocols for image acquisition and processing have been previously detailed (García-García et al., 2018, 2017, 2016). A dual PET/CT (computed tomography) scanner was used (Albira scanner, Bruker NMI, Billerica, Massachusetts, U.S.A.). The radiotracer [18F]FDG was injected into the tail vein (approximately 13 MBq–350 μCi-in 0.2 ml of 0.9%; Curium Pharma, Madrid, Spain). Thirty minutes later, rats were anesthetized by iso- flurane, placed into the tomograph and the images acquired. The tomographic images were reconstructed and processed by PMOD 3.6 software (PMOD Technologies Ltd., Zurich, Switzerland). The steps for quantification of the metabolic activity were the following: (1) co-registration of the acquired images to a magnetic resonance image (MRI) rat brain template; (2) regional [18F]FDG uptake images collec- tion, (3) normalization of the uptake images to the standardized uptake value (SUV) based on the animal BW, the dose injected and the corrected [18F]FDG uptake decay. The MRI rat brain template used for image co-registration and the volumes of interest (VOIs) corresponding to the brain areas for quantitation of metabolic activity are shown in Fig. 2. 2.5. Neurohistochemical assessments Rats were sacrificed by decapitation the day after the PET acquisi- tions. Brains were dissected, cut longitudinally into two halves, frozen on dry ice, and stored at − 80 ◦C. Brain slices (30 μm-thickness) from the left hemibrain were collected using a cryostat (Leica CM1850, Leica Biosystems, Germany). Sections containing the hippocampus (6 slices/ slide) were thaw-mounted onto Superfrost Plus slides (Thermo Scienti- fic, Germany), dried on a hot plate and stored into slide boxes at − 80 ◦C L. García-García et al. http://topics.sciencedirect.com/topics/page/Lithium_chloride http://topics.sciencedirect.com/topics/page/Methylscopolamine_bromide http://topics.sciencedirect.com/topics/page/Methylscopolamine_bromide http://topics.sciencedirect.com/topics/page/Status_epilepticus http://topics.sciencedirect.com/topics/page/Pentobarbital http://topics.sciencedirect.com/topics/page/Magnetic_resonance_imaging http://topics.sciencedirect.com/topics/page/Magnetic_resonance_imaging European Journal of Pharmacology 939 (2023) 175453 3 until the day of the assays. 2.5.1. Neuronal viability and disruption of hippocampal integrity Neuronal viability was evaluated by Nissl staining as previously described (García-García et al., 2018, 2017). Briefly, the slices were fixed in 4% formaldehyde in phosphate buffer pH 7.4 (10 min), washed in phosphate buffer (2 × 1 min) and incubated in 0.5% cresyl violet acetate solution (30 min). Afterwards, the sections were washed, dehydrated in graded ethanol series (70%, 95% and 100%) and cleared in xylene. Slices were cover-slipped with DPX mounting medium (Fluka, Switzerland). The images were captured with a digital camera (Leica DFC425, Leica, Germany) coupled to a microscope (Leica DM 2000 LED, Leica, Germany). 2.5.2. Hippocampal neurodegeneration Neurodegeneration was evaluated by Fluoro-Jade C staining, as previously reported (García-García et al., 2017), following the original protocol with minor modifications ((Schmued et al., 2005). Briefly, The samples were fixed in 4% formaldehyde (10 min), rinsed in basic alcohol, 100% ethanol, distilled water, 0.06% potassium permanganate, 0.1% acetic acid solution containing 0.0001% Fluoro-Jade C (Millipore, Darmstadt, Germany), distilled water, and xylene. Then, the slides were cover-slipped with DPX (Fluka). The images were captured with a digital camera (Leica DFC3000G) coupled to a microscope (Leica DM 2000 LED) by using the FITC filter. At the hippocampal CA1, CA3 and hilus, the fluorescence signal was measured using ImageJ 1.46r software. The average value for each rat was calculated and the results were expressed as percentage vs the control group (VEH + SAL). 2.5.3. Reactive astrogliosis Astrogliosis was evaluated by glial fibrillary acidic protein (GFAP) one-step immunofluorescence as previously reported (García-García et al., 2018, 2017). Briefly, the slices were formaldehyde-fixed, washed, blocked and permeabilized with 3% BSA, 0.1% triton X-100 in TBS (60 min) and incubated overnight with the anti-GFAP-Cy3 antibody (1:500, Sigma Aldrich) in 1% BSA in TBS at 4 ◦C. Afterwards, the slides were washed in 0.1% Tween 20 dissolved in Tween (3x for 5 min each) and cover-slipped with Mowiol. The images were captured and examined using the same optical systems used for Fluoro-Jade C, but in this case using the TRITC filter. For each brain section containing the CA1, CA3 and hilus areas at the anterior (dorsal) hippocampus the fluorescence intensity was measured (ImageJ 1.46r software). As with the Fig. 1. Scheme of the experimental procedure of the study: LiCl (127 mg/kg) was administered 18–20 h before the induction of the SE. To induce the SE, 25 mg/kg pilocarpine (or saline in the control groups) was injected. Five min and 8 h after pilocarpine in- jection, naftidrofuryl (25 mg/kg, i.p.) or vehicle were administered. Three days later, to evaluate the regional brain glucose metabolism, PET/CT scans were carried out. The next day (day 4), the rats were sacrificed, and the brains removed to carry out the histochemical analyses. Fig. 2. The upper row shows the coronal, sagittal and transaxial views corresponding to the MRI brain template used for PET and CT images co-registration. The bottom row shows a sample CT image with the volumes of interest (VOIs) of the brain areas used for quantification of the regional metabolic activity by [18F] FDG PET. L. García-García et al. http://topics.sciencedirect.com/topics/page/Formaldehyde European Journal of Pharmacology 939 (2023) 175453 4 Fluoro-Jade C measurement, the average value for each rat was calcu- lated and the results were expressed as percentage vs the control group (VEH + SAL). 2.5.4. Microglia-mediated neuroinflammation Neuroinflammation mediated by activated microglia was studied by [3H]PK11195 autoradiography as published (Foucault-Fruchard et al., 2017) with minor modifications. The slides were dried on a hot plate at 37 ◦C (10 min), preincubated with 50 mM Tris-HCl pH 7.4 at RT (15 min) and then incubated with 1 nM [3H]PK11195 (Perkin Elmer) in preincubation buffer (60 min). Afterwards, the samples were washed in an ice cold preincubation buffer (2 × 5 min) and dipped in ice-cold distilled water. Once air-dried, the slides were exposed to Kodak Bio- Max MR autoradiography film (Carestream, U.S.A.) in an exposure cassette for approximately 2 months. After manual development, the film was placed onto a light box (Kaiser Prolite 5000, Kaiser Foto- technik, Germany) and the images captured with a camera (Leica DFC425) coupled to a stereomicroscope (Leica MZ6). The optical den- sities of the selected brain regions were used as index of neuro- inflammation degree. 2.6. Statistical analyses Analyses were performed with SigmaPlot 11.0 software (Systat Software Inc., Chicago, IL, U.S.A.). Behavioral markers of SE onset (la- tency) and mortality rate were only analyzed in the lithium-pilocarpine- treated rats (PILO + VEH vs PILO + NAFTI) by unpaired Student t-test and z-test for rates and proportions, respectively. Data from BW, PET neuroimaging and histochemical determinations were analyzed by two- way analysis of variance (ANOVA) followed by the post hoc Tukey test. In all cases, statistical significance was considered when p < 0.05. Data are shown as mean ± standard error of the mean (SEM). 3. Results 3.1. Behavioral and physiological changes Naftidrofuryl administration neither modified the latency to SE, nor the number of seizures (Fig. 3A–B) in response to pilocarpine. In the current study, the mortality rate associated with SE was surprisingly low. In fact, there were no deaths in the PILO +VEH group, and only 2 of 13 rats from the PILO + NAFTI group died (death rate: 15.4%). The z- test for rates and proportions showed no significant differences between both groups (p = 0.466). SE resulted in BW loss in the following 24h (p<0.01), the effect being independent of naftidrofuryl treatment (approximately 12% in PILO + VEH and 14% in PILO +NAFTI rats). The BW loss remained until the end of the experiment (Fig. 3C). When comparing the BW of both groups to their respective control groups (SAL + VEH and SAL + NAFTI) at the different time points, significant differences were also found (Fig. 3C). 3.2. Brain glucose metabolism As depicted in Fig. 4A, glucose hypometabolism (measured as SUV) was detected, 3 days after the SE, in epilepsy-related brain areas. Compared to SAL + VEH, the SUV values in PILO + VEH were signifi- cantly reduced. Values ranged from 33.3% in midbrain (p<0.001) to 46.1% in striatum (p<0.01) (Fig. 4B). Specifically in our main area of interest, the hippocampus, the values went down 42.1% (p<0.001; Fig. 4B). Naftidrofuryl, in the absence of SE (SAL + NAFTI) had no significant effect on glucose brain metabolism. However, naftidrofuryl administration (PILO + NAFTI) significantly ameliorated the SE- Fig. 3. (A) and (B) Naftidrofuryl did not show anti- convulsant effects on the SE triggered by pilocarpine administration, not affecting the latency time to SE (A) nor the number of seizures (B). (C) SE triggered by pilocarpine. (PILO + VEH group) resulted in a significant BW loss that was not prevented by previ- ous administration of naftidrofuryl. Figures show the values as means ± SEM. **p<0.01, ##p<0.01 compared to their respective BW before pilocarpine injection (day 0); $p<0.05, &p<0.05 compared to their respective control groups (SAL + VEH and SAL + NAFTI, respectively); two-way ANOVAs followed by post-hoc Tukey test. L. García-García et al. European Journal of Pharmacology 939 (2023) 175453 5 induced hypometabolism in all brain regions studied, showing statisti- cally significant higher SUV data (p<0.05; Fig. 4B). 3.3. Neurohistochemistry As expected, neither signs of neurodegeneration, glial reactivity nor neuroinflammation were observable in SAL + VEH. Besides, nafti- drofuryl administration in the absence of SE (SAL + NAFTI) had no ef- fects in any of the variables studied. Compared with its respective control group, SE triggered by pilo- carpine (PILO + VEH) resulted in a visual loss of hippocampal neurons at the CA1, being this neuronal death lesser at the level of CA3, as shown by the cresyl violet micrographs (Fig. 5). As expected, there were also signs of hippocampal neurodegeneration, as revealed by a robust in- crease in Fluoro-Jade C labeling, measured as FITC fluorescence signal (Fig. 6A). The increase of fluorescence in CA1, CA3 and hilus areas was 10.8, 4.8 and 5.9 times compared with those found in the control group (p<0.05; Fig. 6B). Astrocytic activation in the different hippocampal areas, measured by GFAP immunofluorescence signal was also statisti- cally significant; (p<0.001 in the three hippocampal areas analyzed; Fig. 7A). This increase in GFAP-TRITC signal ranged from 46% to 73% depending on the area. GFAP immunofluorescence in CA1 is shown in Fig. 7C. Regarding microglia-mediated neuroinflammation, SE in PILO + VEH rats resulted in an increase in the optical density obtained from the [3H]PK11195 brain binding that ranged from 35% in thalamus to 61% in entorhinal cortex (Fig. 8A–B). In all the regions such increases were statistically significant (p<0.01 in hippocampus, entorhinal cortex and amygdala and p<0.05 in thalamus, parietal, and temporal cortices; Fig. 8B). It is noteworthy that brains of the pilocarpine-insulted rats show a marked edema formation at the level the piriform cortex, a typical feature of inflammation triggered by pilocarpine (Kim et al., 2010), especially at the level of the entorhinal and piriform cortices Fig. 4. SE induced by pilocarpine (PILO + VEH) induced a marked reduction of glucose metabolism in key areas involved in epileptogenesis, an effect that was partially ameliorated by naftidrofuryl. Regional brain glucose metabolism was evaluated by [18F]FDG PET 3 days after the SE. (A) Representative CT (upper row), [18F]FDG PET normalized to SUV scale (middle row) and merged PET/CT (bottom row) images in coronal, sagittal and trans-axial views of the 4 experimental groups. (B) Regional brain uptake in the 4 experimental groups is shown as SUV units (mean ± SEM); *p<0.05, **p<0.01 PILO + VEH vs. SAL + VEH and PILO + NAFTI vs. SAL + NAFTI; #p<0.05 PILO + NAFTI vs. PILO + VEH; two-way ANOVAs followed by post-hoc Tukey test. L. García-García et al. European Journal of Pharmacology 939 (2023) 175453 6 (depicted by white arrows in Fig. 8B). Noteworthy is the fact that naftidrofuryl administration failed to statistically modify any of the signs of hippocampal integrity disruption, neurodegeneration, glial reactivity or neuroinflammation induced by the SE (see Figs. 5–8). 4. Discussion Herein, we have explored the effects of acute i.p. administration of naftidrofuryl on brain glucose metabolic dysfunction, hippocampal neurodegeneration and neuroinflammation, typical features of the brain damage associated to the SE in the rat lithium-pilocarpine model (Gar- cía-García et al., 2017; Shiha et al., 2015). We have also studied its potential effects on latency to SE, mortality, and BW change. Naftidrofuryl is a 5-HT2 blocker that leads to vasodilation as well as reduction of platelet aggregation. At brain level, both actions result in enhanced blood flow (Young et al., 1983). Besides, in conditions of hypoxia, naftidrofuryl increases oxygen and energetic mediators’ availability. Further, naftidrofuryl has been shown to improve aerobic glycolysis and to protect cell ATP reserves (Ogawa et al., 1991). Therefore, by reducing brain hypoperfusion-induced hypoxia, nafti- drofuryl preserves metabolism and prevents neuronal death. Therefore, it is plausible that administration of naftidrofuryl might protect against some of the deleterious consequences of the SE. In our study, acute naftidrofuryl treatment neither delayed the la- tency time, to the SE, number of seizures (Fig. 1A–B) nor affected the mortality rate consequence of the severity of SE induced by pilocarpine. In fact, the mortality rate associated to SE in this current study was very low. Based on our experience, using the same exact protocol, the death rate is commonly around 25–30%. However, other times it has been very high (Slowing et al., 2022). In any case it has been shown that the mortality rate associated to the severity of this model is hardly pre- dictable, depending not only on the dose of pilocarpine, but also on factors such as rat strain, sub-strain, age, gender, commercial providers, and the time of purchase of animals (Buckmaster, 2004; Curia et al., 2008). In humans, seizures after parenteral administration of high doses of naftidrofuryl have been reported (Pohlmann-Eden et al., 1991). In animal models of epilepsy, anticonvulsant dose-dependent effects of naftidrofuryl have been reported in pentylenetetrazole (PTZ) kindled rats, but not in amygdala-kindled rats (Schmidt, 1990). In our study using the lithium-pilocarpine rat model of SE and after i.p. administra- tion of 25 mg/kg twice, 5 min and 8 h after pilocarpine injection, naf- tidrofuryl had neither convulsant nor anticonvulsant effects. In this context it is important to bear in mind, that compared with the lithium-pilocarpine model of SE, the kindling models of epileptogenesis: (i) rely on repetitive chemical or electrical stimulation of limbic brain structures instead of on a single chemical insult; (ii) they lead to the onset seizures in a progressive manner instead of a rapid emergence of spontaneous seizures, and (iii) the neuropathological features are less severe and appear in a progressive cumulative therefore, more subtle effects might be detected (Becker, 2018; Samokhina and Samokhin, 2018). Accordingly, the effects of naftidrofuryl on convulsant activity seem to be dependent on the experimental animal model as well as the dose regimen. [18F]FDG PET is one of the most commonly used neuroimaging techniques in epileptic patients that allows for the evaluation of early glucose metabolic changes in relationship to synaptic and neuronal ac- tivity (Rocher et al., 2003). Interictal [18F]-FDG PET features glucose hypometabolism in the epileptogenic region. Furthermore, the severity of seizures seems to relate to the degree of hypometabolism and it has been suggested that reciprocal positive feedback exists between seizures and hypometabolism (Blázquez et al., 2022; Gaillard et al., 2002; Zilberter and Zilberter, 2017). Importantly, interictal brain glucose hypometabolism has been consistently reproduced in the silent latent period of the lithium-pilocarpine model of SE (Bascuñana et al., 2021; García-García et al., 2017, 2016; Shiha et al., 2015). Although the exact mechanisms underpinning interictal glucose hypometabolism still remain unclear, neuronal loss, reduced synaptic density and altered cerebral blood flow have been involved. Our current data corroborates that the SE induced by pilocarpine results in glucose hypometabolism in epilepsy-related brain areas (Fig. 4). Furthermore, acute naftidrofuryl administration effectively ameliorates brain glucose hypometabolism Fig. 5. Disruption of the hippocampal integrity produced by SE as visualized by cresyl violet (Nissl) staining. The micrographs show a severe damage induced by pilocarpine-triggered SE on the anterior hippocampus 72 h after pilocarpine injection both in the vehicle (PILO + VEH) and naftidrofuryl (PILO + NAFTI) treated groups. L. García-García et al. European Journal of Pharmacology 939 (2023) 175453 7 induced by SE (Fig. 4). Regarding cerebral blood flow in epilepsy, single photon emission computed tomography (SPECT) studies in humans using the radiophamaceutical tracer compound 99mTc-Hex- amethylpropyleneamine Oxime (99mTc-HMPAO) have shown either interictal hypoperfusion or normal perfusion in the epileptogenic region (Kumar and Chugani, 2017a, 2017b). Similarly, in the rat lithium-pilocarpine model of SE, a recent 99mTc-HMPAO SPECT neu- roimaging study has reported that brain regional hypoperfusion emerges 15 min after the pilocarpine administration (i.e. before reaching SE) and becomes chronic, been detectable up to, at least, 12 weeks later (Bas- cuñana et al., 2021). In the clinical context, impaired cerebral blood flow and glucose hypometabolism concur in many neurological disor- ders. It has been proposed that the use of vasodilators such as nafti- drofuryl might improve cerebral blood flow and therefore contribute to ameliorate the cognitive decline in these neurological diseases (Dau- latzai, 2017; Emeriau et al., 2000). Herein, it is also important to bear in mind both, that epilepsy patients frequently suffer from cognitive impairment, psychiatric and mood disorders (Jensen, 2011) and that epileptic seizures also occur in many other neurological conditions, with a wide variety of etiologies and risk factors, being commonly concurrent with ischemic stroke and traumatic brain injury (Temkin, 2009). Neuroinflammation, an adaptive physiological response to brain cell damage or loss and, gliosis affecting both astrocytes and microglia have been reported in epileptic disorders. It has been also suggested that brain glucose hypometabolism and neuroinflammation may act in concert, contributing to the establishment of the pathology. Neuroinflammation is also a feature of the lithium-pilocarpine rodent model (Bulduk et al., 2019; García-García et al., 2017) as well as of other animal models of acute seizures (García-García et al., 2018; Jupp et al., 2012). In agree- ment with previously reported studies obtained by our group (García-- García et al., 2017, 2016; Shiha et al., 2015), our present data show that SE resulted in hippocampal neuronal damage as revealed by Nissl his- tological observations (Fig. 5) and increased Fluoro-Jade C signal (Fig. 6). Besides, SE triggered reactive astrogliosis, characterized by increased TRITC-GFAP fluorescence, hypertrophy, and proliferation (Fig. 7) and resulted in microglia-mediated neuroinflammation. [3H] PK11195 autoradiographic studies provide measurements of microglial activation associated with mitochondrial overexpression of the peripheral-type benzodiazepine receptor, currently known as 18 kDa translocator protein (TSPO) (Papadopoulos et al., 2006). We show that SE resulted in increased [3H]PK11195 binding (Fig. 8) which agrees with previous studies reporting increased [18F]GE180 (a TSPO PET Fig. 6. Pilocarpine induced marked hippocampal neurodegeneration, which was not reduced by nafti- drofuryl administration, as detected by Fluoro Jade C labeling. (A) Representative images of the hippo- campus from the 4 experimental groups, 3 days after the SE. (B) Bar plot corresponding to Fluoro-Jade C fluorescence intensity values as marker of neuro- degeneration. Data are expressed as percentage of the signal obtained in the SAL + VEH group and shown as mean ± SEM. *p<0.05, **p<0.01 and ***p<0.001 when compared to their respective control (vehicle treated groups); two-way ANOVAs followed by post- hoc Tukey test. L. García-García et al. European Journal of Pharmacology 939 (2023) 175453 8 tracer) signal when neuronal damage occurs as consequence of seizures and epileptogenesis (Brackhan et al., 2018; García-García et al., 2018, 2017). Besides, the brain slices obtained from pilocarpine-insulted rats showed wide edematous areas, especially at the level of piriform and entorhinal cortices area as previously described (Kim et al., 2010). This edema formation was also evident at the hippocampus of many SE-damaged rats. Although, it has been suggested that counteracting the cerebral hypoperfusion during SE in the lithium-pilocarpine model may result in a reduced neurodegeneration and neuroinflammation (Bascu- ñana et al., 2021), our study on the effects of acute naftidrofuryl administration does not support either neuroprotective or antiin- flammatory effects. Considering that in this model, seizure-induced brain damage is caused by glutamate-mediated excitotoxicity (Cav- alheiro et al., 1994; Costa et al., 2004), reduced brain perfusion and increased brain energy requirements (Farrell et al., 2017), the acute improvement in brain blood perfusion by itself seems to be unable to lessen the SE-induced brain damage. Consequently, other complemen- tary treatments might be necessary to comprehensively tackle the multi-factorial processes underpinning brain damage. 5. Conclusions To sum up, as far as we know, this is the first time that functional neuroimaging PET has been applied to assess the effect of naftidrofuryl Fig. 7. Naftidrofuryl did not reduce astroglial reac- tivity in response to the SE triggered by pilocarpine. (A) Graph showing the astrocyte activation as TRITC fluorescence intensity (% from SAL + VEH group); ***p<0.001 vs. their respective control groups. (B) Representative DAPI-labelled images corresponding to CA1, CA3 and hilus/dentate gyrus used for precise localization and quantification of GFAP immunoflu- orescence in these hippocampal subregions. (C) Representative GFAP immunofluorescence micro- graphs at the level of CA1 of the 4 experimental groups, showing the intense astrocyte reactivity trig- gered by pilocarpine. As shown, naftidrofuryl administration (2 × 25 mg/kg) resulted in no reduc- tion of such glial activation. L. García-García et al. European Journal of Pharmacology 939 (2023) 175453 9 on brain glucose hypometabolism induced by SE in the lithium- pilocarpine rat model. Our study does not support acute i.p. nafti- drofuryl having antiseizure properties, neither neuroprotective nor anti- inflammatory effects. Nonetheless, naftidrofuryl treatment did signifi- cantly reduce brain glucose hypometabolism during the silent period that follows SE. Therefore, further studies are needed to determine whether longer treatments providing chronic improved blood flow in the lithium-pilocarpine model of SE together with other therapeutic approaches aimed to neuroprotection might eventually lead to benefi- cial consequences on neurodegeneration and/or neuroinflammation. Likewise, studies on less severe models of epileptogenesis might be a complementary alternative to further dissect the potential beneficial effects of naftidrofuryl in this neurological disorder. CRediT authorship contribution statement Luis García-García: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Visualization, Writing – original draft, preparation, Writing – review & editing. Francisca Gomez: Supervi- sion, Formal analysis, Data curation, Visualization, Writing – original draft, preparation, Writing – review & editing. Mercedes Delgado: Investigation, Data curation. Rubén Fernández de la Rosa: Investiga- tion, Data curation, Visualization. Miguel Ángel Pozo: Resources, Funding acquisition, Project administration. Declaration of competing interest none. Data availability Data will be made available on request. Fig. 8. Neuroinflammation induced by in SE, measured by the [3H]PK11195 binding, in major brain regions involved in epileptogenesis was not reduced by acute administration of naftidrofuryl. (A) Representative [3H]PK11195 autoradiograms corre- sponding to the 4 experimental groups obtained from the anterior hippocampus. The white arrows point to the edematous area at the piriform and entorhinal cortices by pilocarpine-induced SE. (B) Quantifica- tion of the [3H]PK11195-TSPO binding (expressed in % O.D. change from PILO + VEH group and shown as mean ± SEM). ***p<0.001 PILO + VEH vs. SAL + VEH and PILO + NAFTI vs. SAL + NAFTI; two-way ANOVAs followed by post-hoc Tukey test. L. García-García et al. European Journal of Pharmacology 939 (2023) 175453 10 Acknowledgements This work was financially supported by the Spanish Ministerio de Ciencia e Innovación (Retos PID2019-106968RB-100). References Admani, A.K., 1978. New approach to treatment of recent stroke. Br. Med. 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García-García et al. https://doi.org/10.1016/J.NEUROIMAGE.2003.07.002 https://doi.org/10.1016/J.NEUROIMAGE.2003.07.002 https://doi.org/10.1371/JOURNAL.PONE.0078516 https://doi.org/10.1371/JOURNAL.PONE.0078516 https://doi.org/10.1080/00207454.2018.1481064 https://doi.org/10.1080/00207454.2018.1481064 http://refhub.elsevier.com/S0014-2999(22)00714-2/sref47 http://refhub.elsevier.com/S0014-2999(22)00714-2/sref48 http://refhub.elsevier.com/S0014-2999(22)00714-2/sref48 https://doi.org/10.1016/J.BRAINRES.2004.11.054 https://doi.org/10.1111/J.1528-1167.2008.01491.X https://doi.org/10.1016/J.BRAINRESBULL.2014.12.009 https://doi.org/10.1016/J.BRAINRESBULL.2014.12.009 https://doi.org/10.1111/EPI.12223 https://doi.org/10.1055/a-1948-4378 https://doi.org/10.1007/BF00241407 https://doi.org/10.3389/FNEUR.2017.00301 https://doi.org/10.1155/2012/630853 https://doi.org/10.1155/2012/630853 https://doi.org/10.1111/J.1528-1167.2008.02005.X https://doi.org/10.1111/J.1528-1167.2008.02005.X https://doi.org/10.1016/0166-4328(83)90136-5 https://doi.org/10.1016/0014-4886(88)90010-6 https://doi.org/10.1016/0014-4886(88)90010-6 https://doi.org/10.1097/00005344-199406030-00008 https://doi.org/10.1007/s12264-013-1396-x https://doi.org/10.1007/s12264-013-1396-x https://doi.org/10.1159/000115551 https://doi.org/10.7150/IJMS.10527 https://doi.org/10.7150/IJMS.10527 https://doi.org/10.1002/JNR.24064 The vasodilator naftidrofuryl attenuates short-term brain glucose hypometabolism in the lithium-pilocarpine rat model of st ... 1 Introduction 2 Materials and methods 2.1 Animals 2.2 Lithium-pilocarpine model of SE 2.3 Naftidrofuryl administration 2.4 [18F]FDG PET neuroimaging 2.5 Neurohistochemical assessments 2.5.1 Neuronal viability and disruption of hippocampal integrity 2.5.2 Hippocampal neurodegeneration 2.5.3 Reactive astrogliosis 2.5.4 Microglia-mediated neuroinflammation 2.6 Statistical analyses 3 Results 3.1 Behavioral and physiological changes 3.2 Brain glucose metabolism 3.3 Neurohistochemistry 4 Discussion 5 Conclusions CRediT authorship contribution statement Declaration of competing interest Data availability Acknowledgements References