Calibration standards: arachidonoyl ethanolamide (AEA), 2-arachidonoyl glycerol (2-AG), arachidonic acid (AA), palmitoyl ethanolamide (PEA), 1-arachidonoyl glycerol (1-AG), PGD₂, prostaglandin E₂ (PGE₂), 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE), 8(S)-hydroxyeicosatetraenoic acid (8(S)-HETE), 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), 19(S)-hydroxyeicosatetraenoic acid (19(S)-HETE), 20-hydroxyeicosatetraenoic acid (20-HETE), were obtained from BIOMOL Research Laboratories Incorporation (Plymouth Meeting, PA, USA). Internal standards (ISTD): arachidonoyl ethanolamide-d4 (AEA-d4), 2-arachidonoyl glycerol-d₅ (2-AG-d₅), arachidonic acid−d₈ (AA-d₈), palmitoylethanolamide-d₄ (PEA-d₄), 1-arachidonoylglycerol-d₅ (1-AG-d₅), prostaglandin D₂-d₄ (PGD₂-d₄), prostaglandin E₂-d₉ (PGE₂-d₉), 5(S)-hydroxyeicosatetraenoic acid-d₈ (5(S)-HETE-d₈), 12(S)-hydroxyeicosatetraenoic acid-d₈ (12(S)-HETE-d₈), 20-hydroxyeicosatetraenoic acid-d₆ (20-HETE-d6) were obtained from BIOMOL Research Laboratories Incorporation (Plymouth Meeting, PA, USA).

For multiple reaction monitoring (MRM) analysis and lipid extraction, water, acetonitrile (ACN), and formic acid of LC-MS grade were invariably used (Sigma-Aldrich, St. Louis, MO, USA). HPLC grade methyl tert-butyl ether (MTBE) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethyl acetate, n-hexane, 2-propanol, and methanol were obtained from Honeywell International Incorporation (Morristown, NJ, USA).

KA was purchased from Abcam plc. (Cambridge, UK). Palmitoylethanolamide (PEA) was obtained from Cayman Chemical (Ann Arbor, MI, USA). FAAH inhibitors, URB597 (3′-(aminocarbonyl)[1,1′-biphenyl]-3-yl)-cyclohexylcarbamate and URB937 (N-cyclohexyl-carbamic acid, 3′-(aminocarbonyl)-6-hydroxy[1,1′-biphenyl]-3-yl ester) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Cremophore and indomethacin were obtained from BIOMOL Research Laboratories Incorporation (Plymouth Meeting, PA, USA).

For this study, C57BL/6N male mice (8–10 weeks of age) were ordered from Janvier Labs (Saint-Berthevin, France). Experimental procedures were carried out in accordance with the European Community’s Council Directive of 22 September 2010 (2010/63EU) and approved by the local animal care committee of the German Rhineland-Palatinate (file reference: 23 177-07/G16-1-075).

In order to investigate PEA’s impact on acute epileptic seizures and gain insights into its mechanism of action, mice were treated with freshly prepared KA, as conducted previously (Monory et al., 2006; Lerner et al., 2016) and different pre-treatments were applied (PEA, URB597, URB937) by intraperitoneal injection 7 h and/or 30 min prior to the injection of KA. For an overview see Table 1. All animal experiments were conducted in successive days with intermixed groups. For time course analysis, 24 animals per group were used (receiving a particular treament, vehicle or KA injection according to Table 1), which were subdivided in four groups according to the time point of sacrificing post KA injection. Mice were group-housed (4–5/cage) on a 12-h light/dark schedule. Water and food were available ad libitum. The experimenter assessing the behavioral score was blind to the injected agents. Mice were injected intraperitoneally with either KA at a dose of 30 mg/kg body weight or vehicle (0.9% saline, 10 ml/kg body weight; t₀, Scheme 1). Intraperitoneal pre-treatment injections were conducted with PEA at a dose of 40 mg/kg body weight dissolved in DMSO/chremophor/saline (17:2:1; t_P1+t_P2, t_P2, Scheme 1, Table 1). For combined treatment of PEA with the FAAH-inhibitors URB597 or URB937 (Table 1), the inhibitors were added in the solution (t_P2, Scheme 1).

Animals were monitored and scored at 10/20/40/60/90/120/ 150/180 min post KA injection according to the following scale: 0—no response; 1—immobility and staring; 2—forelimb and/or tail extension, rigid posture; 3—repetitive movements, head bobbing; 4—rearing and falling; 5—continuous rearing and falling; 6—severe clonic-tonic seizures; 7—death (Monory et al., 2006). Behavior was assessed as a score of 5 in case of continuously rearing and falling for at least three consecutive episodes and without break as a score 6 for generalized convulsive tonic-clonic seizures, as well as for the so-called “popcorn” bouncing activity, characterized by intermediate hyperactive periods of running. Occurrence of the latter type of seizure activity was restricted to short durations (2–4 min) and subsequently caused death. Status epilepticus (SE) was defined as a period of seizure activity, characterized by tonic clonic convulsions without intermediate recovery for >30 min. Mice with SE were assigned the score 6. In the present study, mice were referred to as being acute epileptic with an onset of epileptic motor activity corresponding to seizure score 3 or higher.

Animals were scored until they were sacrificed, e.g., 20, 60, 120 or latest 180 min post KA injection, with the last scoring time point immediately prior to sacrificing. Accordingly, number of scores obtained at 10 and 20 min post-KA injection correspond to full size experimental groups. After 40 min, total scores decrease over time in relation to reduction of group size due to sacrificing. Consequently, mice sacrificed at 180 min have a full set of behavior scores over the 3 h time course. To enable appropriate statistical analysis between acute and subchronic PEA treatment, we calculated and assessed averages of all scores obtained within a group/per time point of scoring; 10, 20, 40, 60, 90, 120, 150 and 180 min post KA injection (Figure 1A). The results were analyzed using SPSS Statistics Software for Windows (version 23, IBM, Chicago, IL, USA) or Graphpad (version 7, Prism, San Diego, CA, USA). Repeated-measures analysis of variances (ANOVAs) with experimental group and time as independent variables were used to analyze seizure severity after KA injection. The Greenhouse–Geisser correction was used if the condition of sphericity was not met. Significant group effects were further analyzed using Bonferroni’s post hoc analysis for multiple comparisons, whereas significant interactions were further analyzed using simple effects analysis for the effect of group for each time point, also adjusted for multiple testing using Bonferroni correction. The error bars are presented as standard error of the mean (SEM).

To better illustrate distribution of individual scores, i.e., scores higher than 5 and mortality rates, we included a heat map displaying behavior score distribution given in percentage of animals exhibiting a given score from the total number of scored animals (e.g., total number of animals alive at that time point) per experimental group per time point (Figure 1B). In total, 4% of the untreated epileptic mice exhibited SE that eventually led to death for 2%. The 2% of the mice who survived SE, were housed for 5 more days for the purpose of neurodegenerative and immuno-histochemical staining of brain sections that are representative for most severe seizure state, see “Brain and Plasma Sampling” and “Neurodegenerative and Immunohistochemical Staining” sections.

At 20/60/120/180 (t₁–t₄) minutes after KA injection, mice were shortly anesthetized with isoflurane (Forene R, AbbVie Deutschland GmbH and Co. KG, Wiesbaden, Germany) and sacrificed by decapitation (Nomura et al., 2011; Scheme 1). Brains were isolated and directly frozen on dry ice. Dissection of the brains was carried out according to the protocol described previously (Lerner et al., 2016). All tissue samples were then stored at −80°C until further processing. Blood was collected in ice-cold, 500 μL EDTA plasma extraction tubes (KABE Labortechnik, Nümbrecht-Eisenroth, Germany), containing previously added 10 μM indomethacin to prevent ex-vivo COX-2 activity. This collection procedure took 30 s per mouse. Plasma tubes were directly centrifuged at 4°C, 2000 g for 10 min, and the resulting upper plasma phase was removed and stored at −80°C without delay.

eCBs and eiCs were co-extracted using a method recently developed by our group. The same sampling, extraction and quantification protocol as previously reported was applied in this study for both plasma and hippocampal eCBs and eiCs investigation (Lerner et al., 2017). The eCB and eiC values were normalized to protein amount and plasma volume for tissue (Bindila and Lutz, 2016) and plasma samples, respectively. In this study we focused on the analysis of AEA, 2-AG, PEA, AA, PGE₂ and PGD₂. AEA and 2-AG are eCBs that bind to cannabinoid receptor CB1; PEA is an eCB-like molecule. AA is a precursor for the synthesis of prostaglandins (PGs). We focused our analysis on the PGE₂ and PGD₂ due to their prominent involvement in inflammatory processes in general, and arising from KA-induced excitotoxicity, in particular. For the sake of simplicity, we will refer to PGE₂ and PGD₂ as eiCs.

Lipid levels were analyzed at each time-point separately by means of univariate ANOVA for experimental group. Significant group effects were further analyzed using Bonferroni’s post hoc analysis for multiple comparisons. Statistical significance was defined as P < 0.05. Error bars are presented as SEM.

Eight mice (four mice were subchronically PEA-treated epileptic mice, and four mice were non pre-treated epileptic mice) were kept single housed post KA injection for 5 days. Health conditions of mice were frequently checked, all mice showed normal behavior after acute seizure state (3 h post KA injection) and remained like that even though ongoing neurodegenerative processes are expected. In order to determine effect of subchronic treatment of PEA, Fluoro-Jade C (FJC), silverstaining and immunhistochemical staining were performed. For these purposes, perfusion of the brain is required prior to staining. Therefore, mice were deeply anesthetized using pentobarbital (100 mg/kg) and Buprenorphin (0.1 mg/kg) as narcotic, trans-cardially perfused with phosphate buffered-saline (PBS), followed by perfusion with 4% paraformaldehyde (PFA; ROTH, Carl Roth GmbH+Co KG. Karlsruhe, Deutschland) in PBS. The isolated brains were fixed for 24 h in 4% PFA in PBS solution, treated with 30% sucrose in PBS solution for 48 h and stored at −80°C. Forty micrometer thick free floating coronal brain sections were prepared on a Microm HM560 cryostat (Microm) and stored at 4°C in cryoprotection solution (25% glycerol, 25% ethylene glycol and 50% PBS) until use.

To enable detection of KA-provoked neurodegeneration and evaluation of PEA’s assumed neuroprotective properties we performed neurodegenerative FJC and silver staining, and additionally, immunohistochemical double staining of neuronal nuclei (NeuN) and caspase-3 (CASP3) activation in brain sections of previously perfused brains. FJC staining was conducted following previously reported protocol (Schmued et al., 2005). Briefly, 40 μm coronal brain sections were collected and mounted, rinsed in ddH₂O, treated with 1% NaOH (v/v) in 80% EtOH, washed in 70% EtOH and twice in ddH₂O. Slides were treated with 0.06% KMnO₄, washed twice in ddH₂O, and stained with FJC (Millipore, Schwalbach, Germany). Slides were then rinsed twice in ddH₂O and counterstained with DAPI. The staining was then visualized with a Leica DM5500 fluorescence microscope. For the silver staining, sections were mounted and air dried, then incubated in impregnation solution (saturated LiCO₃ in ddH₂O with 10% AgNO₃ and 25% NH₄OH) and washed six times in ddH₂O. Sections were transferred in developer solution (ddH₂O with 19% formaldehyde (v/v), 14% acetone (v/v), 0.3% hydroquinone (w/v), and 1% Tri-Na-Acetate (w/v)) and washed twice in ddH₂O. The staining was then visualized with a Leica DM5500 bright field microscope. For immunohistochemistry, 30 μm coronal brain sections of perfused brains were blocked for 90 min in PBS containing 5% normal donkey serum (NDS), 2.5% bovine serum albumin (BSA), and 0.3% Triton X-10 (TX) and then incubated over night with primary antibodies: mouse anti-NeuN (1:1000, Abcam) and rabbit anti-cleaved Casp3 (1:200, Cell Signalling) in PBS containing 1% NDS, 0.1% BSA, and 0.% TX. Next day, secondary antibodies: Goat anti-Mouse IgG, Alexa Fluor 488 (Invitrogen 1:1000) and Goat-anti Rabbit IgG, Alexa Fluor 546 (1:1000 Invitrogen) were applied for 2 h for immunofluorescent staining, visualized with a Leica DM5500 fluorescent microscope and evaluated with a Leica Application Suite Advanced Fluorescence (LAS AF) Software.

In order to comparatively assess the extent of neurodegenerative processes in hippocampal areas from untreated vs. subchronically PEA-treated epileptic mice, image comparison was performed using identical channel settings in terms of exposure time, intensity, gain and threshold. FJC-positive signals were semi-quantitatively analyzed in the hilus region of the hippocampus from PEA-treated vs. untreated epileptic mice. For this purpose, green fluorescent signals were manually counted using ImageJ software in the region of interest (ROI), e.g., hilus, on three consecutive sections from the same animal. The three coronal brain sections were sampled at 200 μm distance across the brain. Brightness and contrast settings were set fixed prior to import of the picture and FJC signals were split from DAPI signals using split channel function. Positive FJC signals were counted using multipoint tool and statistically analyzed via Graphpad prism 7 software.

Single injection (acute treatment) of exogenous PEA at 30 min prior to KA injection and double injection of PEA (subchronic administration) at 7 h and 30 min prior to KA injection, rendered significant reduction of seizure intensity (Figure 1A). Indeed, after the acute epileptic state (60 min; Lerner et al., 2016), subchronic PEA administrations exerted significant beneficial effects over the entire 180 min, as compared to single PEA injection, whose effects lasted for 120 min after administration (Figure 1A). Mice undergoing acute PEA treatment (PEA/KA) did not exhibit scores higher than 5, while subchronically treated mice (PEA²/KA) not higher than 4 (Figure 1B). In contrast, 8% of untreated epileptic mice (KA) died, 4% exhibited SE (assigned as score 6; Figure 1B) that eventually led to death for 2%. It is of note that normal behavior (score 0) was observed only for mice with subchronic PEA treatment at 10 min post KA administration and more prominent, at late acute seizure state, 120–180 min.

Intraperitoneal subchronic administration of PEA in controls and vehicle injection in controls was performed to understand whether and how PEA modulates the hippocampal and plasma eCB and eiC levels under healthy physiological conditions. In plasma, in the absence of epilepsy, subchronic PEA treatment significantly increased the AEA levels and decreased 2-AG over the entire time course (Supplementary Figure S3). AA levels in plasma were significantly decreased compared to vehicle-injected mice up to 1 h. Inflammation markers, PGE₂ at 20 and 120 min, and PGD₂ at 20 min post KA injection, respectively, were also significantly decreased upon subchronic PEA administration in controls (Supplementary Figure S3). As expected, plasma PEA levels were significantly increased upon exogenous PEA administration in controls over the entire time-course, e.g., 180 min post-injection. Subchronic administration of PEA in controls significantly augmented in hippocampus only the levels of PEA and AA (Supplementary Figure S4), which suggests an up-take of exogenous PEA in the brain.

Immunohistochemical double staining of NeuN and CASP3 (Figure 3A), as well as neurodegenerative FJC- (Figure 3B) and silver (Figure 3C) staining on brain sections sampled 5 days post KA-seizure induction revealed neurodegenerative processes in mice without subchronic PEA treatment compared to saline injected control mice.

KA-induced SE causes massive loss of NeuN signal and apoptosis in untreated epileptic mice, compared to controls, while subchronic PEA treatment notably preserves NeuN signal without showing indication of apoptotic events. Distribution of FJC- stained neurodegeneration correlates with maximal seizure intensities, that were observed within 180 min post KA injection: SE (score 6) causes widespread FJC-signal in hippocampal areas (Figure 3B; lower middle image), while seizure score of 5 results in less spread FJC-signal, predominately located adjacent to CA3 region (Figure 3B; upper middle image). In comparison, upon subchronic PEA treatment a behavior score of 4 was obtained and FJC-signal occurs to a lower extent (Figure 3B; upper and lower image).

Semiquantitative comparison of positive FJC signals in the hilus region 5 days post KA injection revealed significantly reduced (**P < 0.0016 KA vs. PEA²/KA) levels of FJC-sensitive neurodegeneration upon subchronic PEA treatment (the treated mouse had a max. behavioral score of 4 within the 180 min time-course) compared to FJC-signals in the hilus regions obtained 5 days post KA-induced SE (Supplementary Figure S5).

Silver staining indicates widespread neurodegenerative effects provoked by KA-induced SE (Figure 3C, middle image), while a moderate extent of neurodegeneration was observed on brain section from mice with subchronic PEA treatment (Figure 3C; right image) in relation to brain section from healthy control mice (Figure 3C; left side).

Pharmacological blockade of FAAH has been proposed as an effective anticonvulsant therapy, due to the increases of endogenous levels of the AEA and PEA, whose function as endogenous neuroprotectants is well established. URB597 is an FAAH inhibitor, which acts systemically by irreversible FAAH blockade and has a biphasic effect as an anti- or pro-convulsant agent in a dose-dependent manner. Therefore, we aimed at comparing the effectiveness of exogenous PEA and URB597 as anticonvulsant agents and at gaining insights into how the two therapeutic approaches modulate eCBs and eiCs in brain and periphery. Moreover, by using specific blockade of peripheral FAAH by URB937 we aimed at gaining some insights into the contribution of peripheral increase of AEA and PEA levels to seizure alleviation, hence anticonvulsant effects. Another rationale for this comparison is that PEA reportedly potentiates the AED efficiency (Citraro et al., 2016) and the effect of AEA on the cannabinoid receptor (CBR) and/or vanilloid receptor 1 (TRPV1; Ben-Shabat et al., 1998; De Petrocellis et al., 2001; Costa et al., 2008). We therefore, performed a time course study of the effects of URB597, URB937 and each in combination with exogenous PEA administration on the modulation of AEA and PEA, as well as related eCB and eiCs in brain and plasma, and of the anticonvulsant properties in epileptic mice in comparison with PEA-treated epileptic mice.

Behavioral analysis of seizure intensity over 180 min post-KA injection in mice treated with PEA, URB597, and combination of PEA and URB597 showed no significant differences between the three groups (Figure 4A). This indicates that PEA-induced anticonvulsion is similarly effective as for URB597 and that co-administration with URB597 does not induce any cumulative therapeutic effect due to drug interaction (Figure 4A).

Pharmacological blockade of peripheral FAAH was less effective than PEA treatment in maintaining anticonvulsive effects over the entire time course. This is evident in the pre- and late-acute seizure phase, where URB937-treated mice showed significantly higher convulsion intensity compared to those animals treated with PEA only (Figure 4B). Moreover, the combination of PEA/URB937 fails to be effective in the acute (60 min) and late-acute seizure phase (60–120 min), suggesting some mutually exclusive effects of these combined treatments in the acute seizure state. This also suggests that, upon FAAH blockade, the pharmacological increase of AEA and PEA must occur also in the brain in order to exert an effective therapeutic action for seizure prevention.

In hippocampus, administration of URB597 boosts the production of endogenous AEA and PEA, which occurs already at 20 min post seizure induction and is maintained at a significant high level over the 180 min time course (Figure 5). Combined administration of PEA/URB597 in KA-injected mice had similar effects as URB597 treatment on the hippocampal AEA and PEA levels, indicating that no cumulative effects of exogenous PEA administration and blockage of PEA and AEA degradation by URB597 occur (Figure 5).

URB597 administration maintains in hippocampus at late-acute seizure phase (120–180 min) a higher level of inflammation markers, PGE₂ and PGD₂ compared to PEA administration only. This molecular feature occurs also when PEA and URB597 are co-administered, but is less significant compared to URB597 treatment alone (Figure 5). Similar effects are observed for AA at and after acute seizure state (60–180 min) upon URB597 and URB597/PEA treatment compared to PEA treatment only.

In periphery, URB597-mediated inhibition of AEA degradation (Figure 5) was maintained over the entire time course also when co-administered with PEA, rendering in periphery constant, significantly higher levels of AEA compared to PEA-treated mice. Exogenous administration of PEA and inhibition of endogenous PEA degradation by URB597 had similar effects on the PEA level in plasma. The combined treatment with URB597 and PEA, however, rendered a cumulative effect on the plasma PEA level over the entire time course indicated by the significantly higher levels of plasma PEA compared to those obtained by URB597 or PEA treatment alone (Figure 5). URB597 and PEA/URB597 treatments have a pronounced effect on plasma 2-AG over the entire time course maintaining it at significantly lower levels compared to PEA treatment. Conversely, AA levels are significantly augmented for up to 120 min upon URB597 and URB597/PEA compared to PEA treatment. PEA, URB597 and URB597/PEA treatments modulate the inflammation markers PGE₂ and PGD₂ in a similar manner and extent, except for acute phase where the PGE₂ level is maintained higher upon URB597 compared to PEA treatment (Figure 5).

In plasma, URB937-induced blockade of the peripheral FAAH rendered significantly increased AEA and PEA levels over the entire 180 min time span compared to PEA treatment only (Figure 6). Interestingly, the potency of URB937 in increasing AEA levels (Figure 6) was about two fold less compared to URB597 (Figure 5), but about two fold higher in increasing the PEA levels (Figures 5, 6). Thus, our data suggest that URB937 is more effective in inhibiting PEA degradation than URB597, but URB597 is more potent than URB937 in blocking AEA degradation (Figures 5, 6).

Plasma levels of 2-AG were significantly decreased upon URB937 administration and in co-administration with PEA, which indicates a similar 2-AG modulation as by URB597 and URB597/PEA treatments. PGE₂ is maintained in the late-seizure phase (120–180 min) at higher levels upon URB937 and URB937/PEA treatment compared to PEA treatment (Figure 6).

Interestingly, the URB937 treatment had similar effects as the PEA treatment on the hippocampal response of AEA, 2-AG, AA, PGE₂ and PGD₂ to KA-excitotoxic stimulus (Figure 6). This effect is likely modulated by the high levels of endogenous PEA in hippocampus and plasma resulting from URB937 treatment (Figure 6, see “Discussion” section). In hippocampus, upon URB937 and URB937/PEA treatment the only significant changes compared to PEA treatment were that of PEA elevation over the entire time course and of PGE₂ at the late phase (180 min; Figure 6).

Conclusively, the peripheral effects of URB937 on the AEA levels are less effective compared to PEA treatment to achieve a sustained anticonvulsive effect, and this concurs with significantly stronger epileptic seizure episodes compared to PEA treatment at acute and late-acute seizure phase (Figure 6). This also indicates that pharmacological modulation of peripheral FAAH tone only is not sufficient to sustain anticonvulsive effects.