International Journal of Neuropsychopharmacology (2018) 21(8): 786–795 doi:10.1093/ijnp/pyy033 Advance Access Publication: March 22, 2018 Regular Research Article regular research article Inhibition of Fatty Acid Amide Hydrolase by PF-3845 Alleviates the Nitrergic and Proinflammatory Response in Rat Hippocampus Following Acute Stress Hsiao-Jou Cortina Chen, Jereme G. Spiers, Conrad Sernia, Nickolas A. Lavidis School of Biomedical Sciences, The University of Queensland, St Lucia, Australia (Drs Chen, Spiers, Sernia, and Lavidis); MRC Toxicology Unit, University of Leicester, Leicester, United Kingdom (Dr Spiers). Correspondence: Hsiao-Jou Cortina Chen, PhD, School of Biomedical Sciences, The University of Queensland, St Lucia, 4072, Australia (email@example.com). ABSTRACT Background: Long-term exposure to stress has been demonstrated to cause neuroinflammation through a sustained overproduction of free radicals, including nitric oxide, via an increased inducible nitric oxide synthase activity. We previously demonstrated that inducible nitric oxide synthase activity and mRNA are significantly upregulated in the rat hippocampus following just 4 hours of restraint stress. Similar to nitric oxide, endocannabinoids are synthesized on demand, with preclinical observations suggesting that cannabinoid receptor agonists and endocannabinoid enhancers inhibit nitrergic activity. Specifically, previous work has shown that enhancement of endocannabinoids via inhibition of fatty acid amide hydrolase with PF-3845 reduced inducible nitric oxide synthase-expressing microglia following traumatic brain injury. However, this describes cannabinoid modulation following physical injury, and therefore the present study aimed to examine the effects of PF-3845 in the modulation of nitrergic and inflammatory-related genes within the hippocampus after acute stress exposure. Methods: Following vehicle or PF-3845 injections (5 mg/kg; i.p.), male Wistar rats were exposed to 0 (control), 60, 240, or 360 minutes of restraint stress after which plasma and dorsal hippocampus were isolated for further biochemical and gene expression analysis. Results: The results demonstrate that pretreatment with PF-3845 rapidly ameliorates plasma corticosterone release at 60 minutes of stress. An increase in endocannabinoid signalling also induces an overall attenuation in inducible nitric oxide synthase, tumor necrosis factor-alpha convertase, interleukin-6, cyclooxygenase-2, peroxisome proliferator-activated receptor gamma mRNA, and the transactivation potential of nuclear factor kappa-light-chain-enhancer of activated B cells in the hippocampus. Conclusions: These results suggest that enhanced endocannabinoid levels in the dorsal hippocampus have an overall antinitrosative and antiinflammatory effect following acute stress exposure. Keywords: acute stress, endocannabinoids, fatty acid amide hydrolase inhibitor, inducible nitric oxide synthase, neuroinflammatory response Introduction Glucocorticoids are the final hormonal response of an acti- roles of glucocorticoids along with protecting the host from vated hypothalamic-pituitary-adrenal (HPA) axis, which exerts noxious insults is the regulation of immune homeostasis a plethora of physiological functions through the ubiqui- (Cancedda et al., 2002; Cain and Cidlowski, 2017). Stress and tously expressed glucocorticoid receptors. One of the essential glucocorticoids are generally regarded to be antiinflammatory, Received: October 11, 2017; Revised: March 15, 2018; Accepted: March 21, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 786 provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Downloaded from https://academic.oup.com/ijnp/article-abstract/21/8/786/4951485 by Ed 'DeepDyve' Gillespie user on 07 August 2018 Chen et al. | 787 Significance Statement This study employed an enzyme inhibitor, PF-3845, to pharmacologically enhance endocannabinoid content that demonstrated antiinflammatory properties by decreasing the expression of iNOS and other inflammatory genes following acute psychological stress in rats. as numerous studies have demonstrated their ability to sup- on demand postsynaptically as neuromodulators in response to press gene transcription of several proinflammatory cytokines stress and generally function in opposition to the HPA response. (Barnes, 1998; Coutinho and Chapman, 2011). Nevertheless, in For example, the selective cannabinoid receptor type 1 (CBR) addition to the antiinflammatory actions, it has become increas- antagonist has been reported to modulate corticosterone release ingly apparent that the initial rise in glucocorticoids follow- and exert anxiogenic effects (Wade et al., 2006)Hill . and col- ing stress exposure primes the immune effector cells through leagues (2011) demonstrated that endocannabinoid signaling innate immune signalling pathways such as toll-like receptor plays an important role in suppressing corticosteroid secretion upregulation (de Pablos et al., 2006F ; rank et al., 2007, 2012; following stress through inhibition of GABAergic neurotransmis- Wohleb et al., 2011). This is considered to be an important adap- sion. Furthermore, pharmacological inhibition of fatty acid amide tive action by glucocorticoids, with dissipation of the fight/flight hydrolase (FAAH), an enzyme responsible for rapid physiological response heightening immune defences and vigilance beyond degradation of anandamide, robustly blocked stress-induced basal levels and promoting more vigorous reactions to persist- corticosterone release and anxiety-like behaviors (Patel et al., ing or impending threats (Sapolsky et al., 2000; Johnson et al., 2004; Bluett et al., 2014). In addition to a critical involvement in 2002; Sorrells et al., 2009). Specifically, within the CNS, it has stress modulation, endocannabinoids have demonstrated antiin- been demonstrated that short-term stress can modulate the flammatory properties, as FAAH knockout mice have a reduced microglial immunophenotype to sensitize a neuroinflammatory lipopolysaccharide-induced hyperalgesia and oedema, an effect response that may be exaggerated in subsequent inflamma- mediated mainly through cannabinoid receptor type 2 (Naidu tory challenges (Blandino et al., 2006 Fr ; ank et al., 2007; Sugama et al., 2010). Accumulating evidence has also suggested that endo- et al., 2007, 2009, 2011; Blandino et al., 2009). Activated microglia cannabinoids and their derivatives mediate antiinflammatory upregulate antigen presenting molecules such as major histo- effects by serving as potent agonists for the peroxisome prolif- compatibility complex, increase phagocytic activity, generate erator-activated receptors (PPARs), a subfamily of nuclear recep- proinflammatory cytokines and reactive nitrogen species, and tors (Ulrich-Lai and Ryan, 2013; O’Sullivan, 2016). Interestingly, overall induce a neuroinflammatory response (de Pablos et al., Tchantchou and colleagues (2014) have recently demonstrated 2006; Frank et al., 2007; Colton, 2009; Chen et al., 2016). effective suppression of traumatic brain injury-induced neuroin- Proinflammatory cytokines including tumor necrosis factor flammation, including iNOS and Cox-2 expression, in microglia/ alpha (Tnf-α) and interleukin-1β are among the principle messen- macrophages by PF-3845, an irreversible FAAH inhibitor with high gers responsible for initiating and coordinating the acute phase sensitivity and long duration. However, this represents modula- inflammatory response, with interleukin-1β acting through auto- tion occurring in a condition of chronic injury, while the effects of crine/paracrine signalling to further potentiate interleukin-6 pro- endocannabinoid modulation by FAAH inhibition on nitrergic and duction (Dinarello, 1998). Furthermore, it has been reported that inflammatory indicators following a short-term stress remain following acute immobilization stress, the increase in soluble Tnf- unclear. Therefore, the present study has examined the effects α was released by a zinc-dependent, modular cell surface protein of PF-3845 on nitrergic indicators and inflammatory-related gene known as a disintegrin and metalloproteinase 17 (Adam17), or expression changes in the dorsal hippocampus following differ - commonly as Tnf-α convertase (TACE), in the rat brain cortex ing durations of acute restraint stress. (Madrigal et al., 2002). In recent years, it has become clear that interleukin-1 and Tnf-α triggered the canonical nuclear factor Materials And Methods kappa-light-chain-enhancer of activated B cells (NF- B) κ pathway involving RelA and C-Rel, subsequently initiating downstream Ethical Approval and Experimental Animals inflammatory, oxidative, and nitrosative signaling (Mercurio and Manning, 1999; Albrecht et al., 2007Solt et ; al., 2007). An alterna- All experimental procedures were in accordance with regulations tive NF-κB pathway can be activated by Tnf receptor superfam- and policies outlined by The University of Queensland Animal ily members resulting in activation of RelB and p52 complexes Ethics Committee under AEC approval number SBS/456/14/URG. (Sun, 2011). Another important inflammatory signaling cascade Outbred male Wistar rats (Rattus norvegicus) aged 5 to 6 weeks post- involves cyclooxygenase-2 (Cox-2), a constitutively expressed natal weighing 206.6 ± 2.04 g were sourced from The University of enzyme in the brain that converts arachidonic acid into prosta- Queensland Biological Resources breeding colony. Rats were housed glandin. A number of studies have demonstrated its involvement individually under controlled laboratory conditions (22°C ± 2°C; in HPA axis activation, with inhibition of Cox-2 demonstrating 55 ± 5% humidity) with a 12-hour-light/dark cycle (lights off at 12:30 antidepressant-like properties without affecting major cytokine pm). Standard rat chow and water were available ad libitum. responses following lipopolysaccharide-injection (de Paiva et al., 2010; Teeling et al., 2010; Ma et al., 2013). Experimental Protocol Recently, cannabinoids have received extensive research interest due to their roles in increasing sleep and fat storage Part A while attenuating the inflammatory response and stress-induced To evaluate the effectiveness of PF-3845 on FAAH antagonism fol- behaviors (Pacher et al., 2006). The 2 major endogenous can- lowing acute stress, an experiment was conducted using 5 mg/kg nabinoids, N-arachidonoylethanolamine (commonly known as of PF-3845 dissolved in 2% dimethyl sulfoxide in normal saline anandamide) and 2-arachidonoylglycerol (2-AG), are produced (0.9% sodium chloride) as adapted from Ahn and colleagues Downloaded from https://academic.oup.com/ijnp/article-abstract/21/8/786/4951485 by Ed 'DeepDyve' Gillespie user on 07 August 2018 788 | International Journal of Neuropsychopharmacology, 2018 (2009). Rats were habituated to human handling for 10 min/d, -80 C for later determination of corticosterone concentrations. 6 days prior to experimentation. On the day of the treatment, Rats were then overdosed with 100 mg/kg of sodium pentobar - rats were transported in individual home cages from the colony bital (i.p.). The whole brain was rapidly removed and frozen on room to an experimental room within the same animal facility. powdered dry ice for storage at -80C. Dorsal hippocampus was The FAAH inhibitor PF-3845 was injected i.p. with an injection isolated and stored at -80 C for later relative gene expression volume of 10 mL/kg 1 hour prior to a 60-minute acute restraint analysis. We have included plasma measures for one animal stress (stress starts at 1:30 pm) using wire mesh restrainers. from which we were unable to obtain hippocampal tissue and During this period, rats were acclimatized to the novel experi- hence was excluded from analysis of gene expression. mental room under low light and noise. To isolate the effects of restraint stress, all rats were deprived of food and water during Plasma Corticosterone Assay the 1-hour habituation and stress treatment period. Rats were Corticosterone concentrations were measured by an in-house then randomly allocated to 0 (control) or 60 minute stress groups radioimmunoassay using anti-rat corticosterone polyclonal (n = 4 per group). At the end of each treatment period, rats were antibody (Sapphire Bioscience Pty. Ltd.) and tritiated [1, 2, 6, 7- weighed and killed with sodium pentobarbital (100 mg/kg i.p. H]-corticosterone tracer as previously described in Spiers and injection; Lethabarb, Virbac), and the whole brain was rapidly colleagues (2016). Radioactivity was counted in liquid scintillation removed and snap frozen. Frozen brains were sectioned on a cocktail (Ultima Gold, Perkin Elmer) using a Liquid Scintillation cryostat, and the dorsal hippocampus was cryo-dissected from Spectrometer (Tri-Carb 3100 TR, Perkin Elmer). Dichloromethane sections according to Banasr and colleagues (2006) and a rat extraction recovery was 79.14% and intra- and inter-assay coef- brain atlas (Paxinos and Watson, 2007). The tissue was stored at ficients of variation were 5.79% and 2.12%, respectively. -80 C for FAAH activity assay and markers of NO and nitration including total nitrite and nitrate and 3-nitrotyrosine. Hippocampal tissues were homogenized with 10 volumes Real-Time PCR (w/v) of homogenizing buffer (0.1 M Tris-HCl, 20 µM of aprotinin, Total RNA was extracted from the dorsal hippocampus using a 100 µM of leupeptin, and 1µM of pepstatin A), and an aliquot of RNeasy mini kit (Qiagen) treated with deoxyribonuclease I and whole homogenate was removed for 3-nitrotrosine determin- reversed transcribed into cDNA using iScript Reverse Transcription ation. A commercially available ELISA kit (ab116691, Abcam) was Supermix (Bio-Rad Laboratories) according to the manufacturer’s used for the quantitative measurement of 3-nitrotyrosine accord- instructions. Taqman gene expression assay-on-demand kits (Life ing to the manufacturer’s instructions. The remaining crude hom- Technologies) with optimized primers and FAM-labelled probes ogenate was centrifuged at 10 000 × g for 10 minutes at 4°C, and the were used to detect gene expression of Nos2 (inducible nitric oxide resultant supernatant was used in the measurement of nitrite and synthase; Rn00561646_m1), Nos1 (neuronal nitric oxide synthase; nitrate and FAAH activity. The total NO metabolites, NO , w xere Rn00583793_m1), Tnf (tumor necrosis factor alpha; Rn01525859_ measured in ultra-filtered supernatant using a commercially avail- g1), Adam17 (ADAM metallopeptidase domain 17; Rn00571880_ able total nitrate/nitrite colorimetric assay kit (Cayman Chemical m1), Slc39a14 (solute carrier family 39 (zinc transporter), member Company). A fluorescence-based assay was used to detect FAAH 14; Rn01468336_m1), Il1b (interleukin 1 beta; Rn00580432_m1), activity using a novel substrate for FAAH, arachidonyl 7-amino, Il6 (interleukin 6; Rn01410330_m1), Ptgs2 (prostaglandin-endop- 4-methyl coumarin amide (AAMCA), modified from Ramarao and eroxide synthase 2; Rn01483828_m1), Nfkbia (nuclear factor of colleagues (2005). The enzyme FAAH hydrolyses AAMCA, result- kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; ing in the liberation of the highly fluorescent 7-amino 4-methyl Rn01473657_g1), and Pparγ (peroxisome proliferator activated coumarin (AMC) that can be monitored at an excitation wave- receptor gamma; Rn00440945_m1). The ∆∆C method was used for length of 355 nm and emission wavelength at 460 nm. In brief, all expression assays, which were run in a multiplex reaction with the assay was carried out in black-walled plates containing 50 µL VIC-labelled GAPDH as endogenous control using the QuantStudio of supernatant diluted in 0.05 M Tris-HCl (pH = 9.0) with 50 µL of 6 Flex Real-Time PCR System (Applied Biosystems). 10 µM AAMCA solution in assay buffer (0.05 M Tris-HCl, 2 mM EDTA, 100 mM HEPES, 0.04% Triton X-100, 2 mg/mL BSA; pH = 9.0). The assay plates were incubated at room temperature for 2.5 Statistical Analysis hours with readings taken every 15 minutes using the POLARstar Data were analyzed using statistical software GraphPad Prism OPTIMA microplate reader (BMG Labtechnologies) to kinetically (Version 7.03, GraphPad Software Inc.). All data sets were first monitor AAMCA hydrolysis by FAAH in the sample. Data were checked for normality using the Brown-Forsythe test. Normally finally adjusted with corresponding protein concentrations. distributed data were subsequently analyzed using 2-way ANOVA with Fisher’s Least Significant Difference test to make Part B comparisons between all groups with the vehicle control and Rats were habituated as described earlier and were randomly between vehicle and PF-3845 data at corresponding time-points. allocated to 8 treatment groups (n = 5–7) with control, or Results were expressed as mean ± SEM and P values < .05 were restraint stress (starts at 1:30 PM) for 60, 240, and 360 minutes considered statistically significant. with either vehicle or PF-3845 injection. The FAAH inhibitor, PF-3845 (5 mg/kg; Sigma), dissolved in 2% dimethyl sulfoxide in normal saline (0.9% sodium chloride) was injected 60 minutes Results before stress treatment with an injection volume of 10 mL/kg (i.p.). Following each allocated treatment, a blood sample was Systemic PF-3845 Treatment Decreased FAAH collected in sodium heparin (20 IU/mL blood) via tail-tipping. Activity and 3-Nitrotyrosine Formation in the Dorsal Heparinized blood samples were centrifuged at 2000 x g for 5 Hippocampus minutes immediately after collection, and plasma glucose levels were determined using standard glucometer (FreeStyle Optium Treatment with PF-3845 at a dose of 5 mg/kg was effective at Neo, Abbott). The resulting plasma supernatant was stored at decreasing FAAH activity in the dorsal hippocampus measured by Downloaded from https://academic.oup.com/ijnp/article-abstract/21/8/786/4951485 by Ed 'DeepDyve' Gillespie user on 07 August 2018 Chen et al. | 789 AAMCA hydrolysis when injected 1 hour prior to stress treatment making this a good downstream indicator of stress induction. The (Table 1). Two-way ANOVA demonstrated a significant main effect accompanying changes in plasma glucose following treatments of PF-3845 treatment [F = 25.94, P = .0003] on FAAH activity with demonstrated a time-dependent effect [F = 27.4, P < .0001]. (1, 12) (3, 36) no significant effects of time and the interaction between drug Compared with vehicle controls, posttest analysis showed a sig- treatment and stress duration. There was a significant reduction nificant increase at 60 minutes of restraint in the vehicle-treated in AMC liberation indicative of reduced FAAH activity in PF-3845- group alone, while both vehicle and PF-3845 treated animals treated groups at 0 (P < .01) and 60 (P < .05) minutes of stress com- displayed decreased glucose by 360 minutes of restraint expos- pared with the corresponding vehicle groups. Two-way ANOVA also ure. Moreover, PF-3845 treatment significantly decreased glucose showed a main effect of PF-3845 treatment [F = 19.32, P = .0009] mobilization compared with the corresponding vehicle group fol- (1, 12) on 3-nitrotyrosine levels. Posttest analysis further revealed thatlo wing 60 minutes (P < .05) of restraint stress (Figure 1b). PF-3845 treatment reduced hippocampal 3-nitrotyrosine compared with vehicle-treated groups at 0 (P < .01) and 60 (P < .05) minutes of Upregulation of iNOS Following Exposure to restraint stress exposure. No significant difference was observed in Restraint was Attenuated by PF-3845 total NO betw x een the vehicle- and PF-3845-treated groups. Figure 2a demonstrates a progressive time-dependent [F (3, = 10.17, P < .0001] increase in hippocampal iNOS mRNA levels 40) Inhibition of FAAH Dampens Acute Stress-Induced in both vehicle and PF-3845-treated groups [F = 4.191, P = .047] (1, 40) Corticosterone Release and Glucose Mobilization that became significant following exposure to 360 minutes of To determine whether increased endocannabinoid signaling mod- restraint. Posttest analysis also revealed PF-3845 treatment signifi- ulates HPA output, we determined concentrations of the plasma cantly ameliorated the increase in inducible NOS mRNA expres- stress hormone, corticosterone. Pretreatment with the FAAH sion compared with the corresponding vehicle-treated group at inhibitor, PF-3845, significantly attenuated corticosterone release 360 minutes (P < .05) of restraint exposure. No significant changes [F = 5.863, P = .0201] following restraint stress exposure in a were observed in hippocampal neuronal NOS (Nos1) mRNA levels (1, 36) time-dependent manner [F = 24.42, P < .0001]. Compared with between vehicle- and PF-3845-treated groups (Figure 2b). (3, 36) vehicle controls, restraint effectively elevated plasma cortico- sterone in all groups regardless of drug treatment. However, sig- PF-3845 Alters Tnf-α Expression and Ameliorates nificantly lower corticosterone concentrations were observed in TACE and ZIP14 Upregulation Following Exposure to the PF-3845-treated group (P < .01) compared with the correspond- Restraint Stress ing vehicle group at 60 minutes of stress (Figure 1a). Stress expos- ure triggers physiological processes, including hepatic glycolysis Treatment with FAAH inhibitor altered hippocampal Tnf-α and gluconeogenesis, that acutely increase circulating glucose, mRNA expression [F = 6.963, P = .0122] following 60 and 240 (1, 36) Table 1. The Effect of the Fatty Acid Amide Hydrolase Inhibitor PF-3845 (5 mg/kg, i.p. injection) on Fatty Acid Amide Hydrolase Activity Measured by AMC fluorescence, NOx, and 3-Nitrotyrosine from Control and Stressed Rats (n = 4/group) Treatment Duration of Stress (min) Vehicle PF-3845 Hippocampal AMC fluorescence 0 15.19 ± 0.98 9.70 ± 1.05** (AU/mg protein) 60 14.10 ± 0.85 10.11 ± 0.82* Hippocampal NOx 0 2.95 ± 0.45 2.04 ± 0.40 (µM/mg protein) 60 2.85 ± 0.47 1.72 ± 0.25 Hippocampal 0 43.67 ± 1.67 31.93 ± 3.54** 3-nitrotyrosine 60 38.22 ± 2.45 29.29 ± 0.90* (ng/mg protein) Isolated dorsal hippocampus was collected from rats exposed to 0 (control) and 60 minute of restraint stress. Two-way ANOVA followed by Fisher’s least significant difference test. Data are expressed as mean ± SEM. *P < .05 and **P < .01 vs vehicle-treated group at each respect- ive time-point. Figure 1. The effect of the fatty acid amide hydrolase inhibitor, PF-3845 (5 mg/kg, i.p. injection), on plasma (A) corticosterone and (B) glucose levels from control and stressed rats (n = 5–7/group). Plasma corticosterone and glucose concentration was determined in blood samples collected via tail-tipping from rats exposed to 0 (con- † †† ††† trol), 60, 240, and 360 minutes of acute restraint stress. Data are presented as mean ± SEM. P < .05, P < .01, and P < .001 vs vehicle-treated group at 0 (control) minute; *P < .05 and **P < .01 between vehicle- and PF-3845-treated groups at each respective time-point. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/8/786/4951485 by Ed 'DeepDyve' Gillespie user on 07 August 2018 790 | International Journal of Neuropsychopharmacology, 2018 (P < .05) minutes of restraint exposure compared with the cor - the zinc importer Slc39a14 (Zip14). Posttest analysis revealed responding vehicle groups, which displayed mild but nonsig- vehicle treatment caused a transient increase in Slc39a14 mRNA nificant decreases compared with the vehicle-treated control levels following 60 minutes (P < .05) of restraint stress compared group (Figure 3a). The proinflammatory cytokine, Tnf-α, is syn- with the vehicle controls. This increase at 60 minutes was thesized as a 32-kDa transmembrane anchored precursor that attenuated in PF-3845-treated animals compared with the cor - is cleaved by TACE, yielding a nonglycosylated soluble 17-kDa responding vehicle group (Figure 3c). protein. A 2-way ANOVA demonstrated that administration of PF-3845 significantly reduced Adam17 mRNA levels [F = 15.21, (1, 36) Inhibition of FAAH Constrains Upregulation P = .0004] in the dorsal hippocampus. Posttest analysis revealed of Interleukin-1β and Interleukin-6 Following vehicle treatment alone resulted in a progressive increase in Acute Stress Adam17 mRNA that became significant following 360 minutes of exposure to restraint compared with vehicle controls. This Inflammatory cytokines are rapidly upregulated following increase was attenuated by treatment with PF-3845 at 60 (P < .05), activation of immune cells that reside in the CNS, including 240, and 360 [P < .01] minutes of restraint stress compared with microglia, CNS macrophages, and astrocytes. Previous stud- the corresponding vehicle-treated animals (Figure 3b). It is ies have revealed the antiinflammatory properties exerted by known that proteolytically active ADAM enzymes contain zinc endocannabinoids are partly through attenuation of micro- binding sequences in their catalytic domain and the activity of glial activation. In the present study, there was a significant TACE can be reduced by zinc chelators. Treatment with PF-3845 time-dependent [F = 17.21, P < .0001] and PF-3845 treatment- (3, 36) significantly reduced [F = 4.71, P = .0367] mRNA expression of dependent [F = 4.358, P = .044] effect on Il1b gene expression (1, 36) (1, 36) Figure 2. The effect of the fatty acid amide hydrolase inhibitor, PF-3845 (5 mg/kg, i.p. injection), on hippocampal (A) inducible nitric oxide synthase (Nos2) and (B) neur - onal nitric oxide synthase (Nos1) mRNA expression from control and stressed rats (n = 5–6/gr oup). The relative expression was determined in the dorsal hippocampus † ††† collected from rats exposed to 0 (control), 60, 240, and 360 minutes of acute restraint stress. Data are presented as mean ± SEM P < .05 and P < .001 vs vehicle-treated group at 0 (control) minute; *P < .05 between vehicle- and PF-3845-treated groups at each respective time-point. Figure 3. The effect of the fatty acid amide hydrolase inhibitor, PF-3845 (5 mg/kg, i.p. injection), on hippocampal (A) tumor necrosis factor (Tnf), (B) a disintegrin and metalloprotease 17 (Adam17), and (C) solute carrier family 39 member 14 (Slc39a14) mRNA expression from control and stressed rats (n = 5–6/gr oup). The relative mRNA expression was determined in the dorsal hippocampus from rats exposed to 0 (control), 60, 240, and 360 minutes of acute restraint stress. Data are presented as mean ± SEM. P < .05 vs vehicle-treated group at 0 (control) minute; *P < .05 and **P < .01 between vehicle- and PF-3845-treated groups at each respective time-point. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/8/786/4951485 by Ed 'DeepDyve' Gillespie user on 07 August 2018 Chen et al. | 791 following restraint stress exposure. Posttest analysis showed a PF-3845 Acutely Suppressed Expression of Cox-2 progressive increase in both treatment groups that significantly and Attenuated the Upregulation of IκB-α and PPARγ differed from vehicle controls following 240 (P < .05) and 360 mRNA in the Dorsal Hippocampus (P < .001) minutes of restraint in vehicle-treated and 360 minutes It has been demonstrated that Cox-2 is constitutively expressed of restraint in PF-3845-treated groups (Figure 4a). Treatment in the brain and is primarily responsible for the production of with PF-3845 also reduced interleukin-1β mRNA (P < .05) com- prostanoids, including prostaglandin E , which is involved in pared with the corresponding vehicle-treated group following 2 pathological inflammatory conditions. Notably in Figure 5a, 60 minutes of restraint stress. Furthermore, FAAH inhibition [F (1, PF-3845 treatment [F = 4.476, P = .041] resulted in decreased = 18.95, P = .0001] also attenuated the stress-induced upregu- (1, 36) 36) Cox-2 mRNA levels in animals exposed to 60 minutes of stress lation of interleukin-6 mRNA in a time-dependent manner [F (3, compared with both the vehicle-treated controls (P < .01) and = 6.049, P = .002]. Exposure to restraint robustly upregulated 36) the corresponding vehicle-treated group at 60 minutes (P < .05) interleukin-6 mRNA in vehicle-treated groups at 60 (P < .05), of restraint. The transcription factor NF-кB plays a central role 240, and 360 (P < .001) minutes of restraint compared with the in the proinflammatory signaling pathway by regulating the vehicle controls (Figure 4b). Treatment with PF-3845 effectively expressions of proteins involved in oxidative stress and inflam- attenuated this increase, with further posttest analysis reveal- mation, including iNOS, pro-inflammatory cytokines, and Cox- ing significant reductions compared with corresponding vehi- 2. To examine the NF-кB transactivation potential, expression cle-treated groups at 60 (P < .05), 240, and 360 (P < .01) minutes of of the inhibitory subunit Nfkbia (or IκB-α) was monitored as restraint exposure. Figure 4. The effect of the fatty acid amide hydrolase inhibitor, PF-3845 (5 mg/kg, i.p. injection), on hippocampal (A) interleukin-1β (Il1b) and (B) interleukin-6 (Il6) mRNA expression from control and stressed rats (n = 5–6/group). The relative mRNA expression was determined in the dorsal hippocampus from rats exposed to 0 (control), † †† ††† 60, 240, and 360 minutes of acute restraint stress. Data are presented as mean ± SEM. P < .05, P < .01, and P < .001 vs vehicle-treated group at 0 (control) minute; *P < .05 and **P < .01 between vehicle- and PF-3845-treated groups at each respective time-point. Figure 5. The effect of the fatty acid amide hydrolase inhibitor, PF-3845 (5 mg/kg, i.p. injection), on hippocampal (A) prostaglandin-endoperoxide synthase 2 (Ptgs2; also known as cyclooxygenase-2 or Cox-2), (B) nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (Nfkbia), and (C) peroxisome proliferator activated receptor gamma (Pparγ) mRNA expression from control and stressed rats (n = 5–6/gr oup). The relative mRNA expression was determined in the dorsal hippo- †† ††† campus from rats exposed to 0 (control), 60, 240, and 360 minutes of acute restraint stress. Data are presented as mean ± SEM. P < .01 and P < .001 vs vehicle-treated group at 0 (control) minute; *P < .05, **P < .01, and ***P < .001 between vehicle- and PF-3845-treated groups at each respective time-point. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/8/786/4951485 by Ed 'DeepDyve' Gillespie user on 07 August 2018 792 | International Journal of Neuropsychopharmacology, 2018 described by Bottero and colleagues (2003). Two-way ANOVA AMPA receptor internalization, resulting in long-term depres- demonstrated PF-3845 was highly effective [F = 19.16, P < .001] sion in the basolateral amygdala (Duan et al., 2017). Thus, it is (1, 36) in ameliorating stress-induced IκB-α mRNA expression over the very likely that the attenuation in stress-induced corticosterone time course of the experiment [F = 15.05, P < .001] (Figure 5b). observed in the present study was due to FAAH inhibition within (3, 36) Compared with vehicle-treated controls, posttest analysis the amygdala, which in turn reduced the overall drive through revealed restraint stress induced a sustained increase in vehicle- the HPA axis and consequently decreased plasma glucose at treated animals following 60, 240, and 360 (P < .001) minutes, an 60 minutes of stress. This is in agreement with results from effect only transiently observed in PF-3845-treated animals at 60 Rivera and colleagues (2015) demonstrating decreased levels minutes of restraint. Moreover, PF-3845 treatment significantly of plasma glucose, triglycerides, and total cholesterol following reduced IκB-α mRNA compared with the corresponding vehicle- acute or repeated treatment with the selective FAAH inhibi- treated groups at 60 (P < .001), 240, and 360 (P < .05) minutes of tor [3-(3-Carbamoylphenyl)phenyl] N-cyclohexylcarbamate restraint stress exposure. In addition to preventing the break- (URB597). down of endocannabinoids, inhibitors of FAAH also prevent Previous reports have demonstrated that the expression breakdown of the ligands for PPARγ. Treatment with PF-3845 of the calcium-independent NOS isoform can become rapidly effectively attenuated increases in hippocampal PPARγ mRNA elevated within astrocytes and microglia following stimulation [F = 3.82, P < .0179] following exposure to restraint stress in a by a variety of factors, including proinflammatory cytokines (3, 36) time-dependent manner [F = 15.51, P < 001] (Figure 5c). There (Aktan, 2004; Calabrese et al., 2007; Chen et al., 2015). We have (1, 36) was a progressive increase in vehicle-treated groups that became previously demonstrated a 10.5-fold increase in whole hip- significant following 360 minutes (P < .05) of restraint exposure pocampal iNOS mRNA following 240 minutes of restraint stress, compared with vehicle-treated controls. Posttest analysis also which corresponded well with the 2-fold increase in iNOS revealed PF-3845 treatment reduced PPARγ mRNA expression at activity (Chen et al., 2016). In comparison, the increase in iNOS 60, 240 (P < .05), and 360 (P < .01) minutes of restraint stress com- mRNA was not as robust in the present study using only the pared with the corresponding vehicle-treated groups. dorsal hippocampus, increasing by approximately 3-fold at the equivalent restraint duration and approximately 6-fold at the longest restraint duration tested, and we observed no signifi- Discussion cant changes in expression of the calcium-dependent isoform, This study has demonstrated that administration of the FAAH nNOS. This increase in iNOS mRNA was significantly attenuated by FAAH inhibition at 360 minutes of restraint compared with inhibitor, PF-3845, robustly suppresses acute stress-induced upregulation of nitrergic and proinflammatory responses due to the vehicle group and is likely a combination of the early reduc- tion in corticosterone secretion by PF-3845 administration and blockade of anandamide degradation within the dorsal hippo- campus. The results show that pretreatment with PF-3845 rap- a direct antiinflammatory action of the drug. Previous reports have shown that selective CBR agonists inhibit nitrite produc- idly dampens restraint stress-induced plasma corticosterone release and glucose liberation. Treatment with this FAAH inhibi- tion and iNOS protein expression in an astroglia-derived cell line following lipopolysaccharide and interferon gamma stimu- tor also attenuated iNOS, TACE, interleukin-6, Cox-2, and PPARγ mRNA upregulation and ameliorated the transactivation poten- lation (Esposito et al., 2001). Thus, activation of the cannabinoid receptor that is negatively coupled to adenylate cyclase and sub- tial of NF-κB, suggesting that enhanced endocannabinoid levels in the dorsal hippocampus have an overall antinitrosative and sequent altered cAMP signaling appears to be one likely mech- anism mediating the inhibition of iNOS expression (Esposito antiinflammatory effect following acute stress exposure. Intraperitoneal injection of 5 mg/kg PF-3845 effectively low- et al., 2001; Won et al., 2004; Turu and Hunyady, 2010). The transcriptional activation of iNOS has previously been ered tonic FAAH activity at 0 and 60 minutes of restraint stress (1 and 2 hours postdosing) measured by a selective FAAH substrate shown to rely heavily on Tnf-α-induced NF-κB transactiva- tion. Madrigal and colleagues (2002) demonstrated that 1 hour AAMCA (Ramarao et al., 2005). It has been demonstrated previ- ously that PF-3845-mediated FAAH inhibition rapidly elevated of stress caused an increase in cortical TACE activity and Tnf- α level, leading to translocation of NF-κB into the nucleus by anandamide and other endogenous fatty acid ethanolamides such as oleoylethanolamide and palmitoylethanolamide, which 4 hours and a subsequent increase in iNOS activity by 6 hours of stress exposure. Moreover, as little as 30 minutes of stress peaked at 2 to 3 hours postinjection (Ahn et al., 2009). It was also confirmed that FAAH inhibition with PF-3845 decreased was sufficient to increase TACE activity with no change in cor - responding protein levels. Pharmacological inhibition of TACE nitric oxide signaling compared with vehicle-treated groups using 3-nitrotyrosine as a collective index of protein nitration activity strongly attenuated these changes, indicating that Tnf- α production is involved in NF-κB activation prior to iNOS tran- (Schopfer et al., 2003; Radi, 2004). This occurred in the absence of increased nitric oxide metabolism and irrespective of stress scription. In the present study, stress exposure induced delayed differences in Adam17 mRNA, with PF-3845 treatment-depend- exposure, indicating increased endocannabinoid signalling may potentiate the metabolism of existing nitrated proteins. ent changes in Tnf/Adam17 resulting in differences that related more to the antiinflammatory nature of the drug. Although this The importance of endocannabinoid signalling in regulation of the stress system has been demonstrated by several stud- may seem paradoxical comparison with the results obtained by Madrigal and colleagues (2002) in the cortex, it is likely that the ies (Patel et al., 2004W ; ade et al., 2006; Cota et al., 2007; Steiner et al., 2008). Enhanced FAAH-mediated anandamide hydrolysis hippocampus undergoes a constrained increase in secreted Tnf- α at the protein level via increased TACE-induced cleavage of pro- has been observed in response to acute stress, and therefore the rapid loss of anandamide levels in the basolateral amygdala may Tnf protein already present in the cytosol, and changes at the mRNA level may occur earlier than the time-points examined play an important role in the disinhibition of HPA axis follow- ing stress (Hill et al., 2009). Moreover, due to this physiological in the present study (Madrigal et al., 2002 Gooz, ; 2010). Cleavage of pro-Tnf may be facilitated by increased zinc availability, as it role of anandamide, administration of PF-3845 to mice exposed to chronic corticosterone treatment has anxiolytic effects has been shown previously that TACE is a zinc-dependent met- alloprotease and its catalytic activity can be inhibited by zinc through activation of astroglial CB R, which in turn triggers Downloaded from https://academic.oup.com/ijnp/article-abstract/21/8/786/4951485 by Ed 'DeepDyve' Gillespie user on 07 August 2018 Chen et al. | 793 chelating agents (Moss et al., 2001Udec ; hukwu et al., 2017). In (García-Bueno et al., 2005). Using specific pharmacological tools, this study, we demonstrated that exposure to 60 minutes of García-Bueno and colleagues (2008) further demonstrated that restraint stress increased expression of the zinc transporter PPARγ activation following restraint stress is mediated by the (Zip14) in the dorsal hippocampus of vehicle-treated animals, stress-induced increase in catecholamines, glucocorticoids, and an effect that was ameliorated by PF-3845 treatment. Increased glutamatergic signalling. expression of this cell membrane-localized zinc importer in In conclusion, psychological stress produces a characteris- vehicle-treated animals could function to support TACE in tic temporal profile in several proinflammatory cytokines and terms of providing zinc for structural stabilization and substrate nitrosative activity in the dorsal hippocampus. Reductions in catalysis. In addition to the Tnf/TACE system, stress expos- corticosterone and direct antiinflammatory action of enhanced ure also altered hippocampal interleukin-1β and interleukin-6 endocannabinoid signaling resulted in decreased proinflamma- mRNA in a time-dependent manner. Both interleukin-1β and tory markers following psychological stress exposure. Although interleukin-6 mRNA increased in the hippocampus following this present work was limited to measurements of mRNA lev- stress exposure in vehicle-treated animals. Converging evidence els, we have previously shown iNOS mRNA correlates well with indicates that the immediate cellular source of these proinflam- enzymatic activity and thus, PF-3845-mediated reductions in matory cytokines derives from the sensitization of microglia by iNOS mRNA following stress exposure likely attenuate stress- glucocorticoids and norepinephrine (Blandino et al., 2006 F; rank induced nitrosative stress on a biochemical level. By estab- et al., 2007, 2012; Chen et al., 2016). Furthermore, previous stud- lishing the importance of the endocannabinoid system in the ies have suggested that microglia most likely constitute the neuroinflammatory response following stress, this study opens main cellular source of endocannabinoid under neuroinflam- up potential therapeutic targets for conditions exacerbated or matory conditions (Stella, 2009). In the present study, increased caused by prolonged stress exposure. endocannabinoid signalling by PF-3845 caused an early inhib- ition of the increase in interleukin-1β that was only significantly Funding different from vehicle treatment following 60 minutes of stress. However, PF-3845 markedly attenuated increases in hippocam- This work was supported by a University of Queensland Research pal interleukin-6 mRNA at all durations of stress exposure. The Grant. H.-J.C. Chen and J.G. Spiers were recipients of APA and magnitude of these changes may indicate that the endocannab- UQRS scholarships. These funding bodies have no further role in inoid system preferentially interacts with interleukin-6 during a the study design; in the collection, analysis, and interpretation normal response to stress. However, interleukin-6 also exhibits of data; in the writing of the report; and in the decision to sub- a positive feed-forward expression, and therefore reductions in mit the paper for publication. corticosterone by PF-3845 may prevent transcription and further potentiation of interleukin-6 mRNA in the hippocampus (Bethin Acknowledgments et al., 2000; Spooren et al., 2011). There was a subtle but significant reduction in hippocampal We thank Professor Karen M. Moritz for her continuous support Cox-2 following 60 minutes of stress in the PF-3845-treated group. and sharing of reagents. We also thank the animal technicians While Cox-2 has been heavily linked to the proinflammatory from the University of Queensland Biological Resources animal pathway, it also mediates inactivation of anandamide signaling by house for animal care and husbandry. catalyzing the formation of prostaglandins from arachidonic acid (Hermanson et al., 2014). Several studies have demonstrated that Statement of Interest selective Cox-2 inhibitors can elevate anandamide and 2-AG con- centrations in addition to prolonging the depolarization-induced None. suppression of inhibition representing a protracted action of endocannabinoids (Kim and Alger, 2004; Hermanson et al., 2014). References In the present study, the status of NF- B follo κ wing stress exposure and drug treatment was determined by monitoring the inhibitory Ahn K, Johnson DS, Mileni M, Beidler D, Long JZ, McKinney MK, subunit IκB-α, responsible for binding and inactivating NF-Bκ. 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International Journal of Neuropsychopharmacology – Oxford University Press
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