Additive antinociceptive action of intrathecal anandamide reuptake inhibitor and morphine in the management of post-incisional pain in rats.

Spinal opioids have mixed efficacy and their adverse effects force treatment cessation of postoperative pain. Consequently, there is an ongoing search for new therapeutic strategies. Here, we evaluated the analgesic efficacy of intrathecal UCM707, an anandamide reuptake inhibitor, and morphine combination. Firstly, we assessed the effects of morphine (1, 5 and 10 µg), UCM707 (75 µg) and its combination in the hot plate. Then, morphine + UCM707 at sub-effective doses was evaluated in a rat post-incisional pain model. In addition, µ-, CB1r-, CB2r- and TRPV1-antagonists were pre-administered before the combination. Activation of µ-opioid and CB1r, and Cnr1, Cnr2, Oprm1 and TRPV1 expressions were evaluated in the lumbar sacra and periaqueductal grey by [35 S]-GTPγS binding autoradiography and qPCR studies. In the hot plate, morphine (1 µg) and UCM707 (75 µg) induced a more robust analgesic effect than each drug alone. Morphine plus UCM707 did not modify µ-opioid nor CB1 receptor function in the PAG or LS. Cnr1 and TRPV1 expression increased in the lumbar sacra (LS). Morphine plus UCM707 significantly reduced post-incisional pain at 1 and 4 days after surgery. Cnr1, Cnr2 and TRPV1 expressions increased in the LS. Blockade of µ-opioid receptor reduced combination effects on days 1 and 4. CB1r- and CB2r-antagonism reduced morphine + UCM707 effects on days 1 and 4, respectively. CB1r and TRPV1-antagonism improved their antinociceptive effects on day 4. These results revealed a synergistic/additive analgesic effect of UCM707 and morphine combination controlling postincisional pain. CB1r, CB2r and TRPV1 contribute differently as central sensitization occurs.

Molecular Engineering of Electrosprayed Hydrogel Microspheres to Achieve Synergistic Anti-Tumor Chemo-Immunotherapy with ACEA Cargo.

Molecular engineering of drug delivering platforms to provide collaborative biological effects with loaded drugs is of great medical significance. Herein, cannabinoid receptor 1 (CB1)- and reactive oxygen species (ROS)-targeting electrosprayed microspheres (MSs) are fabricated by loading with the CB1 agonist arachidonoyl 2'-chloroethylamide (ACEA) and producing ROS in a photoresponsive manner. The synergistic anti-tumor effects of ACEA and ROS released from the MSs are assessed. ACEA inhibits epidermal growth factor receptor signaling and altered tumor microenvironment (TME) by activating CB1 to induce tumor cell death. The MSs are composed of glycidyl methacrylate-conjugated xanthan gum (XGMA) and Fe3+, which form dual molecular networks based on a Fe3+-(COO-)3 network and a C═C addition reaction network. Interestingly, the Fe3+-(COO-)3 network can be disassembled instantly under the conditions of lactate sodium and ultraviolet exposure, and the disassembly is accompanied by massive ROS production, which directly injures tumor cells. Meanwhile, the transition of dual networks to a single network boosts the ACEA release. Together, the activities of the ACEA and MSs promote immunogenic tumor cell death and create a tumor-suppressive TME by increasing M1-like tumor-associated macrophages and CD8+ T cells. In summation, this study demonstrates strong prospects of improving anti-tumor effects of drug delivering platforms through molecular design.

ACEA Attenuates Oxidative Stress by Promoting Mitophagy via CB1R/Nrf1/PINK1 Pathway after Subarachnoid Hemorrhage in Rats.

METHOD: Endovascular perforation was performed to establish a SAH model of rats. ACEA was administered intraperitoneally 1 h after SAH. The CB1R antagonist AM251 was injected intraperitoneally 1 h before SAH induction. Adenoassociated virus- (AAV-) Nrf1 shRNA was infused into the lateral ventricle 3 weeks before SAH induction. Neurological tests, immunofluorescence, DHE, TUNEL, Nissl staining, transmission electron microscopy (TEM), and Western blot were performed. RESULTS: The expression of CB1R, Nrf1, PINK1, Parkin, and LC3II increased and peaked at 24 h after SAH. ACEA treatment exhibited the antioxidative stress and antiapoptosis effects after SAH. In addition, ACEA treatment increased the expression of Nrf1, PINK1, Parkin, LC3II, and Bcl-xl but repressed the expression of Romo-1, Bax, and cleaved caspase-3. Moreover, the TEM results demonstrated that ACEA promoted the formation of mitophagosome and maintained the normal mitochondrial morphology of neurons. The protective effect of ACEA was reversed by AM251 and Nrf1 shRNA, respectively. CONCLUSIONS: This study demonstrated that ACEA alleviated oxidative stress and neurological dysfunction by promoting mitophagy after SAH, at least in part via the CB1R/Nrf1/PINK1 signaling pathway.

Assessing the ability of boys with Duchenne muscular dystrophy age 4-7 years to swallow softgel capsules: Clinical trial experience with edasalonexent.

WHAT IS KNOWN AND OBJECTIVE: There is limited information on acceptability of solid dosage forms by young patients with neuromuscular disorders such as Duchenne muscular dystrophy (DMD). Capsule size selection and ability to swallow the NF-κB inhibitor edasalonexent were assessed in males 4-7 years of age with DMD enrolled in clinical trials for a new therapeutic. METHODS: The Phase 3 PolarisDMD randomized, double-blind, placebo-controlled trial enrolled 131 patients from 8 countries. The Phase 2 MoveDMD trial enrolled 31 patients in the United States. As part of enrolment criteria, these trials assessed the ability to swallow softgel 100 mg (~10 mm) or 250 mg (~15 mm) capsules formulated with a phosphatidylcholine-containing coating. Supportive strategies included pill-swallowing techniques and aids. RESULTS: Most (97%; 175/181) patients screened were able to swallow capsules. In Phase 2 and 3, respectively, 77% (24/31) and 61% (80/131) of enrolled patients selected the larger capsule and among those selecting the smaller capsule, most transitioned to the larger capsule. There were no obvious geographical differences in ability to swallow capsules and size selection was not correlated with age. Compliance was high (92%-98%) through 52 weeks of dosing with no discontinuations due to capsule burden. WHAT IS NEW AND CONCLUSION: Swallowing of capsules was not a barrier for drug administration in young patients with DMD. Capsule formulations may be an acceptable alternative to liquid formulations for children as young as 4 years of age.

[Controversy about the critical role of long-chain polyunsaturated fatty acids, arachidonic acid (ARA) and docosahexaenoic acid (DHA), during infancy]. / Controversia actual sobre el papel crítico de los ácidos grasos poliinsaturados de cadena larga, araquidónico (ARA) y docosahexaenoico (DHA), en el lactante.

INTRODUCTION: Long-chain polyunsaturated fatty acids (LC-PUFAs) are critical for infant growth and development, particularly arachidonic acid (ARA, C20:4n-6) and docosahexaenoic acid (DHA, C22:6n-3). ARA and DHA are components of cell membrane phospholipids and play an important role in cell division, differentiation, and signaling; and DHA is the n-3 fatty acid predominant in the developing brain and retina. During the third trimester of pregnancy, LC-PUFAs increase substantially in fetal circulation, and a "biomagnification" process in the fetal brain is observed. Moreover, LC-PUFAs are precursors of eicosanoids and metabolites, which modulate the intensity and duration of the immune response. LC-PUFA synthesis implies complex desaturation and elongation processes on their principal precursors, linoleic acid (LA) (18:3 n-6) (series n-6) and α-linolenic acid (LNA) (20:3 n-3) (series n-3), where fatty acid desaturases (FADS) and elongases (ELOVL) are competing. It is important to notice that during the first months of life, as a consequence of low enzymatic activity, LC-PUFA synthesis from LA and LNA is reduced, especially in those infants carrying variations in the FADS and ELOVL genes, which are involved in LC-PUFA synthesis, and so they are unable to supply their own DHA and ARA needs. Homozygote infants for FADS haplotype A (97 % of the Latinoamerican population) show low levels of ARA (only 43 %) and DHA (only 24 %) when compared to those carrying haplotype D (more prevalent in Europe, Africa and Asia). Human milk is the only source of LA, LNA, ARA, and DHA for the neonate and infant till complementary feeding (CF) is introduced. Infants fed with infant formulas must receive enough amounts of LA, LNA, ARA, and DHA to cover their nutritional requirements. The new guidelines by the European Food Safety Authority (EFSA) (2016) recommend that infant formulas and follow-on formulas must contain 20-50 mg of DHA/100 kcal (0.5-1 % of total fatty acids, which is higher than in human milk and the majority of infant formulas in the market), and it is not necessary to add ARA. This new regulation, which is already applicable since February 2020, has resulted in profound controversy because there is no scientific evidence about its appropriateness and safety for healthy children. Then, different international expert groups have revised the research already published about the effects of ARA and DHA addition to infant formulas, and discussed different emerging questions from this European directive. The expert group led from the University of Granada (Spain) recommends the addition of ARA in similar or higher concentrations than those of DHA, at least equal to those present in human milk (0.3 % of total fatty acids), although preferably 0.5 % and up to around 0.64 % of total fatty acids, since new studies confirm the optimal intake of ARA and DHA during the different developmental stages. This recommendation could be of particular importance for infants carrying the haplotype A of FADS.

INTRODUCCIÓN: Los ácidos grasos poliinsaturados de cadena larga (AGPI-CL) son críticos para el crecimiento y desarrollo infantil, en particular los ácidos araquidónico (ARA, C20:4n-6) y docosahexaenoico (DHA, C22:6n-3). El ARA y el DHA son componentes de los fosfolípidos de las membranas celulares y desempeñan importantes funciones en la división, diferenciación y señalización celular, siendo el DHA el ácido graso de la serie n-3 predominante en el cerebro y la retina en desarrollo. Durante el tercer trimestre de la gestación, los AGPI-CL aumentan de forma sustancial en la circulación fetal, observándose un proceso de "biomagnificación" en el cerebro fetal. Además, los AGPI-CL son precursores de los eicosanoides y metabolitos implicados en la modulación de la intensidad y duración de la respuesta inmunitaria. La síntesis de AGPI-CL implica un complejo proceso de desaturación y elongación desde los precursores principales, el ácido linoleico (18:3 n-6) (LA) (serie n-6) y el ácido α-linolénico (20:3 n-3) (LNA) (serie n-3), por los cuales compiten las enzimas desaturasas (FADS) y elongasas (ELOVL). Es importante indicar que en los primeros meses de vida, como consecuencia de la baja actividad enzimática, la síntesis de AGPI-CL a partir de LA y LNA es reducida, especialmente en los niños con variaciones en los genes que codifican las FADS y ELOVL involucradas en la síntesis de AGPI-CL y que, por tanto, son incapaces de cubrir por sí mismos sus necesidades de ARA y DHA. Los homocigotos para el haplotipo A de las FADS (97 % de la población latinoamericana) muestran niveles de ARA y DHA de tan solo un 43 % y un 24 %, respectivamente, inferiores a los de los individuos con haplotipo D (más frecuente en Europa, África y Asia). La leche humana constituye la única fuente de LA, LNA, ARA y DHA para el recién nacido y el lactante hasta la introducción de la alimentación complementaria (AC). Los niños alimentados con fórmulas infantiles deben recibir las cantidades de LA, LNA, ARA y DHA suficientes para cubrir los requerimientos nutricionales. La nueva normativa de la Autoridad Europea de Seguridad Alimentaria (EFSA) (2016) indica que las fórmulas infantiles de inicio y continuación deben contener entre 20 y 50 mg de DHA/100 kcal (0,5-1 % del total de ácidos grasos: más elevado que en la leche humana y en la mayoría de fórmulas infantiles comercializadas) sin la necesidad de incluir también ARA. Esta nueva regulación, que está vigente desde febrero de 2020, ha despertado una gran controversia, al no existir evidencia científica acerca de su pertinencia y seguridad para los niños sanos. Por ello, diferentes grupos de expertos internacionales han revisado la investigación publicada acerca del ARA y el DHA, y discutido diferentes cuestiones emergentes a partir de esta nueva directiva Europea. El grupo de expertos, liderado desde la Universidad de Granada (España), recomienda la adición de ARA en concentraciones iguales o mayores que las de DHA, alcanzando al menos el contenido presente en la leche humana (0,3 % del total de ácidos grasos), aunque preferiblemente un 0,5 % y hasta alrededor del 0,64 % del total de AG, hasta que nuevos estudios confirmen la ingesta óptima de ARA y DHA durante las distintas etapas del desarrollo. Esta recomendación podría ser de especial importancia para los niños portadores del haplotipo A de las FADS.

Differential Effects of 17,18-EEQ and 19,20-EDP Combined with Soluble Epoxide Hydrolase Inhibitor t-TUCB on Diet-Induced Obesity in Mice.

17,18-Epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) are bioactive epoxides produced from n-3 polyunsaturated fatty acid eicosapentaenoic acid and docosahexaenoic acid, respectively. However, these epoxides are quickly metabolized into less active diols by soluble epoxide hydrolase (sEH). We have previously demonstrated that an sEH inhibitor, t-TUCB, decreased serum triglycerides (TG) and increased lipid metabolic protein expression in the brown adipose tissue (BAT) of diet-induced obese mice. This study investigates the preventive effects of t-TUCB (T) alone or combined with 19,20-EDP (T + EDP) or 17,18-EEQ (T + EEQ) on BAT activation in the development of diet-induced obesity and metabolic disorders via osmotic minipump delivery in mice. Both T + EDP and T + EEQ groups showed significant improvement in fasting glucose, serum triglycerides, and higher core body temperature, whereas heat production was only significantly increased in the T + EEQ group. Moreover, both the T + EDP and T + EEQ groups showed less lipid accumulation in the BAT. Although UCP1 expression was not changed, PGC1α expression was increased in all three treated groups. In contrast, the expression of CPT1A and CPT1B, which are responsible for the rate-limiting step for fatty acid oxidation, was only increased in the T + EDP and T + EEQ groups. Interestingly, as a fatty acid transporter, CD36 expression was only increased in the T + EEQ group. Furthermore, both the T + EDP and T + EEQ groups showed decreased inflammatory NFκB signaling in the BAT. Our results suggest that 17,18-EEQ or 19,20-EDP combined with t-TUCB may prevent high-fat diet-induced metabolic disorders, in part through increased thermogenesis, upregulating lipid metabolic protein expression, and decreasing inflammation in the BAT.

Calcium-independent phospholipase A2 inhibitor produces an analgesic effect in a rat model of neuropathic pain by reducing central sensitization in the dorsal horn.

OBJECTIVE: Phospholipase A2 (PLA2) plays an important role in regulating the production of arachidonic acid and various eicosanoids. The aim of our study was to investigate the analgesic mechanisms of calcium-dependent cytosolic phospholipase A2 and calcium-independent PLA2 (iPLA2) inhibitors in the spinal cord in a rat model of neuropathic pain. METHODS: Lumbar 5 spinal nerve ligation was performed in male Sprague-Dawley rats to develop a peripheral neuropathic pain model. Paw withdrawal thresholds in response to von Frey filaments, brush, pressure, and pinch were measured. Lumbar wide dynamic range neuronal firing rates and iPLA2 subtype expression were measured by in vivo extracellular recording and double immunofluorescence staining, respectively. RESULTS: In our rat models, oral administration of prednisolone, a non-selective PLA2 inhibitor, and intrathecal injection of bromoenolactone, a iPLA2 inhibitor, significantly increased the ipsilateral hindpaw withdrawal thresholds in response to von Frey filament stimulation, but intrathecal injection of arachidonyl trifluoromethyl ketone, a selective cytosolic PLA2 inhibitor, did not show significant changes. In spinal dorsal horn neurons, bromoenolactone reduced neuronal firing rates in response to withdrawal stimulation and spontaneous firing rates in the ipsilateral side of the spinal dorsal horn. In addition, the expression of iPLA2 was co-localized with astrocytes and neurons on the ipsilateral side of the dorsal horn in rats that underwent spinal nerve ligation. DISCUSSION: These data suggest that selective iPLA2 inhibitor produce analgesia in neuropathic rats by reducing central sensitization in the dorsal horn.

Inhibitory effect of intrathecally administered AM404, an endocannabinoid reuptake inhibitor, on neuropathic pain in a rat chronic constriction injury model.

BACKGROUND: The endocannabinoid system modulates a wide variety of pain conditions. Systemically administered AM404, an endocannabinoid reuptake inhibitor, exerts antinociceptive effects via activation of the endocannabinoid system. However, the mechanism and site of AM404 action are not fully understood. Here, we explored the effect of AM404 on neuropathic pain at the site of the spinal cord. METHODS: Male Sprague-Dawley rats were subjected to chronic constriction injury (CCI) of the sciatic nerve. The effects of intrathecal administration of AM404 on mechanical and cold hyperalgesia were examined using the electronic von Frey test and cold plate test, respectively. Motor coordination was assessed using the rotarod test. To understand the mechanisms underlying the action of AM404, we tested the effects of pretreatment with the cannabinoid type 1 (CB1) receptor antagonist AM251, CB2 receptor antagonist AM630, and transient receptor potential vanilloid type 1 (TRPV1) antagonist capsazepine. RESULTS: AM404 attenuated mechanical and cold hyperalgesia with minimal effects on motor coordination. AM251 significantly inhibited the antihyperalgesic action of AM404, whereas capsazepine showed a potentiating effect. CONCLUSIONS: These results indicate that AM404 exerts antihyperalgesic effects primarily via CB1, but not CB2, receptor activation at the site of the spinal cord. TRPV1 receptors appear to play a pronociceptive role in CCI rats. The endocannabinoid reuptake inhibitor may be a promising candidate treatment for neuropathic pain.

Cannabinoid Receptor-2 Activation in Keratinocytes Contributes to Elevated Peripheral ß-Endorphin Levels in Patients With Obstructive Jaundice.

BACKGROUND: Cholestatic diseases are often accompanied by elevated plasma levels of endogenous opioid peptides, but it is still unclear whether central or peripheral mechanisms are involved in this process, and little is known about the change of pain threshold in these patients. The purpose of this study was to determine the preoperative pain threshold, postoperative morphine consumption, and central and peripheral ß-endorphin levels in patients with obstructive jaundice. This study also tests the hypothesis that activation of the cannabinoid receptor-2 (CB2R) in skin keratinocytes by endocannabinoids is the mechanism underlying circulating ß-endorphin elevation in patients with obstructive jaundice. METHODS: The electrical pain thresholds, 48-hour postoperative morphine consumption, concentrations of ß-endorphin in plasma and cerebrospinal fluid, skin and liver ß-endorphin expression, and plasma levels of endocannabinoids were measured in jaundiced (n = 32) and control (n = 32) patients. Male Sprague-Dawley rats and human keratinocytes (human immortalized keratinocyte cell line [HaCaT]) were used for the in vivo and in vitro experiments, respectively. Mechanical and thermal withdrawal latency, plasma level, and skin expression of ß-endorphin were measured in CB2R-antagonist-treated and control bile duct-ligated (BDL) rats. In cultured keratinocytes, the effect of CB2R agonist AM1241-induced ß-endorphin expression was observed and the phosphorylation of extracellular-regulated protein kinases 1/2, p38, and signal transducer and activator of transcription (STAT) pathways were investigated. RESULTS: This study found (1) the plasma level of ß-endorphin (mean ± standard error of the mean [SEM]) was 193.9 ± 9.6 pg/mL in control patients, while it was significantly increased in jaundiced patients (286.6 ± 14.5 pg/mL); (2) the electrical pain perception threshold and the electrical pain tolerance threshold were higher in patients with obstructive jaundice compared with controls, while the 48-hour postoperative morphine consumption was lower in the jaundiced patients; (3) there was no correlation between plasma ß-endorphin levels, electrical pain thresholds, and 48-hour postoperative morphine consumption in patients with obstructive jaundice; (4) the plasma level of the endogenous cannabinoid anandamide was increased in the jaundiced patients; (5) CB2R antagonist treatment of the BDL rats reduced ß-endorphin levels in plasma and skin keratinocytes, while it did not alter the nociceptive thresholds in BDL and control rats; (6) the endocannabinoid anandamide-induced ß-endorphin synthesis and release via CB2R in cultured keratinocytes; and (7) phosphorylation of extracellular-regulated protein kinases 1/2 is involved in the CB2R-agonist-induced ß-endorphin expression in keratinocytes. CONCLUSIONS: CB2R activation in keratinocytes by the endocannabinoid anandamide may play an important role in the peripheral elevation of ß-endorphin during obstructive jaundice.

The endocannabinoid 2-arachidonoylglycerol and dual ABHD6/MAGL enzyme inhibitors display neuroprotective and anti-inflammatory actions in the in vivo retinal model of AMPA excitotoxicity.

The endocannabinoid system has been shown to be a putative therapeutic target for retinal disease. Here, we aimed to investigate the ability of the endocannabinoid 2-arachidonoylglycerol (2-AG) and novel inhibitors of its metabolic enzymes, α/ß-hydrolase domain-containing 6 (ABHD6) and monoacylglycerol lipase (MAGL), a) to protect the retina against excitotoxicity and b) the mechanisms involved in the neuroprotection. Sprague-Dawley rats, wild type and Akt2-/- C57BL/6 mice were intravitreally administered with phosphate-buffered saline or (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA). 2-AG was intravitreally co-administered with AMPA in the absence and presence of AM251 or AM630 (cannabinoid 1 and 2 receptor antagonists, respectively) or Wortmannin [Phosphoinositide 3-Kinase (PI3K)/Akt inhibitor]. Inhibitors of ABHD6 and dual ABHD6/MAGL (AM12100 and AM11920, respectively) were co-administered with AMPA intravitreally in rats. Immunohistochemistry was performed using antibodies raised against retinal neuronal markers (bNOS), microglia (Iba1) and macroglia (GFAP). TUNEL assay and real-time PCR were also employed. The CB2 receptor was expressed in rat retina (approx. 62% of CB1 expression). 2-AG attenuated the AMPA-induced increase in TUNEL+ cells. 2-AG activation of both CB1 and CB2 receptors and the PI3K/Akt downstream signaling pathway, as substantiated by the use of Akt2-/- mice, afforded neuroprotection against AMPA excitotoxicity. AM12100 and AM11920 attenuated the AMPA-induced glia activation and produced a dose-dependent partial neuroprotection, with the dual inhibitor AM11920 being more efficacious. These results show that 2-AG has the pharmacological profile of a putative therapeutic for retinal diseases characterized by neurodegeneration and neuroinflammation, when administered exogenously or by the inhibition of its metabolic enzymes.

Central nervous system, peripheral and hemodynamic effects of nanoformulated anandamide in hypertension.

PURPOSE: Hypertensive lesions induce alterations at hemodynamic, peripheral, and central levels. Anandamide (N-arachidonoylethanolamine; AEA) protects neurons from inflammatory damage, but its free administration may cause central adverse effects. AEA controlled release by nanoformulations could reduce/eliminate its side effects. The present study aimed to evaluate the effects of nanoformulated AEA (nf-AEA) on systolic blood pressure (SBP), behavior, and central/peripheral inflammatory, oxidative, and apoptotic state in spontaneously hypertensive rats (SHR). MATERIALS/METHODS: Male rats were used, both Wistar Kyoto (WKY) and SHR (n â€‹= â€‹10 per group), with/without treatment with nf-AEA (obtained by electrospraying) at a weekly dose of 5 â€‹mg/kg IP for 4 weeks. SBP was measured and behavioral tests were performed. Inflammatory/oxidative markers were quantified at the central (brain cortex) and peripheral (serum) level. RESULTS: SHR showed hyperactivity, low anxiety, and high concentrations of central/peripheral inflammatory/oxidative markers, also higher apoptosis of brain cortical cells compared to WKY. As opposed to this group, treatment with nf-AEA in SHR significantly reduced SBP, peripheral/central inflammatory/oxidative makers, and central apoptosis. Nf-AEA also increased neuroprotective mechanisms mediated by intracellular heat shock protein 70 (Hsp70), which were attenuated in untreated SHR. Additionally, nf-AEA reversed the abnormal behaviors observed in SHR without producing central adverse effects. CONCLUSIONS: Our results suggest protective properties of nf-AEA, both peripherally and centrally, through a signaling pathway that would involve the type I angiotensin II receptor, Wilms tumor transcription factor 1, Hsp70, and iNOS. Considering non-nf-AEA limitations, this nanoformulation could contribute to the development of new antihypertensive and behavioral disorder treatments associated with neuroinflammation.

Effects of systemic endocannabinoid manipulation on social and exploratory behavior in prairie voles (Microtus ochrogaster).

RATIONALE: Anandamide is an endocannabinoid that contributes to certain aspects of social behavior, like play and reward, by binding to cannabinoid receptor type 1 (CB1). Most interesting is the recent discovery that anandamide may be mobilized by oxytocin receptor activation under certain contexts, particularly in the nucleus accumbens. OBJECTIVES: Given the established role of oxytocin and the nucleus accumbens in the neurobiology of pair-bonding, we investigated whether systemic administration of brain-permeable modulators of the endocannabinoid system could alter preferential partner contact in both male and female prairie voles. METHODS: Specifically, we tested whether intraperitoneal administration of the neutral CB1 antagonist AM4113 (4.0-16.0 mg/kg) or the anandamide hydrolysis inhibitor URB597 (5.0-20.0 mg/kg) could prevent or facilitate partner preference formation, respectively. To further investigate the specificity of effects on partner preference, we repeated our URB597 dosing regimen on an additional group of females and tested their anxiety-related behavior in both an elevated-plus maze and a light/dark test. RESULTS: AM4113 administration had no effect on partner preference. But while URB597 also had no effect on partner preference, low-dose females did increase absolute preferential contact with either the partner or the stranger; individual females spent significant contact time with either the partner or the stranger. None of our outcome measures in either anxiety test showed significant effects of treatment. CONCLUSIONS: Our results reveal that experimentally increasing anandamide levels in female prairie voles can increase social contact with both a familiar and novel male via unknown mechanisms that are likely separate from anxiety reduction.

Pharmaco-molecular assessment of the effects of anandamide and its antagonists on hippocampal tissue in Wistar albino rats.

OBJECTIVE: The members of the matrix metalloproteinase (MMP) family and cannabinoids (CBs) are reportedly associated with hippocampus-dependent memory functions. However, the effects of endogenously formed CBs on hippocampal long-term potentiation remain unknown. The present study aimed to investigate the changes in the gene and protein expression levels of matrix metallopeptidase 9 (MMP-9), phosphatase and tensin homolog (PTEN), and NOTCH receptor 1 (NOTCH1) in rat hippocampal tissues treated with anandamide (AEA), AM251, 6-iodopravadolin (AM630), and N-[4-{[(3,4-Dimethyl-5-isoxazolyl)amino]sulfonyl}phenyl] (ML193). MATERIALS AND METHODS: The subjects were divided into 10 groups (n = five per group). The pharmaceuticals were administered via intraperitoneal injection once a day for seven days, except for the control group. The resected hippocampal tissues were then evaluated using a quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analysis. The data obtained were statistically analyzed, and p < 0.01 was considered statistically significant. RESULTS: Contrary to the literature, the changes in MMP-9 expression were not statistically significant, but the changes in PTEN and NOTCH1 were. The findings of this in vivo experimental study revealed that the agonists and antagonists acting on the CB system have significant molecular effects on hippocampal tissue. CONCLUSIONS: The changes in gene and protein expressions may be one of the reasons for the neurodegenerative processes observed in patients using these agonists and antagonists, whose effects on the CB system have not been fully explained yet. Our study can contribute to the literature as it is the first study investigating the MMP-9, PTEN and NOTCH1 gene and protein expression.

Peripheral deficiency and antiallodynic effects of 2-arachidonoyl glycerol in a mouse model of paclitaxel-induced neuropathic pain.

BACKGROUND: Modulation of the endocannabinoid system has been shown to alleviate neuropathic pain. The aim of this study was to evaluate if treatment with paclitaxel, a chemotherapeutic agent that induces neuropathic pain, affects endocannabinoid levels at a time when mice develop paclitaxel-induced mechanical allodynia. We also evaluated the peripheral antiallodynic activity of the endocannabinoid 2-arachidonoyl glycerol (2-AG) and an inhibitor of monoacylglycerol lipase (MAGL), an enzyme responsible for 2-AG hydrolysis. METHODS: Female BALB/c mice were treated intraperitoneally with paclitaxel to induce mechanical allodynia. Levels of the endocannabinoids, N-arachidonoylethanolamine (anandamide, AEA), 2-AG, and the N-acylethanolamines (NAEs), N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), which are structurally-related to AEA, in the brain, spinal cord and paw skin were measured using LC-MS/MS. Protein expression of MAGL in the paw skin was measured using Wes™. The effects of subcutaneous (s.c.) injection of 2-AG and JZL184 (a MAGL inhibitor) into the right hind paw of mice with paclitaxel-induced mechanical allodynia were assessed using the dynamic plantar aesthesiometer. The effects of pretreatment, s.c., into the right hind paw, with cannabinoid type 1 (CB1) receptor antagonist AM251 and CB2 receptor antagonist AM630 on the antiallodynic effects of 2-AG were also evaluated. RESULTS: The levels of 2-AG were reduced only in the paw skin of paclitaxel-treated mice, whilst the levels of AEA, PEA and OEA were not significantly altered. There was no change in the expression of MAGL in the paw skin. Administration of 2-AG and JZL184 produced antiallodynic effects against paclitaxel-induced mechanical allodynia in the injected right paw, but did not affect the uninjected left paw. The antiallodynic activity of 2-AG was antagonized by both AM251 and AM630. CONCLUSION: These results indicate that during paclitaxel-induced mechanical allodynia there is a deficiency of 2-AG in the periphery, but not in the CNS. Increasing 2-AG in the paw by local administration of 2-AG or a MAGL inhibitor, alleviates mechanical allodynia in a CB1 and CB2 receptor-dependent manner.

Arachidonyl-2'-chloroethylamide (ACEA), a synthetic agonist of cannabinoid receptor, increases CB1R gene expression and reduces dyskinesias in a rat model of Parkinson's disease.

l-Dopa is the most effective drug used for Parkinson's disease (PD), but after long-term treatment, the vast majority of PD patients develop abnormal involuntary movements (AIMs) termed l-Dopa-induced dyskinesia (LID). Cannabinoid receptors in the basal ganglia can modulate motor functions, but their role in the treatment of LID is controversial. Therefore, the aim of this study is to evaluate the motor behavior and mRNA expression of the cannabinoid receptor-1 (CB1R), encoded by the Cnr1 gene, in the striatum and globus pallidus of a 6-hydroxydopamine rat model of PD. The evaluated rats had 6-hydroxydopamine-induced injury, LID, and LID treated with arachidonyl-2'-chloroethylamide (ACEA), a cannabinoid receptor agonist. Contralateral turns and AIMs were recorded to assess motor behavior. Gene expression was quantified by reverse transcription coupled with quantitative polymerase chain reaction using TaqMan probes. Behavioral evaluations demonstrated that dyskinetic rats treated with ACEA had a significant reduction in AIMs compared to the dyskinetic group. The expression of CB1R mRNA was significantly decreased in the 6-hydroxydopamine-injured and dyskinetic rats, compared to intact rats. The striata of dyskinetic rats treated with ACEA exhibited highly significant increases in CB1R mRNA expression. Contrary to results in the striatum, a lower CB1R expression was observed in globus pallidus from dyskinetic ACEA-treated group. In summary, significant differences in mRNA expression of CB1R were found between the evaluated groups of rats, suggesting the occurrence of compensatory mechanisms that may result in the ACEA-mediated reduction of dyskinesias in a rat model of PD.

Sciadonic acid derived from pine nuts as a food component to reduce plasma triglycerides by inhibiting the rat hepatic Δ9-desaturase.

Sciadonic acid (Scia) is a Δ5-olefinic fatty acid that is particularly abundant in edible pine seeds and that exhibits an unusual polymethylene-interrupted structure. Earlier studies suggested that Scia inhibited the in vitro expression and activity of the Stearoyl-CoA Desaturase 1 (SCD1), the hepatic Δ9-desaturase involved in the formation of mono-unsaturated fatty acids. To confirm this hypothesis, rats were given 10% Scia in diets balanced out with n-6 and n-3 fatty acids. In those animals receiving the Scia supplement, monoene synthesis in the liver was reduced, which was partly attributed to the inhibition of SCD1 expression. As a consequence, the presence of Scia induced a 50% decrease in triglycerides in blood plasma due to a reduced level of VLDL-secreted triglycerides from the liver. In non-fasting conditions, results showed that Scia-induced inhibition of SCD1 led to a decrease in the proportions of 16:1n-7 and 18:1n-7 in the liver without impacting on the level of 18:1n-9, suggesting that only triglycerides with neosynthesized monoenes are marked out for release. In conclusion, this in vivo study confirms that Scia highly inhibits SCD1 expression and activity. The work was performed on normo-triglyceride rats over six weeks, suggesting promising effects on hyper-triglyceridemic models.

Biophysical and biomechanical properties of neural progenitor cells as indicators of developmental neurotoxicity.

Conventional in vitro toxicity studies have focused on identifying IC50 and the underlying mechanisms, but how toxicants influence biophysical and biomechanical changes in human cells, especially during developmental stages, remain understudied. Here, using an atomic force microscope, we characterized changes in biophysical (cell area, actin organization) and biomechanical (Young's modulus, force of adhesion, tether force, membrane tension, tether radius) aspects of human fetal brain-derived neural progenitor cells (NPCs) induced by four classes of widely used toxic compounds, including rotenone, digoxin, N-arachidonoylethanolamide (AEA), and chlorpyrifos, under exposure up to 36 h. The sub-cellular mechanisms (apoptosis, mitochondria membrane potential, DNA damage, glutathione levels) by which these toxicants induced biochemical changes in NPCs were assessed. Results suggest a significant compromise in cell viability with increasing toxicant concentration (p < 0.01), and biophysical and biomechanical characteristics with increasing exposure time (p < 0.01) as well as toxicant concentration (p < 0.01). Impairment of mitochondrial membrane potential appears to be the most sensitive mechanism of neurotoxicity for rotenone, AEA and chlorpyrifos exposure, but compromise in plasma membrane integrity for digoxin exposure. The surviving NPCs remarkably retained stemness (SOX2 expression) even at high toxicant concentrations. A negative linear correlation (R2 = 0.92) exists between the elastic modulus of surviving cells and the number of living cells in that environment. We propose that even subtle compromise in cell mechanics could serve as a crucial marker of developmental neurotoxicity (mechanotoxicology) and therefore should be included as part of toxicology assessment repertoire to characterize as well as predict developmental outcomes.

Docosahexaenoic and Arachidonic Acid Supplementation of Toddlers Born Preterm Does Not Affect Short-Term Growth or Adiposity.

BACKGROUND: Dietary DHA intake among US toddlers is low. Healthy physical growth is an important objective for the clinical care of children born preterm. OBJECTIVES: The aim of the trial was to examine the effects of supplementing toddlers born preterm with DHA and arachidonic acid (AA) for 180 d on growth and adiposity. METHODS: Omega Tots, a randomized placebo-controlled trial, was conducted between April 2012 and March 2017. Children born at <35 wk gestation who were 10-16 mo in corrected age were assigned to receive daily oral supplements of DHA and AA (200 mg each, "DHA + AA") or corn oil (placebo) for 180 d. Prespecified secondary outcomes included weight, length, head circumference, mid-upper arm circumference, triceps and subscapular skinfolds, BMI, and their respective z scores, and body fat percentage, which were measured at baseline and trial completion. Mixed-effects regression was used to compare the change in outcomes between the DHA + AA and placebo groups, controlling for baseline values. RESULTS: Among 377 children included in the analysis (median corrected age = 15.7 mo, 48.3% female), 348 (92.3%) had growth or adiposity data at baseline and trial end. No statistically significant differences between the DHA + AA and placebo groups in growth or adiposity outcomes were observed. For instance, the change in weight-for-age z scores was 0.1 for the DHA + AA group and 0.0 for the placebo group (effect size = 0.01, P = 0.99). However, post-hoc subgroup analyses revealed a statistically significant interaction between treatment group and sex, suggesting somewhat slower linear growth for females assigned to the DHA + AA group compared with the placebo group. CONCLUSIONS: Among toddlers born preterm, daily supplementation with DHA + AA for 180 d resulted in no short-term differences in growth or adiposity compared with placebo. If DHA supplementation is implemented after the first year of life, it can be expected to have no effect on short-term growth or adiposity. This trial is registered with clinicaltrials.gov as NCT02199808.

Anandamide-nanoformulation obtained by electrospraying for cardiovascular therapy.

Anandamide (AEA), an endogenous cannabinoid, has a relevant antihypertensive effect. However, its cardioprotective role has been barely explored due to unfavorable physico-chemical properties and, sometimes, undesirable psychoactive effects. In this context, drug encapsulation in nanocarriers could overcome the limitations associated with the administration of AEA in free form. The aim of the present study was to encapsulate AEA in poly-ε-caprolactone/Pluronic® F127 nanoparticles (AEA/PCL/PF127 NPs) by means of electrospraying, to characterize their physico-chemical properties and cytocompatibility and to evaluate their effect in an in vivo model of cardiovascular remodeling caused by hypertension. AEA/PCL/PF127 NPs were characterized in terms of morphology, size, polydispersity, Z-potential, hydrophilicity, thermal and spectroscopic properties. Also, the encapsulation and loading efficiencies and in vitro release of AEA were analyzed. AEA/PCL/PF127 NPs (700-1000 nm) showed adequate cytocompatibility. For the cardiovascular remodeling studies, normotensive (WKY) and hypertensive (SHR) male rats were treated or not with AEA/PCL/PF127 NPs (5 mg/Kg, intraperitoneal injection) weekly for 1 month. Inflammatory markers and hemodynamic, structural and cardiac functional parameters were monitored. In SHR, the treatment with AEA/PCL/PF127 NPs reversed all altered cardiovascular markers and parameters (p < 0.05). Overall, nanoformulated AEA obtained by electrospraying proved to be effective for the treatment of hypertension and its comorbidities, especially cardiovascular remodeling.

Epoxyeicosatrienoic acid analog EET-B attenuates post-myocardial infarction remodeling in spontaneously hypertensive rats.

Epoxyeicosatrienoic acids (EETs) and their synthetic analogs have cardiovascular protective effects. Here, we investigated the action of a novel EET analog EET-B on the progression of post-myocardial infarction (MI) heart failure in spontaneously hypertensive rats (SHR). Adult male SHR were divided into vehicle- and EET-B (10 mg/kg/day; p.o., 9 weeks)-treated groups. After 2 weeks of treatment, rats were subjected to 30-min left coronary artery occlusion or sham operation. Systolic blood pressure (SBP) and echocardiography (ECHO) measurements were performed at the beginning of study, 4 days before, and 7 weeks after MI. At the end of the study, tissue samples were collected for histological and biochemical analyses. We demonstrated that EET-B treatment did not affect blood pressure and cardiac parameters in SHR prior to MI. Fractional shortening (FS) was decreased to 18.4 ± 1.0% in vehicle-treated MI rats compared with corresponding sham (30.6 ± 1.0%) 7 weeks following MI induction. In infarcted SHR hearts, EET-B treatment improved FS (23.7 ± 0.7%), markedly increased heme oxygenase-1 (HO-1) immunopositivity in cardiomyocytes and reduced cardiac inflammation and fibrosis (by 13 and 19%, respectively). In conclusion, these findings suggest that EET analog EET-B has beneficial therapeutic actions to reduce cardiac remodeling in SHR subjected to MI.