RESUMEN

This study carried out to investigate the anti-inflammatory and antinociceptive effect of tropane alkaloid (EB7) isolated from E. bezerrae. It evaluated the toxicity and possible involvement of ion channels in the antinociceptive effect of EB7, as well as its anti-inflammatory effect in adult zebrafish (Zfa). Docking studies with EB7 and COX-1 and 2 were also performed. The tested doses of EB7 (4, 20 and 40 mg/kg) did not show any toxic effect on Zfa during the 96h of analysis (LD50>40 mg/kg). They did not produce any alteration in the locomotor behavior of the animals. Furthermore, EB7 showed promising pharmacological effects as it prevented the nociceptive behavior induced by hypertonic saline, capsaicin, formalin and acid saline. EB7 had its analgesic effect blocked by amiloride involving the neuromodulation of ASICs in Zfa. In evaluating the anti-inflammatory activity, the edema induced by κ-carrageenan 3.5 % was reduced by the dose of 40 mg/kg of EB7 observed after the fourth hour of analysis, indicating an effect similar to that of ibuprofen. Molecular docking results indicated that EB7 exhibited better affinity energy when compared to ibuprofen control against the two evaluated targets binding at different sites in the cocrystallized COX-1 and 2 inhibitors.

Asunto(s)

Analgésicos , Simulación del Acoplamiento Molecular , Pez Cebra , Animales , Analgésicos/farmacología , Analgésicos/química , Analgésicos/aislamiento & purificación , Tropanos/farmacología , Tropanos/aislamiento & purificación , Tropanos/química , Edema/tratamiento farmacológico , Edema/inducido químicamente , Carragenina/farmacología , Ciclooxigenasa 2/metabolismo , Ciclooxigenasa 1/metabolismo , Bignoniaceae/química , Relación Dosis-Respuesta a Droga , Relación Estructura-Actividad , Alcaloides/farmacología , Alcaloides/aislamiento & purificación , Alcaloides/química , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/química , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/aislamiento & purificación , Antiinflamatorios/farmacología , Antiinflamatorios/química , Antiinflamatorios/aislamiento & purificación , Estructura Molecular

RESUMEN

Alcohol use disorder (AUD) is characterized by a set of behavioral, cognitive, nutritional, and physiological phenomena derived from the uncontrolled use of alcoholic beverages. There are cases in which AUD is associated with anxiety disorder, and when untreated, it requires careful pharmacotherapy. Blue Calm® (BC) is a food supplement indicated to aid restorative sleep, which has traces of medicinal plant extracts, as well as myo-inositol, magnesium bisglycinate, taurine, and L-tryptophan as its main chemical constituents. In this context, this study aimed to evaluate the potential of the BC in the treatment alcohol withdrawal-induced anxiety in adult zebrafish (aZF). Initially, BC was submitted to antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl radical. Subsequently, the aZF (n = 6/group) were treated with BC (0.1 or 1 or 10 mg/mL; 20 µL; p.o.), and the sedative effect and acute toxicity (96 h) were evaluated. Then, the anxiolytic-like effect and the possible GABAergic mechanism were analyzed through the Light & Dark Test. Finally, BC action was evaluated for treating alcohol withdrawal-induced anxiety in aZF. Molecular docking was performed to evaluate the interaction of the major chemical constituents of BC with the GABAA receptor. BC showed antioxidant potential, a sedative effect, was not toxic, and all doses of BC had an anxiolytic-like effect and showed potential for the treatment of alcohol withdrawal-induced anxiety in aZF. In addition to the anxiolytic action, the main chemical constituents of BC were confirmed in the molecular docking, thus suggesting that BC is an anxiolytic that modulates the GABAergic system and has pharmacological potential for the treatment of alcohol withdrawal-induced anxiety.

Asunto(s)

Alcoholismo , Ansiolíticos , Síndrome de Abstinencia a Sustancias , Animales , Pez Cebra/fisiología , Ansiolíticos/farmacología , Ansiolíticos/uso terapéutico , Ansiedad/inducido químicamente , Ansiedad/tratamiento farmacológico , Ansiedad/psicología , Alcoholismo/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , Receptores de GABA-A , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Trastornos de Ansiedad/tratamiento farmacológico , Suplementos Dietéticos , Hipnóticos y Sedantes

RESUMEN

Drugs used to manage type 2 diabetes mellitus cause adverse effects. Therefore, the search for new drugs as an alternative for the treatment of diabetes increases. The effect of triterpene 3ß-6ß-16ß-trihydroxylup-20(29)-ene isolated from the leaves of C. leprosum (CLF-1) on sucrose-induced hyperglycemia in adult zebrafish (Danio rerio) was evaluated. Initially, adult zebrafish (n = 6/group) underwent hyperglycemia induction by sucrose at 83.25 mM/L for 7 days by immersion. The hyperglycemic groups were treated with CLF-1 (4, 20, and 40 mg/kg), metformin (200 mg/kg), and acarbose (300 mg/kg) for 4 days. The in silico interaction of CLF-1, metformin, and acarbose with the enzyme maltase-glucoamylase (CtMGAM) was investigated. CLF-1 reduced sucrose-induced hyperglycemia after 4 days of treatment, in addition to having better affinity energy with CtMGAM than metformin and acarbose. Thus, CLF-1 may be a new pharmacological alternative as a hypoglycemic agent for the treatment of diabetes.

Asunto(s)

Combretum , Diabetes Mellitus Tipo 2 , Hiperglucemia , Metformina , Triterpenos , Acarbosa/farmacología , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hiperglucemia/inducido químicamente , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/farmacología , Extractos Vegetales/farmacología , Hojas de la Planta , Sacarosa , Triterpenos/farmacología , Triterpenos/uso terapéutico , Pez Cebra

RESUMEN

Turnera subulata Sm. belongs to the family Turneraceae and is found in Brazil. The present study evaluated the antinociceptive, anti-inflammatory, and hypoglycemic potential of T. subulata flower extract (EtFloTsu) in zebrafish (Danio rerio). The total phenol and flavonoid contents of EtFloTsu were determined and identified using the Folin Ciocalteu reagent and aluminum chloride (AlCl3), respectively. The constituents of the extract were identified by HPLC-DAD, and the in vitro antioxidant activity (DPPH) was determined, toxicity in brine shrimp, and acute toxicity of 96 h in adult zebrafish. In addition, adult zebrafish (n = 6/fish) were treated orally with EtFloTsu (4, 20, or 40 mg/kg; vo) and subjected to formalin-induced nociception tests (with its possible mechanism of action with camphor), carrageenan-induced inflammation, and D-glucose-induced hyperglycemia (111 mM). Oxidative stress in the liver and brain tissues was assessed. EtFloTsu showed high levels of phenolic and flavonoid compounds with antioxidant activity. The phytochemicals chlorogenic acid, luteolin-7-o-glucoside, vitexin, and apigenin-7-o-glucoside were also identified in EtFloTsu. The synergism between these constituents was possibly responsible for the antinociceptive (via TRPA1), anti-inflammatory, and hypoglycemic effects of EtFloTsu in adult zebrafish, without causing toxicity in animals. Therefore, T. subulata flowers have therapeutic agents that could treat pain, inflammation, diabetes, and related complications.Communicated by Ramaswamy H. Sarma.

Asunto(s)

Turnera , Pez Cebra , Animales , Antioxidantes/química , Hipoglucemiantes/farmacología , Antiinflamatorios/farmacología , Antiinflamatorios/química , Extractos Vegetales/farmacología , Extractos Vegetales/química , Inflamación/tratamiento farmacológico , Flores , Etanol , Analgésicos/farmacología , Analgésicos/uso terapéutico , Flavonoides

RESUMEN

The aim of this study was to evaluate the anxiolytic-like effect of chrysophanol (CHRY), isolated from hexane extract of Senna cana stem and its possible mechanism of action. CHRY was obtained through chromatographic treatments and its identity was confirmed by uni and bidimensional RMN1H and RMN13C. Adult zebrafish (n = 6/group) were treated (with CHRY (4.0 or 12.0 or 40.0 mg/Kg; 20 µL; intraperitoneally) and submitted to acute toxicity and open field tests. Subsequently, other groups (n = 6/each) received CHRY for the analysis of its effect on the Light & Dark Test. The participation of the GABAergic system was also assessed using the diazepam (GABAA receptor agonist) and flumazenil (GABAA receptor antagonist). CHRY was considered non-toxic, it did not reduce the locomotor activity, and showed an anxiolytic-like effect. This effect was reduced by pre-treatment with flumazenil. The results suggest that CHRY is an anxiolytic-like agent mediated via the GABAergic system.

Asunto(s)

Ansiolíticos , Senna , Animales , Antraquinonas , Ansiolíticos/farmacología , Ansiedad , Flumazenil/farmacología , Receptores de GABA-A , Pez Cebra

RESUMEN

Combretaceae are reported in the literature for presenting neuroprotective and anxiolytic effects in animal models. Combretum lanceolatum Pohl. has few scientific reports on its pharmacological effects. The aim of this study was to evaluate the anxiolytic and anticonvulsant effects of the ethanol extract from the leaves of C. lanceolatum Pohl. (EtFoCl) and its possible mechanism of GABAergic action in adult zebrafish. EtFoCl was subjected to determination of the total phenol concentration, identification of phytochemical flavonoids by HPLC and in vitro antioxidant activity test, open field test and 96-hour acute toxicity in zebrafish. Anxiolytic doses were tested for pentylenetetrazole-induced seizures in adult zebrafish. To study the mechanisms of action, molecular docking simulations were performed between the main phytochemicals and the GABAA receptor (anxiolytic activity) and carbonic anhydrase II (anticonvulsant). The non-toxic doses that caused motor impairment were assessed in acute and chronic anxiety using the light and dark test. EtFoCl had altered the animals' locomotion, presenting an effect similar to the anxiolytic and anticonvulsant. These effects were prevented with flumazenil (GABAA antagonist). The phytochemicals homoorientin and quercetin-3-O-galactoside coupling in a region close to that of the inhibitor diazepam (GABAA receptor). Regarding the anticonvulsant mechanism, Homoorientina and Isovitexina were identified as the most favorable for the complex form with the carbonic anhydrase enzyme. C. lanceolatum has pharmacological potential for the treatment of acute and chronic anxiety and seizures, which can be partially explained by an interaction with the GABAA receptor.Communicated by Ramaswamy H. Sarma.

Asunto(s)

Ansiolíticos , Combretum , Animales , Ansiolíticos/efectos adversos , Pez Cebra , Receptores de GABA-A , Anticonvulsivantes/farmacología , Simulación del Acoplamiento Molecular , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Ansiedad/tratamiento farmacológico , Extractos Vegetales/farmacología

RESUMEN

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.

Asunto(s)

Estructuras Citoplasmáticas/ultraestructura , IMP Deshidrogenasa/química , Proteínas de Pez Cebra/química , Pez Cebra/metabolismo , Animales , Línea Celular , Estructuras Citoplasmáticas/química , Expresión Génica , Guanosina Trifosfato/biosíntesis , Guanosina Trifosfato/metabolismo , Humanos , IMP Deshidrogenasa/genética , IMP Deshidrogenasa/metabolismo , Isoenzimas/química , Isoenzimas/genética , Mutación Puntual , Regulación hacia Arriba , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo

RESUMEN

The action of anxiolytic compounds that act on selective serotonin receptors (SSRIs) have been scarcely evaluated. Serotonergic drugs have been shown to be effective in treating anxiety without presenting adverse effects as benzodiazepines. However, the anxiolytic effects take days to occur. This study aimed to evaluate the anxiolytic effect of the synthetic chalcone, 4'-[(2E) -3- (3-nitrophenyl) -1- (phenyl) prop-2-en-1-one] acetamide (PAAMNBA), and its possible mechanism of action in adult zebrafish (Danio rerio). PAAMNBA was synthesized with a yield of 51.3% and its chemical structure was determined by 1H and 13C NMR. Initially, PAAPMNBA was intraperitoneally administered to zebrafish (n = 6/group) at doses of 4, 12, or 40 mg/kg, and the animals were subsequently subjected to acute and open field toxicity tests. PAAMNBA was administered to the other groups (n = 6/group) for analyzing its effect in the light and dark test. The involvement of the serotonergic (5HT) system was also evaluated using 5-HTR 1, 5-HTR 2A/2C, and 5-HTR 3A/3B receptor antagonists, namely, pizotifeo, granizetron, and ciproeptadina, respectively. Molecular coupling was performed using the 5-HT1 receptor. PAAMNBA was found to be non-toxic, reduced the locomotor activity, and had an anxiolytic effect in adult zebrafish. The effect was reduced by pretreatment with pizotifene and was not reversed by treatment with granizetron and cyproeptadine. A previous in vivo molecular coupling study indicated that chalcones interact with the 5-HT1 receptor. The results suggested that the chalcone, PAAPMNBA, has anxiolytic activity, that is mediated by the serotonergic system via the 5-HT1 receptor. The interaction of PAAPMNBA with the 5-HT1 receptor was confirmed by molecular docking studies.

Asunto(s)

Acetamidas/farmacología , Ansiolíticos/farmacología , Chalcona/farmacología , Serotonina/metabolismo , Acetamidas/química , Animales , Ansiolíticos/química , Ansiedad/tratamiento farmacológico , Ansiedad/metabolismo , Chalcona/análogos & derivados , Descubrimiento de Drogas , Locomoción/efectos de los fármacos , Simulación del Acoplamiento Molecular , Receptores de Serotonina 5-HT1/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismo

RESUMEN

Schinus terebinthifolius Raddi (Anacardiaceae) is popularly known in Brazil as aroeira-da-praia and has pharmacological use as an astringent, antidiarrheal, anti-inflammatory, depurative, diuretic, and antifebrile agent. Although the neuropathic antinociceptive potential of S. terebinthifolius fruits has already been investigated, this study is the first one to analyze the acute antinociceptive effect of the essential oil of S. terebinthifolius (female) leaves (EOFSt) on adult zebrafish. EOFSt was submitted to antioxidant activity evaluation by two methods (ferrous ion-chelating capacity [FIC] and ß-carotene). The animals (n = 6/group) were treated orally (20 µL) with EOFSt (0.1, 0.5, or 1.0 mg/mL) or vehicle (0.9% sodium chloride [NaCl]; 20 µL), and submitted to nociception (formalin, cinnamaldehyde, capsaicin, glutamate, acidic saline, and hypertonic saline). Possible neuromodulation mechanisms, as well motor alterations and toxicity were also evaluated. In the FIC assay, EOFSt showed ferrous ion-chelating capacity in ∼40% to 90%. Regarding the ß-carotene bleaching assay, EOFSt showed inhibition in a 58% to 80% range. Oral administration of EOFSt showed no acute toxicity and did not alter the locomotor system of aZF, and reduced the nociceptive behavior in all tested models. These effects of EOFSt were significantly similar to those of morphine, used as a positive control. The antinociceptive effect of EOFSt was inhibited by naloxone, L-NAME, ketamine, camphor, ruthenium red, and amiloride. The antinociceptive effect of the EOFSt cornea was inhibited by capsazepine. EOFSt has the pharmacological potential for acute pain treatment and this effect is modulated by the opioid system, NMDA receptors, and transient receptor potential ankyrin 1 (TRPA1), transient receptor potential vanilloid 1 (TRPV1), and acid-sensing ion channels. The EOFSt also has the pharmacological potential for corneal pain treatment and this effect is modulated by the TRPV1 channel.

Asunto(s)

Anacardiaceae/química , Analgésicos/farmacología , Aceites Volátiles/farmacología , Pez Cebra/fisiología , Administración Oral , Analgésicos/química , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/metabolismo , Femenino , Masculino , Aceites Volátiles/química , Hojas de la Planta/química

RESUMEN

Studies involving foods associated with pain reversal and anti-inflammatory effects using zebrafish are rarely reported in the literature. This study aimed to evaluate the effect of graviola (Annona muricata L.) fruit bar (GFB) and GFB added with acerola (Malpighia glabra L) seed extract (ASE) on acute nociception and abdominal inflammation in adult zebrafish (Danio rerio). Acute nociception was induced by formalin, capsaicin, cinnamaldehyde, acidic saline, glutamate (cutaneous models), and hypertonic saline (corneal model), and inflammation was induced by carrageenan. Both GFB and ASE exhibited antinociceptive effect modulated by the nitrergic system, guanylate cyclase, and transient receptor potential ankyrin 1 and acid-sensing ion channels. The antinociceptive effect of GFB also appears to be modulated by the opioid system and glutamatergic receptors (N-methyl-D-aspartate receptor). Only ASE presented corneal antinociceptive effect. Both samples showed anti-inflammatory effect, being more significant the effect of GFB. The addition of acerola by-product extract in GFB results in a product with greater biological potential.

Asunto(s)

Analgésicos/farmacología , Annona/química , Antiinflamatorios/farmacología , Inflamación/tratamiento farmacológico , Dolor Nociceptivo/tratamiento farmacológico , Animales , Conducta Animal , Modelos Animales de Enfermedad , Femenino , Frutas/química , Masculino , Malpighiaceae/química , Semillas/química , Pruebas de Toxicidad Aguda , Pez Cebra

RESUMEN

The analysis of behavior in animal models is an important objective in many research fields, including neuroscience, psychology, toxicology, and neuropsychopharmacology. Animal models have been used for many years, and several behavioral paradigms, such as locomotor activity, social interactions, and cognitive behavior, have been studied in animal models to correlate the behaviors with pharmacological or environmental interventions and with molecular, biochemical, and physiological findings. We reviewed the literature looking for open-source, freely available software to analyze animal behavior and found 12 freely available programs: ToxTrack, EthoWatcher, Mouse Behavior Tracker, Mouse Move, JAABA, wrMTrck, AnimalTracker, idTracker, Ctrax, Mousetracker, VideoHacking, and Cowlog, which were developed with different programs, work on different platforms, and have particular types of inputs and outputs and analysis capabilities. We reviewed some examples of their use, tested some of them, and provided several recommendations for the future development of programs for the automated analysis of behavior in animal models. In conclusion, we show freely available software for the automated analysis of behavior in animal models such as adult zebrafish and provide information for researchers and students looking for quick, easy-to-implement, and inexpensive behavior analysis alternatives.

Asunto(s)

Conducta Animal , Etología/métodos , Ciencia de los Animales de Laboratorio/métodos , Programas Informáticos , Pez Cebra , Animales , Etología/instrumentación , Ciencia de los Animales de Laboratorio/instrumentación

RESUMEN

This study aimed to evaluate the antinociceptive effect of oleanolic acid using adult zebrafish models of orofacial pain. Acute nociception was induced by formalin, capsaicin, cinnamaldehyde, menthol, acidified saline or glutamate (cutaneous modes) and hypertonic saline (corneal model). In another set of experiments, animals were pre-treated with naloxone, L-NAME, methylene blue, ketamine, camphor, HC-030031, mefenamic acid, ruthenium red or amiloride to investigate the mechanism of antinociception. The involvement of central afferent C-fibers was also investigated. A molecular docking was performed using the TRPV1 channel. Motor activity was evaluated with the open field test. Pre-treatment with oleanolic acid significantly reduced nociceptive behavior associated with acute pain. Antinociception was effectively inhibited by ruthenium red and capsaicin-induced desensitization. Presence of trpv1 was confirmed by RT-PCR in cerebral tissue of zebrafish. In line with in vivo experiments, docking studies indicated that oleanolic acid may interact with TRPV1. Results confirm the potential pharmacological relevance of oleanolic acid as an inhibitor of orofacial nociception mediated by TRPV1.

Asunto(s)

Analgésicos/farmacología , Conducta Animal/efectos de los fármacos , Ácido Oleanólico/farmacología , Canales Catiónicos TRPV/metabolismo , Proteínas de Pez Cebra/metabolismo , Acetanilidas/farmacología , Analgésicos/uso terapéutico , Animales , Sitios de Unión , Capsaicina/farmacología , Dolor Facial/tratamiento farmacológico , Dolor Facial/etiología , Formaldehído/farmacología , Simulación del Acoplamiento Molecular , Ácido Oleanólico/química , Ácido Oleanólico/uso terapéutico , Estructura Terciaria de Proteína , Purinas/farmacología , Rojo de Rutenio/química , Rojo de Rutenio/metabolismo , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/genética , Termodinámica , Pez Cebra

RESUMEN

The aim of this study was to evaluate the antinociceptive effect of Kaempferol-3-O-rutinoside (KR), isolated from the plant Ouratea fieldingiana, on the orofacial nociception and possible mechanisms of action. Adult zebrafish (Danio rerio) were tested as a behavioral model to study formalin, glutamate, capsaicin, cinnamaldehyde and acidic saline-induced orofacial nociception, using as parameter the number of times the fish crossed the lines between the quadrants of a glass Petri dish during a specific time. Morphine was used as positive control. The effect of KR was tested for modulation by opioid (naloxone), nitrergic (L-NAME), TRPV1 (ruthenium red), TRPA1 (camphor) or ASIC (amiloride) antagonists. The effect of KR on zebrafish locomotor behavior was evaluated with the open field test. KR did not alter the fish's locomotor system and significantly reduced the orofacial nociceptive behavior induced by all noxious agents compared to the control group. The antinociceptive effect of KR was similar to morphine. All antagonists inhibited the antinociceptive effect of KR. KR has pharmacological potential for the treatment of acute orofacial pain and this effect is modulated by the opioid and nitrergic systems as well as TRPV1, TRPA1 and ASIC channels. These results can lead to the development of a new natural product for the treatment of orofacial pain and confirm the popular use of O. fieldingiana leaf for pain relief.

Asunto(s)

Analgésicos/farmacología , Dolor Facial/tratamiento farmacológico , Quempferoles/farmacología , Ochnaceae/química , Analgésicos/aislamiento & purificación , Analgésicos Opioides/farmacología , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Quempferoles/aislamiento & purificación , Masculino , Morfina/farmacología , Naloxona/farmacología , Nocicepción/efectos de los fármacos , Hojas de la Planta , Pez Cebra

RESUMEN

Neem fruit (Azadirachta indica A. Juss.) are popularly used to treat infections, diarrhea, fever, bronchitis, skin diseases, infected burns and hypertension. Although the antinociceptive and anti-inflammatory potential of A. indica has already been investigated in experimental models of pain and inflammation in mice, the current research is the first to report the evaluation of the capacity of A. indica fruit ethanolic extract (EtFrNeem) in acute pain attenuation using the adult zebrafish (Danio rerio) as an alternative model to the use in rodents. EtFrNeem was submitted to antioxidant action, preliminary chemical prospecting, FT-IR and determination of phenol and flavonoid content tests. Subsequently, EtFrNeem was tested for acute nociception and abdominal inflammation, locomotor activity, and acute toxicity in adult zebrafish. Possible neuromodulation mechanisms were also evaluated. EtFrNeem showed low antioxidant activity, but was shown to be rich in flavonoids. EtFrNeem showed no anti-inflammatory action, did not alter the locomotor system, and it was not toxic. However, EtFrNeem significantly reduced the nociceptive behavior induced by formalin, glutamate and acidic saline, when compared to the control group. These effects of EtFrNeem were significantly similar to those of morphine, used as a positive control. The antinociceptive effect of EtFrNeem was inhibited by naloxone, ketamine and amiloride. EtFrNeem has the pharmacological potential for acute pain treatment and this effect is modulated by the opioid system, NMDA receptors and ASICs channels. These results lead us to studies of isolation and characterization of EtFrNeem bioactive principles, using adult zebrafish as an experimental model.

Asunto(s)

Analgésicos Opioides/farmacología , Analgésicos/farmacología , Azadirachta/química , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Frutas/química , Meliaceae/química , Extractos Vegetales/farmacología , Canales Iónicos Sensibles al Ácido/metabolismo , Animales , Antioxidantes/metabolismo , Modelos Animales de Enfermedad , Etanol , Flavonoides/farmacología , Locomoción/efectos de los fármacos , Morfina/farmacología , Dolor/tratamiento farmacológico , Dolor/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Pez Cebra

RESUMEN

Zebrafish is an excellent model that can be utilized as an adjunct to current rodent models for studies of eye diseases because the anatomy and ultrastructural characterization of its cornea show much similarity with the human cornea. Therefore, we developed a behavioral model of corneal nociception using the adult zebrafish (Danio rerio). We analyzed the nociceptive effect of hypertonic saline (0.15-5.0 M sodium chloride [NaCl]) applied to the surface of the right or left cornea, on the animals' gender and locomotor activity through the open-field test. The behavioral model of corneal nociception was characterized by the antinociceptive effect of morphine (8.0 or 16 mg/kg; intraperitoneally [i.p.]), an opioid analgesic, and capsazepine, an antagonist of transient receptor potential vanilloid type 1 channels. We also tested whether the corneal antinociceptive effect of morphine could be modulated by naloxone, an opioid antagonist. Finally, we used the light and dark test to assess the anxiolytic effect of hypertonic saline (5.0 M NaCl; 5 µL) applied to the right or left cornea of the animals. As a result, hypertonic saline significantly increased (p < 0.01 vs. control) the corneal nociceptive behavior of adult zebrafish (D. rerio). Morphine significantly inhibited (p < 0.01 vs. 5.0 M NaCl) the hypertonic saline-induced corneal nociception and this effect was blocked by naloxone. Capsazepine (20 mg/kg; i.p.) significantly inhibited (p < 0.05 vs. control) the corneal nociception induced by hypertonic saline. Hypertonic saline, applied to the surface of the right or left cornea of the animals, induced nociception and did not cause a presumptive anxiolytic effect. Gender and site of application did not affect the profile of response to hypertonic saline. The results suggest that the adult zebrafish can also be used as a behavioral model of corneal nociception, with the advantages of significant homology with the human genome and low cost.

Asunto(s)

Analgésicos/farmacología , Córnea/efectos de los fármacos , Modelos Animales de Enfermedad , Nocicepción/fisiología , Solución Salina Hipertónica/toxicidad , Animales , Conducta Animal/efectos de los fármacos , Locomoción , Nocicepción/efectos de los fármacos , Pez Cebra

RESUMEN

The zebrafish (Danio rerio) has been proposed as a low-cost and simple alternative to the use of higher vertebrates in laboratory research on novel compounds with antinociceptive potential. In this study, we tested adult zebrafish (Danio rerio) as an alternative behavioral model of formalin-induced nociception. We evaluated the nociceptive effect of 0.1% formalin (3 or 5 µL; intramuscularly [i.m.]), applied into the tail or lips, on locomotor activity, using as parameter the number of times the fish crossed the lines between the quadrants of a glass Petri dish during the neurogenic stage (0-5 min) and the inflammatory stage (15-30 min). The behavioral model was validated by testing the antinociceptive effect of morphine and indomethacin (standard analgesic drugs used in the formalin test of rodents). We also tested whether the effect of morphine could be modulated by naloxone, an opioid antagonist. The effect of morphine and indomethacin on zebrafish locomotor behavior was evaluated with the open field test. The white/black test was used to rule out the anxiolytic effect of 0.1% formalin injected into the tail on adult zebrafish. Formalin (0.1%; 3 and 5 µL injected into the tail) increased significantly the nociceptive behavior of the adult zebrafish in both stages (p < 0.001 vs. control). Morphine and indomethacin (both 0.2 mg/mL; 20 µL; intraperitoneally [i.p.]) significantly inhibited nociception induced with formalin (5 µL injected i.m. into the tail) in both stages (p < 0.001). Naloxone blocked the antinociceptive effect of morphine. No influence on locomotion was observed. Locally administered formalin (injected into the tail) induced nociception, but not anxiety. The results suggest that the adult zebrafish behavioral model is a feasible alternative to more conventional laboratory models used in research on novel compounds with antinociceptive potential.

Asunto(s)

Formaldehído/toxicidad , Indometacina/administración & dosificación , Morfina/administración & dosificación , Pez Cebra/fisiología , Analgésicos Opioides/farmacología , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Conducta Animal , Modelos Animales de Enfermedad , Locomoción , Nocicepción/efectos de los fármacos