Bromelain regulates TRP channels to induce orofacial nociception relief in adult zebrafish.

Bromelain, the main protease enzyme found in the pineapple plant (Ananas comosus), has had its antinociceptive effect previously demonstrated. This investigation aimed to appraise the role of TRP (Transient Receptor Potential) channels in the nociception-relieving effects of bromelain in the orofacial region of adult zebrafish. The animals were pretreated with bromelain (3.0, 10.0 or 30.0 mg/mL; gavage) and submitted to open field and acute orofacial (capsaicin - TRPV1 agonist, cinnamaldehyde - TRPA1 agonist or menthol - TRPM8 agonist) nociception tests. The investigation also explored the contribution of central afferent C-fibers. Naive groups were included for comparison. Bromelain did not independently affect the zebrafish movement patterns. However, bromelain decreased the nociceptive responses elicited by all three TRP channel activators. Capsazepine (TRPV1 inhibitor) and AMTB (TRPM8 inhibitor), but not HC-030031 (TRPA1 inhibitor), prevented the antinociceptive effect of bromelain. Moreover, capsaicin-induced desensitization effectively nullified the antinociceptive effect of bromelain. Collectively, these findings corroborate the therapeutic relevance of bromelain as a suppressor of orofacial nociception, which seems to be intricately connected to the modulation of TRP channels.

Pharmacological Potential of cis-jasmone in Adult Zebrafish (Danio rerio).

This study evaluates the pharmacological potential of cis-jasmone (CJ) in adult zebrafish (Danio rerio; aZF). Initially, aZF (n = 6/group) were pretreated (20 µL; p. o.) with CJ (0.1 or 0.3 or 1.0 mg/mL) or vehicle (0.5% Tween 80). The animals were submitted to acute toxicity and locomotion tests, pentylenetetrazole-induced seizure, carrageenan-induced abdominal edema, and cinnamaldehyde-, capsaicin-, menthol-, glutamate-, and acid saline-induced orofacial nociception. The possible mechanisms of anticonvulsant, anxiolytic, and antinociceptive action were evaluated. The involvement of central afferent fibers sensitive to cinnamaldehyde and capsaicin and the effect of CJ on the relative gene expression of TRPA1 and TRPV1 in the brain of aZF were also analyzed, in addition to the study of molecular docking between CJ and TRPA1, TRPV1 channels, and GABAA receptors. CJ did not alter the locomotor behavior and showed pharmacological potential in all tested models with no toxicity. The anticonvulsant effect of CJ was prevented by flumazenil (GABAergic antagonist). The anxiolytic-like effect of CJ was prevented by flumazenil and serotonergic antagonists. The antinociceptive effect was prevented by TRPA1 and TRPV1 antagonists. Chemical ablation with capsaicin and cinnamaldehyde prevented the orofacial antinociceptive effect of CJ. Molecular docking studies indicate that CJ interacted with TRPA1, TRPV1, and GABAA receptors. CJ inhibited the relative gene expression of TRPA1 and TRPV1. CJ has pharmacological potential for the treatment of seizures, anxiety, inflammation, and acute orofacial nociception. These effects are obtained by modulating the GABAergic and serotonergic systems, as well as the TRPs and ASIC channels.

Parkia platycephala Lectin (PPL) Inhibits Orofacial Nociception Responses via TRPV1 Modulation.

Lectins are a heterogeneous group of proteins that reversibly bind to simple sugars or complex carbohydrates. The plant lectin purified from the seed of Parkia platycephala (PPL) was studied. This study aimed to investigate the possible orofacial antinociceptive of PPL lectin in adult zebrafish and rodents. Acute nociception was induced by cinnamaldehyde (0.66 µg/mL), 0.1% acidified saline, glutamate (12.5 µM) or hypertonic saline (5 M NaCl) applied into the upper lip (5.0 µL) of adult wild zebrafish. Zebrafish were pretreated by intraperitoneal injection (20 µL) with vehicle (Control) or PPL (0.025; 0.05 or 0.1 mg/mL) 30 min before induction. The effect of PPL on zebrafish locomotor behaviour was evaluated in the open field test. Naive groups were included in all tests. In one experiment, animals were pre-treated with capsazepine to investigate the mechanism of antinociception. The involvement of central afferent C-fibres was also investigated. In another experiment, rats pre-treated with PPL or saline were submitted to the temporomandibular joint formalin test. Other groups of rats were submitted to infraorbital nerve transection to induce chronic pain, followed by induction of mechanical sensitivity using von Frey. PPL reduced nociceptive behaviour in adult zebrafish, and this is related to the activation of the TRPV1 channels since antinociception was effectively inhibited by capsazepine and by capsaicin-induced desensitization. PPL reduced nociceptive behaviour associated with temporomandibular joint and neuropathic pain. The results confirm the potential pharmacological relevance of PPL as an inhibitor of orofacial nociception in acute and chronic pain.

Topiramate in an Experimental Model of Epilepsy - Similarity between Generic, Similar and Reference Drugs.

Background and Purpose: The literature is still controversial in relation to therapeutic differences between innovative, generic, and similar anti-seizures medications (ASM). Topiramate (TPM) is an ASM used in the treatment of various seizure types and in different epileptic syndromes, as well as in other groups of morbidities, and it is available in many generic and similar forms, besides the innovator. The aim of this translational work was to compare different brands of TPM by using animal models of seizures induced by pentylenetetrazole (PTZ). Methods: Five brands of TPM (one reference, two similar and two generics) were tested in mice. Animals were previously treated with TPM (n=6/brand) and latencies from PTZ injection to onset of manifestations, first seizure and death were measured and compared between groups. Experiment was conducted in two settings: acute seizure model (PTZ 80 mg/kg) and kindling model (PTZ 20, 30, and 40 mg/kg in 8 alternate days). Results: The experiment did not demonstrate significant differences between the TPM brands regarding the protective effect in the acute seizure and kindling models. Conclusions: In conclusion, results can be explained by true therapeutic equivalence or insufficiency of the PTZ model to reveal differences among brands.

Development of a Nanoformulation for Oral Protein Administration: Characterization and Preclinical Orofacial Antinociceptive Effect.

Nanoencapsulation is a valid alternative for the oral administration of peptide drugs and proteins, as nanoparticles protect them from proteolytic degradation in the gastrointestinal tract and promote the absorption of these macromolecules. The orofacial antinociceptive effect of frutalin (FTL), through the intraperitoneal route, has already been proven. This study aimed to develop, characterize, and evaluate the orofacial antinociceptive activity of an oral formulation containing FTL in acute and neuropathic preclinical tests. Nanoencapsulated FTL was administered by oral route. The acute nociceptive behavior was induced by administering capsaicin to the upper lip and NaCl to the right cornea. The nociceptive behavior was also induced by formalin injected into the temporomandibular joint. The neuropathic pain model involved infraorbital nerve transection (IONX), which induced mechanical hypersensitivity and was assessed by von Frey stimulation. Trpv1 gene expression was analyzed in the trigeminal ganglion. The analyzed sample did not show any cytotoxicity; 52.2% of the FTL was encapsulated, and the size of the nanocapsule was less than 200 nm, the polydispersion was 0.361, and the zeta potential was - 5.87 and - 12.8 mV, with and without FTL, respectively. Nanoencapsulated FTL administered by oral route had an orofacial antinociceptive effect in acute and neuropathic rodent models. The antinociceptive effect of FTL was prevented by ruthenium red, but not by camphor. FTL reduced Trpv1 gene expression. FTL promotes orofacial antinociception, probably due to the antagonism of TRPV1 channels, and the nanoformulation represents an effective method for the oral administration of this protein. HIGHLIGHTS: • Nanoformulation for oral protein administration. • Nanocapsule containing FTL prevents orofacial nociceptive acute and neuropathic pain. • Frutalin promotes orofacial antinociception behavior antagonism of TRPV1 channels.

Dioclea Altissima Seed Lectin (DAL) Prevents Anxiety-like Behavioral Responses in Adult Zebrafish (Danio Rerio): Involvement of GABAergic and 5-HT Systems.

BACKGROUND: Plant lectins have shown promising neuropharmacological activities in animal models. OBJECTIVE: This study evaluated the effect of Dioclea altissima seed lectin (DAL) on adult zebrafish behavior. METHOD: Zebrafish (n=6/group) were treated (i.p.; 20 µL) with DAL (0.025; 0.05 or 0.1 mg/mL), vehicle or diazepam (DZP) and submitted to several tests (open field, light/dark preference or novel tank). Flumazenil, pizotifen or granisetron were administered 15 min before DAL (0.05 mg/mL), and the animals were evaluated on light/dark preference test. It was also verified whether the DAL effect depended on its structural integrity and ability to interact with carbohydrates. RESULTS: DAL decreased the locomotor activity of adult zebrafish (0.025; 0.05 or 0.1 mg/mL), increased the time spent in the upper region of the aquarium (0.025 mg/mL), and decreased the latency time of adult zebrafish to enter the upper region on the novel tank test. DAL (0.05 mg/mL) also increased their permanence in the light zone of the light/dark preference test. The effect of DAL was dependent on carbohydrate interaction and protein structure integrity and was prevented by pizotifen, granizetron and flumazenil. CONCLUSION: DAL was found to have an anxiolytic-like effect mediated by the 5-HT and GABAergic receptors.

Impact of sex and environmental conditions on the responses to pain in zebrafish / Impacto do sexo e das condições ambientais nas respostas do peixe-zebra à dor

ABSTRACT BACKGROUND AND OBJECTIVES: Adult zebrafish (Danio rerio) has been proposed as a low-cost and simple alternative to the use of rodents in laboratory research on novel compounds with antinociceptive potential. This study aimed to assess whether there is an influence of animal sex and the test environment on the orofacial nociceptive behavior of the adult zebrafish. METHODS: First, cinnamaldehyde, menthol, capsaicin, acidic saline, or glutamate was applied into the lips of the adult male or female zebrafish. Naive groups were included as control. The orofacial nociception was quantified in terms of locomotor activity. In other series of experiments, it was evaluated whether the apparatus, acclimatization, period of test, temperature of the water and color of the open field would alter the nociceptive response to cinnamaldehyde. RESULTS: The nociceptive behavior did not depend on the sex of the animal, apparatus, time the test was performed or the color of the open field. However, acclimatization promoted nociceptive behavior in naive animals and did not alter the nociceptive response to cinnamaldehyde (p<0.01 vs acclimatized naive). The nociception behavior was presented only when the test was performed at a temperature of 26ºC (p<0.01 vs naive). CONCLUSION: The results suggest the need to control the environment and water temperature as an environmental source of variation during the nociceptive behavior test of the adult zebrafish.

RESUMO JUSTIFICATIVA E OBJETIVOS: O peixe-zebra adulto (Danio rerio) tem sido proposto como uma alternativa simples e de baixo custo ao uso de roedores em pesquisas laboratoriais de novos compostos com potencial antinociceptivo. Este estudo teve como objetivo avaliar se há influência do sexo do animal e do ambiente de teste no comportamento nociceptivo orofacial do peixe-zebra adulto. MÉTODOS: Inicialmente, cinamaldeído, mentol, capsaicina, solução salina ácida ou glutamato foi aplicada nos lábios do peixe-zebra adulto masculino ou feminino. Grupos naive foram incluídos como controle. A nocicepção orofacial foi quantificada em termos de atividade locomotora. Em outra série de experimentos, foi avaliado se o aparato, aclimatação, período de teste, temperatura da água e cor do campo aberto alterariam a resposta nociceptiva ao cinamaldeído. RESULTADOS: O comportamento nociceptivo não dependeu do sexo do animal, do equipamento de teste, do horário em que o teste foi realizado ou da cor do campo aberto. No entanto, a aclimatação promoveu comportamento nociceptivo em animais naive e não alterou promoveu comportamento nociceptivo em animais naive e não alterou a resposta nociceptiva ao cinamaldeído (p<0,01 vs naive aclimatado). O comportamento nociceptivo foi verificado apenas quando o teste foi executado a uma temperatura de 26ºC (p<0,01 vs naive). CONCLUSÃO: Os resultados sugerem a necessidade de controlar o ambiente e a temperatura da água como fonte de variação ambiental durante o teste de comportamento nociceptivo do peixe-zebra adulto.

Is the orofacial antinociceptive effect of lectins intrinsically related to their specificity to monosaccharides?

Lectins are proteins of non-immunological origin that may play several biological applications, of which we can highlight the anti-inflammatory and antinociceptive activities. In this work, we evaluated the possible effect of orofacial antinociceptive activity of three plant lectins, Dioclea violacea (DVL - Man/Glc-binding), Vatairea macrocarpa (VML - Gal-binding) and PPL (Parkia platycephala - Man/Glc-binding) in adult zebrafish. Acute nociception was induced by menthol (1.2 µM), or capsaicin (4.93 µM) applied into in the upper lip (5.0 µL) of adult wild zebrafish. Zebrafish were pretreated by intraperitoneal injection (20 µL) with vehicle (Control) or lectins (0.025; 0.05 or 0.1 mg/mL) 30 min before induction. The effect of lectins on zebrafish locomotor behavior was evaluated with the open field test. Naive groups (n = 8) were included in all tests. Our results indicate that only PPL presented antinociceptive induced by capsaicin, suggesting the potential clinical application of PPL as inhibitor of orofacial nociception and that this effect may be due to the modulation of TRPV1 channel. In conclusion, lectins that exhibit affinity to the same or different carbohydrates do not necessarily have an antinociceptive effect on the orofacial nociception model, indicating that the glycan carbohydrate binding pattern may be related to the effect on nociception inhibition.

Orofacial antinociceptive effect of sulphated polysaccharide from the marine algae Hypnea pseudomusciformis in rodents.

This study aimed to evaluate the antinociceptive effect of sulphated polysaccharide from the marine algae Hypnea pseudomusciformis (PLS) using rodent models of orofacial pain. Acute pain was induced by formalin, capsaicin, cinnamaldehyde, acidified saline or glutamate (cutaneous modes) and hypertonic saline (corneal model). In one experiment, animals were pretreated with ruthenium red, glibenclamide, naloxone, L-NAME, methylene blue or ketamine to investigate the mechanism of antinociception. In another experiment, animals pretreated with PLS or saline were submitted to the temporomandibular joint formalin test. In yet another, animals were submitted to craniofacial pain induced by mustard oil. Motor activity was evaluated with the open-field test. Cytotoxicity and antioxidant activities were also assessed. Pre-treatment with PLS significantly reduced nociceptive behavior associated with acute pain. Antinociception was effectively reduced, but not inhibited, by ruthenium red and ketamine. L-NAME and glibenclamide enhanced the PLS effect. PLS antinociception was resistant to methylene blue, naloxone and heating. PLS presented no cytotoxicity or antioxidant properties. Our results confirm the potential pharmacological relevance of PLS as an inhibitor of orofacial nociception in acute pain probably mediated by glutamatergic, nitrergic, TRPs and K + ATP pathways.

The orofacial antinociceptive effect of Kaempferol-3-O-rutinoside, isolated from the plant Ouratea fieldingiana, on adult zebrafish (Danio rerio).

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.

Adult Zebrafish (Danio rerio) As a Model for the Study of Corneal Antinociceptive Compounds.

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.

Neuropharmacological characterization of frutalin in mice: Evidence of an antidepressant-like effect mediated by the NMDA receptor/NO/cGMP pathway.

In this study we evaluated the effect of frutalin (FTL) on mouse behavior. Mice (n=6/group) were treated (i.p.) with FTL (0.25; 0.5 or 1mg/kg) or vehicle and submitted to several tests (hole-board/HBT, elevated plus maze/PMT, open field/OFT, tail suspension/TST, or forced swimming/FST). Yohimbine, ketamine, l-NAME, aminoguanidine, 7-NI, methylene blue, l-arginine or dl-serine was administered 30min before FTL (0.5mg/kg). To evaluate the subchronic effect, animals were injected with FTL or vehicle for 7days and submitted to the FST. Molecular docking was simulated using FTL against NOS and the NMDA receptor. No changes were observed in the HBT or the OFT. FTL (0.25mg/kg) increased the number of entries into enclosed arms in the PMT. FTL reduced immobility in the TST (0.25 and 0.5mg/kg) and the FST (0.25mg/kg; 0.5mg/kg). The effect of FTL was dependent on carbohydrate interaction and protein structure integrity and was reduced by ketamine, l-NAME, aminoguanidine, 7-NI and methylene blue, but not by l-arginine, yohimbine or dl-serine. The antidepressant-like effect remained after subchronic treatment. The molecular docking study revealed a strong interaction between FTL and NOS and NMDA. FTL was found to have an antidepressant-like effect mediated by the NMDA receptor/NO/cGMP pathway.

Adult Zebrafish (Danio rerio): An Alternative Behavioral Model of Formalin-Induced Nociception.

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.

Acute and neuropathic orofacial antinociceptive effect of eucalyptol.

Terpenes have a wide range of pharmacological properties, including antinociceptive action. The anti-inflammatory and antinociceptive effects of eucalyptol are well established. The purpose of this study was to evaluate the antinociceptive effect of eucalyptol on acute and neuropathic orofacial pain in rodent models. Acute orofacial and corneal nociception was induced with formalin, capsaicin, glutamate and hypertonic saline in mice. In another series, animals were pretreated with capsazepine or ruthenium red to evaluate the involvement of TRPV1 receptors in the effect of eucalyptol. In a separate experiment, perinasal tissue levels of IL-1ß, TNF-α and IFN-γ were measured. Rats were pretreated with eucalyptol before induction of temporomandibular joint pain with formalin or mustard oil. In another experiment, rats were submitted to infraorbital nerve transection (IONX) to induce chronic pain, followed by induction of mechanical hypersensitivity using Von Frey hairs. Locomotor performance was evaluated with the open-field test, and molecular docking was conducted on the TRPV1 channel. Pretreatment with eucalyptol significantly reduced formalin-induced nociceptive behaviors in all mouse strains, but response was more homogenous in the Swiss strain. Eucalyptol produced antinociceptive effects in all tests. The effect was sensitive to capsazepine but not to ruthenium red. Moreover, eucalyptol significantly reduced IFN-γ levels. Matching the results of the experiment in vivo, the docking study indicated an interaction between eucalyptol and TRPV1. No locomotor activity changes were observed. Our study shows that eucalyptol may be a clinically relevant aid in the treatment of orofacial pain, possibly by acting as a TRPV1 channel antagonist.