Molecular Aspects Involved in the Mechanisms of Bothrops jararaca Venom-Induced Hyperalgesia: Participation of NK1 Receptor and Glial Cells.

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

Molecular aspects involved in the mechanisms of Bothrops jararaca venom-induced Hyperalgesia: participation of NK1 Receptor and glial cells

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

Role of 5-HT1A receptors in the ventral hippocampus in the regulation of anxiety- and panic-related defensive behaviors in rats.

Changes in 5-HT1A receptor (5-HT1AR)-mediated neurotransmission in the hippocampus have been associated with anxiety, depression and in the mode of action of antidepressant drugs. It has been commonly accepted that whereas the dorsal pole of the hippocampus (DH) is involved in cognitive processing, the ventral pole (VH) is associated with emotional regulation. However, to date, only a few studies have directly addressed the role played by VH 5-HT1ARs in anxiety and panic processing, and their results are conflicting. Here we report that intra-VH administration of the 5-HT1A receptor agonist 8-OH-DPAT, the endogenous agonist serotonin (5-HT), or the standard anxiolytic benzodiazepine midazolam impaired the acquisition of inhibitory avoidance in the elevated T-maze (ETM) of male Wistar rats, indicating an anxiolytic effect. Conversely, local injection of the 5-HT1AR antagonist WAY-100635 caused the opposite effect. These results were equally found in the Vogel conflict test. None of these drugs interfered with locomotor activity in the open-field test, nor did they alter the expression of the escape response in the ETM, a defensive behavior associated with panic. Pre-injection of a sub-effective dose of WAY-100635 in the VH blocked the anxiolytic effect of 5-HT or 8-OH-DPAT in the Vogel test, confirming the involvement of 5-HT1AR for this behavioral effect. The effect in this test was anxiety-selective as none of the drugs affected water consumption or nociception. In conclusion, our results suggest that 5-HT1ARs in the VH play a tonic inhibitory role in anxiety processing. These receptors, however, are not involved in the regulation of panic-related escape behavior.

Participação das células gliais na hiperalgesia induzida pelo veneno de Bothrops jararaca e pelo conteúdo plaquetário: efeito da depleção de plaquetas circulantes

Pain has a protective and adaptive role in the body, and is essential to alert about harmful stimuli in the environment. In this context, the increased response to painful stimuli is called hyperalgesia, in which astrocytes and microglia, presente in the spinal cord, act in the central mediation of this effect. Studies from our group demonstrated that platelets are involved in hyperalgesia induced by carrageenan and Bothrops jararaca venom (Bjv). Furthermore, it was observed that whole platelets or platelet releasate (PR) also induce hyperalgesia, suggesting that these cells play a crucial role in the genesis of inflammatory pain. Considering this information, the aim of this project is evaluate the participation of microglia and astrocytes, presente in the spinal cord, during the peak of hyperalgesia evoked by Bjv or by PR, as weel as the involvement of platelets in changes observed in glial cells. Hyperalgesia was assessed by analgesy-meter 2 hours after intraplantar (i.pl) injection of Bjv and PR, associated or not with different treatments, such as anti-rat platelet antibody (which causes depletion of circulating platelets) and minocycline (inhibit microglial activation). The results showed that both Bjv and PR induced hyperalgesia, wich was abolished by depletion of platelets, reinforcing the importance of these cells in the development of hyperalgesia. Microglial blockade also inhibited hyperalgesia induced by Bjv and PR. To confirm the involvement of glial cells, ipsilateral side of spinal cord samples of vBj and CSP, obtained from animals submitted to the nociceptive test, were quantified in western blotting (WB) analysis for microglia (Iba-1) and astrocyte (GFAP). The expression of Iba-1 and GFAP was up-regulated in both groups, Bjv and PR. However, platelet depletion only inhibited microglia marking in Bjv group, not interfering with the glial markers in the PR group, indicating that there is a relationship between circulating platelets and microglial activation during hyperalgesia evoked by Bjv. The role of NK1 receptors in hyperalgesia evoked by Bjv was also evaluated. The data obtained demonstrated that the NK1 receptor antagonist (GR 82334) inhibited the venom hyperalgesia. Furthermore, an increase in the expression of NK1 receptors was detected in the spinal cord of animals injected with Bjv by WB, suggesting that NK1 receptors participate in the central mediation of this effect. Platelet depletion did not interfere with this effect. Taken together, these datas confirm the participation of circulating platelets in the genesis of hyperalgesia and demonstrate the involvement of microglial and astrocytic cells in the central mediation of hyperalgesia induced by Bjv and PR. Furthermore, the results suggest that the increase in microglial expression induced by Bjv is dependent on circulating platelets. In addition, suggest that the hyperalgesic effect of Bjv is mediated, at least in part, by NK1 receptors.

A dor tem um papel protetor e adaptativo no organismo, além de ser essencial para alertar sobre estímulos nocivos no ambiente. Neste contexto, é chamada de hiperalgesia a resposta aumentada aos estímulos dolorosos, em que microgliais e astrócitos, presentes na medula espinal, participam da mediação central desse efeito. Estudos do nosso grupo demonstraram que as plaquetas circulantes estão envolvidas na hiperalgesia induzida pela carragenina e pelo veneno de serpentes Bothrops jararaca (vBj). Ainda, foi observado que plaquetas íntegras e o conteúdo de secreção plaquetária (CSP) também induzem hiperalgesia, sugerindo que estas células desempenham papel primordial na gênese da dor inflamatória. Considerando estas informações, o objetivo deste projeto é determinar a participação da microglia e do astrócito, presentes na medula espinal, no desencadeamento da hiperalgesia induzida pelo vBj ou pelo CSP, bem como o envolvimento das plaquetas circulantes nas alterações observadas nas células gliais. A hiperalgesia foi avaliada pelo teste de pressão de pata de ratos após 2 horas da injeção intraplantar (i.pl) do vBj ou do CSP, pico da hiperalgesia, associadas ou não aos tratamentos prévios com anticorpo anti-plaqueta de rato, que causa a depleção de plaquetas circulantes, ou com minociclina (bloqueador da microglia). Os resultados obtidos demonstraram que tanto o vBj quanto o CSP induziram hiperalgesia, a qual foi abolida pela depleção de plaquetas circulantes, confirmando a importância dessas células no desencadeamento desse efeito. O bloqueio da microglia também inibiu a hiperalgesia causada pelo vBj e CSP. Para verificar a participação das células gliais na hiperalgesia observada, amostras da medula espinal no lado ipsilateral a injeção i.pl. do vBj e do CSP, obtidas de animais submetidos ao teste nociceptivo, foram avaliadas em ensaio de western blotting (WB) para marcação de microglia (Iba-1) e astrócito (GFAP). Os resultados demonstraram maior expressão de ambos os marcadores tanto no grupo vBj quanto no CSP. Entretanto, a depleção de plaquetas inibiu apenas a marcação da microglia no grupo vBj, não interferindo com a marcação dos astrócitos ou com os marcadores gliais no grupo CSP, indicando que existe uma relação entre as plaquetas circulantes e a ativação da microglia durante a hiperalgesia induzida pelo vBj. Também foi investigado o envolvimento dos receptores NK1 para taquicininas na hiperalgesia acarretada pelo vBj. Os dados obtidos demostraram que o antagonista de receptores NK1 (GR 82334) inibiu a hiperalgesia induzida pelo veneno. Além disso, foi observado por WB aumento da expressão desses receptores na medula espinal de ratos injetados com o vBj, sugerindo que os receptores NK1 participam da mediação da hiperalgesia causada pelo veneno. A depleção de plaquetas não interferiu com esse efeito. Em conjunto, os dados apresentados confirmam a participação das plaquetas circulantes na gênese da hiperalgesia e demonstram o envolvimento das células microgliais e astrocitárias na mediação central da hiperalgesia induzida pelo vBj e pelo CSP. Ademais, sugerem que a o aumento da expressão da microglia induzida pelo vBj é dependente das plaquetas circulantes. Ainda, que o efeito hiperalgésico do vBj é mediado, pelo menos em parte, por receptores NK1.