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Platelet-rich plasma (PRP) is a biological blood-derived therapeutic obtained from whole blood that contains higher levels of platelets. PRP has been primarily used to mitigate joint degeneration and chronic pain in osteoarthritis (OA). This clinical applicability is based mechanistically on the release of several proteins by platelets that can restore joint homeostasis. Platelets are the primary source of brain-derived neurotrophic factor (BDNF) outside the central nervous system. Interestingly, BDNF and PRP share key biological activities with clinical applicability for OA management, such as anti-inflammatory, anti-apoptotic, and antioxidant. However, the role of BDNF in PRP therapeutic activities is still unknown. Thus, this work aimed to investigate the implications of BDNF in therapeutic outcomes provided by PRP therapy in vitro and in-vivo, using the MIA-OA animal model in male Wistar rats. Initially, the PRP was characterized, obtaining a leukocyte-poor-platelet-rich plasma (LP-PRP). Our assays indicated that platelets activated by Calcium release BDNF, and suppression of M1 macrophage polarization induced by LP-PRP depends on BDNF full-length receptor, Tropomyosin Kinase-B (TrkB). OA animals were given LP-PRP intra-articular and showed functional recovery in gait, joint pain, inflammation, and tissue damage caused by MIA. Immunohistochemistry for activating transcriptional factor-3 (ATF-3) on L4/L5 dorsal root ganglia showed the LP-PRP decreased the nerve injury induced by MIA. All these LP-PRP therapeutic activities were reversed in the presence of TrkB receptor antagonist. Our results suggest that the therapeutic effects of LP-PRP in alleviating OA symptoms in rats depend on BDNF/TrkB activity.

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BACKGROUND: Pannexin1 (Panx1) is a membrane channel expressed in different cells of the nervous system and is involved in several pathological conditions, including pain and inflammation. At the central nervous system, the role of Panx1 is already well-established. However, in the periphery, there is a lack of information regarding the participation of Panx1 in neuronal sensitization. The dorsal root ganglion (DRG) is a critical structure for pain processing and modulation. For this reason, understanding the molecular mechanism in the DRG associated with neuronal hypersensitivity has become highly relevant to discovering new possibilities for pain treatment. Here, we aimed to investigate the role of Panx1 in acute nociception and peripheral inflammatory and neuropathic pain by using two different approaches. METHODS: Rats were treated with a selective Panx1 blocker peptide (10Panx) into L5-DRG, followed by ipsilateral intraplantar injection of carrageenan, formalin, or capsaicin. DRG neuronal cells were pre-treated with 10Panx and stimulated by capsaicin to evaluate calcium influx. Panx1 knockout mice (Panx1-KO) received carrageenan or capsaicin into the paw and paclitaxel intraperitoneally. The von Frey test was performed to measure the mechanical threshold of rats' and mice's paws before and after each treatment. RESULTS: Pharmacological blockade of Panx1 in the DRG of rats resulted in a dose-dependent decrease of mechanical allodynia triggered by carrageenan, and nociception decreased in the second phase of formalin. Nociceptive behavior response induced by capsaicin was significantly lower in rats treated with Panx1 blockade into DRG. Neuronal cells with Panx1 blockage showed lower intracellular calcium response than untreated cells after capsaicin administration. Accordingly, Panx1-KO mice showed a robust reduction in mechanical allodynia after carrageenan and a lower nociceptive response to capsaicin. A single dose of paclitaxel promoted acute mechanical pain in wildtype (WT) but not in Panx1-KO mice. Four doses of chemotherapy promoted chronic mechanical allodynia in both genotypes, although Panx1-KO mice had significant ablation in the first eight days. CONCLUSION: Our findings suggest that Panx1 is critical for developing peripheral inflammatory pain and acute nociception involving transient receptor potential vanilloid subtype 1 (TRPV1) but is not essential for neuropathic pain chronicity.

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Several recent studies have established the efficacy of photobiomodulation therapy (PBMT) in painful clinical conditions. Diabetic neuropathy (DN) can be related to activating mitogen-activated protein kinases (MAPK), such as p38, in the peripheral nerve. MAPK pathway is activated in response to extracellular stimuli, including interleukins TNF-α and IL-1ß. We verified the pain relief potential of PBMT in streptozotocin (STZ)-induced diabetic neuropathic rats and its influence on the MAPK pathway regulation and calcium (Ca2+) dynamics. We then observed that PBMT applied to the L4-L5 dorsal root ganglion (DRG) region reduced the intensity of hyperalgesia, decreased TNF-α and IL-1ß levels, and p38-MAPK mRNA expression in DRG of diabetic neuropathic rats. DN induced the activation of phosphorylated p38 (p-38) MAPK co-localized with TRPV1+ neurons; PBMT partially prevented p-38 activation. DN was related to an increase of p38-MAPK expression due to proinflammatory interleukins, and the PBMT (904 nm) treatment counteracted this condition. Also, the sensitization of DRG neurons by the hyperglycemic condition demonstrated during the Ca2+ dynamics was reduced by PBMT, contributing to its anti-hyperalgesic effects.

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Inflammation is a vital process for the injured tissue restoration and one of its hallmarks is inflammatory hyperalgesia. The cyclooxygenase (COX) pathway is strongly related to the inflammatory and painful process. Usually, the COX-1 isoform is described as homeostatic, while COX-2 is characterized as inducible in inflammatory conditions. Although it is well known that neutrophil cells are the first to arrive at the inflamed site and the major source of COX-2 is still unknown, the specific role of neutrophil-derived COX-2 in the pain process is. Thus, in the present study, we demonstrate for the first time that neutrophil-derived COX-2 plays a key role in peripheral inflammatory hyperalgesia. Conditional knockout mice for COX-2 in neutrophils (COX-2 fl/fl: Mrp8cre±) exhibited higher pain sensitivity after carrageenan (CG) injection and long-lasting IL-1ß-induced hyperalgesia compared with the control group (COX-2 fl/fl). Also, CG-induced inflammation in COX-2 fl/fl: Mrp8cre± mice showed COX-1 overexpression, and increased neutrophil migration and pro-inflammatory cytokines (e.g., IL-1ß and CXCL1). These findings revealed that neutrophil COX-2 has an important role in the regulation of inflammatory hyperalgesia.

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Previous data indicate that the diabetogenic substance streptozotocin might act in nociceptive neurons changing the sensory signal, regardless of hyperglycemia. In the present article the effects of streptozotocin were compared with another diabetogenic drug, alloxan, for diabetes induction in rats. A possible direct effect of these drugs was tested by means of in vivo experiments and in vitro assays using cultured primary nociceptive neurons. Streptozotocin (17.5 and 35 mg/kg), alloxan (15 and 30 mg/kg) or vehicle were injected in adult male rats and the animal groups were separated according to glycemic levels. Body mass, glycemia and paw mechanical sensitivity were evaluated for 5 weeks. Streptozotocin caused an increase in mechanical sensitivity in both hyperglycemic and normoglycemic rats, while alloxan induced mechanical sensitization only in hyperglycemic animals. Injection of both substances induced local inflammation at rat paws; however, only streptozotocin caused significant mechanical sensitization when injected near to sensory neurons at the dorsal root ganglia. Also, streptozotocin treatment induced a reduction in intracellular calcium levels and inhibited capsaicin induced calcium transients and membrane depolarization. Alloxan did not affect calcium levels or membrane potential in primary nociceptive neurons. These findings suggest that alloxan might be a better option for animal studies regarding painful diabetic neuropathy as streptozotocin directly affects nociceptive neurons, probably by modulating TRPV1 channel activation.

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The most frequently used local anesthetics (LA) for local infiltration have an ionizable amine in the range of pH 7.6-8.9. Effective anesthesia of inflamed tissues is a great challenge, especially because the induced local acidosis decreases the fraction of the neutral (more potent) LA species in situ. To solve this limitation, the butyl-substituted benzocaine analogue butamben (BTB) - that has no ionizable amine group close to the physiological pH - could be useful if it was not for its low solubility. To overcome the solubility problem, an optimized formulation for BTB using nanostructured lipid carriers (NLC) was developed by a factorial design and characterized using DLS, XRD, DSC and cryo-EM. The release kinetics and cytotoxicity of the new formulation were measured in vitro, while the in vivo tests assessed its effectiveness on healthy and inflamed tissues, in rats. The optimized NLCBTB formulation showed desirable physicochemical properties (size = 235.6 ± 3.9 nm, polydispersity = 0.182 ± 0.006 and zeta potential = -23.6 ± 0.5 mV), high (99.5%) encapsulation efficiency and stability during 360 days of storage at room temperature. NLCBTB prolonged the release of butamben and decreased its in vitro cytotoxicity without inducing any in vivo toxic alteration. In the inflammatory hyperalgesia model, the NLCBTB formulation showed potential for the management of inflammatory pain, displaying greater analgesic effectiveness (40%) and a prolonged effect.

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BACKGROUND AND PURPOSE: While dipyrone is a widely used analgesic, its mechanism of action is not completely understood. Recently, we have reported that the dipyrone metabolite 4-aminoantipyrine (4-AA) reduces PGE2 -induced pain-related behaviour through cannabinoid CB1 receptors. Here, we ascertained, in naive and PGE2 -induced "inflamed" conditions, both in vivo and in vitro, the molecular mechanisms involved in the 4-AA-induced analgesic effects. EXPERIMENTAL APPROACH: The effect of local administration of 4-AA (160 µg per paw) on capsaicin (0.12 µg per paw) injection-induced pain-related behaviour and 4-AA's effect on 500-nM capsaicin-induced changes in intracellular calcium concentration ([Ca2+ ]i ) in cultured primary sensory neurons were assessed in vivo and in vitro, respectively. KEY RESULTS: 4-AA reduced capsaicin-induced nociceptive behaviour in naive and inflamed conditions through CB1 receptors. 4-AA (100 µM) reduced capsaicin-induced increase in [Ca2+ ]i in a CB1 receptor-dependent manner, when PGE2 was not present. Following PGE2 application, 4-AA (1-50 µM) increased the [Ca2+ ]i . Although 4-AA activated both TRPV1 and TRPA1 channels, increased [Ca2+ ]i was mediated through TRPV1 channels. Activation of TRPV1 channels resulted in their desensitisation. Blocking CB1 receptors reduced both the excitatory and desensitising effects of 4-AA. CONCLUSION AND IMPLICATIONS: CB1 receptor-mediated inhibition of TRPV1 channels and TRPV1-mediated Ca2+ -influx- and CB1 receptor-dependent desensitisation of TRPV1 channels contribute to the anti-nociceptive effect of 4-AA in naive and inflamed conditions respectively. Agonists active at both CB1 receptors and TRPV1 channels might be useful as analgesics, particularly in inflammatory conditions.

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Recently the voltage-gated sodium (Nav) channels began to be studied as possible targets for analgesic drugs. In addition, specific Nav 1.8 blockers are currently being used to treat some types of chronic pain pathologies such as neuropathies and fibromyalgia. Nav 1.8+ fibers convey nociceptive information to brain structures belonging to the limbic system, which is involved in the pathophysiology of major depressive disorders. From this, using a model of chronic social defeat stress (SDS) and intrathecal injections of Nav 1.8 antisense, this study investigated the possible involvement of Nav 1.8+ nociceptive fibers in SDS- induced hyperalgesia in C57/BL mice. Our results showed that SDS induced a depressive-like behavior of social avoidance and increased the sensitivity to mechanical (electronic von Frey test) and chemical (capsaicin test) nociceptive stimuli. We also showed that intrathecal injection of Nav 1.8 antisense reversed the SDS-induced hyperalgesia as demonstrated by both, mechanical and chemical nociceptive tests. We confirmed the antisense efficacy and specificity in a separate no-defeated cohort through real-time PCR, which showed a significant reduction of Nav 1.8 mRNA and no reduction of Nav 1.7 and Nav 1.9 in the L4, L5 and L6 dorsal root ganglia (DRG). The present study advances the understanding of SDS-induced hyperalgesia, which seems to be dependent on Nav 1.8+ nociceptive fibers.

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The aim of this study was to determine whether maternal diabetes induced by alloxan injection in the first gestational day of female Wistar rats interferes with the development of the nociceptive peripheral system of the offspring. Behavioral and histologic analysis was performed using the adult offspring of diabetic and control rats. It was found that the offspring of diabetic rats were more sensitive to thermal stimulation and showed an altered response to carrageenan-induced inflammatory hyperalgesia. The histological analysis showed an increased proportion of nociceptive neurons, while the population of non-nociceptive myelinated neurons was reduced. Therefore, exposition to hyperglycemia and/or hyperinsulinemia in uterus, caused by a diabetic mother, might result in altered nociceptive sensations in the offspring throughout life.

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Cumulating data suggests that ion channel alterations in nociceptive neurons might be involved in the development of diabetic painful neuropathy. In the present study we investigated the involvement of ATP-sensitive potassium (K+ATP) channels in the acute effect of high glucose solution in vitro and in vivo. High glucose concentrations depolarized cultured nociceptive neurons and depolarization was blocked by the K+ATP opener, diazoxide or by insulin. Glucose injection at the rat dorsal root ganglia (L5) resulted in acute mechanical hyperalgesia that was blocked by diazoxide. Mannitol injection indicates that osmolarity changes are not responsible for glucose effect. Therefore, this study suggests that K+ATP channels expressed in peripheral sensory neurons might be involved in the development of diabetic painful neuropathy. Since sulfonylureas, that act by blocking K+ATP are used for diabetes treatment, it is important to evaluate the possible side effects of such drugs at primary sensory neurons.

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