Unveiling Targets for Treating Postoperative Pain: The Role of the TNF-α/p38 MAPK/NF-κB/Nav1.8 and Nav1.9 Pathways in the Mouse Model of Incisional Pain.

Although the mouse model of incisional pain is broadly used, the mechanisms underlying plantar incision-induced nociception are not fully understood. This work investigates the role of Nav1.8 and Nav1.9 sodium channels in nociceptive sensitization following plantar incision in mice and the signaling pathway modulating these channels. A surgical incision was made in the plantar hind paw of male Swiss mice. Nociceptive thresholds were assessed by von Frey filaments. Gene expression of Nav1.8, Nav1.9, TNF-α, and COX-2 was evaluated by Real-Time PCR in dorsal root ganglia (DRG). Knockdown mice for Nav1.8 and Nav1.9 were produced by antisense oligodeoxynucleotides intrathecal treatments. Local levels of TNF-α and PGE2 were immunoenzymatically determined. Incised mice exhibited hypernociception and upregulated expression of Nav1.8 and Nav1.9 in DRG. Antisense oligodeoxynucleotides reduced hypernociception and downregulated Nav1.8 and Nav1.9. TNF-α and COX-2/PGE2 were upregulated in DRG and plantar skin. Inhibition of TNF-α and COX-2 reduced hypernociception, but only TNF-α inhibition downregulated Nav1.8 and Nav1.9. Antagonizing NF-κB and p38 mitogen-activated protein kinase (MAPK), but not ERK or JNK, reduced both hypernociception and hyperexpression of Nav1.8 and Nav1.9. This study proposes the contribution of the TNF-α/p38/NF-κB/Nav1.8 and Nav1.9 pathways to the pathophysiology of the mouse model of incisional pain.

Bone marrow-derived mesenchymal stem/stromal cells reverse the sensorial diabetic neuropathy via modulation of spinal neuroinflammatory cascades.

BACKGROUND: Diabetic neuropathy (DN) is a frequent and debilitating manifestation of diabetes mellitus, to which there are no effective therapeutic approaches. Mesenchymal stem/stromal cells (MSC) have a great potential for the treatment of this syndrome, possibly through regenerative actions on peripheral nerves. Here, we evaluated the therapeutic effects of MSC on spinal neuroinflammation, as well as on ultrastructural aspects of the peripheral nerve in DN-associated sensorial dysfunction. METHODS: C57Bl/6 mice were treated with bone marrow-derived MSC (1 × 106), conditioned medium from MSC cultures (CM-MSC) or vehicle by endovenous route following the onset of streptozotocin (STZ)-induced diabetes. Paw mechanical and thermal nociceptive thresholds were evaluated by using von Frey filaments and Hargreaves test, respectively. Morphological and morphometric analysis of the sciatic nerve was performed by light microscopy and transmission electron microscopy. Mediators and markers of neuroinflammation in the spinal cord were measured by radioimmunoassay, real-time PCR, and immunofluorescence analyses. RESULTS: Diabetic mice presented behavioral signs of sensory neuropathy, mechanical allodynia, and heat hypoalgesia, which were completely reversed by a single administration of MSC or CM-MSC. The ultrastructural analysis of the sciatic nerve showed that diabetic mice exhibited morphological and morphometric alterations, considered hallmarks of DN, such as degenerative changes in axons and myelin sheath, and reduced area and density of unmyelinated fibers. In MSC-treated mice, these structural alterations were markedly less commonly observed and/or less pronounced. Moreover, MSC transplantation inhibited multiple parameters of spinal neuroinflammation found in diabetic mice, causing the reduction of activated astrocytes and microglia, oxidative stress signals, galectin-3, IL-1ß, and TNF-α production. Conversely, MSC increased the levels of anti-inflammatory cytokines, IL-10, and TGF-ß. CONCLUSIONS: The present study described the modulatory effects of MSC on spinal cord neuroinflammation in diabetic mice, suggesting new mechanisms by which MSC can improve DN.

Antinociceptive properties of physalins from Physalis angulata.

Pain is the most common reason a patient sees a physician. Nevertheless, the use of typical painkillers is not completely effective in controlling all pain syndromes; therefore further attempts have been made to develop improved analgesic drugs. The present study was undertaken to evaluate the antinociceptive properties of physalins B (1), D (2), F (3), and G (4) isolated from Physalis angulata in inflammatory and centrally mediated pain tests in mice. Systemic pretreatment with 1-4 produced dose-related antinociceptive effects on the writhing and formalin tests, traditional screening tools for the assessment of analgesic drugs. On the other hand, only 3 inhibited inflammatory parameters such as hyperalgesia, edema, and local production of TNF-α following induction with complete Freund's adjuvant. Treatment with 1, 3, and 4 produced an antinociceptive effect on the tail flick test, suggesting a centrally mediated antinociception. Reinforcing this idea, 2-4 enhanced the mice latency reaction time during the hot plate test. Mice treated with physalins did not demonstrate motor performance alterations. These results suggest that 1-4 present antinociceptive properties associated with central, but not anti-inflammatory, events and indicate a new pharmacological property of physalins.

Different mechanisms guide the antinociceptive effect of bone marrow-mononuclear cells and bone marrow-mesenchymal stem/stromal cells in trigeminal neuralgia.

AIMS: Trigeminal neuralgia (TN) is a type of chronic orofacial pain evoked by trivial stimuli that manifests as episodes of excruciating and sudden, recurrent paroxysmal pain. Most patients are refractory to pharmacological therapy used for the treatment of TN. Mononuclear cells (MNC) and mesenchymal stem/stromal cells (MSC) have shown therapeutic potential in painful neuropathies, but their mechanism of action is not fully understood. The present work aimed to investigate the antinociceptive effect and mechanism of action of MNC and MSC in experimental TN. MATERIALS AND METHODS: Mice submitted to the chronic constriction injury of the infraorbital nerve (CCI-ION) mouse model of TN received a single intravenous injection of saline, MNC, or MSC (1 × 106 cells/mouse). The effect of the treatments on the behavioral signs of painful neuropathy, morphological aspects of the infraorbital nerve, and inflammatory and oxidative stress markers in the infraorbital nerve were assessed. KEY FINDINGS: MNC and MSC improved behavioral painful neuropathy, activated key cell signaling antioxidant pathways by increasing Nrf2 expression, and reduced the proinflammatory cytokines IL-1ß and TNF-α. However, treatment with MSC, but not MNC, was associated with a sustained increase of IL-10 and with the re-establishment of the morphometric pattern of the infraorbital nerve, indicating a difference in the mechanism of action between MNC and MSC. In line with this result, in IL-10 knockout mice, MSC transplantation did not induce an antinociceptive effect. SIGNIFICANCE: Importantly, these data suggest an IL-10-induced disease-modifying profile related to MSC treatment and reinforce cell therapy's potential in treating trigeminal neuralgia.

High-Content Imaging-Based Assay for SARS-CoV-2-Neutralizing Antibodies.

The COVID-19 pandemic and the consequent emergence of new SARS-CoV-2 variants of concern necessitates the determination of populational serum potency against the virus. Here, we standardized and validated an imaging-based method to quantify neutralizing antibodies against lentiviral particles expressing the spike glycoprotein (pseudovirus). This method was found to efficiently quantify viral titers based on ZsGreen-positive cells and detect changes in human serum neutralization capacity induced by vaccination with up to two doses of CoronaVac, Comirnaty, or Covishield vaccines. The imaging-based protocol was also used to quantify serum potency against pseudoviruses expressing spikes from Delta, Omicron BA.1.1.529, and BA.4/5. Our results revealed increases in serum potency after one and two doses of the vaccines evaluated and demonstrated that Delta and Omicron variants escape from antibody neutralization. The method presented herein represents a valuable tool for the screening of antibodies and small molecules capable of blocking viral entry and could be used to evaluate humoral immunity developed by different populations and for vaccine development.

IGF-1 overexpression improves mesenchymal stem cell survival and promotes neurological recovery after spinal cord injury.

BACKGROUND: Survival and therapeutic actions of bone marrow-derived mesenchymal stem cells (BMMSCs) can be limited by the hostile microenvironment present during acute spinal cord injury (SCI). Here, we investigated whether BMMSCs overexpressing insulin-like growth factor 1 (IGF-1), a cytokine involved in neural development and injury repair, improved the therapeutic effects of BMMSCs in SCI. METHODS: Using a SCI contusion model in C57Bl/6 mice, we transplanted IGF-1 overexpressing or wild-type BMMSCs into the lesion site following SCI and evaluated cell survival, proliferation, immunomodulation, oxidative stress, myelination, and functional outcomes. RESULTS: BMMSC-IGF1 transplantation was associated with increased cell survival and recruitment of endogenous neural progenitor cells compared to BMMSC- or saline-treated controls. Modulation of gene expression of pro- and anti-inflammatory mediators was observed after BMMSC-IGF1 and compared to saline- and BMMSC-treated mice. Treatment with BMMSC-IGF1 restored spinal cord redox homeostasis by upregulating antioxidant defense genes. BMMSC-IGF1 protected against SCI-induced myelin loss, showing more compact myelin 28 days after SCI. Functional analyses demonstrated significant gains in BMS score and gait analysis in BMMSC-IGF1, compared to BMMSC or saline treatment. CONCLUSIONS: Overexpression of IGF-1 in BMMSC resulted in increased cell survival, immunomodulation, myelination, and functional improvements, suggesting that IGF-1 facilitates the regenerative actions of BMMSC in acute SCI.

Conditioned Medium of Bone Marrow-Derived Mesenchymal Stromal Cells as a Therapeutic Approach to Neuropathic Pain: A Preclinical Evaluation.

Neuropathic pain is a type of chronic pain caused by injury or dysfunction of the nervous system, without effective therapeutic approaches. Mesenchymal stromal cells (MSCs), through their paracrine action, have great potential in the treatment of this syndrome. In the present study, the therapeutic potential of MSC-derived conditioned medium (CM) was investigated in a mouse model of neuropathic pain induced by partial sciatic nerve ligation (PSL). PSL mice were treated by endovenous route with bone marrow-derived MSCs (1 × 106), CM, or vehicle. Gabapentin was the reference drug. Twelve hours after administration, neuropathic mice treated with CM exhibited an antinociceptive effect that was maintained throughout the evaluation period. MSCs also induced nonreversed antinociception, while gabapentin induced short-lasting antinociception. The levels of IL-1ß, TNF-α, and IL-6 were reduced, while IL-10 was enhanced on sciatic nerve and spinal cord by treatment with CM and MSCs. Preliminary analysis of the CM secretome revealed the presence of growth factors and cytokines likely involved in the antinociception. In conclusion, the CM, similar to injection of live cells, produces a powerful and long-lasting antinociceptive effect on neuropathic pain, which is related with modulatory properties on peripheral and central levels of cytokines involved with the maintenance of this syndrome.

The anti-inflammatory and immunomodulatory potential of braylin: Pharmacological properties and mechanisms by in silico, in vitro and in vivo approaches.

Braylin belongs to the group of natural coumarins, a group of compounds with a wide range of pharmacological properties. Here we characterized the pharmacological properties of braylin in vitro, in silico and in vivo in models of inflammatory/immune responses. In in vitro assays, braylin exhibited concentration-dependent suppressive activity on activated macrophages. Braylin (10-40 µM) reduced the production of nitrite, IL-1ß, TNF-α and IL-6 by J774 cells or peritoneal exudate macrophages stimulated with LPS and IFN-γ. Molecular docking calculations suggested that braylin present an interaction pose to act as a glucocorticoid receptor ligand. Corroborating this idea, the inhibitory effect of braylin on macrophages was prevented by RU486, a glucocorticoid receptor antagonist. Furthermore, treatment with braylin strongly reduced the NF-κB-dependent transcriptional activity on RAW 264.7 cells. Using the complete Freund's adjuvant (CFA)-induced paw inflammation model in mice, the pharmacological properties of braylin were demonstrated in vivo. Braylin (12.5-100 mg/kg) produced dose-related antinociceptive and antiedematogenic effects on CFA model. Braylin did not produce antinociception on the tail flick and hot plate tests in mice, suggesting that braylin-induced antinociception is not a centrally-mediated action. Braylin exhibited immunomodulatory properties on the CFA model, inhibiting the production of pro-inflammatory cytokines IL-1ß, TNF-α and IL-6, while increased the anti-inflammatory cytokine TGF-ß. Our results show, for the first time, anti-inflammatory, antinociceptive and immunomodulatory effects of braylin, which possibly act through the glucocorticoid receptor activation and by inhibition of the transcriptional activity of NF-κB. Because braylin is a phosphodiesterase-4 inhibitor, this coumarin could represent an ideal prototype of glucocorticoid receptor ligand, able to induce synergic immunomodulatory effects.

Avaliação do efeito do transplante de células-tronco mesenquimais derivadas de medula óssea em modelo murino de neuropatia periférica diabética / Evaluation of the effect of mesenchymal stem cells derived from bone marrow transplantation in a murine model of diabetic peripheral neuropathy

O diabetes é uma doença de alta prevalência que, frequentemente, induz o comprometimento do sistema nervoso periférico. Na neuropatia diabética periférica, os sintomas mais encontrados são os sensitivos, no qual a dor neuropática, condição crônica caracterizada por alodinia e hiperalgesia, é a mais debilitante. Esta, prejudica a qualidade de vida do paciente, sendo muitas vezes não responsiva aos métodos farmacológicos convencionais de tratamento. Diante desse panorama, o desenvolvimento de novas abordagens terapêuticas que possuam ação efetiva neste tipo de dor é de grande relevância. O uso da terapia celular no tratamento de lesões do sistema nervoso tem demonstrado resultados promissores e o potencial terapêutico de células-tronco na neuropatia experimental tem sido proposto. Neste estudo, avaliou-se o efeito de células-tronco mesenquimais derivadas da medula óssea (CMsMO) na neuropatia diabética periférica estabelecida em modelo experimental de diabetes induzido por estreptozotocina (ETZ)...

Diabetes is a highly prevalent disease which frequently compromises the peripheral nervous system. In peripheral diabetic neuropathy, the most frequent symptoms are sensitive, in which the neuropathic pain, chronic condition characterized by allodynia and hyperalgesia, is the most debilitating. Neuropathic pain affects the quality of patients’ lives, and is often not responsive to pharmacological conventional treatment methods. Against this background, the development of new therapeutic approaches that have an effective action in this type of pain is of great importance. The use of cell therapy in the treatment of lesions in the nervous system has shown promising results and the therapeutic potential of stem cells in experimental neuropathy has been proposed. In this study, we evaluated the effect of mesenchymal stem cells derived from bone marrow (CMsMO) in peripheral diabetic neuropathy established in experimental model of streptozotocin (STZ) induced diabetes in mice. Four weeks after the induction of the model by administration of STZ...