Abatacept inhibits Th17 differentiation and mitigates α-synuclein-induced dopaminergic dysfunction in mice.

Parkinson's disease (PD) is a multifaceted disease characterized by degeneration of nigrostriatal dopaminergic neurons, which results in motor and non-motor dysfunctions. Accumulation of α-synuclein (αSYN) in Lewy bodies is a key pathological feature of PD. Although the exact cause of PD remains unknown, accumulating evidence suggests that brain infiltration of T cells plays a critical role in the pathogenesis of disease, contributing to neuroinflammation and dopaminergic neurodegeneration. Here, we used a mouse model of brain-infused aggregated αSYN, which recapitulates motor and non-motor dysfunctions seen in PD patients. We found that αSYN-induced motor dysfunction in mice is accompanied by an increased number of brain-residing Th17 (IL17+ CD4+) cells, but not CD8+ T cells. To evaluate whether the modulation of T cell response could rescue αSYN-induced damage, we chronically treated animals with abatacept (8 mg/kg, sc, 3x per week), a selective T-cell co-stimulation modulator. We found that abatacept treatment decreased Th1 (IFNƔ+ CD4+) and Th17 (IL17+ CD4+) cells in the brain, rescued motor function and prevented dopaminergic neuronal loss in αSYN-infused mice. These results highlight the significance of effector CD4+ T cells, especially Th17, in the progression of PD and introduce novel possibilities for repurposing immunomodulatory drugs used for arthritis as PD-modifying therapies.

Innate immune memory mediates increased susceptibility to Alzheimer's disease-like pathology in sepsis surviving mice.

Sepsis survivors show long-term impairments, including alterations in memory and executive function. Evidence suggests that systemic inflammation contributes to the progression of Alzheimers disease (AD), but the mechanisms involved in this process are still unclear. Boosted (trained) and diminished (tolerant) innate immune memory has been described in peripheral immune cells after sepsis. However, the occurrence of long-term innate immune memory in the post-septic brain is fully unexplored. Here, we demonstrate that sepsis causes long-lasting trained innate immune memory in the mouse brain, leading to an increased susceptibility to Aß oligomers (AßO), central neurotoxins found in AD. Hippocampal microglia from sepsis-surviving mice shift to an amoeboid/phagocytic morphological profile when exposed to low amounts of AßO, and this event was accompanied by the upregulation of several pro-inflammatory proteins (IL-1ß, IL-6, INF-γ and P2X7 receptor) in the mouse hippocampus, suggesting that a trained innate immune memory occurs in the brain after sepsis. Brain exposure to low amounts of AßO increased microglial phagocytic ability against hippocampal synapses. Pharmacological blockage of brain phagocytic cells or microglial depletion, using minocycline and colony stimulating factor 1 receptor inhibitor (PLX3397), respectively, prevents cognitive dysfunction induced by AßO in sepsis-surviving mice. Altogether, our findings suggest that sepsis induces a long-lasting trained innate immune memory in the mouse brain, leading to an increased susceptibility to AßO-induced neurotoxicity and cognitive impairment.

Promiscuous G-Protein-Coupled Receptor Inhibition of Transient Receptor Potential Melastatin 3 Ion Channels by Gßγ Subunits.

Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gßγ subunits liberated following activation of Gαi/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gßγ released from Gαs and Gαq Activation of the Gs-coupled adenosine 2B receptor and the Gq-coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the Gßγ sink ßARK1-ct (C terminus of ß-adrenergic receptor kinase-1) was coexpressed with TRPM3. In neurons isolated from mouse dorsal root ganglion (DRG), native TRPM3 channels were inhibited by activating Gs-coupled prostaglandin-EP2 and Gq-coupled bradykinin B2 (BK2) receptors. The Gi/o inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gαi/o or through the major protein kinases activated downstream of G-protein-coupled receptor (GPCR) activation. When DRG neurons were dialyzed with GRK2i, which sequesters free Gßγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in WT mice and did not develop in Trpm3-/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gßγ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.SIGNIFICANCE STATEMENT The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gαi/o-coupled GPCRs inhibit TRPM3 through a direct interaction between Gßγ subunits and TRPM3. Since Gßγ proteins can be liberated from other Gα subunits than Gαi/o, we examined whether activation of Gs- and Gq-coupled receptors also influence TRPM3 via Gßγ. Our results demonstrate that activation of Gs- and Gq-coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gßγ liberation. We also demonstrated that Gs- and Gq-coupled receptors inhibit TRPM3 in vivo, thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gßγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits.

Toxicological Evaluation of Anti-Scrapie Trimethoxychalcones and Oxadiazoles.

An altered form of the cellular prion protein, the PrPScor PrPRes, is implicated in the occurrence of the still untreatable transmissible spongiform encephalopathies. We have previously synthesized and characterized aromatic compounds that inhibit protease-resistant prion protein (PrPRes) accumulation in scrapie-infected cells. These compounds belong to different chemical classes, including acylhydrazones, chalcones and oxadiazoles. Some of the active compounds were non-toxic to neuroblastoma cells in culture and seem to possess drugable properties, since they are in agreement with the Lipinski´s rule of 5 and present desirable pharmacokinetic profiles as predicted in silico. Before the evaluation of the in vivo efficacy of the aromatic compounds in scrapie-infected mice, safety assessment in healthy mice is needed. Here we used Swiss mice to evaluate the acute toxicity profile of the six most promising anti-prionic compounds, the 2,4,5-trimethoxychalcones (J1, J8, J20 and J35) and the 1,3,4-oxadiazoles (Y13 and Y17). One single oral administration (300 mg/kg) of J1, J8, J20, J35, Y13 and Y17 or repeated intraperitoneal administration (10 mg/kg, 3 times a week, for 4 weeks) of J1, J8 and J35, did not elicit toxicity in mice. We strongly believe that the investigated trimethoxychalcones and oxadiazoles are interesting compounds to be further analyzed in vivo against prion diseases.

Mitotherapy prevents peripheral neuropathy induced by oxaliplatin in mice.

Oxaliplatin (OXA) is an antineoplastic agent used for the treatment of cisplatin-resistant tumours, presenting lower incidence of nephrotoxicity and myelotoxicity than other platinum-based drugs. However, OXA treatment is highly associated with painful peripheral neuropathy, a well-known and relevant side effect caused by mitochondrial dysfunction. The transfer of functional exogenous mitochondria (mitotherapy) is a promising therapeutic strategy for mitochondrial diseases. We investigated the effect of mitotherapy on oxaliplatin-induced painful peripheral neuropathy (OIPN) in male mice. OIPN was induced by i.p. injections of oxaliplatin (3 mg/kg) over 5 consecutive days. Mechanical (von Frey test) and cold (acetone drop test) allodynia were evaluated between 7 and 17 days after the first OXA treatment. Mitochondria was isolated from donor mouse livers and mitochondrial oxidative phosphorylation was assessed with high resolution respirometry. After confirming that the isolated mitochondria were functional, the organelles were administered at the dose of 0.5 mg/kg of mitochondrial protein on days 1, 3 and 5. Treatment with OXA caused both mechanical and cold allodynia in mice that were significant 7 days after the initial injection of OXA and persisted for up to 17 days. Mitotherapy significantly prevented the development of both sensory alterations, and attenuated body weight loss induced by OXA. Mitotherapy also prevented spinal cord ERK1/2 activation, microgliosis and the increase in TLR4 mRNA levels. Mitotherapy prevented OIPN by inhibiting neuroinflammation and the consequent cellular overactivity in the spinal cord, presenting a potential therapeutic strategy for pain management in oncologic patients undergoing OXA treatment.

A partial agonist of PPARγ prevents paclitaxel-induced peripheral neuropathy in mice, by inhibiting neuroinflammation.

BACKGROUND AND PURPOSE: Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of paclitaxel, affecting 30-50% of patients. Increased survival and concern with patients' quality of life have encouraged the search for new tools to prevent paclitaxel-induced neuropathy. This study presents the glitazone 4-[(Z)-(2,4-dioxo-1,3-thiazolidin-5-ylidene)methyl]-N-phenylbenzene-sulfonamide (TZD-A1) as a partial agonist of peroxisome proliferator-activated receptor γ (PPARγ), its toxicological profile and effects on paclitaxel-induced CIPN in mice. EXPERIMENTAL APPROACH: Interactions of TZD-A1 with PPARγ were analysed using in silico docking and in vitro reporter gene assays. Pharmacokinetics and toxicity were evaluated using in silico, in vitro and in vivo (C57Bl/6 mice) analyses. Effects of TZD-A1 on CIPN were investigated in paclitaxel-injected mice. Axonal and dorsal root ganglion damage, mitochondrial complex activity and cytokine levels, brain-derived neurotrophic factor (BDNF), nuclear factor erythroid 2-related factor 2 (Nrf2) and PPARγ, were also measured. KEY RESULTS: Docking analysis predicted TZD-A1 interactions with PPARγ compatible with partial agonism, which were corroborated by in vitro reporter gene assays. Good oral bioavailability and safety profile of TZD-A1 were shown in silico, in vitro and in vivo. Paclitaxel-injected mice, concomitantly treated with TZD-A1 by i.p. or oral administration, exhibited decreased mechanical and thermal hypersensitivity, effects apparently mediated by inhibition of neuroinflammation and mitochondrial damage, through increasing Nrf2 and PPARγ levels, and up-regulating BDNF. CONCLUSION AND IMPLICATIONS: TZD-A1, a partial agonist of PPARγ, provided neuroprotection and reduced hypersensitivity induced by paclitaxel. Allied to its safety profile and good bioavailability, TZD-A1 is a promising drug candidate to prevent and treat CIPN in cancer patients.

P2X7 receptor contributes to long-term neuroinflammation and cognitive impairment in sepsis-surviving mice.

Introduction: Sepsis is defined as a multifactorial debilitating condition with high risks of death. The intense inflammatory response causes deleterious effects on the brain, a condition called sepsis-associated encephalopathy. Neuroinflammation or pathogen recognition are able to stress cells, resulting in ATP (Adenosine Triphosphate) release and P2X7 receptor activation, which is abundantly expressed in the brain. The P2X7 receptor contributes to chronic neurodegenerative and neuroinflammatory diseases; however, its function in long-term neurological impairment caused by sepsis remains unclear. Therefore, we sought to evaluate the effects of P2X7 receptor activation in neuroinflammatory and behavioral changes in sepsis-surviving mice. Methods: Sepsis was induced in wild-type (WT), P2X7-/-, and BBG (Brilliant Blue G)-treated mice by cecal ligation and perforation (CLP). On the thirteenth day after the surgery, the cognitive function of mice was assessed using the novel recognition object and Water T-maze tests. Acetylcholinesterase (AChE) activity, microglial and astrocytic activation markers, and cytokine production were also evaluated. Results: Initially, we observed that both WT and P2X7-/- sepsis-surviving mice showed memory impairment 13 days after surgery, once they did not differentiate between novel and familiar objects. Both groups of animals presented increased AChE activity in the hippocampus and cerebral cortex. However, the absence of P2X7 prevented partly this increase in the cerebral cortex. Likewise, P2X7 absence decreased ionized calcium-binding protein 1 (Iba-1) and glial fibrillary acidic protein (GFAP) upregulation in the cerebral cortex of sepsis-surviving animals. There was an increase in GFAP protein levels in the cerebral cortex but not in the hippocampus of both WT and P2X7-/- sepsis-surviving animals. Pharmacological inhibition or genetic deletion of P2X7 receptor attenuated the production of Interleukin-1ß (IL-1ß), Tumor necrosis factor-α (TNF-α), and Interleukin-10 (IL-10). Conclusion: The modulation of the P2X7 receptor in sepsis-surviving animals may reduce neuroinflammation and prevent cognitive impairment due to sepsis-associated encephalopathy, being considered an important therapeutic target.

SARS-CoV-2 Spike protein induces TLR4-mediated long-term cognitive dysfunction recapitulating post-COVID-19 syndrome in mice.

Cognitive dysfunction is often reported in patients with post-coronavirus disease 2019 (COVID-19) syndrome, but its underlying mechanisms are not completely understood. Evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein or its fragments are released from cells during infection, reaching different tissues, including the CNS, irrespective of the presence of the viral RNA. Here, we demonstrate that brain infusion of Spike protein in mice has a late impact on cognitive function, recapitulating post-COVID-19 syndrome. We also show that neuroinflammation and hippocampal microgliosis mediate Spike-induced memory dysfunction via complement-dependent engulfment of synapses. Genetic or pharmacological blockage of Toll-like receptor 4 (TLR4) signaling protects animals against synapse elimination and memory dysfunction induced by Spike brain infusion. Accordingly, in a cohort of 86 patients who recovered from mild COVID-19, the genotype GG TLR4-2604G>A (rs10759931) is associated with poor cognitive outcome. These results identify TLR4 as a key target to investigate the long-term cognitive dysfunction after COVID-19 infection in humans and rodents.

Taxane-induced neurotoxicity: Pathophysiology and therapeutic perspectives.

Taxane-derived drugs are antineoplastic agents used for the treatment of highly common malignancies. Paclitaxel and docetaxel are the most commonly used taxanes; however, other drugs and formulations have been used, such as cabazitaxel and nab-paclitaxel. Taxane treatment is associated with neurotoxicity, a well-known and relevant side effect, very prevalent amongst patients undergoing chemotherapy. Painful peripheral neuropathy is the most dose-limiting side effect of taxanes, affecting up to 97% of paclitaxel-treated patients. Central neurotoxicity is an emerging side effect of taxanes and it is characterized by cognitive impairment and encephalopathy. Besides impairing compliance to chemotherapy treatment, taxane-induced neurotoxicity (TIN) can adversely affect the patient's life quality on a long-term basis. Despite the clinical relevance, not many reviews have comprehensively addressed taxane-induced neurotoxicity when they are used therapeutically. This article provides an up-to-date review on the pathophysiology of TIN and the novel potential therapies to prevent or treat this side effect.

Pharmacological Treatment of Chemotherapy-Induced Neuropathic Pain: PPARγ Agonists as a Promising Tool.

Chemotherapy-induced neuropathic pain (CINP) is one of the most severe side effects of anticancer agents, such as platinum- and taxanes-derived drugs (oxaliplatin, cisplatin, carboplatin and paclitaxel). CINP may even be a factor of interruption of treatment and consequently increasing the risk of death. Besides that, it is important to take into consideration that the incidence of cancer is increasing worldwide, including colorectal, gastric, lung, cervical, ovary and breast cancers, all treated with the aforementioned drugs, justifying the concern of the medical community about the patient's quality of life. Several physiopathological mechanisms have already been described for CINP, such as changes in axonal transport, mitochondrial damage, increased ion channel activity and inflammation in the central nervous system (CNS). Another less frequent event that may occur after chemotherapy, particularly under oxaliplatin treatment, is the central neurotoxicity leading to disorders such as mental confusion, catatonia, hyporeflexia, etc. To date, no pharmacological therapy has shown satisfactory effect in these cases. In this scenario, duloxetine is the only drug currently in clinical use. Peroxisome proliferator-activated receptors (PPARs) belong to the class of nuclear receptors and are present in several tissues, mainly participating in lipid and glucose metabolism and inflammatory response. There are three PPAR isoforms: α, ß/δ and γ. PPARγ, the protagonist of this review, is expressed in adipose tissue, large intestine, spleen and neutrophils. This subtype also plays important role in energy balance, lipid biosynthesis and adipogenesis. The effects of PPARγ agonists, known for their positive activity on type II diabetes mellitus, have been explored and present promising effects in the control of neuropathic pain, including CINP, and also cancer. This review focuses largely on the mechanisms involved in chemotherapy-induced neuropathy and the effects of the activation of PPARγ to treat CINP. It is the aim of this review to help understanding and developing novel CINP therapeutic strategies integrating PPARγ signalling.

The selective TRPV4 channel antagonist HC-067047 attenuates mechanical allodynia in diabetic mice.

Painful diabetic neuropathy (PDN) is a serious symptom that compromises quality of life and remains without effective pharmacological treatment. The transient receptor vanilloid 4 (TRPV4) is a cation-permeable channel implicated in sensory transduction and pain signalling. Therefore, drugs that act on TRPV4 may have therapeutic applications to treat PDN. In the present work, we assessed the effect of the selective TRPV4 channel antagonist HC-067047 on painful neuropathy associated with streptozotocin (STZ)-induced diabetes in mice. STZ-treated animals presented both mechanical and cold allodynia at 6 weeks after diabetes induction. Notably, HC-067047 (1 mg/kg, s.c.) given daily between 2 and 6 weeks after diabetes induction significantly prevented the development of mechanical allodynia. Additionally, both single and repeated treatments with HC-067047 (10 mg/kg, s.c.) significantly reverted established mechanical allodynia induced by STZ. However, HC-067047 was not capable of affecting either thermal cold allodynia or hyperglycemia. Similarly, HC-067047 treatments showed no effect on body weight, temperature, locomotor activity or motor coordination of control mice. Immunohistochemistry assay showed that TRPV4 expression was not different in sciatic nerve, dorsal root ganglia (DRG) or hind paw plantar skin from diabetic and non-diabetic mice, suggesting that HC-067047 acts on constitutive receptors to inhibit mechanical allodynia. Taken together, the data generated in the present study show the potential relevance of using TRPV4 antagonists to treat painful neuropathy associated with diabetes.

Spinal blockage of CXCL1 and its receptor CXCR2 inhibits paclitaxel-induced peripheral neuropathy in mice.

Painful peripheral neuropathy is the most dose-limiting side effect of paclitaxel (PTX), a widely used anti-cancer drug to treat solid tumours. The understanding of the mechanisms involved in this side effect is crucial to the development of new therapeutic approaches. CXCL1 chemokine and its receptor CXCR2 have been pointed as promising targets to treat chronic pain. Herein, we sought to evaluate the possible involvement of CXCL1 and CXCR2 in the pathogenesis of PTX-induced neuropathic pain in mice. PTX treatment led to increased levels of CXCL1 in both dorsal root ganglion and spinal cord samples. Systemic treatment with the anti-CXCL1 antibody (10 µg/kg, i.v.) or the selective CXCR2 antagonist (SB225002, 3 mg/kg, i.p.) had minor effect on PTX-induced mechanical hypersensitivity. On the other hand, the intrathecal (i.t.) treatment with anti-CXCL1 (1 ng/site) or SB225002 (10 µg/site) consistently inhibited the nociceptive responses of PTX-treated mice. Similar results were obtained by inhibiting the PI3Kγ enzyme a downstream pathway of CXCL1/CXCR2 signalling with either the selective AS605240 (5 µg/site, i.t.) or the non-selective wortmannin PI3K inhibitor (0.4 µg/site, i.t.). Overall, the data indicates that the up-regulation of CXCL1 is important for the development and maintenance of PTX-induced neuropathic pain in mice. Therefore, the spinal blockage of CXCL1/CXCR2 signalling might be a new innovative therapeutic approach to treat this clinical side effect of PTX.

Kinin Receptors Sensitize TRPV4 Channel and Induce Mechanical Hyperalgesia: Relevance to Paclitaxel-Induced Peripheral Neuropathy in Mice.

Kinin B1 (B1R) and B2 receptors (B2R) and the transient receptor potential vanilloid 4 (TRPV4) channel are known to play a critical role in the peripheral neuropathy induced by paclitaxel (PTX) in rodents. However, the downstream pathways activated by kinin receptors as well as the sensitizers of the TRPV4 channel involved in this process remain unknown. Herein, we investigated whether kinins sensitize TRPV4 channels in order to maintain PTX-induced peripheral neuropathy in mice. The mechanical hyperalgesia induced by bradykinin (BK, a B2R agonist) or des-Arg9-BK (DABK, a B1R agonist) was inhibited by the selective TRPV4 antagonist HC-067047. Additionally, BK was able to sensitize TRPV4, thus contributing to mechanical hyperalgesia. This response was dependent on phospholipase C/protein kinase C (PKC) activation. The selective kinin B1R (des-Arg9-[Leu8]-bradykinin) and B2R (HOE 140) antagonists reduced the mechanical hyperalgesia induced by PTX, with efficacies and time response profiles similar to those observed for the TRPV4 antagonist (HC-067047). Additionally, both kinin receptor antagonists inhibited the overt nociception induced by hypotonic solution in PTX-injected animals. The same animals presented lower PKCε levels in skin and dorsal root ganglion samples. The selective PKCε inhibitor (εV1-2) reduced the hypotonicity-induced overt nociception in PTX-treated mice with the same magnitude observed for the kinin receptor antagonists. These findings suggest that B1R or B2R agonists sensitize TRPV4 channels to induce mechanical hyperalgesia in mice. This mechanism of interaction may contribute to PTX-induced peripheral neuropathy through the activation of PKCε. We suggest these targets represent new opportunities for the development of effective analgesics to treat chronic pain.

Brain-Defective Insulin Signaling Is Associated to Late Cognitive Impairment in Post-Septic Mice.

Sepsis survivors frequently develop late cognitive impairment. Because little is known on the mechanisms of post-septic memory deficits, there are no current effective approaches to prevent or treat such symptoms. Here, we subjected mice to severe sepsis induced by cecal ligation and puncture (CLP) and evaluated the sepsis-surviving animals in the open field, novel object recognition (NOR), and step-down inhibitory avoidance (IA) task at different times after surgery. Post-septic mice (30 days post-surgery) failed in the NOR and IA tests but exhibited normal performance when re-evaluated 45 days after surgery. Cognitive impairment in post-septic mice was accompanied by reduced hippocampal levels of proteins involved in synaptic plasticity, including synaptophysin, cAMP response element-binding protein (CREB), CREB phosphorylated at serine residue 133 (CREBpSer133), and GluA1 phosphorylated at serine residue 845 (GluA1pSer845). Expression of tumor necrosis factor α (TNF-α) was increased and brain insulin signaling was disrupted, as indicated by increased hippocampal IRS-1 phosphorylation at serine 636 (IRS-1pSer636) and decreased phosphorylation of IRS-1 at tyrosine 465 (IRS-1pTyr465), in the hippocampus 30 days after CLP. Phosphorylation of Akt at serine 473 (AktpSer473) and of GSK3 at serine 9 (GSK3ßpSer9) were also decreased in hippocampi of post-septic animals, further indicating that brain insulin signaling is disrupted by sepsis. We then treated post-septic mice with liraglutide, a GLP-1 receptor agonist with insulinotropic activity, or TDZD-8, a GSK3ß inhibitor, which rescued NOR memory. In conclusion, these results establish that hippocampal inflammation and disrupted insulin signaling are induced by sepsis and are linked to late memory impairment in sepsis survivors.

Transient Receptor Potential Canonical Channels 4 and 5 Mediate Escherichia coli-Derived Thioredoxin Effects in Lipopolysaccharide-Injected Mice.

Thioredoxin plays an essential role in bacterial antioxidant machinery and virulence; however, its regulatory actions in the host are less well understood. Reduced human Trx activates transient receptor potential canonical 5 (TRPC5) in inflammation, but there is no evidence of whether these receptors mediate bacterial thioredoxin effects in the host. Importantly, TRPC5 can form functional complexes with other subunits such as TRPC4. Herein, E. coli-derived thioredoxin induced mortality in lipopolysaccharide- (LPS-) injected mice, accompanied by reduction of leukocyte accumulation, regulation of cytokine release into the peritoneum, and impairment of peritoneal macrophage-mediated phagocytosis. Dual TRPC4/TRPC5 blockade by ML204 increased mortality and hypothermia in thioredoxin-treated LPS mice but preserved macrophage's ability to phagocytose. TRPC5 deletion did not alter body temperature but promoted additional accumulation of peritoneal leukocytes and inflammatory mediator release in thioredoxin-administered LPS mice. Thioredoxin diminished macrophage-mediated phagocytosis in wild-type but not TRPC5 knockout animals. TRPC5 ablation did not affect LPS-induced responses. However, ML204 caused mortality associated with exacerbated hypothermia and decreased peritoneal leukocyte numbers and cytokines in LPS-injected mice. These results suggest that bacterial thioredoxin effects under LPS stimuli are mediated by TRPC4 and TRPC5, shedding light on the additional mechanisms of bacterial virulence and on the pathophysiological roles of these receptors.

Antiallodynic effect of ß-caryophyllene on paclitaxel-induced peripheral neuropathy in mice.

Painful peripheral neuropathy is a common side effect of paclitaxel (PTX). The use of analgesics is an important component for management of PTX-induced peripheral neuropathy (PINP). However, currently employed analgesics have several side effects and are poorly effective. ß-caryophyllene (BCP), a dietary selective CB2 agonist, has shown analgesic effect in neuropathic pain models, but its role in chemotherapy-induced neuropathic pain has not yet been investigated. Herein, we used the mouse model of PINP to show the therapeutic effects of BCP in this neuropathy. Male Swiss mice receiving PTX (2 mg kg-1, ip, four alternate days) were treated with BCP (25 mg kg-1, po, twice a day) either during or after PTX administration. Some groups were also pretreated with AM630 (CB2 antagonist, 3 mg kg-1, ip) or AM251 (CB1 antagonist, 1 mg kg-1, ip). Spinal cord samples were collected in different time points to perform immunohistochemical analysis. BCP attenuated the established mechanical allodynia induced by PTX (p < 0.0001) in a CB2-dependent manner. Of note, when given concomitantly with PTX, BCP was able to attenuate the development of PINP (p < 0.0001). Spinal cord immunohistochemistry revealed that preventive treatment with BCP reduced p38 MAPK and NF-κB activation, as well as the increased Iba-1 and IL-1ß immunoreactivity promoted by PTX. Our findings show that BCP effectively attenuated PINP, possibly through CB2-activation in the CNS and posterior inhibition of p38 MAPK/NF-κB activation and cytokine release. Taken together, our results suggest that BCP could be used to attenuate the establishment and/or treat PINP.

Effect of novel selective non-peptide kinin B(1) receptor antagonists on mouse pleurisy induced by carrageenan.

Two novel selective non-peptide kinin B(1) receptor antagonists, the benzodiazepine antagonist and SSR240612, were evaluated in carrageenan-induced mouse pleurisy. The peptide R-715 (0.5 mg/kg, i.p.) and the non-peptide benzodiazepine (3 mg/kg, i.p.) antagonists significantly decreased cellular migration (predominantly neutrophils), without altering plasma exudation. SSR240612 (1 mg/kg, i.p.) diminished total cells and neutrophils, besides exudation. Oral administration of SSR240612 (10 mg/kg) also reduced total cell and neutrophil counts. Only the benzodiazepine antagonist inhibited the lung myeloperoxidase activity. No tested antagonist significantly altered the lung and pleural TNFalpha and IL-1beta production. We provide interesting evidence on the anti-inflammatory in vivo effects of non-peptide B(1) receptor antagonists.

Evaluation of antimetastatic activity and systemic toxicity of camptothecin-loaded microspheres in mice injected with B16-F10 melanoma cells.

PURPOSE: The aim of this work was to evaluate the pulmonary antimetastatic activity and the systemic toxicity of camptothecin-loaded microspheres. METHODS: PCL microspheres containing camptothecin (CPT) were prepared by the emulsion solvent/evaporation method and characterized according to their encapsulation efficiency, particle size, morphology, and drug release. The ability of CPT to inhibit the lung metastasis was verified using an experimental mouse model intravenously injected with metastatic B16- F10 melanoma cells. The microspheres and the free drug were given intraperitoneally at a dose of 7 mg/kg at intervals of three or five days for 24 days. The systemic toxicity of CPT was evaluated by weight measurements, survival and hemograms of the animals. RESULTS: The encapsulation efficiency was nearly 80%. The drug release was complete after 72 hours, but the burst effect increased from 7% to 35% with the increase in CPT content in the particles. It was observed during the in vivo essays that all groups treated with CPT had a decrease of nearly 70% in the number of lung metastases. However, systemic toxicity was verified in animals that received the free drug. CONCLUSION: Camptothecin-loaded microspheres demonstrated similar therapeutic efficacy when compared to those of the free drug, but the toxicity was significantly reduced.

Temporal and Regional Expression of Glucose-Dependent Insulinotropic Peptide and Its Receptor in Spinal Cord Injured Rats.

Spinal cord injury (SCI) results in loss of movement, sensibility, and autonomic control at the level of the lesion and at lower parts of the body. Several experimental strategies have been used in attempts to increase endogenous mechanisms of neuroprotection, neuroplasticity, and repair, but with limited success. It is known that glucose-dependent insulinotropic peptide (GIP) and its receptor (GIPR) can enhance synaptic plasticity, neurogenesis, and axonal outgrowth. However, their role in the injury has never been studied. The aim of this study was to evaluate the changes in expression levels of both GIP and GIPR in acute and chronic phases of SCI in rats. Following SCI (2 to 24 h after damage), the rat spinal cord showed a lesion in which the epicenter had a cavity with hemorrhage and necrosis. Furthermore, the lesion cavity also showed ballooned cells 14 and 28 days after injury. We found that SCI induced increases in GIPR expression in areas neighboring the site of injury at 6 h and 28 days after the injury. Moreover, higher GIP expression was observed in these regions on day 28. Neuronal projections from the injury epicenter showed an increase in GIP immunoreactivity 24 h and 14 and 28 days after SCI. Interestingly, GIP was also found in progenitor cells at the spinal cord canal 24 h after injury, whereas both GIP and GIPR were present in progenitor cells at the injury epicenter 14 days after in SCI animals. These results suggest that GIP and its receptor might be implicated with neurogenesis and the repair process after SCI.

Cinnamaldehyde modulates LPS-induced systemic inflammatory response syndrome through TRPA1-dependent and independent mechanisms.

Cinnamaldehyde is a natural essential oil suggested to possess anti-bacterial and anti-inflammatory properties; and to activate transient receptor potential ankyrin 1 (TRPA1) channels expressed on neuronal and non-neuronal cells. Here, we investigated the immunomodulatory effects of cinnamaldehyde in an in vivo model of systemic inflammatory response syndrome (SIRS) induced by lipopolysaccharide. Swiss mice received a single oral treatment with cinnamaldehyde 1 h before LPS injection. To investigate whether cinnamaldehyde effects are dependent on TRPA1 activation, animals were treated subcutaneously with the selective TRPA1 antagonist HC-030031 5 min prior to cinnamaldehyde administration. Vehicle-treated mice were used as controls. Cinnamaldehyde ameliorated SIRS severity in LPS-injected animals. Diminished numbers of circulating mononuclear cells and increased numbers of peritoneal mononuclear and polymorphonuclear cell numbers were also observed. Cinnamaldehyde augmented the number of peritoneal Ly6C(high) and Ly6C(low) monocyte/macrophage cells in LPS-injected mice. Reduced levels of nitric oxide, plasma TNFα and plasma and peritoneal IL-10 were also detected. Additionally, IL-1ß levels were increased in the same animals. TRPA1 antagonism by HC-030031 reversed the changes in the number of circulating and peritoneal leukocytes in cinnamaldehyde-treated animals, whilst increasing the levels of peritoneal IL-10 and reducing peritoneal IL-1ß. Overall, cinnamaldehyde modulates SIRS through TRPA1-dependent and independent mechanisms.