Blockage of the fractalkine pathway reduces hyperalgesia and prevents morphological glial alterations-Comparison between inflammatory and neuropathic orofacial pain in male rats.

This study aimed to evaluate the effects of inhibitors of the fractalkine pathway in hyperalgesia in inflammatory and neuropathic orofacial pain in male rats and the morphological changes in microglia and satellite glial cells (SGCs). Rats were submitted to zymosan-induced arthritis of the temporomandibular joint or infraorbital nerve constriction, and treated intrathecally with a P2 X7 antagonist, a cathepsin S inhibitor or a p-38 mitogen-activated protein kinase (MAPK) inhibitor. Mechanical hyperalgesia was evaluated 4 and 6 h following arthritis induction or 7 and 14 days following nerve ligation. The expression of the receptor CX3 CR1 , phospho-p-38 MAPK, ionized calcium-binding adapter molecule-1 (Iba-1), and glutamine synthetase and the morphological changes in microglia and SGCs were evaluated by confocal microscopy. In both inflammatory and neuropathic models, untreated animals presented a higher expression of CX3 CR1 and developed hyperalgesia and up-regulation of phospho-p-38 MAPK, which was prevented by all drugs (p < .05). The number of microglial processes endpoints and the total branch length were lower in the untreated animals, but the overall immunolabeling of Iba-1 was altered only in neuropathic rats (p < .05). The mean area of SGCs per neuron was significantly altered only in the inflammatory model (p < .05). All morphological alterations were reverted by modulating the fractalkine pathway (p < .05). In conclusion, the blockage of the fractalkine pathway seemed to be a possible therapeutic strategy for inflammatory and neuropathic orofacial pain, reducing mechanical hyperalgesia by impairing the phosphorylation of p-38 MAPK and reverting morphological alterations in microglia and SGCs.

Oral carcinogenesis triggers a nociceptive behavior and c-Fos expression in rats' trigeminal pathway.

OBJECTIVE: To recognize changes that occur along the trigeminal pathway in oral cancer in order to establish an effective approach to pain control. METHODS: Wistar rats were divided into control and 4-NQO groups for 8, 12, 16, or 20 weeks. 4-NQO suspension was administered on the animals' tongues. Mechanical hyperalgesia, assessment of facial expressions, and an open-field test were performed. After euthanasia, the animals' tongues were removed for macro- and microscopic analysis. c-Fos expression was analyzed in the trigeminal pathway structures. RESULTS: 4-NQO induced time-dependent macroscopic lesions that were compatible with neoplastic tumors. Histopathological analysis confirmed oral squamous cell carcinoma in 50% of the animals on the 20th week. There was a significant nociceptive threshold reduction during the first two weeks, followed by a threshold return to the baseline levels, decreasing again from the 12th week. Facial nociceptive expression scores were observed on the 20th week, while increased grooming and exploratory activity were observed on the 8th week. Trigeminal ganglion showed an increased c-Fos immunoexpression on the 20th week, and in the trigeminal subnucleus caudalis, it occurred on the 16th and 20th. The long-term carcinogenic exposure caused changes in the nociceptive behavior and c-Fos expression in the rats' trigeminal pathway.

Neurotoxic effect of oxaliplatin: Comparison with its oxalate-free analogue cis-[PtII(1R,2R-DACH)(3-acetoxy-1,1-cyclobutanedicarboxylato)] (LLC-1402) in mice.

Studies suggest that oxalate is involved in the development oxaliplatin-induced peripheral sensory neuropathy (OPSN). This study aimed to compare the neurotoxic effects of oxaliplatin with its oxalate-free cytotoxic analogue cis-[PtII(1R,2R-DACH)(3-acetoxy-1,1-cyclobutanedicarboxylato)] (LLC-1402) in mice. Oxaliplatin and LLC-1402 were intravenously injected in male Swiss mice with a total of nine injections. Oxalate was intraperitoneally injected in other animals. The development of OPSN was evaluated using mechanical and thermal sensitivity tests. Dorsal root ganglia of the mice were removed to evaluate c-Fos, ATF3 and iNOS expression and a sample of blood was collected for leukocyte count and hepatic and renal biochemical function tests. Oxaliplatin and LLC-1402 decreased the mechanical and thermal nociceptive threshold, whilst oxalate lead to a partial and later increase in the mechanical sensitivity (P<0.05). c-Fos, ATF3 and iNOS expressions were increased in neuronal cells during and after the end of the injections in animals treated with oxaliplatin and LLC-1402 (P<0.05), even though oxaliplatin lead to an earlier increase. Only c-Fos expression was elevated during the period of injections in the oxalate group (P<0.05), but this expression reduced after the end of the treatment. c-Fos expression was also shown in glial satellite cells only in the oxaliplatin-treated animals. Oxaliplatin and LLC-1402 reduced leukocyte count (P<0.05), but did not change renal and liver functions. In conclusion, oxalate may contribute to an earlier development of peripheral sensory neuropathy. However, the antitumor cytotoxic mechanism of oxaliplatin seems to be the main responsible by its neurotoxic effect.

The antioxidant effects of the flavonoids rutin and quercetin inhibit oxaliplatin-induced chronic painful peripheral neuropathy.

BACKGROUND: Oxaliplatin, the third-generation platinum compound, has evolved as one of the most important therapeutic agents in colorectal cancer chemotherapy. The main limiting factor in oxaliplatin treatment is painful neuropathy that is difficult to treat. This side effect has been studied for several years, but its full mechanism is still inconclusive, and effective treatment does not exist. Data suggest that oxaliplatin's initial neurotoxic effect is peripheral and oxidative stress-dependent. A spinal target is also suggested in its mechanism of action. The flavonoids rutin and quercetin have been described as cell-protecting agents because of their antioxidant, antinociceptive, and anti-inflammatory actions. We proposed a preventive effect of these agents on oxaliplatin-induced painful peripheral neuropathy based on their antioxidant properties. METHODS: Oxaliplatin (1 mg/kg, i.v.) was injected in male Swiss mice, twice a week (total of nine injections). The development of sensory alterations, such as thermal and mechanical allodynia, was evaluated using the tail immersion test in cold water (10°C) and the von Frey test. Rutin and quercetin (25-100 mg/kg, i.p.) were injected 30 min before each oxaliplatin injection. The animals' spinal cords were removed for histopathological and immunohistochemical evaluation and malondialdehyde assay. RESULTS: Oxaliplatin significantly increased thermal and mechanical nociceptive response, effects prevented by quercetin and rutin at all doses. Fos immunostaining in the dorsal horn of the spinal cord confirmed these results. The oxidative stress assays mainly showed that oxaliplatin induced peroxidation in the spinal cord and that rutin and quercetin decreased this effect. The flavonoids also decreased inducible nitric oxide synthase and nitrotyrosine immunostaining in the dorsal horn of the spinal cord. These results suggest that nitric oxide and peroxynitrite are also involved in the neurotoxic effect of oxaliplatin and that rutin and quercetin can inhibit their effect in the spinal cord. We also observed the preservation of dorsal horn structure using histopathological analyses. CONCLUSIONS: Oxaliplatin induced painful peripheral neuropathy in mice, an effect that was prevented by rutin and quercetin. The mechanism of action of oxaliplatin appears to be, at least, partially oxidative stress-induced damage in dorsal horn neurons, with the involvement of lipid peroxidation and protein nitrosylation.

Temporomandibular joint arthritis increases canonical Wnt pathway expression in the articular cartilage and trigeminal ganglion in rats.

The canonical Wnt pathway participates in inflammatory diseases and it is involved in neuropathic pain. This study evaluated the immunoexpression of the canonical Wnt signaling pathway in the articular cartilage of the temporomandibular joint (TMJ) and along the nociceptive trigeminal pathway in arthritic rats. For this, male Wistar rats were divided into Control (C) and Arthritic (RA) groups. Arthritis induction was performed through subcutaneous injection of methylated bovine serum albumin (mBSA) and complete Freund Adjuvant (CFA)/ Incomplete Freund Adjuvant (IFA) on the first 14 days (once a week), followed by 3 weekly intra-articular injections of mBSA (10 µl/joint; left TMJ). The following parameters were evaluated: nociceptive threshold, inflammatory infiltrate, type I and III collagen birefringence, immunohistochemistry for IL-1ß, TNF-α, IL-6, Wnt10b, ß-catenin, cyclin-D1 in articular cartilage, c-Myc in synovial membrane, and immunofluorescence analysis for c-Fos, Wnt-10b and ß-catenin in the trigeminal ganglion and the trigeminal subnucleus caudalis. The RA group showed intense articular cartilage damage with proliferation of type III collagen, increased immunoexpression of proinflammatory cytokines and Wnt-10b, ß-catenin and cyclin-D1 in the articular cartilage and c-Myc in the synovial membrane. In the RA group, a reduction in the nociceptive threshold was observed, followed by a significant increase in the expression of Wnt-10b in neurons and ß-catenin in satellite cells of the trigeminal ganglion. c-Fos immunoexpression was observed in neurons, peripherally and centrally, in arthritic rats. Our data demonstrated that TMJ arthritis in rats causes articular cartilage damage and nociceptive behavior, with increased immunoexpression of canonical Wnt pathway in the articular cartilage and trigeminal ganglion.

TLR4 deficiency upregulates TLR9 expression and enhances irinotecan-related intestinal mucositis and late-onset diarrhoea.

BACKGROUND AND PURPOSE: Severe diarrhoea, a common gastrointestinal manifestation of anticancer treatment with irinotecan, might involve single nucleotide polymorphisms (SNPs) of toll-like receptors (TLRs), described as critical bacterial sensors in the gut. Here, colorectal cancer patients carrying missense TLR4 A896G (rs4986790) or C1,196T (rs4986791) SNPs and Tlr4 knockout (Tlr4-/-) mice were given irinotecan to investigate the severity of the induced diarrhoea. EXPERIMENTAL APPROACH: Forty-six patients treated with irinotecan-based regimens had diarrhoea severity analysed according to TLR4 genotypes. In the experimental setting, wild-type (WT) or Tlr4-/- mice were given irinotecan (45 or 75 mg·kg-1 , i.p.) or saline (3 ml·kg-1 ). Diarrhoea severity was evaluated by measuring intestinal injury and inflammatory markers expression after animals were killed. KEY RESULTS: All patients with TLR4 SNPs chemotherapy-treated presented diarrhoea, whereas gastrointestinal toxicity was observed in 50% of the wild homozygous individuals. Mice injected with irinotecan presented systemic bacterial translocation and increased TLR4 immunostaining in the intestine. In line with the clinical findings, Tlr4 gene deficiency enhanced irinotecan-related diarrhoea and TLR9 expression in mice. An increased myeloperoxidase activity and Il-18 expression along with IL-10 decreased production in Tlr4-/- mice also indicated an intensified intestinal damage and inflammatory response. CONCLUSION AND IMPLICATIONS: TLR4 deficiency upregulates TLR9 expression and enhances intestinal damage and the severity of late-onset diarrhoea during irinotecan-based treatment. Identifying patients genetically predisposed to chemotherapy-associated diarrhoea is a strategy toward precision medicine.

Endocannabinoid System Attenuates Oxaliplatin-Induced Peripheral Sensory Neuropathy Through the Activation of CB1 Receptors.

Oxaliplatin-induced neurotoxicity is expressed as a dose-limiting peripheral sensory neuropathy (PSN). Cannabinoid substances have been investigated for the analgesic effect. This study aimed to investigate the role of cannabinoid receptors in oxaliplatin-associated PSN. Swiss male mice received nine oxaliplatin injections (2 mg/kg, i.v.). Mechanical and thermal nociceptive tests were performed for 56 days. CB1, CB2, and c-Fos expression were assessed in dorsal root ganglia (DRG), spinal cord (SC), trigeminal ganglia (TG), spinal trigeminal nucleus caudalis (Sp5C), and periaqueductal gray (PAG). Iba-1 expression was assessed in DRG and ATF3 in TG. Cannabidiol (10 mg/kg, p.o.) or a CB1/CB2 non-selective agonist (WIN 55,212-2; 0.5 mg/kg, s.c.) or AM251 (CB1 antagonist) or AM630 (CB2 antagonist) (3 mg/kg, i.p.) were injected before oxaliplatin. Oxaliplatin increased CB1 in DRG, SC, TG, Sp5C, and ventrolateral PAG, with no interference in CB2 expression. Cannabidiol increased CB1 in DRG, reduced mechanical hyperalgesia and c-Fos expression in DRG and SC. Additionally, WIN 55,212-2 increased CB1 in DRG, reduced mechanical hyperalgesia, cold allodynia and c-Fos expression in DRG and SC. CB1 blockage hastened the cold allodynia response, but the CB2 antagonist failed to modulate the oxaliplatin-induced nociceptive behavior. Oxaliplatin also increased Iba-1 in DRG, suggesting immune response modulation which was reduced by cannabidiol and enhanced by AM630. The modulation of the endocannabinoid system, through the CB1 receptor, attenuates the oxaliplatin-associated PNS. The activation of the endocannabinoid system could be considered as a therapeutic target for controlling oxaliplatin-associated neuropathy.

Cytoplasmic CCR7 (CCR7c) immunoexpression is associated with local tumor recurrence in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of cancer, which tests negative for estrogen receptors, progesterone receptors, and lacks overexpression of the human epidermal growth factor 2 (C-erbB2, HER2/neu) gene. The expression of chemokines and their receptors, including CCR7, has been described in several types of cancer, contributing to tumor progression. AIM OF THE STUDY: This study investigated the association between the membrane and cytoplasmic CCR7 expression and the prognosis of TNBC. MATERIALS AND METHODS: Surgical paraffin histopathology blocks and clinico-pathological data were assessed from 133 patients. Samples were analyzed by immunohistochemistry and immunofluorescence using the Tissue Microarray technique for scoring the intensity of CCR7 expression. RESULTS: TNBC patients in which the CCR7 labeling was predominantly in the cytoplasm of tumor cells presented increased local tumor recurrence (P = 0.033). Conversely, there was no statistical difference in five-year overall survival between the patients with low (77%) versus high (80%) cytoplasmic CCR7 expression (P = 0.7104). Additionally, the risk of death between these groups was 1.19 (95% CI = 0.48-2.91). CONCLUSION: The cytoplasmic CCR7 expression associates with an increased incidence of tumor relapse in TNBC, not affecting patients survival. Consequently, the cell compartment in which the CCR7 localizes could serve as a prognostic marker in this cancer subtype.

Involvement of Endothelin Receptors in Peripheral Sensory Neuropathy Induced by Oxaliplatin in Mice.

The aim of this study was to evaluate the participation of the endothelin ETA and ETB receptors and the effects of bosentan in oxaliplatin-induced peripheral sensory neuropathy (OIN) in mice. Adult male Swiss mice received 1 mg/kg of oxaliplatin intravenously, twice a week for 5 weeks. Dorsal root ganglia (DRG) and spinal cords were removed for evaluation of the endothelin ETA and ETB receptor expression. Afterwards, selective (BQ-123 and BQ-788; 10 nmol in 30 µL, intraplantarly) and non-selective (bosentan, 100 mg/kg, orally) antagonists were administered in order to evaluate the involvement of the endothelin receptors in OIN. Mechanical and thermal nociception tests were performed once a week for 56 days. Oxaliplatin induced mechanical and thermal hypersensitivity and increased the endothelin ETA receptor expression in both the DRG and spinal cord (P < 0.05). Endothelin ETB receptor expression was increased in the DRG (P < 0.05) but not in the spinal cord. Both endothelin ETA and ETB receptor selective antagonists partially prevented mechanical hyperalgesia in mice with OIN (P < 0.05). Moreover, bosentan prevented mechanical and thermal hypersensitivity in oxaliplatin-treated mice (P < 0.05). In conclusion, both endothelin ETA and ETB receptors seem to be involved in the OIN in mice and they should be considered possible targets for the management of this clinical feature.

Metformin reduces c-Fos and ATF3 expression in the dorsal root ganglia and protects against oxaliplatin-induced peripheral sensory neuropathy in mice.

Oxaliplatin is a third-generation platinum drug commonly used as the first line treatment of metastatic colorectal cancer. Oxaliplatin-based anticancer regimens course with dose-limiting neurotoxicity. The pharmacological strategies used to manage such side effect are not totally effective. Metformin is an anti-diabetic drug that is described to negatively modulate painful diabetic neuropathy. Then, this study aimed to assess the effect of metformin in the oxaliplatin-induced peripheral sensory neuropathy in mice. For that purpose, Swiss male mice were injected with oxaliplatin (1, 2 or 4 mg/kg, i.v., twice a week with a total of nine injections) alone or in combination with daily administration of metformin (250 mg/kg, p.o.). Thermal and mechanical nociceptive tests were performed once a week for five weeks. Then, the animals were euthanized on day 35 post-first injection of oxaliplatin and the dorsal root ganglia were harvested for the assessment of c-Fos and ATF3 expressions. Oxaliplatin caused a nociceptive response accompanied by the increased expression of c-Fos and ATF3 in the dorsal root ganglia and spinal cord. In addition, the oxaliplatin-associated nociception was significantly attenuated by metformin (P < 0.05), which also reduced the expression of c-Fos and ATF3 (P < 0.05). Therefore, metformin protected from the peripheral sensory neuropathy induced by oxaliplatin, which was confirmed by the reduction of c-Fos and ATF3 expression, two known neuronal activation and damage markers, respectively.

Antinociceptive and anti-inflammatory effects of Caryocar coriaceum Wittm fruit pulp fixed ethyl acetate extract on zymosan-induced arthritis in rats.

The ethyl acetate extract from the fruit pulp of Caryocar coriaceum Wittm (Caryocaraceae), popularly known as pequi, has wide applications in popular medicine. Preclinical tests have demonstrated the therapeutic properties of the oil. We investigated the antinociceptive and anti-inflammatory effects of Pequi C. coriaceum Wittm ethyl acetate extract (PCCO) on zymosan-induced arthritis in rat knee joint. The animals were pretreated with PCCO for 7 consecutive days or with a single dose. Paw elevation time (PET), leukocyte infiltration, myeloperoxidase activity (MPO) and cytokine levels were assessed 4h after zymosan injection. Synovial tissue was harvested for immunohistochemical analysis, edema and vascular permeability. We observed a significant decrease in PET with PCCO pretreatment. PCCO showed a significant reduction of leukocyte migration and a decrease in MPO. Decreases were observed in cytokine release in the synovial fluid and TNF-α and cyclooxygenase-1 immunostaining in synovial tissue. Edema was inhibited by treatment with all doses of PCCO. The data suggest that PCCO exerts antinociceptive and anti-inflammatory effects on arthritis in rats.