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The aim of this study is to investigate effects of cisplatin preadministration on oral ulcerative mucositis-induced nociception by using an experimental model of rats.After two rounds of cisplatin administration, oral ulcers developed with topical acetic acid treatment in rats. Spontaneous mouth rubbing behavior was observed as spontaneous nociceptive behavior in a plastic cage. Head-withdrawal behavior was observed as mechanical allodynia by using von Frey test in the oral mucosa of conscious rats. Bacterial invasion and inflammatory cell infiltration into oral ulcerative region and systemic leukocyte phagocytic activity were assessed.Following cisplatin preadministration, oral ulcerative mucositis-induced spontaneous nociceptive behavior was not observed in the model. The preadministration enhanced leukocyte phagocytic activity, leading to reduce bacterial invasion and inflammatory cell infiltration in the oral ulcerative region. In contrast, oral ulcerative mucositis-induced mechanical allodynia was induced. The exaggerated mechanical allodynia in the oral ulcerative region was largely inhibited by topical treatment with the antioxidative drug, ɑ-lipoic acid, or the blocker of N-formyl peptide receptor 1, N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine.These results suggest that cisplatin preadministration suppresses spontaneous nociception in oral ulcerative region, due to antiinflammatory effects by enhancement of leukocyte phagocytic activity, but exaggerates mechanical allodynia due to oxidative stress with N-formyl peptide receptor 1 activation. The suppression of spontaneous nociception is one of the advantages of cisplatin treatment for head and neck cancer patients although the exaggerated allodynia is a serious symptom.

Objective Cisplatin, a platinum-based anticancer drug, produces reactive oxygen species (ROS) in many cell types and induces mechanical allodynia in the hands and/or feet (chemotherapy-induced painful neuropathy: CIPN). In this study, we examined the possibility of inducing neuropathy in the oral region using oral keratinocytes and rats. Methods Human oral keratinocytes (HOKs) were used to evaluate ROS generation after cisplatin application by a ROS-reactive fluorescent assay. In rats, after cisplatin administrations (two times), the trigeminal ganglion (TG) was investigated by electron microscopy and quantitative RT-PCR. Using our proprietary assay system, oral pain-related behaviors were observed in cisplatin-treated rats. Results In rats, cisplatin administration reduced food intake and body weight. In electron microscopic analysis, glycogen granules in the TG were depleted following administration, although organelles were intact. In HOK cells, cisplatin significantly increased ROS generation with cell death, similar to glycolysis inhibitors. Cisplatin administration did not show any effects on Trpa1 mRNA levels in the TG. However, the same procedure induced hypersensitivity to mechanical stimulation and the TRPA1 agonist allyl isothiocyanate in the oral mucosa. Mechanical hypersensitivity was inhibited by the antioxidative drug α-lipoic acid and the TRPA1 antagonist HC-030031, similar to that of the hind paw. Conclusion The present findings suggest that cisplatin induces TRPA1-mediated CIPN due to ROS generation in the oral region. This study will provide a better understanding of persistent oral pain in cancer patients.

Objective Recent studies have demonstrated that mouthwash made with the traditional Japanese medicine hangeshashinto exhibits anti-inflammatory action and alleviates oral mucositis scores, including pain complaints, in patients undergoing chemoradiotherapy. However, no study has demonstrated the mechanism underlying how hangeshashinto provides pain relief in oral ulcers. Design The analgesic effects on pain-related behaviors following the topical application of hangeshashinto were evaluated in an oral ulcer rat model treated with acetic acid using recently developed methods. Indomethacin, the representative anti-inflammatory agent, was intraperitoneally administered. The tissue permeability of the oral mucosa was histologically evaluated after applying the fluorescent substance FluoroGold. Results The topical application of hangeshashinto in ulcerative oral mucosa suppressed mechanical pain hypersensitivity over 60 min, without any effects on healthy mucosa. The same drug application also inhibited oral ulcer-induced spontaneous pain. Indomethacin administration failed to block the mechanical pain hypersensitivity, though it did largely block spontaneous pain. Topical anesthesia with lidocaine showed hyposensitivity to mechanical stimulation in healthy mucosa. In the ulcer regions in which the oral epithelial barrier was destroyed, deep parenchyma was stained with FluoroGold, in contrast to healthy oral mucosa, in which staining was limiting to the superficial site. Conclusions Hangeshashinto leads to long-lasting analgesic effects, specifically in the ulcer region by destroying the epithelial barrier. Hangeshashinto alleviates oral ulcer-induced pain in inflammation-dependent and/or independent manner.

In many patients with cancer, chemotherapy-induced severe oral ulcerative mucositis causes intractable pain, leading to delays and interruptions in therapy. However, the pain mechanism in oral ulcerative mucositis after chemotherapy has not been extensively studied. In this study, we investigated spontaneous pain and mechanical allodynia in a preclinical model of oral ulcerative mucositis after systemic administration of the chemotherapy drug 5-fluorouracil, using our proprietary pain assay system for conscious rats. 5-Fluorouracil caused leukopenia but did not induce pain-related behaviors. After 5-fluorouracil administration, oral ulcers were developed with topical acetic acid treatment. Compared with saline-treated rats, 5-fluorouracil-exposed rats showed more severe mucositis with excessive bacterial loading due to a lack of leukocyte infiltration, as well as enhancements of spontaneous pain and mechanical allodynia. Antibacterial drugs, the lipid A inhibitor polymyxin B and the TRPV1/TRPA1 channel pore-passing anesthetic QX-314, suppressed both the spontaneous pain and the mechanical allodynia. The cyclooxygenase inhibitor indomethacin and the TRPV1 antagonist SB-366791 inhibited the spontaneous pain, but not the mechanical allodynia. In contrast, the TRPA1 antagonist HC-030031 and the N-formylmethionine receptor FPR1 antagonist Boc MLF primarily suppressed the mechanical allodynia. These results suggest that 5-fluorouracil-associated leukopenia allows excessive oral bacterial infection in the oral ulcerative region, resulting in the enhancement of spontaneous pain through continuous TRPV1 activation and cyclooxygenase pathway, and mechanical allodynia through mechanical sensitization of TRPA1 caused by neuronal effects of bacterial toxins. These distinct pain mechanisms explain the difficulties encountered with general treatments for oral ulcerative mucositis-induced pain in patients with cancer and suggest more effective approaches.

Background Stomatitis induces severe and painful hypersensitivity to pungency and physical contact during meals. Many studies have used anesthetized animals to examine evoked nociception in the oral mucosa, but no reports have used traditional behavioral assays to evaluate nociception in conscious animals. New methods We developed two new methods of applying chemical or mechanical stimulation directly to the oral mucosa of the mandibular vestibule of conscious rats. Nociceptive evaluations were performed by measuring facial grooming time and the head withdrawal threshold to von Frey stimulations. (1) For the intraoral dropping method, rat mucosa was transiently exposed by hand, and a drop of a pungent solution was applied. (2) For the stable intraoral opening method, rat mucosa was long-term exposed following piercing surgery of the mental skin after habitual training for 2–3 weeks. Results In the intraoral dropping method, the application of 100 μM capsaicin or 100 mM allyl isothiocyanate prolonged mouth-rubbing time. Capsaicin-induced mouth-rubbing time was further enhanced following the development of an acetic acid-induced ulcer. The stable intraoral opening method enabled stable measurements of the mechanical withdrawal threshold in the oral mucosa of conscious rats. Ulcer development decreased the mechanical threshold, whereas topical lidocaine treatment increased the threshold. Comparison with existing methods These new methods enable the evaluations of motivational nocifensive behaviors in response to intraoral stimulations without any anesthetic effects. Conclusions The intraoral dropping and stable intraoral opening methods can be used in combination with traditional behavioral assays to evaluate nociception in the oral mucosa of conscious rats.

The traditional Japanese herbal medicine hangeshashinto (HST) has beneficial effects for the treatment of oral ulcerative mucositis (OUM) in cancer patients. However, the ingredient-based mechanism that underlies its pain-relieving activity remains unknown. In the present study, to clarify the analgesic mechanism of HST on OUM-induced pain, we investigated putative HST ingredients showing antagonistic effects on Na+ channels in vitro and in vivo. A screen of 21 major ingredients using automated patch-clamp recordings in channel-expressing cells showed that [6]-gingerol and [6]-shogaol, two components of a Processed Ginger extract, considerably inhibited voltage-activated Na+ currents. These two ingredients inhibited the stimulant-induced release of substance P and action potential generation in cultured rat sensory neurons. A submucosal injection of a mixture of [6]-gingerol and [6]-shogaol increased the mechanical withdrawal threshold in healthy rats. In a rat OUM model, OUM-induced mechanical pain was alleviated 30min after the swab application of HST despite the absence of anti-bacterial and anti-inflammatory actions in the OUM area. A swab application of a mixture of [6]-gingerol and [6]-shogaol induced sufficient analgesia of OUM-induced mechanical or spontaneous pain when co-applied with a Ginseng extract containing abundant saponin. The Ginseng extract demonstrated an acceleration of substance permeability into the oral ulcer tissue without an analgesic effect. These findings suggest that Na+ channel blockage by gingerol/shogaol plays an essential role in HST-associated analgesia of OUM-induced pain. This pharmacological mechanism provides scientific evidence supporting the use of this herbal medicine in patients suffering from OUM-induced pain.

Purpose We examined the effects of sweet taste stimulation on pain tolerance threshold (PTT) of oral mucosa in children. Subjects and methods Subjects comprised 10 children (mean age, 7.3±1.2 years) for whom PTT of oral mucosa was measured 2min after oral administrations of sucrose or xylitol and water. Sine wave current stimulation (2kHz, 250Hz and 5Hz, SWCS) which can stimulate sensory nerve fiber selectively (A β , A δ and C) was used to measure PTT. Results Sweet taste stimulation with sucrose or xylitol increased oral mucosa PTT in children, but not in adults. No difference in the increased PTT was seen between sucrose and xylitol. Conclusions Oral administration of sucrose and xylitol may potentially relieve pain associated with local anesthetic injections in children.

Purpose: The effect of mechanical stretch was examined on cultured retinal pigment epithelial (RPE) cells in order to observe changes in their production of tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF) in response to physiological strain. Methods: Bovine RPE cells in near-confluent culture were exposed to mechanical stretch of the bottom of a 6-cm petri dish at the maximum magnitude of 4500 microstrain and at a cycle of 30 seconds for 72 hours. TIMP-1 and VEGF levels in the medium following 24, 48, and 72 hours of cyclic stretch were measured by enzyme immunoassay. Results: The growth of RPE cells during the 72-hour period of stretching did not show a significant difference from that of nonstretched control cells. RPE cells in the stretched group produced a significantly larger amount of TIMP-1 at 48 and 72 hours after stretch, compared with nonstretched control ( P = .044 and P = .027, respectively, Student t -test). The levels of VEGF produced by RPE cells were not significantly different between the stretched group and nonstretched control group. Conclusions: The secretion of TIMP-1 by bovine RPE cells was enhanced by cyclic mechanical stretch. Mechanical strain is one factor in regulating the secretion of TIMP-1 by RPE cells.

OBJECTIVE—The first complement component C1s was reported to have novel functions to degrade matrix components, besides its activities in the classic complement pathway. This study explores participation of C1s in articular cartilage degradation in rheumatoid arthritis (RA). METHODS—Normal articular cartilage (n=6) and cartilage obtained from joints with RA (n=15) and osteoarthritis (OA, n=10) were immunostained using anti-C1s monoclonal antibodies PG11, which recognises both active and inactive C1s, and M241, which is specifically reactive to activated C1s. The effects of inflammatory cytokines on C1s production by human articular chondrocytes were also examined by sandwich ELISA. RESULTS—In normal articular cartilage, C1s was negative in staining with both PG11 and M241. In contrast, degenerating cartilage of RA was stained with PG11 (14 of 15 cases), and in most of the cases (13 of 15 cases) C1s was activated as revealed by M241 staining. In OA, C1s staining was restricted in severely degrading part of cartilage (5 of 10 cases), and even in that part C1s was not activated. In addition, C1s production by chondrocytes in vitro was increased by an inflammatory cytokine, tumour necrosis factor α. CONCLUSION—These results suggest that C1s activated in degenerative cartilage matrix of RA but not in that of OA. C1s is thought to participate in the pathogenesis of RA through its collagenolytic activity in addition to the role in the classic cascade. Keywords: complement C1s; articular cartilage; rheumatoid arthritis

During dental treatments, intraoral appliances frequently induce traumatic ulcers in the oral mucosa. Such mucosal injury-induced mucositis leads to severe pain, resulting in poor quality of life and decreased cooperation in the therapy. To elucidate mucosal pain mechanisms, we developed a new rat model of intraoral wire-induced mucositis and investigated pain mechanisms using our proprietary assay system for conscious rats. A thick metal wire was installed in the rats between the inferior incisors for one day. In the mucosa of the mandibular labial fornix region, which was touched with a free end of the wire, traumatic ulcer and submucosal abscess were induced on day 1. The ulcer was quickly cured until next day and abscess formation was gradually disappeared until five days. Spontaneous nociceptive behavior was induced on day 1 only, and mechanical allodynia persisted over day 3. Antibiotic pretreatment did not affect pain induction. Spontaneous nociceptive behavior was sensitive to indomethacin (cyclooxygenase inhibitor), ONO-8711 (prostanoid receptor EP1 antagonist), SB-366791, and HC-030031 (TRPV1 and TRPA1 antagonists, respectively). Prostaglandin E2 and 15-deoxyΔ12,14-prostaglandin J2 were upregulated only on day 1. In contrast, mechanical allodynia was sensitive to FSLLRY-NH2 (protease-activated receptor PAR2 antagonist) and RN-1734 (TRPV4 antagonist). Neutrophil elastase, which is known as a biased agonist for PAR2, was upregulated on days 1 to 2. These results suggest that prostanoids and PAR2 activation elicit TRPV1- and TRPA1-mediated spontaneous pain and TRPV4-mediated mechanical allodynia, respectively, independently of bacterial infection, following oral mucosal trauma. The pathophysiological pain mechanism suggests effective analgesic approaches for dental patients suffering from mucosal trauma-induced pain.

Hamster complement C1s cDNA was inserted into expression plasmid BCMGSNeo, and transfected to SEA7 cells, A31 mouse fibroblasts transformed by polyoma virus. The transfectant secreted a large amount of recombinant C1s that was activated in the serum free culture medium and hydrolyzed acetyl-Gly- l -Lys-naphthyl ester (AGLNE). C1s was purified to a homogeneity from the culture medium of the transfectant by DEAE-Sephadex, Dymatrex orange A and size-exclusion HPLC. Purified hamster C1s consumed human complement in hemolytic assay and hydrolyzed gelatin in enzymography. To investigate the enzymic action of C1s at molecular levels, several antibodies were prepared against hamster C1s. One peptide (amino-acid residues 379–391) and two peptides (amino-acid residues 478–496 and 560–583) corresponding to the heavy and the light chain, respectively, were synthesized. The amino-acid sequences of these regions is not conserved between hamster and human C1s. Antibodies against these peptides were raised in rabbits. The anti-peptide antibodies bound specifically to hamster serum and recombinant C1s but not to human C1s. They inhibited the esterase activity of recombinant C1s to varying degrees depending on each antibody's binding site.

Antibody independent activation of complement Cls was examined by immunoblot analysis using an antibody against a synthetic peptide of hamster Cls L chain. Approx. 50% of Cls secreted from hamster embryo malignant fibroblasts Nil2C2 was functionally active in its two-chain form in the serum free culture medium. In contrast, no active Cls was round in a culture medium of hamster embryo fibroblasts (HEF). Active Cls was detectable, however, in the culture medium after HEF became a cell line. The immune complex independent activation of Cls was also observed in rat cell lines but not in secondary rat embryo fibroblasts. Cls in a membrane fraction of Nil2C2 was a proenzyme form and was not activated by incubation of the membrane itself suggesting that Cls was activated after secretion. The activation of Cls was not inhibited by human C1 inhibitor (C1-INH), benzamidine or soy bean trypsin inhibitor (SBTI) but was inhibited by leupeptin, nitrophenyl guanidinobenzoate and DFP. Our results suggest that Cls is activated either by a serine proteinase(s) other than those reported to cleave Cls or by an activator which directly stimulates autoactivation of Cls.

Background: Ulcerative oral mucositis is frequently observed in cancer patients undergoing chemoradiotherapy; it induces intractable, severe pain during meals and leads to a deterioration of the patient's quality of life. Recently, hangeshashinto (HST), a traditional Japanese medicine consisting of seven crude herbal drugs, has been reported to alleviate gastric cancer chemotherapy-induced oral mucositis in a randomized, double-blind, placebo-controlled clinical trial. In this study, we investigated the efficacy of HST on oral ulcer (OU)-induced mucosal pain in rats using a new behavioral technique, the stable intraoral opening (SIO) method. Methods: A piece of filter paper was soaked in a 50%-acetic acid solution and placed in the labial fornix region of the inferior incisors of anesthetized rats for 30 seconds. OU-induced mucosal pain was evaluated using the SIO method that enables measurement of intraoral mechanical pain threshold in conscious rats by stimulation to the exposed oral mucosa with von Frey filaments. The effects of HST, the extract of crude drugs or ingredients contained in HST after application on the OU region of the OU model, were evaluated using the SIO method. Tissue penetration in the OU was examined by application of a retrograde tracer fluorogold. For possible target evaluation for analgesic effects, the typical 21 ingredients contained in HST were screened by measurement of channel currents or intracellular calcium concentrations in human Nav1.8, TRPV1, or TRPA1-expressing CHO or HEK293 cells. Results: Treatment with acetic acid caused obvious ulcers in the labial fornix region, characterized by severe infiltration of inflammatory cells and epidermolysis. Mechanical threshold in the ulcerated oral mucosa was significantly reduced compared to that of the vehicle-treated mucosa. HST recovered the mechanical threshold reduction to naive levels, beginning at 30 m following topical application until 2 h. In the ion channel screening, (6)shogaol and (6)-gingerol in Ginger and isoliquiritigenin in Licorice demonstrated a strong antagonistic effect on Nav1.8 activation. The analgesic effects of isoliquiritigenin and (6)shogaol/(6)-gingerol were slower and/or weaker than that of HST, while they were enhanced by the co-application of a Ginseng extract, containing abundant surfactant saponins, which showed acceleration of tissue penetration in the ulcer region. Conclusions: Mechanical allodynia develops in the OU of acetic acid-treated rats. Direct application of HST in the early stages ameliorates OU-induced mechanical allodynia, and several active ingredients, such as isoliquiritigenin, (6)-shogaol and (6)-gingerol with saponins, may synergistically contribute to the analgesic effects of HST. These results suggest that HST is a useful drug for ameliorating OU-induced pain in patients undergoing chemoradiotherapy.

The activated first component of human complement, C1̄s, was shown to cleave type I and II collagen and gelatin. The proteolytic activity was heat labile and was inhibited by a monoclonal antibody (M241) which recognized light chain of active human C1s or by a serine protease inhibitor, DFP, but not by a chelating agent.

Expression of the first complement component (C1s) has been examined in chondrocytes of hamster epiphyseal cartilage during development and fracture healing. C1s is immunostained with anti-hamster C1s monoclonal antibody, PG11. The C1s staining increases in accordance with chondrocyte differentiation and reaches a maximal level in hypertrophic chondrocytes. This change is observed at both the tibia ossification center and at the callus in which the replacement of cartilage by bone marrow takes place. The concomitant increase of C1s and chondrocyte hypertrophy has been confirmed by RNA blot and by in situ hybridization. These results, in addition to previous findings on C1s collagenolytic and gelatinolytic activities, suggest C1s participation in cartilage remodeling.