Virgínia Cláudia Carneiro Girão

Tadalafil analgesia in experimental arthritis involves suppression of intra-articular TNF release

BACKGROUND AND PURPOSE We investigated the effect of the phosphodiesterase‐5 inhibitor, tadalafil, on the acute hypernociception in rat models of arthritis.

Effects of nitric oxide on neutrophil influx depends on the tissue: role of leukotriene B4 and adhesion molecules

Background and purpose:  We investigated the effect of nitric oxide synthase (NOS) inhibition on polymorphonuclear cell (PMN) influx in zymosan or lipopolysaccharide (LPS)‐induced arthritis and peritonitis.

ANTI‐INFLAMMATORY AND ANTI‐NOCICEPTIVE ACTIVITY OF RISEDRONATE IN EXPERIMENTAL PAIN MODELS IN RATS AND MICE

1 The antinociceptive effect of risedronate in experimental pain models in rats and mice was investigated in the present study. 2 Rats received zymosan intra‐articularly (i.art.) into the right knee joint and the nociceptive response was assessed using the articular incapacitation test. Joint washouts were used for determining cell influx, tumour necrosis factor (TNF)‐α and leukotriene (LT) B4 levels. 3 Mice received either zymosan (1 mg) or acetic acid (0.6%) i.p. and the nociceptive response was measured as the number of writhings between 0 and 30 min after the stimuli. Control animals received i.p. injections of saline. 4 Groups were pretreated with risedronate (5–500 µg/kg, s.c.) and compared with vehicle (saline)‐treated (NT) animals. One group of rats was cotreated with the µ‐opioid receptor antagonist naloxone (2 mg/kg, s.c.) prior to risedronate, followed by 1 mg zymosan i.art. 5 Risedronate, at 100 and 500 µg/kg, significantly and dose‐dependently inhibited the nociceptive response in the writhings test (P < 0.05), inhibiting responses to acetic acid by 65.4 and 49.2%, respectively, and to zymosan by 72.9 and 71.9%, respectively. 6 Pretreatment with risedronate also significantly (P < 0.05) and dose‐dependently inhibited the articular incapacitation in zymosan‐arthritis. 7 Risedronate, at 50 µg/kg, significantly inhibited TNF‐α release as compared with the NT group (39.4 ± 9.8 vs 145.6 ± 43.3 pg/mL TNF‐α, respectively). 8 Risedronate, at 50 and 100 µg/kg, significantly inhibited LTB4 release into the joints compared with the NT group (2883.1 ± 73.2, 1911.5 ± 205.3 and 4709.9 ± 237.2 pg/mL, respectively). These effects of risedronate were associated with a significant reduction in the inflammatory cell infiltration. 9 Cotreatment with risedronate and naloxone did not reverse the antinociceptive effects of risedronate in zymosan‐arthritis. 10 This is the first demonstration that risedronate displays intrinsic antihypernociceptive activity. This effect is associated with reduced cell infiltration and inhibition of TNF‐α and LTB4 release and is not linked to an endogenous opioid‐release mechanism.

Topical continuous use of Lippia sidoides Cham. essential oil induces cutaneous inflammatory response, but does not delay wound healing process

ETHNOPHARMACOLOGICAL RELEVANCE The essential oil of Lippia sidoides (EOLS) has been used in Brazilian folk medicine as a topical antiseptic agent in skin for treatment of wounds and superficial infections of the body. The aim of this study was to investigate the effects of EOLS on intact and damaged skin, including its action on expression of mediators, COX-2 and VEGF, involved in healing full-thickness cutaneous lesions in vivo. MATERIAL AND METHODS EOLS was analyzed chemically and used at different concentrations to dose-response experiments in skin mice. Skin irritation tests by one-dosage and multiple-dosages and irritation to damaged skin were assessed by macroscopy, morphometry and histological and immunohistochemical analyses. To evaluate the effects of EOLS on wound healing, excision wounds were surgically created on the dorsum of rats, and the ointments at 6% and 12% were applied daily to the wound area. Cutaneous lesions were assessed by planimetric (wound contraction) and macroscopic parameters. RESULTS Skin irritation tests showed that topical application of EOLS promoted cutaneous inflammation in varying degrees, which was demonstrated by increase of skin thickness and formation of cutaneous edema and erythema. Topical administration of EOLS in high concentrations presented an irritant response to skin, but this irritation is lighter when low concentrations this oil were used. Histological evaluation supported the outcome of these models, which revealed accentuated presence of inflammatory cells infiltration. In wound healing process, the lesions treated with EOLS showed intense edema and exsudation up to day 5, but there were not significant differences in the wound contraction on days 14 and 21. No immunohistochemical staining was verified to COX-2 and VEGF mediators in skin treated with EOLS 12%. CONCLUSION The continuous application of EOLS in adequate concentrations on cutaneous wounds increases inflammatory response without delay the lesions closure. The association of these results with antimicrobial action previously related to EOLS allows its indication as an alternative therapeutic modality for topical treatment of infected cutaneous wound. Nevertheless, further studies need to be performed to determine the mechanism of action and support its application in clinical practice.

Meniscal transection rather than excision increases pain behavior and structural damage in experimental osteoarthritis in mice

OBJECTIVE To evaluate pain behavior and structural damage in mice subjected to either meniscal transection or removal. METHODS Mice (10/group) were subjected to transection of the medial collateral and anterior cruciate ligaments (ACLT/MCLT) followed by either transection (meniscotomy) or removal (meniscectomy) of the medial meniscus. A control group was subjected only to transection of the ligaments. Pain was assessed using the electronic pressure-meter paw test. Cell influx, measured in joint exudates, and joint histopathology were assessed after 49 days. Four other groups subjected to meniscotomy received indomethacin, the inducible nitric oxide synthase (iNOS) inhibitor 1400W, morphine or the vehicles. RESULTS Both meniscotomy and meniscectomy groups displayed persistent and significant increase in pain behavior as compared to controls, being significantly more severe in the former. Cell influx was more intense in the meniscotomy as compared to the meniscectomy group. Structural damage at the tibia, but not at the femur, was also more severe in the meniscotomy group. Indomethacin and 1400W partially but significantly reduced pain whereas morphine abrogated pain behavior in meniscotomized mice. CONCLUSION Meniscal transection rather than resection promotes more severe pain and structural damage in mice. Administration of opioids, cyclooxygenase and nitric oxide (NO) synthase inhibitors provide analgesia in this model. Careful description of the structures damaged is crucial when reporting experimental osteoarthritis (OA).

Tumor necrosis factor prevents Candida albicans biofilm formation

Candida species are commensals but some develop biofilms in prosthetic materials and host surfaces that may represent up to 30% of deaths related to infections, particularly in immunosuppressed patients. Tumor necrosis factor (TNF) exhibits a plethora of functions in host defense mechanisms whereas excessive release of TNF in inflammation promotes tissue damage. Cytokines released in an inflammatory milieu may influence the development of microorganisms either by promoting their growth or displaying antimicrobial activity. In protozoa, TNF may affect growth by coupling through a lectin-like domain, distinct from TNF receptors. TNF was also shown to interact with bacteria via a mechanism that does not involve classical TNF receptors. Using an in vitro C. albicans biofilm model, we show that TNF dose-dependently prevents biofilm development that is blocked by incubating TNF with N,N’-diacetylchitobiose, a major carbohydrate component of C. albicans cell wall. This finding represents a relevant and hitherto unknown mechanism that adds to the understanding of why TNF blockade is associated with opportunistic C. albicans infections.

Effect of ethyl acetate extract from husk fiber water of Cocos nucifera in Leishmania braziliensis infected hamsters

The objective of this study was to evaluate the treatment with ethyl acetate extract (EAE) from husk fiber water of Cocos nucifera L., Arecaceae, in L. braziliensis (Lb) infected hamsters. Twelve male hamsters were randomly allocated in three groups (n=4): G1 received only EAE; G2 was infected with Lb only and G3 received EAE after Lb infection. The infection was carried 28 days prior to the treatment with EAE, which was administrated (0.2 mL, 300 mg.kg-1) for 21 consecutive days. Infection was evaluated through skin lesions and infected footpad edema. Haematological evaluation was done on -28th, 0 and 21st days. Imprint footpad and lymph node weight were evaluated on 21st day. Lb infection significantly inhibited the peripheral leukocytes blood. However, neutrophils and lymphocytes values did not have significant alterations. G3 presented eosinophilia in relation to G2. The treatment with EAE did not reduce edema of infected footpad neither weight of drainage lymph node. Infected footpad imprints revealed amastigotes forms and cellular infiltration. Animals from G3 presented skin lesions on 7th day, shown a reduction of these lesions in day 14. Therefore, the treatment with EAE did not alter the etiological agent elimination in these conditions. However, EAE presents a healing activity in this experimental model.

AB0068 Structural Characteristics of Polysaccharides Influence Viscosupplementation Efficacy in Osteoarthritis

Background Intra-articular (i.a) injection of a purified Guar gum (GG) solution or hydrogel provided analgesia similar to that of Hylan G-F20 in experimental osteoarthritis (eOA) suggesting that rheology is not crucial to viscosupplements. Objectives Evaluate the effect of purified GG (DGG), sulfated (DGGSU) of oxidized (DGGOX) GG on pain and cartilage damage in eOA Methods GG was oxidated with a n-oxil-2, 2, 6, 6-tetra metilpiperidina (TEMPO) reagent or subjected to sulfation to produce carboxylated and sulphate groups. Rats subjected to anterior cruciate ligament transection (ACLT) of the knee received 100 μg GG, sulfated or oxidized GG or vehicle (NT) i.a. at day 4 to 7 after ACLT. Joint pain was recorded using the articular incapacitation test. A group received GG weekly or vehicle from day 7 to 70 and joint damage assessed by histology and biochemically, measuring hondroitin-sulfate (CS) cartilage content (μg/mg) and Mw of CS through PAGE. Results FT-InfraRed spectra of DDGSU and DDGOX were different from protein free DGG. DGGSU has a new intense band at 1262 cm–1, due to S=O. Spectral difference occurs in DGGOX but not DGG and a C=O stretching is present in DGGOX spectrum (COOH group). 13C NMR spectrum of DGGOX and DGG revealed absence of the signal at 63.7 ppm present in DGG and two new signals at 177.5 and 177.8 ppm. Absence of the signal from C-6 of free mannose indicates it was substituted in the oxidation. The new signals are from C=O group from COOH. 1H-13C HSQC spectra exhibited a shift to a lower d value of the proton H-1 from mannose residue in DGGOX, indicating substitution at C-6 from mannose. New signals at 70.3, 75.6, and 100.5 ppm were observed in 13C NMR spectrum of sulfated gum due to the insertion of sulfate group in the primary carbon (C-6). All derivatives show lower molar mass than the original guar gum. DGG Mw =3.9 x 106 g/mol, DGGOX and DGGSU, 2.6 x 106 and 2.8 x 106 g/mol, respectively. DGGOX has the lowest intrinsic (2.2 dL/g) and apparent viscosity (9.0 mPa s).DGG exhibits the highest values ([h] =6.2 dL/g and happ =58 mPa s). Intermediate values are obtained for DGGSU ([h] =4.9 dL/g and happ =39 mPa s). GG significantly reduced joint pain, as compared to vehicle whereas oxidat ion or sulfation of GG abrogated GG analgesia. GG also prevented the increase in both CS content (43.8±4.7 μg/mg) and Mw (mobility: 1.18 ±0.03), as compared to NT (p<0.001). GG also significantly prevented joint damage at histopathology (median =7; range 2.5 - 18), as compared to NT (p<0.05). Conclusions The structural integrity of GG is crucial to its analgesia. Intra-articular polysaccharides can be analgesic and chondroprotective regardless of being water-soluble. Acknowledgement Supported by grants 302218/2014–9 and 459334/2014–0 from CNPq-Brasil). Disclosure of Interest None declared

Preconditioning with mono and polyunsaturated fatty acids and low-intensity electrical stimulation. Effects on skin repair in rats

PURPOSE To evaluate the effects of preconditioning with oils mixes containing ω3/ω6/ω9 associated with micro-currents on skin repair in rats. METHODS One-hundred and eight Wistar rats randomized into G-1, G-2 and G-3 groups were treated with saline (0.9%), mix 1 (corn+soybean oils) and mix 2 (olive+canola+flaxseed oils), respectively, in a single dose (0.01ml/g) by gavage. Next, each group was subdivided into sham and stimulated subgroups. Pulsed-wave microcurrents (0.5 µA, 0.5 Hz) were applied to stimulated subgroups for 20 min. One hour later anesthetized rats were subjected to surgery. A dorsal incision (6 cm long) was carried out and closed with interrupted nylon sutures. Samples (1 cm2) were harvested from the mid-portion of the incision on the 7, 14, 21 post-operative (P.O.) days. Variables were analyzed using Mann-Whitney/Dunn tests Significance level was set to 5 % (p<0.05). RESULTS Micro-currents promoted increase of exudate and reduction of epithelialization on day 7 in G1 rats. Mixes 1/2 reduced vascularization on 7/14th days P.O. Both 1/2 mixes reduced fibrosis on day 14. Preconditioning with mix 1 led to increased expression of NF-kB on the 7th day. CONCLUSION Preconditioning with microcurrents has pro-inflammatory effects while oil mixes 1 and 2 decrease fibrosis and vascularization in the proliferative phase of cicatrization.

Anti-inflammatory and Immunomodulatory Effect of an Extract of Coccidioides posadasii in Experimental Arthritis

Trying to surpass host defenses, fungal infections alter the immune response. Components from nonpathogenic fungi present therapeutic anti-inflammatory and immunomodulating activities. This study reveals that proteins present in a Coccidioides posadasii extract provide anti-inflammatory benefit in experimental arthritis. Zymosan was given intra-articularly to rats and mice, and groups were pretreated with C. posadasii extract either per os or intraperitoneally. Controls received the vehicle. Acute hypernociception was evaluated using articular incapacitation and von Frey methods. Cell influx and cytokine levels were assessed in joint exudates. Joint damage was evaluated by histopathology and determination of glycosaminoglycan content of the cartilage. Synovia was evaluated for cell death and inducible nitric oxide synthase (iNOS) expression using TUNEL and immunohistochemistry, respectively. Pretreatment with C. posadasii extract significantly inhibited acute and chronic cell influx, hypernociception, and provoked reduction of glycosaminoglycan loss while reducing chronic synovitis, cell death, and iNOS expression. Reduction/alkylation of C. posadasii extract abrogated these effects. C. posadasii administration did not alter TNF-α, IL-1β, IL-17, and γ-interferon levels, whereas IL-10 levels were significantly reduced. Data reveal that a C. posadasii extract reduces iNOS expression that is associated with inhibition of synovial apoptosis and decrease in IL-10 levels released into zymosan-inflamed joints. Characterization of active components excluded charged carbohydrates while pointing to a protein as responsible for these effects. In summary, systemic administration of components from a pathogenic fungus provides anti-inflammatory effects, being species-independent and orally active. Besides adding to understand host response against fungi, the results may lead to therapeutic implications.