Francisco Airton Castro da Rocha

Low incidence of hip fractures in an equatorial area

Hip fractures in patients older than 60 years of age represent a serious morbidity linked to osteoporosis that contributes to its mortality rates. Both genetic and environmental influences have been reported as important factors related to the epidemiology of osteoporosis. Sobral, a city located in the northeast (equatorial zone) of Brazil (3ºS/40ºE) has 138,565 inhabitants, who are mostly white Portuguese and native Brazilian descendants. We evaluated the occurrence of hip fractures in Sobral between July 1996 and June 2000. This was a retrospective cohort-based study. Data were obtained from the medical records of the Santa Casa de Sobral, which is the reference hospital in this area. All chart records of patients aged >20 years with a diagnosis of femoral or hip fracture were revised. A total of 79 fractures were identified. Ten cases (12.6%) that occurred in patients <50 years of age were excluded from further analysis. Sixty-nine cases (87.3%) occurred in patients aged >50 years, comprising 19 (27.5%) men and 50 (72.4%) women. This gives an age-adjusted annual incidence rate of 5.59/10,000 per year in men and 12.4/10,000 per year in women, respectively, for people >50 years of age. Two patients, aged 79 and 82, died 9 and 6 days, respectively, after hospital admission. The average time of hospitalization was 7.5 days (range 4–19 days). As expected, there was a significant rise in hip fractures in people >50 years old. Interestingly, the hip fracture rate in this population is considerably lower (around 4 times less) than the reported rates in white Caucasians.

Involvement of LTB4 in zymosan-induced joint nociception in mice: participation of neutrophils and PGE2

Leukotriene B4 (LTB4) mediates different inflammatory events such as neutrophil migration and pain. The present study addressed the mechanisms of LTB4‐mediated joint inflammation‐induced hypernociception. It was observed that zymosan‐induced articular hypernociception and neutrophil migration were reduced dose‐dependently by the pretreatment with MK886 (1–9 mg/kg; LT synthesis inhibitor) as well as in 5‐lypoxygenase‐deficient mice (5LO−/−) or by the selective antagonist of the LTB4 receptor (CP105696; 3 mg/kg). Histological analysis showed reduced zymosan‐induced articular inflammatory damage in 5LO−/− mice. The hypernociceptive role of LTB4 was confirmed further by the demonstration that joint injection of LTB4 induces a dose (8.3, 25, and 75 ng)‐dependent articular hypernociception. Furthermore, zymosan induced an increase in joint LTB4 production. Investigating the mechanism underlying LTB4 mediation of zymosan‐induced hypernociception, LTB4‐induced hypernociception was reduced by indomethacin (5 mg/kg), MK886 (3 mg/kg), celecoxib (10 mg/kg), antineutrophil antibody (100 μg, two doses), and fucoidan (20 mg/kg) treatments as well as in 5LO−/− mice. The production of LTB4 induced by zymosan in the joint was reduced by the pretreatment with fucoidan or antineutrophil antibody as well as the production of PGE2 induced by LTB4. Therefore, besides reinforcing the role of endogenous LTB4 as an important mediator of inflamed joint hypernociception, these results also suggested that the mechanism of LTB4‐induced articular hypernociception depends on prostanoid and neutrophil recruitment. Furthermore, the results also demonstrated clearly that LTB4‐induced hypernociception depends on the additional release of endogenous LTs. Concluding, targeting LTB4 synthesis/action might constitute useful therapeutic approaches to inhibit articular inflammatory hypernociception.

Antihyperalgesic effect of pentoxifylline on experimental inflammatory pain

The antihyperalgesic effect of pentoxifylline was investigated in three experimental pain models. Pentoxifylline (0.5–1.6 mg kg−1) given 30 min before the stimulus significantly inhibited the writhing response induced by the intraperitoneal (i.p.) administration of either acetic acid (−90%) or zymosan (−83%), but not that of iloprost, in mice, as well as the zymosan‐induced articular hyperalgesia in the zymosan arthritis in rats (−50%). Pentoxifylline also inhibited the mechanical hypernociception in rats induced by the intraplantar injection of either carrageenin (−81%), bradykinin (−56%) or tumor necrosis factor α (TNF‐α; −46%), but not that induced by interleukin‐1β (IL‐1β) or prostaglandin E2 (PGE2). Pentoxifylline did not inhibit the nociceptive response in the hot plate test in mice. Further, the antinociceptive effect of pentoxifylline in the writhing test in mice and the zymosan‐induced articular hyperalgesia were not reversed by the coadministration of the opioid receptor antagonist naloxone. Thus, pentoxifylline antinociceptive effect is probably not mediated at a central level. Pentoxifylline significantly reduced TNF‐α (−43%) and IL‐1β (−42%) concentrations in the joint exudates of rats stimulated by intra‐articular injection of zymosan and the production of both cytokines (−66 and −86%, respectively) by mouse peritoneal macrophages stimulated in vivo with zymosan as well as the expression of TNF‐α at the tissue level in carrageenin‐injected rat paws. In conclusion, the antinociceptive activity of pentoxifylline is associated with the inhibition of the release of both TNF‐α and IL‐1β.

Dual effect of nitric oxide in articular inflammatory pain in zymosan-induced arthritis in rats

The contribution of nitric oxide (NO) to articular pain in arthritis induced by zymosan (1 mg, intra articular) in rats was assessed by measuring articular incapacitation (AI). Systemic treatment with the non‐selective NO synthase (NOS) inhibitor L‐NAME (10–100 mg kg−1 i.p.) or with the selective iNOS inhibitors aminoguanidine (AG; 10–100 mg kg−1 i.p.) or 1400W (0.5–1 mg kg−1 s.c.) inhibited the AI induced by injection of zymosan 30 min later. Local (intra articular) treatment with the NOS inhibitors (L‐NAME or AG, 0.1–1 μmol; 1400W, 0.01 (μmol) 30 min before zymosan also inhibited the AI. Systemic or local treatment with the NOS inhibibitors (L‐NAME; AG, 100 mg kg−1 i.p. or 0.1 μmol joint−1; 1400W, 1 mg kg−1 s.c. or 0.01 μmol joint−1), 2 h after zymosan did not affect the subsequent AI. Local treatment with the NO donors SNP or SIN‐1, 2 h after zymosan did inhibit AI. L‐NAME and AG, given i.p. inhibited nitrite but not prostaglandin E2 (PGE2) levels in the joints. L‐NAME (100 mg kg−1) but not AG (100 mg kg−1) increased mean arterial blood pressure. Neither L‐NAME, AG nor the NO donor SIN‐1 altered articular oedema induced by zymosan. In conclusion, inhibitors of iNOS decrease pain in zymosan arthritis only when given before the zymosan. This was not due to inhibition of articular PGE2 release or oedema. NO donors also promoted antinociception in zymosan arthritis without affecting oedema.

Protective Effect of an Extract from Ascaris suum in Experimental Arthritis Models

ABSTRACT We investigated the effect of an extract from a helminth (Ascaris suum) in zymosan-induced arthritis (ZYA) or collagen-induced arthritis (CIA). Rats and mice, respectively, received 1 mg and 0.1 mg zymosan intra-articularly (i.a.). Test groups received an A. suum extract either per os (p.o.) or intraperitoneally (i.p.) 30 min prior to i.a. zymosan. Controls received saline. Hypernociception was measured using the articular incapacitation test. Cell influx, nitrite, and cytokine levels were assessed in joint exudates. The synovia and distal femoral extremities were used for histopathology. Cartilage damage was assessed through determining glycosaminoglycan (GAG) content. DBA/1J mice were subjected to CIA. The test group received A. suum extract i.p. 1 day after CIA became clinically detectable. Clinical severity and hypernociception were assessed daily. Neutrophil influx was determined using myeloperoxidase activity. The A. suum extract, either i.p. or p.o., significantly and dose-dependently inhibited cell influx and hypernociception in ZYA in addition to reducing GAG loss and ameliorating synovitis. The A. suum extract reduced i.a. levels of NO, interleukin-1β (IL-1β), and IL-10 but not tumor necrosis factor alpha (TNF-α) in rats subjected to ZYA while reducing i.a. IL-10, but not IL-1β or TNF-α, levels in mice. Clinically, mice subjected to CIA treated with the A. suum extract had less severe arthritis. Hypernociception, myeloperoxidase activity, and synovitis severity were significantly reduced. These data show that a helminth extract given p.o. protects from arthritis severity in two classical arthritis models. This A. suum effect is species independent and functions orally and parenterally. The results show clinical and structural benefits when A. suum extract is given either prophylactically or therapeutically.

Effects of Tumor Necrosis Factor-α Inhibitors Pentoxifylline and Thalidomide on Alveolar Bone Loss in Short-Term Experimental Periodontal Disease in Rats

BACKGROUND Pentoxifylline (PTX) and thalidomide (TLD) have been shown to inhibit cytokine synthesis, mainly tumor necrosis factor (TNF)-α, in different inflammatory conditions. We studied the effects of these cytokine inhibitors in an experimental model of periodontitis. METHODS Wistar rats were subjected to a ligature placement around the second upper right molars. Alveolar bone loss was evaluated by the sum of the distances between the cusp tip and the alveolar bone along the axis of each molar root, subtracting from the contralateral side. Histopathological analysis was based on cell influx, amount of alveolar bone, and cementum integrity. Animals were weighed daily, and total and differential peripheral white blood cell counts were performed at 6 hours and 1, 7, and 11 days after induction of periodontitis. Groups were treated with saline (positive control), PTX, or TLD 1 hour before and daily up to 11 days after induction of periodontitis. RESULTS Alveolar bone loss was inhibited 42%, 54%, and 69% by PTX at 5, 15, and 45 mg/kg, and 25%, 25%, 42%, and 54% by TLD at 5, 15, 45, and 90 mg/kg, respectively, as compared to the control (P <0.05; analysis of variance). Histological analysis showed that PTX and TLD reduced cell influx and alveolar bone and cementum destruction. PTX and TLD also reversed peripheral lymphomonocytosis but not weight loss, as compared to controls. These data showed that both PTX and TLD reduced alveolar bone loss in periodontitis. CONCLUSION The data showed a protective effect of PTX and TLD on experimental periodontitis, suggesting a role for TNF-α in the pathophysiology of periodontitis. J Periodontol 2004;75:162-168.

Hypernociception elicited by tibio-tarsal joint flexion in mice: A novel experimental arthritis model for pharmacological screening

Mice have been used as animal model to study mechanisms underlying inflammatory and immune diseases. The present study describes a model of joint inflammation-induced hypernociception to discriminate pharmacological tests in mice. A polypropylene tip probe with a large area (4.15 mm2) applied on the plantar surface of the hind paw was used to produce a dorsal flexion of tibio-tarsal joint. Experiments were performed to demonstrate that the probe application did not provoke cutaneous nociception. The decrease in the withdrawal threshold of inflamed joint was used as nociceptive parameter. Administration of zymosan in the tibio-tarsal joint induced a dose and time-dependent hypernociception elicited by articular dorsal flexion movement. Maximal joint hypernociception was detected between 7 and 24 h after zymosan injection, and matched maximal inflammation score as determined by histopathology and neutrophil migration assay. In agreement with the inflammatory hypernociceptive paradigm, flexion-elicited hypernociception induced by zymosan was dose-dependently inhibited by morphine (2-8 mg/kg) and by an effective dose of indomethacin (5 mg/kg). The present study demonstrated that the tibio-tarsal flexion reflex is a behavioral nociceptive model that allows a quantitative evaluation of inflammatory joint hypernociception in mice and its pharmacological modulation.

Low-dose doxycycline prevents inflammatory bone resorption in rats

Matrix metalloproteinases (MMP) are considered to be key initiators of collagen degradation, thus contributing to bone resorption in inflammatory diseases. We determined whether subantimicrobial doses of doxycycline (DX) (< or =10 mg kg-1 day-1), a known MMP inhibitor, could inhibit bone resorption in an experimental periodontitis model. Thirty male Wistar rats (180-200 g) were subjected to placement of a nylon thread ligature around the maxillary molars and sacrificed after 7 days. Alveolar bone loss (ABL) was measured macroscopically in one hemiarcade and the contralateral hemiarcade was processed for histopathologic analysis. Groups of six animals each were treated with DX (2.5, 5 or 10 mg kg-1 day-1, sc, 7 days) and compared to nontreated (NT) rats. NT rats displayed significant ABL, severe mononuclear cell influx and increase in osteoclast numbers, which were significantly reduced by 5 or 10 mg kg-1 day-1 DX. These data show that DX inhibits inflammatory bone resorption in a manner that is independent of its antimicrobial properties.

Reactive nitrogen species scavenging, rather than nitric oxide inhibition, protects from articular cartilage damage in rat zymosan‐induced arthritis

The contribution of nitric oxide (NO) and peroxynitrite (PN) to inflammation in a zymosan‐induced (1 mg, intra‐articular, i.art.) rat model of arthritis was assessed by histopathology and by measuring the glycosaminoglycan (GAG) content of the articular cartilage. Progression of the chronic synovitis in zymosan‐induced arthritis (ZYA) was associated with increased nitrite and nitrotyrosine (3‐NT) levels in the joint exudates that paralleled a progressive loss of the GAG content. An increase in 3‐NT was also observed after i.art. PN. The nonselective nitric oxide synthase (NOS) inhibitor L‐NG‐nitroarginine methyl ester (25–75 mg kg−1day−1) or the selective inducible NOS inhibitor aminoguanidine (50–100 mg kg−1day−1) given 1 h before (prophylactic) or 3 days after (therapeutic) injection of the zymosan ameliorated the synovitis, but worsened the GAG loss, as measured at the end of the experiment (day 7). The PN scavenger uric acid (100–250 mg kg−1 i.p. four times daily) given prophylactically until the end of the experiment (day 14), in a dose compatible with its PN scavenging activity, significantly decreased both the synovitis and the GAG loss. In conclusion, PN formation is associated with cartilage damage in addition to proinflammatory activity in ZYA. NOS inhibitors and a PN scavenger were able to reduce the cellular infiltration, while displaying opposite effects on cartilage homeostasis either by enhancing or ameliorating the damage, respectively.

Periarthritis promotes gait disturbance in zymosan-induced arthritis in rats

Abstract.Objective and Design: We studied the contribution of periarthritis and synovitis to gait disturbance in zymosan (Zy)-induced arthritis.¶Methods: Sixty Wistar rats were subjected to injection of Zy (1mg) into their right knee joints. A first group of animals (GI) had Zy injected through the intact skin. A second group (GII) had Zy injected directly into the articular cavity after excision of the skin and subcutaneous tissue surrounding the joint. Gait disturbance was evaluated using the rat-knee joint incapacitation test. Increase in vascular permeability and cell influx were assessed in joint fluids and joint histology was performed.¶Results: Zy injection induced a dose-dependent gait disturbance which was maximal at the third/fourth hour of arthritis, being significantly greater in GI rats, whereas cell influx (neutrophils ≤80%) was maximal at the sixth hour. Cell influx and increase in vasopermeability did not differ between both groups. Histology revealed no significant difference between GI and GII. A third group (GIII), subjected to immune-complex arthritis, that received anti-bovine serum albumin (BSA) antibodies intra-articularly and BSA i.v., did not present gait disturbance, despite the increase in cell counts.¶Conclusions: Vascular permeability increase and cell influx are phenomena independent of gait disturbance. Neutrophils do not seem to contribute to development of gait disturbance in Zy arthritis. Sensitization of specific pain receptors in periarticular rather than in synovial tissue is responsible for gait disturbance in Zy-induced arthritis.