David Cremonezzi

Evaluation of inflammatory biomarkers associated with oxidative stress and histological assessment of low-level laser therapy in experimental myopathy

The objective of the present work was to study the effect of helium–neon (He–Ne) and gallium–arsenide (Ga–As) laser upon inflammatory biomarkers associated with oxidative stress: fibrinogen, nitric oxide (NO), L‐citrulline, and superoxide dismutase (SOD). These were evaluated through histological assessment, in rats with experimental myopathy. Materials and Methods: The groups studied were: (A) control, (B) injured, (C) injured and treated with He–Ne laser, (D) injured and treated with Ga–As laser, (E) irradiated with He–Ne; and (F) irradiated with Ga–As laser. Myopathy was induced by injecting 0.05 mg/rat/day of adrenaline in the left posterior limb muscle at the same point on 5 consecutive days, in groups B, C, and D. Low‐level laser therapy (LLLT) was applied with 9.5 J/cm2 daily for 7 consecutive days with each laser. The determination of the biomarkers was made by spectrophotometry. The muscles (5/8, single blinded) were stained with Gomori Trichrome and examined by optic microscopy. The quantitative variables were statistically analyzed by the Fishers test and categorical data by the Axionvision 4.8 program. Pearsons chi‐squared test was applied, setting significant difference at P < 0.05 for all cases. Results: In group B, the biomarkers were significantly increased compared to the other groups (P < 0.001), except for NO which in group B decreased significantly (P < 0.001). In group B, there was a higher inflammatory infiltration level (80.67%) in relation to destroyed fibers. Conclusions: LLLT caused significant changes in inflammatory biomarkers and oxidative stress: decreased levels of fibrinogen, L‐citrulline and SOD as opposed to the increase of NO in rats with experimental myopathies and significant muscle recovery. Lasers Surg. Med. 42:577–583, 2010.

Helium-Neon Laser Reduces the Inflammatory Process of Arthritis

OBJECTIVE A histological study of the anti-inflammatory effect of helium-neon laser in models of arthropathies induced by hydroxyapatite and calcium pyrophosphate in rats. BACKGROUND Crystal deposition diseases are inflammatory pathologies induced by cellular reaction to the deposit of crystals in the joints. METHODS Fifty-six Suquia strain rats were distributed in seven groups. Two mg of each crystal diluted in 0.05 ml physiologic solution were injected six times in each back limb joint, during two weeks on alternate days. Eight J/cm(2) were applied daily to the crystal-injected joints on five consecutive days. The joints were cut and put in 10% formaldehyde, stained with hematoxylin-eosin and observed by light microscopy. The percentage of area with inflammatory infiltrates was determined in five optical microscopy photographs (100X) for each group and analyzed using the Axionvision 4.6 program. A Pearsons Chi Squared test was applied, with significance level set at p < 0.05. RESULTS Both crystals produced an inflammatory process in the osteoarticular structures, consisting of predominantly mononuclear infiltration, fibrosis, and granulomas of foreign body-type giant cells containing phagocytosed remains of crystals. In the arthritic joints treated with laser, a marked decrease (p < 0.0001) was found in the percentage of area with inflammatory infiltrates, although the granulomas remained in a less ostensible form, with adipose tissue cells, fibrosis bands with light residual inflammation, and an absence of or very few crystals. Laser alone or physiologic solution injection did not produce histological changes. CONCLUSIONS Helium-neon laser reduced the intensity of the inflammatory process in the arthritis model induced by hydroxyapatite and calcium pyrophosphate crystals.

Clomipramine and benznidazole association for the treatment of acute experimental Trypanosoma cruzi infection.

Alternative strategies are being designed to identify candidates among drugs already available on the market that could be used in combination to improve the efficacy of Chagas disease treatment. This work evaluates the effect of the association of clomipramine (CLO) with benznidazole (BZN) for the treatment of experimental Chagas disease in the acute stage, in Swiss albino mice infected with Trypanosoma cruzi Tulahuen strain. Infected mice were treated with CLO 5mg/kg/day and BZN 50 and 100mg/kg/day, each separately or together. Efficacy of the treatment was evaluated through parasitemia, survival, electrocardiography, histopathological studies, serological and PCR assays at 90 days post-infection (dpi). All treatments significantly (P<0.05) reduced mortality and decreased parasitemia. Histopathological analysis of liver and kidneys of mice treated with CLO and the drug combination showed less injury than mice treated only with BZN. The lower dose of BZN (50mg/kg/day) combined with CLO showed the same efficacy as the habitual dose of BZN (100mg/kg/day) combined with CLO. The therapeutic results from the combination of BZN with CLO presented lesser side effects than the treatment with BZN.

Use of clomipramine as chemotherapy of the chronic phase of Chagas disease

Chagas infection is a major endemic disease affecting Latin American countries. The persistence of Trypanosoma cruzi generates a chronic inflammatory reactivity that induces an immune response directed to the hosts tissues. The effectiveness of the treatment in the chronic phase is still unsatisfactory due, amongst other reasons, to the collateral effects of the drugs used. We investigated the effect of clomipramine, a tricyclic antidepressant that, when used as a treatment of T. cruzi-chronically infected mice, inhibits trypanothione reductase, an exclusive and vital enzyme of T. cruzi. Clomipramine improved survival (P<0.05) by diminishing the parasite intensity as demonstrated by PCR studies in the heart and skeletal muscle, and significantly prevented the evolution to fibrosis of the inflammatory infiltrates. Clomipramine could be a good candidate for the treatment of chronic Chagas disease.

Effects of dietary lipids on cell proliferation of murine oral mucosa.

BackgroundThe lack of certain essential polyunsaturated fatty acids (PUFAs) induces perturbation in cell proliferation, apoptosis and dedifferentiation that could be linked to an increased protumorigenic trend. Contrarily, n-3 essential fatty acids (EFAs) arrest cell proliferation in several tumor models. According to the concept of field cancerization, multiple patches of abnormal epithelial proliferation may coexist in the vicinity of oropharyngeal neoplasms.The purpose of the present study is to determine whether certain dietary PUFAs differentially modulate the patterns of cell proliferation and apoptosis at non-tumoral sites of the oral mucosa in mice bearing DMBA induced salivary tumors.After weaning, BALB/c mice were assigned to four diets: Control (C), Corn Oil (CO), Fish (FO) and Olein (O). Two weeks later, DMBA was injected into the submandibular area. The animals were sacrificed between 94 and 184 days at 4–6 PM. Fixed samples of lip, tongue and palate were stained using H-E and a silver technique. A quantification of AgNORs in the basal (BS) and suprabasal stratum (SBS) of the covering squamous epithelia as well as of mitosis and apoptosis was performed.ResultsAnalysis of Variance showed greater proliferation in tongue than in palate or lip. According to the diet, a significant difference was found in the Fish Oil, in which palate exhibited fewer AgNOR particles than that of the control group, both for BS and SBS (p < 0.05 and 0.152, respectively), indicating a reduced cell proliferation.ConclusionsThese results corroborate and reaffirm that the patterns of cell proliferation, apoptosis and differentiation of the oral stratified squamous epithelium may be differentially modulated by dietary lipids, and arrested by n-3 fatty acids, as shown in several other cell populations.

Evaluation of inflammatory biomarkers associated with oxidative stress and histological assessment of magnetic therapy on experimental myopathy in rats.

The effect of pulsed electromagnetic field (PEMF) therapy, also called magnetic therapy, upon inflammatory biomarkers associated with oxidative stress plasma fibrinogen, nitric oxide (NO), L-citrulline, carbonyl groups, and superoxide dismutase (SOD) was evaluated through histological assessment, in rats with experimental myopathy. The groups studied were: (A) control (intact rats that received PEMF sham exposures); (B) rats with myopathy and sacrificed 24 h later; (C) rats with myopathy; (D) rats with myopathy and treated with PEMF; and (E) intact rats treated with PEMF. Groups A, C, D, and E were sacrificed 8 days later. Myopathy was induced by injecting 50 μl of 1% carrageenan λ (type IV) once sub-plantar. Treatment was carried out with PEMF emitting equipment with two flat solenoid disks for 8 consecutive days in groups D and E, at 20 mT and 50 Hz for 30 min/day/rat. The biomarkers were determined by spectrophotometry. The muscles (5/8) were stained with Hematoxylin-Eosin and examined by optic microscopy. Quantitative variables were statistically analyzed by the Fisher test, and categorical applying Pearsons Chi Squared test at p < 0.05 for all cases. In Groups B and C, the biomarkers were significantly increased compared to A, D, and E groups: fibrinogen (p < 0.001); NO, L-citrulline and carbonyl groups (p < 0.05); SOD (p < 0.01) as well as the percentage of area with inflammatory infiltration (p < 0.001). PEMF caused decreased levels of fibrinogen, L-citrulline, NO, SOD, and carbonyl groups and significant muscle recovery in rats with experimental myopathies.

Combined calcitriol and menadione reduces experimental murine triple negative breast tumor

BACKGROUND Calcitriol (D) or 1,25(OH)2D3 inhibits the growth of several tumor cells including breast cancer cells, by activating cell death pathways. Menadione (MEN), a glutathione-depleting compound, may be used to potentiate the antiproliferative actions of D on cancer cells. We have previously shown in vitro that MEN improved D-induced growth arrest on breast cancer cell lines, inducing oxidative stress and DNA damage via ROS generation. Treatment with MEN+D resulted more effective than D or MEN alone. OBJECTIVE To study the in vivo effect of calcitriol, MEN or their combination on the development of murine transplantable triple negative breast tumor M-406 in its syngeneic host. METHODS Tumor M-406 was inoculated s.c., and when tumors reached the desired size, animals were randomly assigned to one of four groups receiving daily i.p. injections of either sterile saline solution (controls, C), MEN, D, or both (MEN+D). Body weight and tumor volume were recorded three times a week. Serum calcium was determined before and at the end of the treatment, at which time tumor samples were obtained for histological examination. RESULTS None of the drugs, alone or in combination, affected mice body weight in the period studied. The combined treatment reduced tumor growth rate (C vs. MEN+D, P<0.05) and the corresponding histological sections exhibited small remaining areas of viable tumor only in the periphery. A concomitant DNA fragmentation was observed in all treated groups and MEN potentiated the calcitriol effect on tumor growth. CONCLUSIONS As previously observed in vitro, treatment with MEN and D delayed tumor growth in vivo more efficiently than the individual drugs, with evident signals of apoptosis induction. Our results propose an alternative protocol to treat triple negative breast cancer, using GSH depleting drugs together with calcitriol, which would allow lower doses of the steroid to maintain the antitumor effect while diminishing its adverse pharmacological effects.