Monica Moya

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.

Inflammatory and oxidative stress markers in experimental crystalopathy: their modification by photostimulation.

Crystalopathies are inflammatory pathologies caused by cellular reactions to the deposition of crystals in the joints. The anti-inflammatory effect of the helium-neon (He-Ne) laser and that of the nonsteroidal anti-inflammatory drugs (NSAIDs) diclofenac, meloxicam, celecoxib, and rofecoxib was studied in acute and chronic arthritis produced by hydroxyapatite and calcium pyrophosphate in rats. The presence of the markers fibrinogen, L-citrulline, nitric oxide, and nitrotyrosine was determined. Crystals were injected into the posterior limb joints of the rats. A dose of 8 J/cm(2) of energy from an He-Ne laser was applied for 3 d in some groups and for 5 d in other groups. The levels of some of the biomarkers were determined by spectrophotometry, and that of nitrotyrosine was determined by ELISA. For statistical analysis, Fishers exact test was used, and p +/- 0.05 was considered significant. In arthritic rats, the fibrinogen, L-citrulline, nitric oxide, and nitrotyrosine levels increased in comparison to controls and to the laser-treated arthritic groups (p +/- 0.001), (p +/- 0.001), (p +/- 0.02), and (p +/- 0.01), respectively. When comparing fibrinogen from arthritic rats with disease induced by hydroxyapatite with undiseased and arthritic rats treated with NSAIDs, the He-Ne laser decreased levels to values similar to those seen in controls (p +/- 0.01). Inflammatory and oxidative stress markers in experimental crystalopathy are positively modified by photobiostimulation.

Simvastatin: pharmacological response in experimental hyperfibrinogenaemias.

Through a disorder in the endothelial haemostatic balance, hyperfibrinogenaemia could generate endothelial dysfunction. Statins would have antiinflammatory effects on injured endothelium. Objective — Simvastatin pharmacological response in rats with hyperfibrinogenaemias induced by laparotomies was studied. Methods and results — Rats were subjected to multiple injuries (MI) for 30 days (1 laparotomy/week) and for 60 days (1 laparotomy/2 weeks). Simvastatin (0.035 mg/kg) was administered orally to the 30-day multiple injuries group after the third injury for a period of 10 days. A similar dose was administered to the 60-day multiple injuries group after the second injury for a period of 45 days. Blood samples of all the groups were obtained 72 hours after the last injury. In the 30 and 60-day multiple injuries groups, a statistically significant fibrinogen increase was observed (336.6 ± 7.5 and 358.7 ± 9.9, respectively) compared with the control group (207.0 ± 3.0) (p < 0.001).There were no significant differences in the plasmatic fibrinogen (PF) levels between the control and simvastatin treated groups (224.9 ± 1.4 and 216.3 ± 4.3, respectively).There were significant differences between the 30 or 60-day MI untreated groups compared with the 30 or 60-day multiple injuries + simvastatin treated group (p < 0.001). Endothelial denudation and intima widening were observed in the untreated injured groups, whereas in the 60 day multiple injuries group + simvastatin, a regression of histopathological lesions was observed. Conclusions — the decrease of the inflammatory component that would accompany early atherogenesis processes and the regression of the histopathological lesions after treatment could be attributed to the decreased plasmatic fibrinogen.

Effects of Atorvastatin on Oxidative Stress Biomarkers and Mitochondrial Morphofunctionality in Hyperfibrinogenemia-Induced Atherogenesis.

Relationship between hyperfibrinogenemia (HF), oxidative stress, and atherogenesis was established. Effect of atorvastatin (Ator) was assessed. Wistar male (6 months) rats were studied: Ctr, control, without HF induction; Ctr-Ator, without HF treated with atorvastatin; AI, atherogenesis induced, and AI-Ator, atherogenesis induced and treated with atorvastatin. Atherogenesis was induced by daily adrenaline injection (0.1 mL/day/rat) for 90 days; treatment started 15 days after induction. Fibrinogen (mg/dL) and nitric oxide (NO) were measured in plasma (mM) and superoxide dismutase (SOD) (U/mL) in red cell lysate by spectrophotometry. Slices of aorta were analyzed by electron microscopy (EM). ANOVA and chi-square test were used; P < 0.05 was established. There were no significant differences between Ctr and Ctr-Atorv in fibrinogen, NO, and SOD values. Comparing Ctr with AI an increase of fibrinogen is observed (P < 0.001), but it decreased after administration of atorvastatin in AI-Ator (P < 0.001). NO diminished in AI relative to Ctr and increased in AI-Ator (P < 0.001). SOD showed an increase in AI and AI-Ator compared to Ctr (P < 0.001). EM revealed expansion of intermembrane space and disorganization of crests in AI. In AI-Ator mitochondrial areas and diameters were similar to control. Atorvastatin normalizes HF, stabilizes NO, increases SOD, and produces a partial regression of mitochondrial lesions.

Mitochondrial Morphofunctional Alterations in Smooth Muscle Cells of Aorta in Rats

In an experimental model of atherogenesis induced by hyperfibrinogenemia (HF), the pharmacological response of vitamin E was studied in order to assess its antioxidant effect on the mitochondrial morphofunctional alterations in aortic smooth muscle cells. Three groups of male rats were used: (Ctr) control, (AI) atherogenesis induced for 120 days, and (AIE) atherogenesis induced for 120 days and treated with vitamin E. HF was induced by adrenalin injection (0.1 mg/day/rat) for 120 days. AIE group was treated with the administration of 3.42 mg/day/rat of vitamin E for 105 days after the first induction. Mitochondria morphology was analyzed by electronic microscopy (EM) and mitochondrial complexes (MC) by spectrophotometry. In group AI the total and mean number of mitochondria reduced significantly, the intermembranous matrix increased, and swelling was observed with respect to Ctr and AIE (P < 0.01). These damages were related to a significant decrease in the activity of citrate synthase and complexes I, II, III, and IV in group AI in comparison to Ctr (P < 0.001). Similar behavior was presented by group AI compared to AIE (P < 0.001). These results show that vitamin E produces a significative regression of inflammatory and oxidative stress process and it resolved the morphofunctional mitochondrial alterations in this experimental model of atherogenic disease.

Myelo peroxidase as an Indicator of Oxidative Stress in Metabolic Syndrome

Background: Increased myeloperoxidase (MPO) activity would be the link between the rise of the inflammatory response and oxidative stress (OS) in metabolic syndrome (MS). Objective: The aim of this study was to determine the enzymatic activity of MPO associated with OS in animals with MS and establish their relationship with probable cardiovascular injury. Methods: Male Wistar rats were divided into two groups: Group A, control (n=12) and Group B, induced MS (n=12). Metabolic syndrome was produced by 6-week administration of 10% fructose diluted in the drinking water. Insulin (mU/ml), glucose (mg/dl), lipid panel (mg/dl), HOMA (homeostatic model assessment), MPO (IU/ml) and superoxide dismutase (SOD) activity (U/ml) were measured. Light microscopy was used for the histological study of the heart and thoracic aorta. Results: Group B showed significantly increased levels of plasma glucose (176±17.3 mg/dl), insulin (29.5±4.52 mU/ml), HOMA (11±1.3), total cholesterol (133±9.6 mg/dl) and triglycerides (75±12.9 mg/dl) compared with Group A: plasma glucose (115±1.1 mg/dl), insulin (4±0.82 mU/ml), HOMA (3±0.38), total cholesterol (69.7±1.6 mg/dl) and triglycerides (46.2±6 mg/dl), (p<0.001 for all variables). A significant decrease in HDL (28.3±1.14 mg/dl) in Group B vs. Group A (61±1.0 mg/dl) (p<0.001) validated the experimental MS model. Myeloperoxidase activity increased significantly in Group B (181.3±15.7 IU/ml) vs. Group A (116.07±4.2 IU/ml) (p<0.001). A similar behavior was seen with SOD antioxidant activity in Group B (181±6 U/ml) vs. Group A (138±3.6 U/ml) (p <0.01). Light microscopy of the heart and thoracic aorta revealed histopathological changes in animals with induced MS. Conclusion: Increased MPO and SOD in Group B would indicate the presence of OS in MS, with consequences at the vascular level.

Inflammatory and oxidative stress markers as indicator of atherogenesis in rats: antioxidants as preventive pharmacological methods.

OBJECTIVE The oxidative process in atherogenesis generated by proinflammatory induction and response to antioxidants vitamins in an experimental model were analyzed. METHODS Male rats were used: (A)Control, (B)Control+vitamin E plus C, (C)Hyperfibrinogenemia and (D)Hyperfibrinogenemia+vitamins E plus C. Hyperfibrinogenemia induced by daily injection of adrenaline (0.1mg/day/rat) for 120 days. TREATMENT 3.42 mg/kg of vitamin E plus 2.14 mg/kg of vitamin C, fifteen days after induction. Vascular histology analyzed by optical microscopy. Fibrinogen, nitrites and superoxide dismutase analyzed by spectrophotometry. STATISTICS MANOVA, Hotelling test for post testing, significance level p<0.05. RESULTS (C) group showed higher fibrinogen than (A) and (B)(p<0.001). Compared to (C) group, (D) showed a decrease of fibrinogen (p<0.001). A marked increase in nitrites was found in (C) versus (A), (B) and (D) groups (p<0.001). Superoxide dismutase activity increased in (C) group compared to groups (A) and (B) (p<0.001). In the group (D) an increase of the activity of this enzyme was observed in comparison to groups (C)(p<0.001), (A) and (B) (p<0.0001 in both). The (C) group shown endothelial denudation, thickening of the vascular intima and extracellular matrix enlargement with foam cells(p<0.001). CONCLUSION These results strongly suggest that vitamins E plus C produce regression of inflammatory and oxidative stress processes in this experimental model.