Hyperbaric oxygen therapy mitigates left ventricular remodeling, upregulates MMP-2 and VEGF, and inhibits the induction of MMP-9, TGF-ß1, and TNF-α in streptozotocin-induced diabetic rat heart.

AIMS: Hyperbaric oxygen (HBO) therapy has been widely used for the adjunctive treatment of diabetic wounds, and is currently known to influence left ventricular (LV) function. However, morphological and molecular repercussions of the HBO in the diabetic myocardium remain to be described. We aimed to investigate whether HBO therapy would mitigate adverse LV remodeling caused by streptozotocin (STZ)-induced diabetes. MAIN METHODS: Sixty-day-old Male Wistar rats were divided into four groups: Control (n = 8), HBO (n = 7), STZ (n = 10), and STZ + HBO (n = 8). Diabetes was induced by a single STZ injection (60 mg/kg, i.p.). HBO treatment (100% oxygen at 2.5 atmospheres absolute, 60 min/day, 5 days/week) lasted for 5 weeks. LV morphology was evaluated using histomorphometry. Gene expression analyzes were performed for LV collagens I (Col1a1) and III (Col3a1), matrix metalloproteinases 2 (Mmp2) and 9 (Mmp9), and transforming growth factor-ß1 (Tgfb1). The Immunoexpression of cardiac tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) were also quantified. KEY FINDINGS: HBO therapy prevented LV concentric remodeling, heterogeneous myocyte hypertrophy, and fibrosis in diabetic rats associated with attenuation of leukocyte infiltration. HBO therapy also increased Mmp2 gene expression, and inhibited the induction of Tgfb1 and Mmp9 mRNAs caused by diabetes, and normalized TNF-α and VEGF protein expression. SIGNIFICANCE: HBO therapy had protective effects for the LV structure in STZ-diabetic rats and ameliorated expression levels of genes involved in cardiac collagen turnover, as well as pro-inflammatory and pro-angiogenic signaling.

CmyoSize: An ImageJ macro for automated analysis of cardiomyocyte size in images of routine histology staining.

BACKGROUND: Cardiomyocyte size histomorphometry is widely required in myocardial remodeling research, but it relies on subjective, time-consuming manual tracing. We aimed to present the CmyoSize, a Fiji/ImageJ macro that reproducibly measures the transnuclear cross-sectional size of cardiomyocytes in multiple H&E images. METHODS: CmyoSize output per image are: mean cross-sectional area (CSA), CSA's local standard deviation (LSD), mean width, and width's LSD. CmyoSize was validated against measurements of an expert examiner, using myocardium micrographs (400-fold magnification) from rats treated with hyperbaric oxygen (HBO), streptozotocin (STZ), as well as control rats (five images/heart, five rats/group). RESULTS: CmyoSize achieved high cardiomyocyte detection precision (95.17 ± 1.11%) and it analyzed each image at least 30-times faster than expert examiner (8.44 ± 0.17 s vs. 266.70 ± 25.15 s). Bland-Altman analyses showed no meaningful bias ( ± 95% confidence interval) between both methods: mean CSA = 1.88 ± 6.83 µm2, CSA's LSD = -4.55 ± 5.07 µm2; mean width = -0.02 ± 0.22 µm; width's LSD = -0.16 ± 0.17 µm. Pearson's r ranged from 0.85 to 0.94 (P < 0.0001). CmyoSize revealed homogenous cardiomyocyte hypertrophy in HBO-treated animals and STZ-induced heterogeneous hypertrophy. CONCLUSION: Fully automated, standardized cardiomyocyte size quantification in H&E images using CmyoSize is feasible, accurate, and time-efficient.

Complete mid-portion rupture of the rat achilles tendon leads to remote and time-mismatched changes in uninjured regions.

PURPOSE: To examine healing adaptations over 17 weeks post Achilles tendon (AT) rupture in the injured region (IR) compared to an uninjured region (UIR) of the AT. METHODS: Twenty-four rats were subjected to a complete right-sided AT rupture, while the left side served as a control. ATs were harvested at 1, 2, 8 and 17 weeks post-rupture and stained with antibodies specific to Collagen type I (Col I) and II (Col II) as well as Alcian Blue and Picrosirius Red staining techniques. Histopathological changes, proteoglycan content, collagen alignment and immunoexpression were assessed. RESULTS: Both regions examined, IR and UIR, exhibited over weeks 1-17 similar healing adaptations of increasing collagen alignment, decreasing Col I immunoexpression, as well as increasing proteoglycan content and Col II occurrence. Increased proteoglycan content was found already at week 2 in the UIR, while it first increased at week 8 in the IR. The area positive to Col II was increased compared to controls at week 8 in the UIR, whereas it first raised at week 17 in the IR. Collagen disorganization successively declined to reach control levels at week 17 in the UIR, but was still higher in the IR. CONCLUSION: This study demonstrated that uninjured areas of the AT remote from the rupture site also undergo pronounced remodeling, although with time-span differences relative to injured AT portions. These changes including the pathologic heterotopic mineralization and chondrogenic differentiation observed in both regions may have implications in the choice of rehabilitation regimes in order to prevent secondary rupture.

Thirty-day experimental diabetes impairs contractility and increases fatigue resistance in rat diaphragm muscle associated with increased anti-oxidative activity.

Diabetes mellitus is a metabolic disorder that can generate tissue damage through several pathways. Alteration and dysfunction of skeletal muscle are reported including respiratory muscles, which may compromise respiratory parameters in diabetic patients. We have aimed to evaluate the diaphragm muscle contractility, tissue remodeling, oxidative stress, and inflammatory parameters from 30 day streptozotocin-treated rats. The diaphragm contractility was assessed using isolated muscle, tissue remodeling using histology and zymography techniques, and tissue oxidative stress and inflammatory parameters by enzyme activity assay. Our data revealed in the diabetes mellitus group an increase in maximum tetanic force (4.82 ± 0.13 versus 4.24 ± 0.18 N/cm2 (p = 0.015)) and fatigue resistance (139.16 ± 10.78 versus 62.25 ± 4.45 s (p < 0.001)), reduction of 35.4% in muscle trophism (p < 0.001), increase of 32.6% of collagen deposition (p = 0.007), reduction of 21.3% in N-acetylglucosaminidase activity (p < 0.001), and increase of 246.7% of catalase activity (p = 0.002) without changes in reactive oxygen species (p = 0.518) and tissue lipid peroxidation (p = 0.664). All observed changes are attributed to the poor glycemic control (471.20 ± 16.91 versus 80.00 ± 3.42 mg/dL (p < 0.001)), which caused defective tissue regeneration and increased catalase activity as a compensatory mechanism.

Ethanol extract of Cissampelos sympodialis ameliorates lung tissue damage in streptozotocin-induced diabetic rats

Diabetes Mellitus (DM) is a metabolic syndrome characterized by hyperglycemia. Chronic complications affect a number of organs, including the lungs. Cissampelos sympodialis Eichl (Menispermaceae) is a plant used to treat respiratory diseases. The aim of this study was to evaluate the effect of Cissampelos sympodialis extract (CSE) in lungs of diabetic rats. We used 30 Wistar rats divided into three groups: control group (CG), diabetic group (DG) and diabetic Cissampelos sympodialis treatment group (DTG). Diabetes was induced by streptozotocin (40 mg/kg i.v.). The CSE (400 mg/kg, po) was administered daily, during four weeks, beginning one week after the onset of DM. The treatment with CSE was not able to reduce blood glucose levels after streptozotocin injection. However, it was able to decrease cholesterol and triglycerides and prevent damage on pancreatic islets morphology. Additionally, morphological alterations such as alveolar septa loss, inflammatory infiltrate and fibrosis were seen in lung tissue of rats with DM, and treatment with CSE apparently reversed these histopathological findings. Thus, CSE treatment reduced the lipid profile and restored the lung architecture of diabetic animals by a mechanism independent of glycemia and which might be associated with the reduction of the damage on the pancreatic islets.

Effect of photobiomodulation and exercise on early remodeling of the Achilles tendon in streptozotocin-induced diabetic rats.

The aim of this study was to compare the treatment effects of laser photobiomodulation (LPBM) therapy and aerobic exercise on the biomechanical properties, tissue morphology and the expression of tendon matrix molecules during early remodeling of Achilles tendon (AT) injury in diabetic rats. Animals were randomly assigned to five groups: injured non diabetic (I, n = 15), injured diabetic (ID, n = 15), injured diabetic plus LPBM (IDL, n = 16), injured diabetic plus aerobic exercise (IDE, n = 16) and injured diabetic plus aerobic exercise and LPBM (IDEAL, n = 17). Type 1 diabetes was induced via a single intravenous injection of Streptozotocin at a dose of 40 mg/kg. A partial tenotomy was performed in the right AT. LPBM was performed with an indium-gallium-aluminum-phosphide 660 nm 10 mW laser device (spot size 0.04 cm2, power density 250 mW/cm2, irradiation duration 16 s, energy 0.16 J, energy density 4 J/cm2) on alternate days for a total of 9 sessions over 3 weeks (total energy 1.44 J), using a stationary contact technique to a single point over the dorsal aspect of the AT. Moderate aerobic exercise was performed on a motorized treadmill (velocity 9 m/min for 60 minutes). At 3 weeks post-injury, biomechanical analyzes as well as assessment of fibroblast number and orientation were performed. Collagen 1 (Col1) and 3 (Col3) and matrix metalloproteinases (MMPs) -3 and 13 protein distributions were studied by immunohistochemistry; while Col1 and Col3 and MMP-2 and 9 gene expression were assessed by quantitative RT-PCR (qRT-PCR). IDEAL exhibited significant increases in several biomechanical parameters in comparison to the other groups. Moreover, IDEAL presented stronger Col1 immunoreactivity when compared to ID, and weaker Col3 immunoreactivity than IDE. Both IDL and IDEAL demonstrated weaker expression of MMP-3 in comparison to I, while IDL presented no expression of MMP-13 when compared to ID. ID, IDL and IDE showed an increased number of fibroblasts in comparison to I, while IDEAL decreased the number of these cells in comparison to ID and IDE. IDL and IDEAL groups exhibited decreased angular dispersion among the fibroblasts when compared to I. The gene expression results showed that IDE demonstrated a downregulation in Col1 mRNA expression in comparison to I and ID. IDEAL demonstrated upregulation of Col1 mRNA expression when compared to IDL or IDE alone and increased MMP-2 expression when compared to IDL and IDE. MMP-9 expression was upregulated in IDEAL when compared to I, IDL and IDE. Our results suggest a beneficial interaction of combining both treatment strategies i.e., aerobic exercise and LPBM, on the biomechanical properties, tissue morphology and the expression of matrix molecules in diabetic tendons.

Anabolic Effect of Insulin Therapy on the Bone: Osteoprotegerin and Osteocalcin Up-Regulation in Streptozotocin-Induced Diabetic Rats.

Type 1 diabetes mellitus (T1DM) is associated with several skeletal alterations, particularly in conditions of poor glycaemic control. Insulin therapy is the major conservative treatment for T1DM; however, the effects of this hormone on bone markers of T1DM rats are limited, and the regulatory mechanisms remain elusive. Therefore, the evaluation of molecular and non-molecular parameters in a chronic animal model of T1DM-induced bone loss, treated with and without insulin, may help in elucidating the insulin mechanisms. Male Wistar rats were assigned into three groups: control, T1DM (T1DM rats induced with streptozotocin [STZ] at 40 mg/kg intravenously) and T1DM plus insulin therapy (T1DMI). After 8 weeks, we evaluated the serum biochemical, tibia histomorphometric and biomechanical parameters, as well as the gene expression of the receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and osteocalcin (OC) of femur mRNA. Compared with T1DM, the T1DMI group showed less bone loss, which was revealed by the increased trabecular width (TbWi, p < 0.001) and trabecular bone area (BAr, p < 0.01), reduced trabecular separation (TbSp, p < 0.01) and increased Young's modulus (p < 0.05). Moreover, molecular analyses indicated that the expression of OPG and OC was up-regulated (p < 0.001 and p < 0.05, respectively). In summary, the up-regulation of OPG and OC in the T1DMI group supports an anabolic effect of insulin, which was demonstrated by the maintenance of bone architecture and flexibility. These results suggest that insulin therapy may prevent T1DM-induced bone loss via the effects on the bone formation.

Protection against T1DM-Induced Bone Loss by Zinc Supplementation: Biomechanical, Histomorphometric, and Molecular Analyses in STZ-Induced Diabetic Rats.

Several studies have established an association between diabetes and alterations in bone metabolism; however, the underlying mechanism is not well established. Although zinc is recognized as a potential preventive agent against diabetes-induced bone loss, there is no evidence demonstrating its effect in chronic diabetic conditions. This study evaluated the effects of zinc supplementation in a chronic (90 days) type 1 diabetes-induced bone-loss model. Male Wistar rats were distributed in three groups: control, type 1 diabetes mellitus (T1DM), and T1DM plus zinc supplementation (T1DMS). Serum biochemical analysis; tibia histomorphometric, biomechanical, and collagen-content analyses; and femur mRNA expression were evaluated. Relative to T1DM, the zinc-supplemented group showed increased histomorphometric parameters such as TbWi and BAr and decreased TbSp, increased biomechanical parameters (maximum load, stiffness, ultimate strain, and Young's modulus), and increased type I collagen content. Interestingly, similar values for these parameters were observed between the T1DMS and control groups. These results demonstrate the protective effect of zinc on the maintenance of bone strength and flexibility. In addition, downregulation of OPG, COL1A, and MMP-9 genes was observed in T1DMS, and the anabolic effects of zinc were evidenced by increased OC expression and serum ALP activity, both related to osteoblastogenesis, demonstrating a positive effect on bone formation. In contrast, T1DM showed excessive bone loss, observed through reduced histomorphometric and biomechanical parameters, characterizing diabetes-associated bone loss. The bone loss was also observed through upregulation of OPG, COL1A, and MMP-9 genes. In conclusion, zinc showed a positive effect on the maintenance of bone architecture and biomechanical parameters. Indeed, OC upregulation and control of expression of OPG, COL1A, and MMP-9 mRNAs, even in chronic hyperglycemia, support an anabolic and protective effect of zinc under chronic diabetic conditions. Furthermore, these results indicate that zinc supplementation could act as a complementary therapy in chronic T1DM.

Efeito do ultrassom terapêutico sobre as propriedades mecânicas do gastrocnêmio em ratos / Effect of therapeutic ultrasound on mecnanical properties of gastrocnemius in rats / Efecto del ultrasounido terapéutico sobre las propiedades mecánicas del gastrocnemio en ratones

INTRODUÇÃO: Apesar de algumas controvérsias quanto à sua utilização, o ultrassom terapêutico (UST) é um recurso comumente aplicado na reabilitação desportiva para aceleração do reparo tecidual de lesões musculares. Sabe-se que lesões musculares influenciam negativamente as propriedades mecânicas da musculatura estriada esquelética e algumas evidências demonstram que o UST poderia ter efeitos benéficos sobre o reparo muscular e, consequentemente, sobre suas propriedades mecânicas. OBJETIVO: Analisar o efeito do UST no reparo tecidual por meio das propriedades mecânicas musculares de ratos após trauma por criolesão. MÉTODOS: Foram utilizados no estudo 30 ratos da linhagem Wistar, os quais foram divididos em três grupos: grupo controle intacto (GC), grupo lesionado sem tratamento (GL), e grupo lesionado e estimulado com UST (frequência de 1 MHz; intensidade de 0,5 W/cm2; ciclo de trabalho de 50%; por quatro minutos diários), durante sete dias consecutivos (GLUST). As propriedades mecânicas obtidas através de ensaio mecânico de tração foram avaliadas em uma máquina universal de ensaios. RESULTADOS: Foram analisados estatisticamente, com nível de significância de 95% (P<0,05). Após sete sessões de tratamento, houve melhora estatisticamente significativa nas propriedades mecânicas de carga no limite de proporcionalidade, carga no limite máximo e resiliência para o GLUST em relação ao GL (p<0,05). CONCLUSÃO: O UST foi eficaz no processo de reparo tecidual, conferindo ao tecido muscular maior resistência à tração e absorção de energia. .

INTRODUCTION: Despite controversial, ultrasonic therapy (UST) is commonly applied in sports rehabilitation practice to accelerate tissue repair after muscle damage. Muscle injuries have a negative influence on the mechanical properties of skeletal striated muscles and some scientific evidence shows that UST could have a positive effects on tissue repair and, therefore, on its mechanical properties. OBJECTIVE: To analyze the effect of UST on muscle repair by evaluation of the mechanical properties of rat muscles after cryolesion. METHODS: We used 30 adult Wistar rats which were divided into three groups: CG (control group), IG (injured group) and IGUST (injured plus UST) - (frequency of 1 MHz; intensity of 0.5 W/cm2; pulsed to 50%; four minutes daily) for seven consecutive days. The mechanical properties obtained through mechanical tests were evaluated in a universal testing machine. RESULTS: Data were statistically analyzed with a significance level of 95% (p<0.05). After seven treatment sessions, we found significant improvement in the mechanical properties of load at the proportionality limit, load at maximum limit and resilience in IGUST compared to IG (p<0.05). CONCLUSION: The UST was effective in the tissue repair process, giving higher tensile strength and energy absorption to the muscle tissue. .

INTRODUCCIÓN: A pesar de algunas controversias cuanto a su utilización, el ultrasonido terapéutico (UST) es un recurso aplicado comúnmente en la rehabilitación deportiva para aceleración de la reparación tecidual de lesiones musculares. Se sabe que las lesiones musculares influyen negativamente en las propiedades mecánicas de la musculatura estriada esquelética y algunas evidencias demuestran que el UST podría tener efectos sobre la reparación muscular y, en consecuencia, con respecto a sus propiedades mecánicas. OBJETIVO: Analizar el efecto del UST en la reparación tecidual por medio de las propiedades mecánicas musculares de ratones después de traumatismo por criolesión. MÉTODOS: Se utilizaron, en el estudio, 30 ratones de la raza Wistar, los cuales fueron divididos en tres grupos: grupo de control intacto (GC), grupo lesionado sin tratamiento (GL) y grupo lesionado y estimulado con UST (frecuencia de 1 MHz; intensidad de 0,5 W/cm2; ciclo de trabajo de 50%, durante cuatro minutos diarios) a lo largo de siete días consecutivos (GLUST). Las propiedades mecánicas obtenidas, mediante el ensayo mecánico de tracción, fueron evaluadas en una máquina universal de ensayos. RESULTADOS: Se analizaron estadísticamente, con nivel de significancia de 95% (P<0,05). Después de siete sesiones de entrenamiento, hubo mejora estadísticamente significativa en las propiedades mecánicas de carga en el límite de proporcionalidad, carga en el límite máximo y resiliencia para el GLUST en relación con el GL (p<0,05). CONCLUSIÓN: El UST fue eficaz en el proceso de reparación tecidual, dando al tejido muscular más resistencia cuanto a la tracción y la absorción de energía. .

Overview of ultrasound usage trends in orthopedic and sports physiotherapy.

BACKGROUND: The purpose of this study is to examine current beliefs about the use, the clinical importance, the theoretical fundamentals and the utilization criteria of therapeutic ultrasound (TUS) among physical therapists on the clinical practice in orthopedic and sports physiotherapy in Brazil. METHODS: A brief survey was developed based on previous studies and was sent to 55 physical therapists with advanced competency in orthopedics and sports physiotherapy. The questions addressed general topics about the professional profile and ultrasound usage and dosage. RESULTS: Our data show the wide availability and frequent use of TUS in this sample of physical therapists. TUS is used in distinct musculoskeletal injuries and/or disorders in both acute and chronic conditions. Muscles, tendons and ligaments represented the major structures where TUS is used. Questions on the basic theory of TUS demonstrated a lack of knowledge of the ultrasound physiological effects as well as its interaction with biological tissues and TUS absolute contraindication. CONCLUSION: A Brazilian profile about the US usage and dosage in orthopedic and sports physiotherapy is presented and highlights the need for a continuous upgrading process and further research into its effects.