Effects of protein-calorie restriction on mechanical function of hypertrophied cardiac muscle

OBJECTIVE: To assess the effect of food restriction (FR) on hypertrophied cardiac muscle in spontaneously hypertensive rats (SHR). METHODS: Isolated papillary muscle preparations of the left ventricle (LV) of 60-day-old SHR and of normotensive Wistar-Kyoto (WKY) rats were studied. The rats were fed either an unrestricted diet or FR diet (50% of the intake of the control diet) for 30 days. The mechanical function of the muscles was evaluated through monitoring isometric and isotonic contractions. RESULTS: FR caused: 1) reduction in the body weight and LV weight of SHR and WKY rats; 2) increase in the time to peak shortening and the time to peak developed tension (DT) in the hypertrophied myocardium of the SHR; 3) diverging changes in the mechanical function of the normal cardiac muscles of WKY rats with reduction in maximum velocity of isotonic shortening and of the time for DT to decrease 50% of its maximum value, and increase of the resting tension and of the rate of tension decline. CONCLUSION: Short-term FR causes prolongation of the contraction time of hypertrophied muscles and paradoxal changes in mechanical performance of normal cardiac fibers, with worsening of the shortening indices and of the resting tension, and improvement of the isometric relaxation.

Amitriptyline aggravates the fibrosis process in a rat model of infravesical obstruction

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mitochondrial alterations and apoptosis in smooth muscle from aged rats

We studied changes in mitochondrial morphology and function in the smooth muscle of rat colon. Under confocal microscopy, tissues loaded with potentiometric dye displayed rapid and spontaneous depolarization. Cyclosporin A (CsA), inhibitor of the permeability transition pore (PTP), caused an increase in mitochondrial membrane potential (DeltaPsi(m)) in tissues from adult young animals. In aged rats these changes were not observed. This suggests that physiological activation of PTP in aged rats is reduced. Electron microscopy showed alterations of the mitochondrial ultrastructure in tissues from aged rats involving a decreased definition of the cristae and fragmentation of the mitochondrial membranes. We also detected an increase in apoptotic cells in the smooth muscle from aged animals. Our results show that the aging process changes PTP activity, the ability to maintain DeltaPsi(m) and mitochondrial morphology. It is suggested that these can be associated with mitochondrial damage and cell death. (C) 2004 Elsevier B.V. All rights reserved.

Morphometric and morphological analysis of neuromuscular junction alterations in the denervated rat diaphragm

The morphological and structural alterations that occur in the neuromuscular junctions of the denervated rat diaphragm were studied. Fifteen adult male albino rats (Rattus norvegicus) aged about 60 days and with a mean weight of 200 g were used. Chronically denervated diaphragms were obtained and the animals were sacrificed after 4, 8 and 12 weeks of denervation. The left antimere of the diaphragm was denervated by section of the phrenic nerve and the right antimere was used as control. Each antimere was divided into three fragments: one was used for histochemical (nonspecific esterase) and morphometric study of neuromuscular junctions, and the other two were used for transmission and scanning electron microscopy (SEM) analysis. Histochemical analysis of the diaphragm neuromuscular junctions after denervation showed only small changes in junction morphology. However, these junctions became smaller and elongated and presented less visible contours with increasing time of denervation. Ultrastructural analysis of neuromuscular junctions after 12 weeks showed more or less organized junctional folds on the muscle fiber surface. The junctional cytoplasm exhibited important alterations such as mitochondrial degeneration and the presence of numerous filaments. SEM revealed the presence of deep primary synaptic grooves with peripheral excavations which housed the nerve terminal boutons and exhibited internally the secondary synaptic clefts present among the junctional folds of the sarcolemma. This study showed that some of the morphological changes demonstrated in other denervated striated skeletal muscles are not repeated at the same intensity or in the same temporal pattern in the rat diaphragm.

Cardiac remodeling in a rat model of diet-induced obesity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies

This was a study on the oxidative stress due to ischaemia (I) and reperfusion (R) in skeletal muscle tissue. Using a tourniquet, groups of rats were submitted to ischaemia for 4 h, followed by different reperfusion periods. The animals were divided in four groups: control; 4 h of ischaemia (IR); 4 h of ischaemia plus 1 h reperfusion (IR-1 h); 4 h of ischaemia plus 24 h reperfusion (IR-24 h); and 4 h of ischaemia plus 72 h reperfusion (IR-72 h). At the end of the procedures, samples of soleus muscle were collected and frozen in n-hexane at -70 degrees C. Cryostat sections were submitted to haematoxylin-eosin, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stains. An additional muscle sample was processed for electron microscopy. No alterations were found in control animals. IR group showed fibres had normal aspect besides some round, acidophilic and hypertrophic fibres. There were several fibres with angular outlines and smaller diameters in this group compared with control group. NADH-TR/SDH reaction was moderately intense in most fibres. In some fibres, cytoplasm showed areas without activity and other fibres had very intense reactivity. IR-1 h group showed oedema hypercontracted fibres with disorganized myofibrils, mitochondria with focal lesions and dilated sarcoplasmic reticulum. NADH-TR/SDH reaction was moderate to weak. IR-24 h showed intense inflammatory infiltrate in the endomysium and perimysium. NADH-TR/SDH reaction was similar to IR-1 h. IR-72 h showed necrotic fibres, areas with inflammatory infiltrate, reduced muscle fibres at different stages of necrosis and phagocytosis, and many small round and basophilic fibres characterizing a regeneration process. NADH-TR/SDH reaction was weak to negative. Our results suggest that ischaemia and the subsequent 1-, 24- and 72-h reperfusions induced progressive histological damage. Although progressive, it may be reversible because there were ultrastructural signs of recovery after 72-h reperfusion. This recovery could in part be due to the low oxidative stress identified by the morphological and histochemical analysis.

Remodeling of rat ventral prostate after castration involves heparanase-1

Androgen deprivation causes the rat ventral prostate to reduce to 10% of its original size by 21 days after castration. The regressive changes result from the loss of epithelial cells by apoptosis and marked reorganization of the stroma. We have investigated whether these changes are accompanied by variations in heparanase expression. The ventral prostate of castrated rats was collected and processed for the quantification of heparan sulfate (HS), for the measurement of heparanase expression and its localization by reverse transcription/polymerase chain reaction, Western blotting, and immunohistochemistry, and for transmission electron microscopy (TEM). Absolute HS content decreased significantly as early as day 7 after surgery. Heparanase mRNA peaked 7 days after castration. The heparanase proenzyme (65 kDa) and the active form (50 kDa) were identified and peaked on day 7 after castration; this coincided with maximum HS-degrading activity. Heparanase was located to the basolateral surface of epithelial cells and in the adjacent stroma. After castration, staining for heparanase was reduced in the epithelium and increased in the stroma. TEM revealed that the peak of heparanase expression at day 7 after castration was associated with extensive changes in the basement membrane of the epithelium, endothelium and smooth muscle cells involving cell shrinkage and/or deletion by apoptosis. These results suggest that heparanase expression increases after castration and correlates with a decreased amount of HS. This variation in heparanase expression is involved in tissue remodeling and in the control of the regressive pattern after 1 week of androgen deprivation.

Doxazosin Treatment Alters Stromal Cell Behavior and Increases Elastic System Fibers Deposition in Rat Prostate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of phonophoresis with Arnica montana onto acute inflammatory process in rat skeletal muscles: An experimental study

This study aimed at verifying the effects of phonophoresis associated with Arnica montana on the acute phase of an inflammatory muscle lesion. Forty Wistar male rats (300 +/- 50 g), of which the Tibialis Anterior muscle was surgically lesioned, were divided into four groups (n = 10 each): control group received no treatment; the ultrasound group (US) was treated in pulsed mode with 1-MHz frequency, 0.5 W/cm(2) intensity (spatial and temporal average - SATA), duty cycle of 1: 2 (2 ms on, 4 ms off, 50%), time of application 3 min per session, one session per day, for 3 days; the phonophoresis or ultrasound plus arnica (US+A) group was treated with arnica with the same US parameters plus arnica gel; and the arnica group (A) was submitted to massage with arnica gel, also for 3 min, once a day, for 3 days. Treatment started 24 h after the surgical lesion. on the 4th day after lesion creation, animals were sacrificed and sections of the lesioned, inflamed muscle were removed for quantitative (mononuclear and polymorphonuclear cell count) and qualitative histological analysis. Collected data from the 4 groups were statistically analyzed and the significance level set at p < 0.05. Results show higher mononuclear cell density in all three treated groups with no significant difference between them, but values were significantly different (p < 0.0001) when compared to control group's. As to polymorphonuclear cell density, significant differences were found between control group (p = 0.0134) and US, US+A and A groups; the arnica group presented lesser density of polymorphonuclear cells when compared (p = 0.0134) to the other groups. No significant difference was found between US and US+A groups. While the massage with arnica gel proved to be an effective anti-inflammatory on acute muscle lesion in topic use, these results point to ineffectiveness of Arnica montana phonophoresis, US having seemingly checked or minimized its anti-inflammatory effect. (C) 2008 Elsevier B. V. All rights reserved.

Differential distribution of some extracellular matrix fibers in an experimentally denervated rat megaileum

Absence of enteric neurons is associated with thickening of the intestinal muscularis externa in Chagas' disease. The thickening is due to hyperplasia and hypertrophy of the smooth muscle cells and increased extracellular matrix components. The influence of the nervous system on the structure of the smooth muscle cells and its associated matrix has been poorly investigated. An experimental model of denervation of the ileum in rats was performed by application of the surfactant agent benzalkonium chloride that selectively destroys the myenteric plexus. Three months later, ileal tissue samples were obtained and studied by histochemistry and transmission electron microsocopy. Sham operated rats were used as controls. The diameter of collagen fibrils was evaluated in electron micrographs. The histopathological analysis showed thickening of the muscular layer. The thin and weakly arranged collagen and reticulin fibers surrounding the smooth muscle cells, observed in control cases by Picrosirius polarization (PSP) stain method, corresponded to a population of loosely packed thin collagen fibrils of uniform diameters (mean = 29.16 nm) at the ultrastructural level. In contrast, the thick and strongly birefringent fibers around the muscle cells, observed in the treated group, stained by PSP, corresponded to densely packed thicker fibrils with large variation in diameter (mean = 39.41 nm). Comparison of the data demonstrated statistically significant difference between the groups suggesting that the replacement of loosely arranged reticulin fibers by fibrous tissue (with typical collagen fiber), may alter the biomechanical function resulting in impairment of muscular contraction. (c) 2007 Elsevier Ltd. All rights reserved.

Short-term stromal alterations in the rat ventral prostate following alloxan-induced diabetes and the influence of insulin replacement

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Localized matrix metalloproteinase (MMP)-2 and MMP-9 activity in the rat ventral prostate during the first week of postnatal development

The initial events in prostatic morphogenesis involve cell proliferation, epithelial canalization and outgrowth toward the stroma. We have hypothesized that stromal rearrangement takes place at the sites of epithelial growth and branching and that this rearrangement involves the action of gelatinases matrix metalloproteinase (MMP)-2 and MMP-9. Thus, the purpose of the present study was to characterize structural aspects of epithelial growth during the first week of postnatal development of the rat ventral prostate and to investigate the expression, localization and activity of MMP-2 and MMP-9 during this period by histological, ultrastructural and immunocytochemical analysis, in addition to gel zymography, in situ zymography and Western blotting. An increasing complexity of prostatic architeture was observed within the first postnatal week. Concurrently, the stroma became more organized and some cells differentiated into smooth muscle cells. Reticulin fibers appeared in a basket-like arrangement around both growing tips and epithelial sprouts, associated with a fainter staining for laminin. MMP-2 and MMP-9 activities were detected. MMP-2/MMP-9 expression decreased during the first week. Developing epithelial cords showed strong and difuse gelatinolytic activity. This activity coincided with the distribution of MMP-2 as determined by immunocytochemistry. on the other hand, MMP-9 was rather concentrated at the epithelial tips. These results suggest that gelatinolytic activity (with contribution of both MMP-2 and MMP-9) in the epithelium and at the epithelium-stroma interface are at least in part responsible for the tissue remodeling that allows epithelial growth and its projection into the surrounding stroma.

Diabetes induces stromal remodelling and increase in chondroitin sulphate proteoglycans of the rat ventral prostate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

High fat-induced obesity associated with insulin-resistance increases FGF-2 content and causes stromal hyperplasia in rat ventral prostate

Obesity affects sex hormone secretion, which can negatively influence prostatic structure, homeostasis, and disease. This investigation aimed to evaluate the repercussions of obesity induced by a high-fat diet on the rat prostate, with or without treatment with the aromatase inhibitor, Letrozole. Adult Wistar rats were fed a high-fat diet (20% saturated fat, O) for 15 weeks to induce obesity or received a balanced diet (4% fat, C). Then, a group of C and O rats were daily treated with Letrozole (1 mg/kg b.w. per day) for 2 weeks (CL and OL, respectively). Subsequently, ventral prostate was processed for analysis by transmission electron microscopy, immunohistochemistry, and Western blotting. Obesity decreased 70% of the testosterone plasma level. The prostate showed epithelial atrophy and dilated acini in the intermediate portion and epithelial wrinkling in the distal tips. The relative frequency of smooth muscle alpha-actin in the O group increased by 67%. Ultrastructurally, epithelial cells in obese animals presented altered secretory organelles, lipid droplets, and thicker subjacent fibromuscular layer. Letrozole treatment caused a partial restoration of the prostatic changes caused by obesity. Obesity increased the prostatic content of fibroblast growth factor-2 (FGF-2) by 150%, and Letrozole treatment increased this protein even more in the control and obese groups. This investigation shows that obesity provokes structural and ultrastructural changes in the epithelium of rat prostate; these changes might affect gland homeostasis and physiology. The epithelial and smooth muscle cell hyperplasia and increased FGF-2 expression observed in this experimental model of obesity/insulin-resistance might explain the high frequency of benign prostatic hyperplasia in insulin-resistant men.

DISTRIBUTION OF DIFFERENT FIBER TYPES OF M-EXTENSOR CARPI RADIALIS LONGUS OF THE RAT

As revealed by the NADH-diaphorase and myosine ATPase, the M. extensor carpi radialis longus of the rat possesses at least 3 main kinds of fibres, with different distribution on the superficial and deep portions of the muscle. The superficial portion revealed that 67.68 % are FG (fast-twitch-glycolytic) fibres, 14.72 % are FOG (fast-twitch-oxidative) fibres and 17.60 % are SO (slow-twitch-glycolytic) fibres. Already the deep portion revealed that 71.29 % are SO (slow-twitch-glycolytic) fibres, 17.46 % are FOG (fast-twitch-oxidative-glycolytic) fibres and 11.25 % are FG (fast-twitch-glycolytic) fibres. The miosine ATPase reaction was used to demonstrate contracting characteristics. These findings suggest that the movements of fast contraction of the M. extensor carpi radialis longus are predominant.