Unacylated ghrelin normalizes skeletal muscle oxidative stress and prevents muscle catabolism by enhancing tissue mitophagy in experimental chronic kidney disease.

Unacylated ghrelin (UnAG) may lower skeletal muscle oxidative stress, inflammation, and insulin resistance in lean and obese rodents. UnAG-induced autophagy activation may contribute to these effects, likely involving removal of dysfunctional mitochondria (mitophagy) and redox state maintenance. In chronic kidney disease (CKD) oxidative stress, inflammation and insulin resistance may negatively influence patient outcome by worsening nutritional state through muscle mass loss. Here we show in a 5/6 nephrectomy (Nx) CKD rat model that 4 d s.c. UnAG administration (200 µg twice a day) normalizes CKD-induced loss of gastrocnemius muscle mass and a cluster of high tissue mitochondrial reactive oxygen species generation, high proinflammatory cytokines, and low insulin signaling activation. Consistent with these results, human uremic serum enhanced mitochondrial reactive oxygen species generation and lowered insulin signaling activation in C2C12 myotubes while concomitant UnAG incubation completely prevented these effects. Importantly, UnAG enhanced muscle mitophagy in vivo and silencing RNA-mediated autophagy protein 5 silencing blocked UnAG activities in myotubes. UnAG therefore normalizes CKD-induced skeletal muscle oxidative stress, inflammation, and low insulin signaling as well as muscle loss. UnAG effects are mediated by autophagy activation at the mitochondrial level. UnAG administration and mitophagy activation are novel potential therapeutic strategies for skeletal muscle metabolic abnormalities and their negative clinical impact in CKD.-Gortan Cappellari, G., Semolic, A., Ruozi, G., Vinci, P., Guarnieri, G., Bortolotti, F., Barbetta, D., Zanetti, M., Giacca, M., Barazzoni, R. Unacylated ghrelin normalizes skeletal muscle oxidative stress and prevents muscle catabolism by enhancing tissue mitophagy in experimental chronic kidney disease.

In vivo visualization of gold-loaded cells in mice using x-ray computed tomography.

The ability to perform cell tracking using x-ray computed tomography combined with gold nanoparticles has been demonstrated recently on ex vivo samples using different malignant and nonmalignant cell lines. Here we proved the concept of the method for in vivo assessment in a small-animal model of malignant brain tumors. The limitations of the method due to radiation dose constraints were investigated using Monte Carlo simulations. Taking into consideration different x-ray entrance doses and the spatial resolution, the visibility of the cell clusters was evaluated. The results of the experiments conducted on mice implanted with F98 tumor cells confirmed the prediction of the Monte Carlo calculations. Small clusters of cells exogenously loaded with gold nanoparticles could be visualized using our in vivo method. FROM THE CLINICAL EDITOR: This article discusses the use of CT-based detection of gold nanoparticle loaded cells of interest in small-animal models of malignant brain tumors, where small clusters of cells loaded with gold nanoparticles could be visualized.

n-3 PUFA dietary lipid replacement normalizes muscle mitochondrial function and oxidative stress through enhanced tissue mitophagy and protects from muscle wasting in experimental kidney disease.

INTRODUCTION AND METHODS: Skeletal muscle mitochondrial dysfunction may cause tissue oxidative stress and consequent catabolism in chronic kidney disease (CKD), contributing to patient mortality. We investigated in 5/6-nephrectomized (Nx) rats the impact of n3-polyunsaturated fatty-acids (n3-PUFA) isocaloric partial dietary replacement on gastrocnemius muscle (Gm) mitochondrial master-regulators, ATP production, ROS generation and related muscle-catabolic derangements. RESULTS: Nx had low Gm mitochondrial nuclear respiratory factor-2 and peroxisome proliferator-activated receptor gamma coactivator-1alpha, low ATP production and higher mitochondrial fission-fusion protein ratio with ROS overproduction. n3-PUFA normalized all mitochondrial derangements and pro-oxidative tissue redox state (oxydized to total glutathione ratio). n3-PUFA also normalized Nx-induced muscle-catabolic proinflammatory cytokines, insulin resistance and low muscle weight. Human uremic serum reproduced mitochondrial derangements in C2C12 myotubes, while n3-PUFA coincubation prevented all effects. n3-PUFA also enhanced muscle mitophagy in-vivo and siRNA-mediated autophagy inhibition selectively blocked n3-PUFA-induced normalization of C2C12 mitochondrial ROS production. CONCLUSIONS: In conclusion, dietary n3-PUFA normalize mitochondrial master-regulators, ATP production and dynamics in experimental CKD. These effects occur directly in muscle cells and they normalize ROS production through enhanced mitophagy. Dietary n3-PUFA mitochondrial effects result in normalized catabolic derangements and protection from muscle wasting, with potential positive impact on patient survival.

The "Gastrocnemius-Achilles Tendon-Calcaneus Complex": Different Responses after Percutaneous versus Vulpius Achilles Tendon Lengthening in New Zealand White Rabbits.

BACKGROUND: This study aimed to describe the clinical, radiological, biomechanical, electromyographic, and histoenzymologic modifications in the "Gastrocnemius-Achilles Tendon-Calcaneus complex" caused by percutaneous Achilles tendon lengthening (PATL) versus Vulpius Achilles tendon lengthening (VATL) in New Zealand White (NZW) rabbits. MATERIALS AND METHODS: Eight female NZW rabbits were used at 7 months of age. Two rabbits were euthanized before surgery for anatomical dissection, three underwent PATL (two bilateral and one unilateral), and the three others underwent VATL (two bilateral and one unilateral). Clinical examination, biomechanics, electromyography, standard radiographs and magnetic resonance imaging (MRI), and histology and histoenzymology were assessed after surgery. RESULTS: At the end of the experiment, the subjects showed good clinical status but different functional outcomes of surgery: rabbits submitted to PATL developed permanent limp and lost their capacity to jump compared to rabbits submitted to VATL which remained able to ambulate and jump normally. Standard radiographs and MRI showed that PATL led to significantly greater increase in dorsal or anterior flexion of the tibiotarsal angle (TT angle) compared to VATL, whereas electromyographic and histoenzymologic observations of muscle unit showed little or no variation between the two groups of operated rabbits. CONCLUSIONS: Although PATL leads to greater improvement in dorsal or anterior flexion (TT angle) of the rabbit ankle compared to VATL, it has negative effects on functional outcome as it reduces the contractile capacity of the rabbit muscle unit, ultimately impairing the ability to ambulate and jump.

Omega 3 Polyunsaturated Fatty Acids Improve Endothelial Dysfunction in Chronic Renal Failure: Role of eNOS Activation and of Oxidative Stress.

BACKGROUND: Endothelial dysfunction is a key vascular alteration in chronic kidney disease (CKD). Omega 3 (n-3) polyunsaturated fatty acids (PUFA) reduce vascular oxidative stress and inflammation. We investigated whether n-3 PUFA could reverse endothelial dysfunction in CKD by improving endothelial nitric oxide synthase (eNOS) function and oxidative stress. METHODS: 5/6 nephrectomized male Wistar rats (CKD; n = 10) and sham operated animals (SHAM; n = 10) were treated for 6 weeks with standard diet. An additional group of CKD rats were fed an n-3 PUFA enriched diet (CKD + PUFA; n = 10). We then measured endothelium-dependent (EDD) and -independent vasodilation, markers of endothelial function and of oxidative stress in thoracic aortas. RESULTS: Compared to SHAM, in CKD aortas EDD and eNOS expression were reduced (p < 0.05) and 3-nitrotyrosine levels were increased, while expression of NADPH oxidase subunits NOX4 and p22phox was similar. In-vitro incubation with Tiron failed to reverse endothelial dysfunction in CKD. In CKD + PUFA, EDD improved (p < 0.05) compared with CKD rats, while blockade of eNOS by L-NAME worsened EDD. These effects were accompanied by increased (p < 0.05) eNOS and reduced (p < 0.05) expression of NOX4 and 3-nitrotyrosine levels. CONCLUSION: Collectively, these findings indicate that n-3 PUFA improve endothelial dysfunction by restoring NO bioavailability in CKD.

Radiologic and histological observations in experimental T1-T12 dorsal arthrodesis: A qualitative description of T1-T12 segment and other body parts involved, between prepubertal age and skeletal maturityxs.

BACKGROUND: This experimental study provides a qualitative description and the morpho-structural features of the fusions taking place in the thoracic spine between prepubertal age and skeletal maturity. There is a lack of informations regarding the influence of partial or total dorso-thoracic vertebral arthrodesis on the development of the thoracic cage as well as its potential effects on different intra and extra-thoracic organs. This study admits the hypothesis that vertebral arthrodesis may have influence on other body areas and so, it intends to verify the possible secondary involvement of other body parts, such as intervertebral discs, cervical and thoracic spinal ganglia, sternocostal cartilage, ovaries and lungs. MATERIALS AND METHODS: Fifty-four female New Zealand white rabbits were submitted to dorsal arthrodesis. The radiologic imaging and light microscopy histological pictures were taken and studied in all. Computed tomography (CT) scan measurements were performed in operated and sham operated rabbits at different time. Similarly, histological specimens of intervertebral discs, cervical and thoracic spinal ganglia, sternocostal cartilage, ovaries and lungs were analyzed at different times. The study ended at the age of 17-18 months. RESULTS: Most rabbits had formed a fusion mass, which was only fibrous at first, then osteofibrous and finally, in the older subjects, structured in lamellar-osteon tissue. Intervertebral foramens were negatively involved in vertebral arthrodesis, as shown by CT scans. Intervertebral discs showed irregular aspects. The increase of atresic follicles and the reduction of primordial follicles in operated rabbits led to the hypothesis of a cause-effect relationship between arthrodesis and modified hormonal status. Dorsal root ganglia showed microscopic alterations in operated rabbits especially. CONCLUSIONS: The process of fusion mass and bone formation, associated with the arthrodesis, involves at different degrees of the vertebral bodies, discs and intervertebral foramens, ganglia and spinal nerve roots.

Effect of thoracic arthrodesis in prepubertal New Zealand white rabbits on cardio-pulmonary function.

BACKGROUND: This experimental study was aimed at evaluating the type of cardiac and pulmonary involvement, in relation to changes of the thoracic spine and cage in prepubertal rabbits with nondeformed spine following dorsal arthrodesis. The hypothesis was that T1-T12 arthrodesis modified thoracic dimensions, but would not modify cardiopulmonary function once skeletal maturity was reached. MATERIALS AND METHODS: The study was conducted in 16 female New Zealand White (NZW) rabbits. Nine rabbits were subjected to T1-T12 dorsal arthrodesis while seven were sham-operated. Echocardiographic images were obtained at 12 months after surgery and parameters for 2-dimensional and M-mode echocardiographic variables were assessed. One week before echocardiographic examination, blood samples were withdrawn from the animals' central artery of the left ear to obtain blood gas values. One week after echocardiographic assessment, a thoracic CT scan was performed under general anesthesia. Chest depth (CD) and width (CW), thoracic kyphosis (ThK) and sternal length (StL) were measured; thoracic index (ThI), expressed as CD/CW ratio. All subjects were euthanized after the CT scan. Heart and lungs were subsequently removed to measure weight and volume. RESULTS: The values for 2-dimensional and M-mode echocardiographic variables were found to be uniformly and significantly higher, compared to those reported in anesthetized rabbits. CD, ThK, and StL were considerably lower in operated rabbits, as compared to the ones that were sham-operated. Similarly, the ThI was lower in operated rabbits than in sham-operated ones. CONCLUSION: Irregularities in thoracic cage growth resulting from thoracic spine arthrodesis did not alter blood and echocardiographic parameters in NZW rabbits.