Cleavage of caspases-1, -3, -6, -8 and -9 substrates by proteases in skeletal muscles from mice undergoing cancer cachexia

A prominent feature of several type of cancer is cachexia. This syndrome causes a marked loss of lean body mass and muscle wasting, and appears to be mediated by cytokines and tumour products. There are several proteases and proteolytic pathways that could be responsible for the protein breakdown. In the present study, we investigated whether caspases are involved in the proteolytic process of skeletal muscle catabolism observed in a murine model of cancer cachexia (MAC16), in comparison with a related tumour (MAC13), which does not induce cachexia. Using specific peptide substrates, there was an increase of 54% in the proteolytic activity of caspase-1, 84% of caspase-8, 98% of caspase-3 151% to caspase-6 and 177% of caspase-9, in the gastrocnemius muscle of animals bearing the MAC16 tumour (up to 25% weight loss), in relation to muscle from animals bearing the MAC13 tumour (1-5% weight loss). The dual pattern of 89 kDa and 25 kDa fragmentation of poly (ADP-ribose) polymerase (PARP) occurred in the muscle samples from animals bearing the MAC16 tumour and with a high amount of caspase-like activity. Cytochrome c was present in the cytosolic fractions of gastrocnemius muscles from both groups of animals, suggesting that cytochrome c release from mitochondria may be involved in caspase activation. There was no evidence for DNA fragmentation into a nucleosomal ladder typical of apoptosis in the muscles of either group of mice. This data supports a role for caspases in the catabolic events in muscle involved in the cancer cachexia syndrome. © 2001 Cancer Research Campaign.

Initiation and progression of ossification of the posterior longitudinal ligament of the cervical spine in the hereditary spinal hyperostotic mouse (twy/twy)

Ossification of the posterior longitudinal ligament (OPLL) is a significantly critical pathology that can eventually cause serious myelopathy. Ossification commences in the vertebral posterior longitudinal ligaments, and intensifies and spreads with the progression of the disease, resulting in osseous projections and compression of the spinal cord. However, the paucity of histological studies the underlying mechanisms of calcification and ossification processes remain obscure. The pathological process could be simulated in the ossifying process of the ligament in mutant spinal hyperostotic mouse (twy/twy). The aim of this study is to observe that enlargement of the nucleus pulposus followed by herniation, disruption and regenerative proliferation of annulus fibrosus cartilaginous tissues participated in the initiation of ossification of the posterior longitudinal ligament of twy/twy mice.

Apoptosis of neurons and oligodendrocytes in the spinal cord of spinal hyperostotic mouse (twy/twy):possible pathomechanism of human cervical compressive myelopathy

Cervical compressive myelopathy is the most serious complication of cervical spondylosis or ossification of the posterior longitudinal ligament (OPLL) and the most frequent cause of spinal cord dysfunction. There is little information on the exact pathophysiological mechanism responsible for the progressive loss of neural tissue in the spinal cord of such patients. In this study, we used the spinal hyperostotic mouse (twy/twy) as a suitable model of human spondylosis, and OPLL to investigate the cellular and molecular changes in the spinal cord. Mutant twy/twy mouse developed ossification of the ligamentum flavum at C2-C3 and exhibited progressive paralysis.

The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice

STUDY DESIGN: The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. OBJECTIVE: To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. SUMMARY OF BACKGROUND DATA: Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. METHODS: AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of ß-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS.: Four weeks after injection, the retrograde delivery of the LacZ marker gene was identified in cervical spinal neurons and some glial cells, including oligodendrocytes in the white matter of the spinal cord, in both the twy/twy mouse and the heterozygous Institute of Cancer Research mouse (+/twy). In the compressed spinal cord of twy/twy mouse, AdV-BDNF gene transfection resulted in a significant decrease in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells present in the spinal cord and a downregulation in the caspase apoptotic pathway compared with AdV-LacZ (control) gene transfection. There was a marked and significant increase in the areas of the spinal cord of AdV-BDNF-injected mice that were NF- and NG2-immunopositive compared with AdV-LacZ-injected mice, indicating the increased presence of neurons and oligodendrocytes in response to BDNF transfection. CONCLUSION: Our results demonstrate that targeted retrograde BDNF gene delivery suppresses apoptosis in neurons and oligodendrocytes in the chronically compressed spinal cord of twy/twy mouse. Further work is required to establish whether this method of gene delivery may provide neuroprotective effects in other situations of compressive spinal cord injury.

The prevalence and phenotype of activated microglia/macrophages within the spinal cord of the hyperostotic mouse (twy/twy) changes in response to chronic progressive spinalcord compression:implications for human cervical compressive myelopathy

Background:Cervical compressive myelopathy, e.g. due to spondylosis or ossification of the posterior longitudinal ligament is a common cause of spinal cord dysfunction. Although human pathological studies have reported neuronal loss and demyelination in the chronically compressed spinal cord, little is known about the mechanisms involved. In particular, the neuroinflammatory processes that are thought to underlie the condition are poorly understood. The present study assessed the localized prevalence of activated M1 and M2 microglia/macrophages in twy/twy mice that develop spontaneous cervical spinal cord compression, as a model of human disease.Methods:Inflammatory cells and cytokines were assessed in compressed lesions of the spinal cords in 12-, 18- and 24-weeks old twy/twy mice by immunohistochemical, immunoblot and flow cytometric analysis. Computed tomography and standard histology confirmed a progressive spinal cord compression through the spontaneously development of an impinging calcified mass.Results:The prevalence of CD11b-positive cells, in the compressed spinal cord increased over time with a concurrent decrease in neurons. The CD11b-positive cell population was initially formed of arginase-1- and CD206-positive M2 microglia/macrophages, which later shifted towards iNOS- and CD16/32-positive M1 microglia/macrophages. There was a transient increase in levels of T helper 2 (Th2) cytokines at 18 weeks, whereas levels of Th1 cytokines as well as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and macrophage antigen (Mac) -2 progressively increased.Conclusions:Spinal cord compression was associated with a temporal M2 microglia/macrophage response, which may act as a possible repair or neuroprotective mechanism. However, the persistence of the neural insult also associated with persistent expression of Th1 cytokines and increased prevalence of activated M1 microglia/macrophages, which may lead to neuronal loss and demyelination despite the presence of neurotrophic factors. This understanding of the aetiopathology of chronic spinal cord compression is of importance in the development of new treatment targets in human disease. © 2013 Hirai et al.

Fiber-type transitions and satellite cell activation in low-frequency-stimulated muscles of young and aging rats

We examined satellite cell content and the activity of satellite cell progeny in tibialis anterior muscles of young (15 weeks) and aging (101 weeks) Brown Norway (BN) rats, after they were exposed for 50 days to a standardized and highly reproducible regime of chronic low-frequency electrical stimulation. Chronic low-frequency electrical stimulation was successful in inducing fast-to-slow fiber-type transformation, characterized by a 2.3-fold increase in the proportion of IIA fibers and fourfold and sevenfold decreases in the proportion of IID/X and IIB fibers in both young and aging BN rats. These changes were accompanied by a twofold increase in the satellite cell content in both the young and aging groups; satellite cell content reached a level that was significantly higher in the young group (p < .04). The total muscle precursor cell content (i.e., satellite cells plus progeny), however, did not differ between groups, because there was a greater number of satellite cell progeny passing through the proliferative and differentiative compartments of the aging group. The resulting 1.5-fold increase in myonuclear content was similar in the young and aging groups. We conclude that satellite cells and satellite cell progeny of aging BN rats possess an unaltered capacity to contribute to the adaptive response.

Leg muscles motion during whole body linear frequency sweep vibration

Whole body vibration (WBV) aims to mechanically activate muscles by eliciting stretch reflexes. Mechanical vibrations are usually transmitted to the patient body standing on a oscillating plate. WBV is now more and more utilized not only for fitness but also in physical therapy, rehabilitation and in sport medicine. Effects depend on intensity, direction and frequency of vibration; however, the training frequency is one of the most important factors involved. A preliminary vibratory session can be dedicated to find the best vibration frequency for each subject by varying, stepwise, the stimulation frequency and analyzing the resulting EMG activity. This study concentrates on the analysis of muscle motion in response to a vibration frequency sweep, while subjects held two different postures. The frequency of a vibrating platform was increased linearly from 10 to 60 Hz in 26 s, while platform and single muscles (Rectus Femoris, Biceps Femoris - long head and Gastrocnemius Lateralis) motions were monitored using tiny, lightweight three-axial MEMS accelerometers. Displacements were estimated integrating twice the acceleration data after gravity contribution removal. Mechanical frequency response (amplitude and phase) of the mechanical chains ending at the single muscles was characterized. Results revealed a mechanical resonant-like behavior at some muscles, very similar to a second-order system in the frequency interval explored; resonance frequencies and dumping factors depended on subject and its positioning onto the vibrating platform. Stimulation at the resonant frequency maximizes muscle lengthening, and in turn muscle spindle solicitation, which produce muscle activation. © 2009 Springer-Verlag.

A lumped parameter model for the analysis of the motion of the muscles of the lower limbs under whole-body vibration

Through a lumped parameter modelling approach, a dynamical model, which can reproduce the motion of the muscles of a human body standing in different postures during Whole Body Vibrations (WBVs) treatment, has been developed. The key parameters, associated to the dynamics of the motion of the muscles of the lower limbs, have been identified starting from accelerometer measurements. The developed model can be usefully applied to the optimization of WBVs treatments which can effectively enhance muscle activation. © 2013 IEEE.

Measurement of intervertebral cervical motion by means of dynamic X-ray image processing and data interpolation

Accurate measurement of intervertebral kinematics of the cervical spine can support the diagnosis of widespread diseases related to neck pain, such as chronic whiplash dysfunction, arthritis, and segmental degeneration. The natural inaccessibility of the spine, its complex anatomy, and the small range of motion only permit concise measurement in vivo. Low dose X-ray fluoroscopy allows time-continuous screening of cervical spine during patient's spontaneous motion. To obtain accurate motion measurements, each vertebra was tracked by means of image processing along a sequence of radiographic images. To obtain a time-continuous representation of motion and to reduce noise in the experimental data, smoothing spline interpolation was used. Estimation of intervertebral motion for cervical segments was obtained by processing patient's fluoroscopic sequence; intervertebral angle and displacement and the instantaneous centre of rotation were computed. The RMS value of fitting errors resulted in about 0.2 degree for rotation and 0.2 mm for displacements. © 2013 Paolo Bifulco et al.

Analysis and modelling of muscles motion during whole body vibration

The aim of the study is to characterize the local muscles motion in individuals undergoing whole body mechanical stimulation. In this study we aim also to evaluate how subject positioning modifies vibration dumping, altering local mechanical stimulus. Vibrations were delivered to subjects by the use of a vibrating platform, while stimulation frequency was increased linearly from 15 to 60Hz. Two different subject postures were here analysed. Platform and muscles motion were monitored using tiny MEMS accelerometers; a contra lateral analysis was also presented. Muscle motion analysis revealed typical displacement trajectories: motion components were found not to be purely sinusoidal neither in phase to each other. Results also revealed a mechanical resonant-like behaviour at some muscles, similar to a second-order system response. Resonance frequencies and dumping factors depended on subject and his positioning. Proper mechanical stimulation can maximize muscle spindle solicitation, which may produce a more effective muscle activation. © 2010 M. Cesarelli et al.

Anticancer effects of Curcuma C20-dialdehyde against colon and cervical cancer cell lines

Background: Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. Materials and Methods: Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. Results: Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of 65.4±1.74 μg/ml, 58.4±5.20 μg/ml and 72.0±0.03 μg/ml, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. Conclusions: Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.