scholarly journals MyoBio: An automated bioreactor system technology for standardized perfusion-decellularization of whole skeletal muscle

Paul Ritter ◽  
Aijia Cai ◽  
Barbara Reischl ◽  
Maren Fiedler ◽  
Gerhard Prol ◽  
D. E. Philpott ◽  
A. Takahashi

Two month, eight month and two year old rats were treated with 10 or 20 mg/kg of E. Coli endotoxin I. P. The eight month old rats proved most resistant to the endotoxin. During fixation the aorta, carotid artery, basil arartery of the brain, coronary vessels of the heart, inner surfaces of the heart chambers, heart and skeletal muscle, lung, liver, kidney, spleen, brain, retina, trachae, intestine, salivary gland, adrenal gland and gingiva were treated with ruthenium red or alcian blue to preserve the mucopolysaccharide (MPS) coating. Five, 8 and 24 hrs of endotoxin treatment produced increasingly marked capillary damage, disappearance of the MPS coating, edema, destruction of endothelial cells and damage to the basement membrane in the liver, kidney and lung.

Joachim R. Sommer ◽  
Nancy R. Wallace

After Howell (1) had shown that ruthenium red treatment of fixed frog skeletal muscle caused collapse of the intermediate cisternae of the sarcoplasmic reticulum (SR), forming a pentalaminate structure by obi iterating the SR lumen, we demonstrated that the phenomenon involves the entire SR including the nuclear envelope and that it also occurs after treatment with other cations, including calcium (2,3,4).From these observations we have formulated a hypothesis which states that intracellular calcium taken up by the SR at the end of contraction causes the M rete to collapse at a certain threshold concentration as the first step in a subsequent centrifugal zippering of the free SR toward the junctional SR (JSR). This would cause a) bulk transport of SR contents, such as calcium and granular material (4) into the JSR and, b) electrical isolation of the free SR from the JSR.

A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

Joachim R. Sommer ◽  
Teresa High ◽  
Betty Scherer ◽  
Isaiah Taylor ◽  
Rashid Nassar

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.

C. Uphoff ◽  
C. Nyquist-Battie ◽  
T.B. Cole

Ultrastructural alterations of skeletal muscle have been observed in adult chronic alcoholic patients. However, no such study has been performed on individuals prenatally exposed to ethanol. In order to determine if ethanol exposure in utero in the latter stages of muscle development was deleterious, skeletal muscle was obtained from newborn guinea pigs treated in the following manner. Six Hartly strain pregnant guinea pigs were randomly assigned to either the ethanol or the pair-intubated groups. Twice daily the 3 ethanol-treated animals were intubated with Ensure (Ross Laboratories) liquid diet containing 30% ethanol (6g/Kg pre-pregnant body weight per day) from day 35 of gestation until parturition at day 70±1 day. Serum ethanol levels were determined at 1 hour post-intubation by the Sigma alcohol test kit. For pair-intubation the Ensure diet contained sucrose substituted isocalorically for ethanol. Both food and water intake were monitored.

S.L. White ◽  
C.B. Jensen ◽  
D.D. Giera ◽  
D.A. Laska ◽  
M.N. Novilla ◽  

In vitro exposure to LY237216 (9-Deoxo-11-deoxy-9,11-{imino[2-(2-methoxyethoxy)ethylidene]-oxy}-(9S)-erythromycin), a macrolide antibiotic, was found to induce cytoplasmic vacuolation in L6 skeletal muscle myoblast cultures (White, S.L., unpubl). The present study was done to determine, by autoradiographic quantitative analysis, the subcellular distribution of 3H-LY237216 in L6 cells.L6 cells (ATCC, CRL 1458) were cultured to confluency on polycarbonate membrane filters (Millipore Corp., Bedford, MA) in M-199 medium (GIBCO® Labs) with 10% fetal bovine serum. The cells were exposed from the apical surface for 1-hour to unlabelled-compound (0 μCi/ml) or 50 (μCi/ml of 3H-LY237216 at a compound concentration of 0.25 mg/ml. Following a rapid rinse in compound-free growth medium, the cells were slam-frozen against a liquid nitrogen cooled, polished copper block in a CF-100 cryofixation unit (LifeCell Corp., The Woodlands, TX). Specimens were dried in the MDD-C Molecular Distillation Drier (LifeCell Corp.), vapor osmicated and embedded in Spurrs low viscosity resin. Ultrathin sections collected on formvar coated stainless steel grids were counter-stained, then individually mounted on corks. A monolayer of Ilford L4 nuclear emulsion (Polysciences, Inc., Warrington, PA) was placed on the sections, utilizing a modified “loop method”. The emulsions were exposed for 7-weeks in a light-tight box at 4°C. Autoradiographs were developed in Microdol-X developer and examined on a Philips EM410LS transmission electron microscope. Quantitative analysis of compound localization employed the point and circle approach of Williams; incorporating the probability circle method of Salpeter and McHenry.

I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

J.P. Schroeter ◽  
M.A. Goldstein ◽  
J.P. Bretaudiere ◽  
L.H. Michael ◽  
R.L. Sass

We have recently established the existence of two structural states of the Z band lattice in cross section in cardiac as well as in skeletal muscle. The two structural states are related to the contractile state of the muscle. In skeletal muscle at rest, the Z band is in the small square (ss) lattice form, but tetanized muscle exhibits the basket weave (bw) form. In contrast, unstimu- lated cardiac muscle exhibits the bw form, but cardiac muscles exposed to EGTA show the ss form.We have used two-dimensional computer enhancement techniques on digitized electron micrographs to compare each lattice form as it appears in both cardiac and skeletal muscle. Both real space averaging and fourier filtering methods were used. Enhanced images were displayed as grey-scale projections, as contour maps, and in false color.There is only a slight difference between the lattices produced by the two different enhancement techniques. Thus the information presented in these images is not likely to be an artifact of the enhancement algorithm.

2005 ◽  
Vol 41 ◽  
pp. 173-186 ◽  
Didier Attaix ◽  
Sophie Ventadour ◽  
Audrey Codran ◽  
Daniel Béchet ◽  
Daniel Taillandier ◽  

The ubiquitin–proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin–protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients.

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