Rat Thigh Research Articles

Satellite cells surrounding axotomised rat dorsal root ganglion cells increase expression of a GFAP-like protein.

Segments of sciatic nerve, 8-10 mm long, were removed from the left thigh of adult Sprague-Dawley rats. One day to 6 weeks after operation the animals were killed and the 4th and 5th lumbar dorsal root ganglia dissected out on both the operated and the unoperated side. A monoclonal and two polyclonal antibodies to glial fibrillary acidic protein (GFAP) were used to localize GFAP-like molecules in cryostat sections of the ganglia by means of indirect immunofluorescence. Expression of GFAP in satellite cells, demonstrable by the binding of polyclonal antibodies, had increased noticeably by three days post operation and remained at a high level throughout the remaining period of the experiment. Whereas on the unoperated side only about 15% of the neurons in the ganglia were surrounded by GFAP-positive satellite cells, on the operated side about 88% of the neurons were surrounded by GFAP-positive cells. Satellite cells could not be labelled with the monoclonal antibodies to GFAP.

歯由来骨形成因子に関する実験的研究

A bone morphogenetic protein (BMP) that induces differentiation of mesenchymaltype cell into cartilage and bone was partially purified from bovine tooth. In this study, the activity of BMP was assayed by its ability to induce enchondral ossification after injections of samples into rat thigh muscles.Frozen teeth were ground to fine particles and demineralized in 0.6 N HCl. The demineralized tooth matrix was treated with 4 M guanidine hydrochloride to extract BMP. Three volumes of cold ethanol were added to the supernatant obtained by filtration and the insoluble materials formed were collected by centrifugation. These materials were redissolved in 4 M guanidine hydrochloride and subjected to gel filtration using a Superose 12 column. The precipitate formed by adding three volumes of cold ethanol to each fraction was collected by centrifugation and BMP activity was assayed.The molecular weight of BMP of bovine tooth analyzed by electrophoresis on a SDS polyacrylamide gel was 27, 000. The combination of partially purified BMP with 0.5% atelocollagen (in HCl, pH 3) more effectively enlarged the volumes of cartilage and bone than did BMPTalone, indicating usefulness of atelocollagen as a carrier. BMP induced enchondral ossification in the periosteum of the femur as well as in the muscle, suggesting that the periosteum includes cells that differentiate into osteoblasts in response to BMP.

A novel method for labelling of leukocytes with technetium-99m and its comparative evaluation for abscess scintigraphy

A new method, based on the pretreatment of leukocytes with glucoheptonate prior to treating with reduced 99mTc, has been developed for the preparation of 99mTc labelled leukocytes. The leukocytes labelled with a 99mTc concentration (5.59%/g tissue) similar to that of 111In-leukocytes (6.27%/g tissue), in the experimental abscess were in rat thigh. Concentration of 99mTc-leukocytes in blood at 24 h was only about 35% as compared to that of 111In-leukocytes. Biodistribution in the rat organs was similar in both cases, except in the liver where 99mTc-leukocytes exhibited about 4-fold greater concentration. Images of experimental abscess in rat by using 99mTc-leukocytes were comparable to those obtained with 111In-leukocytes.

Experimental study on mechanism of absorption of cryonecrotized tumor antigens

On Day 7 of subcutaneous MRMT-1 tumor inoculation in the thigh of Sprague-Dawley rats, the tumor was treated cryosurgically by contact method. Postoperative changes in local blood circulation were observed by colloidal carbon perfusion and hydrogen-clearance methods, and the pathway and time course of tumor antigen absorption were observed by measuring uptake in serum and lymph nodes of [ 3H]thymidine injected intratumorally. Results were: (1) At 0.5 to 1 hr after cryosurgery, vascular stasis, dilatation, and tortuosity were observed. There was no inflow of carbon and almost no uptake of [ 3H]thymidine. (2) At 6 hr, vascular stasis and sludging become more marked. There was no inflow of carbon, while uptake of [ 3H]thymidine markedly increased. (3) At 24 hr, some carbon inflow in fin vessels and partial recovery of blood circulation in the peritumoral region were observed. Rate of [ 3H]thymidine uptake was further increased. (4) At 72 hr, carbon inflow and blood circulation increased further, while the cryonecrotized tumer showed hyaline degeneration. (6) At 168 hr, increase of newly formed vessels and recovery of blood circulation were remarkable, but uptake of [ 3H]thymidine was decreased in parall with increased demarcation of the Cryonecrotized tumor. From these results, it was suggested that the absorption of Cryonecrotized tumor antigens started through lymphatic channels in the early period and then took place also through newly formed capillaries surrounding the tumor at 24 hr after cryosurgery and continued until 72 hr after cryosurgery.

Use of He-Ne Laser for Treatment of Soft Tissue Trauma: Evaluation by Gallium-67 Citrate Scanning

Sequential quantitative Gallium-67 Citrate (Ga-67) scanning was used to evaluate the resolution of inflammation in standardized bruises in rat thighs with and without Helium Neon (He-Ne) laser treatment. The severity of inflammation in the injured tissue was assessed as the ratio of Ga-67 localized in the bruise to the Ga-67 localized in the noninjured area of the same geometry. During the first 12 days after injury, both (He-Ne laser) treated and nontreated rats healed rapidly with no observable differences in Ga-67 localization. J Orthop Sports Phys Ther 1986;8(2):93-96.

DNA binding proteins of rat thigh muscle: Purification and characterization of an endonuclease

Two major DNA binding proteins of molecular weights 34,000 and 38,000 have been identified in the 30,000 g supernatant (S-30) fraction of rat thigh muscle extracts. The presence of 38 KD DNA binding protein in the muscle S-30 could be demonstrated only if Triton X-100 treated extracts were used for Afinity chromatography suggesting that this protein may be a membrane associated DNA binding protein. The 38 KD DNA binding protein differed from the 34 KD DNA binding protein also in its chromatographic behaviour in DE-52 columns in which the 38 KD protein was retained, while the 34 KD protein came out in the flow-through in an electrophoretically pure form. The 34 KD DNA binding protein can also be purified by precipitation with MgCl2. Incubation of 0 15 M NaCl eluates (containing the 38 KD and/or 34 KD DNA binding protein) in the presence of 100 mM Mg2+ resulted in the specific precipitation of the 34 KD protein. Prolonged incubation (30 days) of the 0.15 M NaCl eluates containing the two DNA binding proteins at 4°C led to the preferential degradation of the 34 KD DNA binding protein. Nitrocellulose filter binding assays indicated selective binding of purified 34 KD protein to ss DNA. Purified 34 KD DNA binding protein cleaved pBR 322 supercoiled DNA, and electrophoresis of the cleavage products in agarose gels revealed a major DNA band corresponding to the circular form of DNA.

Disposition of ipriflavone (TC-80) in rats and dogs.

Oral 14C-ipriflavone was absorbed by rats to give a maximum plasma 14C level at 1.5 h and a half-life of 5.8 h. In dogs, after po dosing, the plasma 14C peaked at 0.5 h, followed by gradual decline. The plasma of both animals contained mostly metabolites, with small amounts of unchanged ipriflavone. In rats, 14C was distributed widely in tissues, with relatively high concns. in the liver, kidney and gut. Distribution in rat thigh bone of unmetabolized ipriflavone was also demonstrated. 14C-Ipriflavone was eliminated mostly as metabolites within 48 and 72 h, respectively, in rats and dogs. Rats excreted more 14C in urine than in feces, whereas the reverse was noted in dogs. Biliary excretion and reabsorption of 14C were also obvious in both animals.

Tumour oxygenation under normobaric and hyperbaric conditions.

Tumour oxygenation during exposure to normobaric and hyperbaric oxygen was assessed by means of a cryophotometric micromethod and a specially constructed pressure chamber. The tumours investigated were DS-carcinosarcomas implanted subcutaneously into the thigh of Sprague-Dawley rats. The animals were anaesthetised and put on a heating pad. Mean arterial blood pressure was monitored throughout the entire time of pressurisation. Cryobiopsies were removed from the tumours after exposure to O2 for 30 min at 1 bar (atmospheric pressure), 2, 3, or 4 bar. Oxyhaemoglobin saturation (HbO2) values of single red blood cells in tumour microvessels were determined photometrically in the frozen tissue samples as a quantitative measure for tumour oxygenation. Frequency distribution curves of HbO2 showed that there was a marked improvement of the O2 supply to tumour tissue in O2 atmospheres at 1 bar compared with exposure to air at 1 bar. Pressurisation in O2 atmospheres up to 2 and 3 bar did not substantially alter the distribution curve of the HbO2 values in comparison with the data sampled at 1 bar O2 exposure, yet led to a significant drop in the occurrence of HbO2 values below 5% saturation. Thus, pressurisation particularly raises those tissue O2 partial pressures (pO2) that are in a range where the radiosensitivity is critically influenced by the pO2. Pressurisation up to 4 bar caused a shift of the HbO2 distribution curve to values significantly higher than those from tumours exposed to O2 at 1 bar, with no values below 35% saturation. Using these data and a previously published model for computing tissue pO2 values it can be shown that radiobiological hypoxia is unlikely to occur in DS-carcinosarcomas exposed to an O2 atmosphere at 4 bar under the conditions chosen. Hyperbaric oxygenation is recommended as an efficient adjuvant of X irradiation, particularly in superficial tumours.

Nucleotide concentrations in cellular tissues during potassium and magnesium deficiency

1. 1. Potassium deficiency reduced the concentrations of ATP and GTP in rat thigh muscle hut not in liver. 2. 2. The concentrations of UTP. CTP and the UDP-sugar derivatives with glucose, galactose and N-acetylglucosamine in liver were all increased. 3. 3. Magnesium deficiency, however, had little effect on the nucleotide concentrations in liver.

Changes in cellular and subcellular composition during potassium deficiency

1. 1. Cellular tissues vary in their susceptibility to potassium depletion. 2. 2. Potassium is lost from the supernatant fractions of rat thigh muscle, heart and kidney, but not from liver or brain. 3. 3. Sodium accumulates in these same fractions and is accompanied by additional magnesium in thigh muscle. 4. 4. The soluble cytoplasm contains about 80% of the total cellular potassium and it is present entirely in the ionic form.

Changes in myosin and myosin light chain kinase during myogenesis.

Myosins and myosin light chain kinases have been isolated from a cloned line of myoblasts (L5/A10) as this cell line undergoes differentiation toward adult muscle. At least three myosin isozymes were obtained during this developmental process. Initially a nonmuscle type of myosin was found in the myoblasts. The molecular weights of the myoblast light chains were 20 000 and 15 000. Myosin isolated from early myotubes had light chains with molecular weights of 20 000 and 19 500. Myosin isolated from myotubes which contained sarcomeres had light chains with molecular weights of 23 000, 18 500, and 16 000. This last myosin was similar in light chain complement to adult rat thigh muscle. Two forms of the myosin light chain kinase activity were detected: a calcium-independent kinase in the myoblasts and a calcium-dependent kinase in the myotubes with sarcomeres. No myosin light chain kinase activity was detected in the early myotubes.

Localisation of experimental staphylococcal abscesses by 99MTC-technetium-labelled liposomes.

The accumulation of 99mTc-technetium-labelled liposomes in abscesses was studied. Abscesses were produced in the thighs of albino rats by intramuscular injection of Staphylococcus aureus. After 4 days these abscesses were used to determine the localisation of 99mTc-technetium-labelled anionic, cationic and neutral liposomes in the abscess area. This was achieved by radionuclide images produced by a gamma camera and an associated data-processing system. There was a pronounced uptake of 99mTc-technetium-labelled anionic liposomes in the abscess area compared with the corresponding unaffected thigh. Similar uptake was not shown by the 99mTc-technetium-labelled cationic and neutral liposomes. Abscess uptake of anionic liposomes was maximal at or before 30 min after injection and was not enhanced by prior opsonisation with aggregated rat immunoglobulin.

Use of monoclonal antibodies in the analysis of myoblast development

Purified rabbit immunoglobulin reactive with the myogenic E63 subclone of L8 rat myoblasts, was used to distinguish determinants on these cells and on the developmentally defective nonfusing fu-1 subclone. Indirect immunofluorescence indicated that the distribution of antigens on E63 and fu-1 cells was quite different. Repeated adsorption of this antibody with fu-1 cells removed all immunoglobulins reactive with common determinants, leaving only antibody reactive with the differentiating E63 myoblasts. Antibody adsorbed onto fu-1 cells was eluted, repurified, and reacted with the E63 cells. As a refinement of this immunologic approach to studying myoblast interaction and development, monoclonal antibodies were prepared by the method of hybridoma formation. Spleen cells of Balb C mice immunized with E63 myoblasts were fused with SP 2/0-Ag 14 myeloma cells. Of the 209 clones obtained, 49 produced antibody reactive with E63 cells as determined by radioimmunoassay and immunofluorescence. These monoclonal antibodies produced at least seven distinct patterns of immunofluorescence on E63 cells. The presence, localization, and distribution of six antigenic determinants were investigated on E63 myoblasts and myotubes, developmentally defective fu-1 myoblasts, primary newborn rat thigh myoblasts, myotubes, liver, spleen, brain, and lung cells and chick embryo myoblasts and myotubes. Our results show: (1) Five of the six determinants were localized fully or in part on the myoblast cell surface. (2) E63 myoblasts contain at least four determinants which are absent or greatly reduced on differentiated myotubes. Three of these determinants are also almost totally absent on the developmentally defective fu-1 cells. (3) The presence and/or distribution of several E63 determinants are altered in the nonfusing fu-1 cells. Several determinants are greatly reduced on fu-1 cells whereas others are maintained; some have different topographic distributions. (4) Some determinants do not undergo quantitative or topographic alterations as the myoblast differentiates. (5) The distribution and developmental changes detected with E63 cells were also found with primary rat myoblasts. All six determinants were also present on rat tissues of nonmuscle origins. (6) Chick embryo myoblasts contain three of these six determinants and these undergo analogous changes in distribution to that seen with the differentiating rat cells.

Selective tumor heating by shortwave radiofrequency (RF).

Experimental solid tumors were treated in vivo with external high frequency dielectric heating to observe any heat selectivity between the tumor mass on one hand, and the subcutaneous tissue, muscle, and systemic temperatures on the other. Methylcholantrene-induced sarcoma cells were inoculated into the subcutaneous tissue or muscles of the posterior thigh of isologous Fischer rats. When the tumor mass reached the desired size, dielectric heating with a fixed frequency of 13.56 megahertz (MHz) was applied locally to the tumor-bearing area. All the periods of treatment were kept constant at 1 hour. Temperature was measured with thermocouple probes inserted directly into the tumor mass and the tissues lying within the electromagnetic field. Systemic temperature was monitored via a clinical mercury thermometer inserted into the rectum. Temperature recordings were taken at 5-minute intervals during which time the power was turned off in order to avoid the RF interference and to allow thermal equilibrium between the probes and the tissue. The results showed a high selective temperature gradient for the tumor mass as compared to the subcutaneous and muscle tissues when tumor masses were greater than 1.0 cu cm. No selectivity was detected in small tumors or in nontumor-bearing controls. Systemic temperature did not rise by these treatments. No tumor regression was observed at this dosage. Burns were noted in those animals in which normal tissue temperature rose above 43 C.

Acetylcholine-induced current in perfused rat myoballs.

Spherical "myoballs" were grown under tissue culture conditions from striated muscle of neonatal rat thighs. The myoballs were examined electrophysiologically with a suction pipette which was used to pass current and perfuse internally. A microelectrode was used to record membrane potential. Experiments were performed with approximately symmetrical (intracellular and extracellular) sodium aspartate solutions. The resting potential, acetylcholine (ACh) reversal potential, and sodium channel reversal potential were all approximately 0 mV. ACh-induced currents were examined by use of both voltage jumps and voltage ramps in the presence of iontophoretically applied agonist. The voltage-jump relaxations had a single exponential time-course. The time constant, tau, was exponentially related to membrane potential, increasing e-fold for 81 mV hyperpolarization. The equilibrium current-voltage relationship was also approximately exponential, from -120 to +81 mV, increasing e-fold for 104 mV hyperpolarization. The data are consistent with a first-order gating process in which the channel opening rate constant is slightly voltage dependent. The instantaneous current-voltage relationship was sublinear in the hyperpolarizing direction. Several models are discussed which can account for the nonlinearity. Evidence is presented that the "selectivity filter" for the ACh channel is located near the intracellular membrane surface.

Sarcoplasmic reticulum-bound pyruvate kinase in rat skeletal muscl

1. 1. Sarcoplasmic reticulum isolated from rat thigh muscle was found to contain a small amount (0.8%) of M 1-type pyruvate kinase which could be dissociated by a broad spectrum of substances. 2. 2. This binding was maximal at neutral or slightly acidic pH and mediated through non-specific ionic interaction. 3. 3. It is not probable that the binding plays any physiological role in regulating the activity of the enzyme.

Changes in NMR relaxation times of adjacent muscle after implantation of malignant and normal tissue.

In separate experiments, normal foreign tissue and malignant tumour were implanted s.c. into the rat thigh. NMR T1 values of the adjacent normal muscle, resulting from local inflammatory reactions or from malignant invasion, were measured. Elevations in T1 of the underlying muscle occurred within 24 h in both experiments, and it is believed these were caused by rapid inflammatory and immunological reactions to the implants. However the T1 values of muscle samples adjacent to the non-malignant implants decreased during the 11 days after implantation, dropping to values within the normal range. In the second experiment there was progressive malignant invasion into the normal adjacent tissue and the elevated T1 values were maintained throughout the 12-day period. The effects of the implantation on tissue water content are discussed in relation to NMR T1 relaxation times, and the relevance to whole-body NMR imaging of elevated T1 values due to nonmalignant pathological states is considered.

A comparative study of the myosin light chain kinases from myoblast and muscle sources. Studies on the kinases from proliferative rat myoblasts in culture, rat thigh muscle, and rabbit skeletal muscle.

Myosin light chain kinases have been isolated from rat thigh and rabbit skeletal muscle and cultured rat myoblasts. From these preparations, two types of kinases can be distinguished: calcium-dependent and calcium-independent. Both types of kinases can phosphorylate isolated P-light chains of myosin from several sources (skeletal muscle, cardiac muscle, and platelet). Data are shown which support the phosphorylation of the same site on the non-muscle P-light chains by both types of kinases. The rates of these reactins are, however, different for the two types of kinases. Kinetic analysis of the myoblast kinase shows differing affinities for various P-light chains (non-muscle greater than cardiac greater than skeletal). In the proliferative rat myoblast, phosphorylation of myosin is a prerequisite for actin activation of the myosin ATPase activity.

Ultrastructural probing of β-adrenoreceptors on cell surfaces

Establishment of several myogenic cell lines from rat skeletal muscle was achieved by serial passages in vitro of myoblasts from day 2 rat thigh muscles [l-3]. Rat skeletal muscle cell lines L6D [5 1, H6 [5] and L84 [6] were shown to be highly responsive to stimulation by (-)adrenaline (NA) to produce high levels of cyclic AMP (CAMP). Cyclic AMP is an important participant in the life cycle of the cell and its level is a direct function of the various stages of growth. A significant decrease in the level of CAMP was observed [5] after fusion to myotubules. The large increase in intracellular CAMP levels subsequent to the noradrenergic stimulation of L6 cells [4] indicates the presence of a large population of /3-adrenergic receptor sites on the cell surface. Indeed, direct binding studies with 1251-labeled +hydroxybenzylpindolol ([ 12’1] +HYP) confirmed the presence of a large number of f3-adrenergic receptor sites on L6P cells [7]. Therefore, the L6P cells were used to identify the /I-adrenergic receptor subunit with a tritiated irreversible P-blocker [8]. An attempt to study the distribution of the P-adrenoreceptors on the cell surface was carried out by the use of the fluorescent P-blocker, 9-amino-acridino-propranolol(9-AAP), on L6P cells [9]. Although a non-random distribution of fl-adrenoreceptor sites was visualized by fluorescence microscopy using 9-AAP, no information could be obtained as to their ultrastructural distribution. Autoradiographic studies using 12SI-labeled cu-bungarotoxin on embryonic chick skeletal muscle, grown in culture, show a homogeneous distribution of a-bungarotoxin binding sites in newly fused myotubules but during subsequent differentiation,

ACUTE RENAL FAILURE (ARF) FOLLOWING EXPERIMENTAL RHABDOMYOLYSIS

While ARF following rhabdomyolysis or crush syndrome is well documented, its pathophysiology has not been defined. Myoglobin, thought to be responsible for the pathogenesis of ARF, is not toxic unless dehydration or acidosis is already present. To elucidate the pathophysiology of ARF, a new experimental model was established using a crude muscle extract(ME) prepared by homo-genization of saline perfused rat thigh muscle followed by centrifugation and filtration. Experimental rats were injected i.v. with ME and control groups with saline, boiled ME, and myoglobin in normal rat serum. ME caused death at doses >10 mg ME protein/100 g and ARF at 5-10 mg. Oliguria, proteinuria, hemepigmenturia with an active urine sediment, hypocomplementemia, leucopenia and thrombocytopenia developed shortly after ME injection. These findings were not present in the control groups;transient harmless myoglobinuria was present in the control rats given myoglobin. Involvement of the coagulation system suggested by experimental data led to studies using heparin as an anticoagulant. Ten of 10 rats pretreated with heparin before ME injection lived whereas 9 of 10 controls died. This experimental model closely resembles clinical ARF secondary to rhabdomyolysis.Our data also suggest that other biological systems are activated and that muscle constituents other than myoglobin are involved in the pathogenesis of ARF following muscle injury.