Primary human muscle satellite cell culture: variations of cell yield, proliferation and differentiation rates according to age and sex of donors, site of muscle biopsy, and delay before processing.

The present study was performed to determine the influence on human satellite cell yield, proliferation, and differentiation rates of: 1) sex and age of donors; 2) site of the muscle biopsy; and 3) delay before processing of the muscle biopsy sample. We used a standardized primary muscle cell culture procedure on 206 normal muscle samples obtained from different muscle groups of patients aged from 20 to 88 years, at time of orthopedic surgery. Sex of donors did not influence muscle culture parameters. In contrast, aging tended to affect muscle cell yield (age group 50-59 years vs 70-79 years, P < 0.08), but not myogenic cell abilities to proliferate and to fuse into myotubes. The anatomic origin of muscle samples used for culture appeared to influence culture parameters. In contrast with other tested muscles, the tensor fasciae muscle gave both a good cell yield (174 +/- 25 10(3) cells per gram) and homogeneous proliferation and differentiation rates. Storage of the muscle sample at 4 degrees C in transport medium was associated with a very high cell yield when processing was done in early hours after biopsy (277 +/- 50 10(3) cells/g), a high and stable cell yield when processing was done from day 1 to day 3 after biopsy (185 +/- 15 10(3) cells/g), and a poor cell yield when processing was done after day 4 (111 +/- 13 10(3) cells/g). Storage of muscle biopsy samples at 4 degrees C for 1 to 4 days was associated with good proliferation and fusion rates. In conclusion, these data validate a convenient procedure of primary human muscle cell culture, using tensor fasciae muscle biopsy, which is easily done at time of orthopedic surgery, obtained from men and women of all ages (if possible less than 70 years to obtain good cell yield), and allowing of 1-3 days of storage before processing that may compensate uncertainty of the exact time of availability of muscle samples for the scientist.

Modulation of activities and RNA level of the components of the plasminogen activation system during fusion of human myogenic satellite cells in vitro.

Primary cultures of human myogenic stem cells (satellite cells) mimic myogenic differentiation. During this process, the expression of the components of the plasminogen activation system underwent modulation. Activities and mRNA levels of tissue-type and urokinase-type plasminogen activator were increased in a reproducible pattern during differentiation. A modulation of the mRNA level of PAI-2 was also observed. Human satellite cells expressed a urokinase receptor and also the mRNA level of this component underwent modulation. With the exception of PAI-1 mRNA, the level of all mRNAs increased from Day 4 to Day 8, i.e., just before myoblasts fusion, and then remained high at later stages. The modulation of the plasminogen activating activity indicates that this system is directly involved in the fusion process of myogenic differentiation.

DNA excision repair in spontaneously diabetic BB Wistar rats.

BB Wistar rats develop a syndrome characterized by spontaneous diabetes mellitus as well as a wide variety of autoimmune, neoplastic, and degenerative disorders which do not occur in the outbred Wistar strain from which they were derived. This syndrome also includes elements of premature ageing (i.e., a markedly shortened lifespan and premature occurrence of diseases associated with ageing). Excision DNA repair capacity which has been reported to be directly proportional to maximal achievable life span was estimated in neonatal BB Wistar and outbred Wistar rats. Excision repair was assayed autoradiographically by determining unscheduled DNA synthesis following UV radiation of passage 3 cultured skin fibroblasts. No difference in excision repair capacity could be demonstrated between the two strains.

Androgen sensitivity of skeletal muscle: nondependence on the motor nerve in the frog forearm.

The effects of castration and of subsequent androgen administration on fiber size were investigated in several frog skeletal muscles. Four months after castration, cross-sectional cell area decreased by 70% and 14%, respectively, in the flexor carpi radialis and flexor carpi centralis muscles of the forearm and only by 2% in the ileo fibularis muscle of the thigh. Injection of testosterone propionate induced a hypertrophic response that reversed the effects of androgen deprivation; after 6 weeks, complete recovery to the control value was observed in all muscles selected. This sensitivity to the exogenous androgen was not altered by denervation; a similar hypertrophic evolution was seen in the denervated right muscles and in the homologous intact left muscles of the forearms. Using the myosin ATPase reaction, the muscle histochemical patterns were unchanged in all conditions tested. These results suggest that (i) a gradient of sensitivity to androgens exists in different frog muscles; (ii) androgens control the myofiber size but not the nerve-muscle organization as can be seen from the myofibrillar ATPase pattern; and (iii) the androgen sensitivity is not dependent on the motor nerve.

Histopathological lesions in the pancreas of the BB Wistar rat as a function of age and duration of diabetes.

Pancreatic histopathology was studied in 121 BBWd, 43 BBWnd, and 33 Wistar rats. Insulitis was the most common inflammatory lesion in both BBW and BBWnd rats. The incidence was inversely associated with age and with duration of diabetes in BBWd rats, but there was no age-related pattern in BBWnd rats. Small end-stage islets were typical of BBWd rats but were not seen in BBWnd rats. Several BBWd rats showed hyperplastic islets months after the onset of diabetes, a pattern that is also seen in a small percentage of human JOD patients. Several non-specific exocrine inflammatory lesions occurred in both BBWd and BBWnd rats: acute and/or chronic pancreatitis, eosinophilic infiltrates, granulomatous lesions and acute and/or chronic interstitial inflammation. Only chronic interstitial inflammation was seen in outbred Wistar rats.

Serum lipids and lipoprotein composition in spontaneously diabetic BB Wistar rats.

Serum lipid and lipoprotein composition in spontaneously diabetic BB Wistar rats, nondiabetic littermates, and control Wistar rats was studied to elucidate diabetes-related abnormalities of lipoprotein composition. Serum total triglycerides and pre-beta-lipoprotein concentrations of insulin-treated spontaneously diabetic BB and nondiabetic littermate rats were significantly higher than those of control Wistar rats. Serum cholesterol and HDL cholesterol concentrations of spontaneously diabetic BB and nondiabetic littermate rats did not differ from controls. Concentrations of very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) of spontaneously diabetic BB and nondiabetic littermate rats were higher than those of normal rats. With sodium dodecylsulfate-polyacrylamide gel electrophoresis it was observed that the spontaneously diabetic BB and nondiabetic littermate rat VLDL contained higher percentages of apoE relative to total apoC when compared with control Wistar rats. With isoelectric focusing, apoC-II relative percentages in VLDL and HDL of both spontaneously diabetic BB and nondiabetic littermate rats were higher than apoC-II proportions in VLDL and HDL of controls. Apolipoprotein A-I of the control rat HDL showed four isoforms that focused at pI 5.8 (17.3%), 5.75 (30.6%), 5.65 (31.8%), and 5.55 (20.5%); however, the spontaneously diabetic BB and nondiabetic littermate rat HDL apoA-I was mainly represented by two isoforms that focused at pI 5.8 and 5.75. VLDL of both diabetic and nondiabetic BB rats contained higher levels of acidic apoE isoforms compared to their counterparts in control Wistar rats. Although HDL cholesterol concentrations of spontaneously diabetic BB rats remained normal, protein concentrations were higher resulting in a low cholesterol/protein ratio in HDL suggesting that the cholesterol-carrying capacity of spontaneously diabetic BB rat HDL could be less than normal and may be due to an abnormal apoA-I composition. Quantitative alterations of lipid and lipoprotein composition appear in the BB Wistar rat when compared to the Wistar rat, but some of the changes are more pronounced in the spontaneously diabetic BB Wistar rat.

Abnormal bile acid pool and composition in neonates of spontaneously diabetic Wistar BB rats and its change during development.

Streptozotocin-induced diabetes during pregnancy in rats causes a decrease in primary bile acid pool in neonates. To rule out direct drug effect on the fetus as the basis for this change, studies of bile acid pool and composition at birth and during subsequent development was carried out in neonates of spontaneously diabetic Wistar BB rats and compared to control neonates. The cholic acid pool in neonates of diabetic rats was lower when compared to control neonates at birth. The pool of secondary bile acids was markedly increased in neonates of diabetic rats, with increases in lithocholic and 3 beta,12 alpha-dihydroxycholanoic acid. With age, the cholic acid pool of neonates from diabetic rats was increased and at 3 months of age it was actually higher than in control neonates. The pool of chenodeoxycholic at diabetes onset age was lower in neonates of diabetic rats. HDL-cholesterol was lower in neonates of diabetic rats at 1 week, but this reversed at 3 months of age. These studies firmly establish that neonates of diabetic rats have abnormal bile acid pool and composition at birth which changes to adult diabetic pattern with age.

Central pontine myelinolysis following saline treatment of a diabetic rat for dehydration.

Central pontine myelinolysis (CPM) was observed at necropsy in an emaciated 333-day-old BB Wistar insulin-dependent diabetic rat that had been treated for dehydration by subcutaneous injection of normal saline. There was a large area of nearly complete demyelination involving most of the basal portion of the pons, part of the tegmentum, and the base of the middle cerebellar peduncles. There was a relative sparing of neurons and axons, but some unidentifiable large cells with degenerative changes in the cytoplasm were present. No oligodendrocytes or reactive astrocytes were seen and there was no evidence of inflammation or infarction. Although diffuse demyelination can be induced in rats by rapid fluctuation of serum electrolytes, no studies have experimentally induced central pontine myelinolysis nor has it been observed incidentally in animals previously.

Spontaneous thyroiditis in BB Wistar diabetic rats.

Spontaneous lymphocytic thyroiditis was observed at necropsy in 36 BB Wistar diabetic rats (63.2%) and in eight of their nondiabetic siblings (42.1%). The incidence of thyroiditis decreased both with age and with longer duration of diabetes. All rats with pancreatic insulitis (a manifestation of the onset of diabetes) also had thyroiditis. BB Wistar rats with insulitis had more severe lymphocytic thyroiditis, characterized by lymphocytic, plasmacytic, and macrophage infiltration of thyroid interstitium and follicles. A milder, mostly perivascular and interstitial lymphocytic thyroiditis was characteristic of lesions in rats which did not have insulitis. The histological appearance of the thyroiditis suggests that these rats may be subject to autoimmune disease at the onset of diabetes which involves sites other than just the pancreas.

Glomerular manifestations of diabetes in the BB rat.

Morphologic characteristics of the renal glomeruli and tubules of BB rats with spontaneous diabetes mellitus were studied at 30 weeks' duration of diabetes. Whereas the glomerular basement membrane (GBM) was significantly thickened, no changes in the diabetic glomeruli were seen in the peripheral capillary wall area and in the fractional volumes of the mesangial cells or of the mesangial matrix. Light microscopy of the diabetic kidneys were normal, and immunofluorescent examination of diabetic glomeruli showed no increased accumulation of albumin, C3, or IgG. Diabetic rats had increased renal blood flow and glomerular filtration rates. Diabetic rats at 7, 17, and 30 weeks excreted normal amounts of urinary albumin. Thus kidneys of the BB diabetic rat differ from other experimental models of diabetes in that GBM thickening occurs in the absence of mesangial changes and of increased albuminuria. These studies suggest that the mesangium may influence glomerular permeability in diabetes, while thickening of the GBM in diabetes does not necessarily coincide with increased urinary albumin excretion. Furthermore, these results are consonant with the hypothesis that genetic factors may influence the pathological expression of diabetic nephropathy in rats.

Cyclosporine: action, pharmacokinetics, and effect in the BB rat model.

Cyclosporine (Cy) was given to BB rats in an attempt to prevent the onset of diabetes. A dose of 15 to 20 mg/kg/d given orally or subcutaneously was associated with high Cy trough serum levels and caused nephrotoxicity and severe weight loss. Ten mg/kg/d of Cy was tolerated well. Forty rats were treated with Cy starting at 34 days of age. No Cy-treated rats developed diabetes by day 121, compared with 70% of the control rats. Once Cy was discontinued on day 121, 38% of female and 13% of male rats developed diabetes by day 169. Transient, spontaneously remitting hyperglycemia developed in nine rats. This occurred both in control rats and in rats on Cy, but it was more common in females than in males. Thus, Cy prevents diabetes mellitus in the BB rat when trough Cy serum levels greater than 100 ng/mL are achieved. Diabetes occurs in some rats after Cy is discontinued. In all treatment subgroups, diabetes occurred more frequently in females than in males.

Pathological lesions in the spontaneously diabetic BB Wistar rat: a comprehensive autopsy study.

A total of 145 BB Wistar diabetic rats, 46 of their nondiabetic siblings, and 43 outbred Wistar rats were autopsied and the frequency of lesions in all organ systems were determined. Common strain-related lesions included pulmonary infections, granulomas, lymphoid hyperplasia, lymphomas, lymphocytopenia, eosinophilia, supradiaphragmatic accessory lobes of the liver, and prostatic atrophy. These suggest some basic strain-related abnormalities of the immune system that were selected by the process of inbreeding. Diabetes-related lesions were insulitis, testicular atrophy, cataracts, hepatic fatty change, pancreatitis, lymphocytic thyroiditis, hypoglycemic brain damage, central pontine myelinolysis, stomach erosions, and idiopathic megacolon. Many of these are sequelae of human juvenile-onset diabetes and support the validity of the BB Wistar rat as an animal model for human diabetes mellitus. The absence of several important sequelae of the human disease (i.e., diabetic nephropathy, atherosclerosis, and severe microangiopathy) suggests a degree of infidelity as a model for human diabetes mellitus. Nonspecific lesions occurring in all three groups of rats included myocardial degeneration and fibrosis, splenic extramedullary hematopoiesis, and chronic progressive glomerulonephropathy.

Supradiaphragmatic accessory lobe of the liver in BB Wistar rats.

Supradiaphragmatic accessory livers were observed in two closely related rats in a series of 172 necropsies on BB Wistar rats. The gross and histological appearance of both accessory lobes are described. This abnormality has been reported in only one other inbred strain of rats where it also arose with a very low incidence. As in the previous report, the pattern of occurrence of these accessory lobes suggests a mode of inheritance that is either polygenic or autosomal recessive with low penetrance.

Hematological Characteristics of the BB Wistar Rat.

Complete blood counts, differential white blood cell and platelet counts were performed on male and female BB Wistar diabetic rats (BBWd), their nondiabetic siblings (BBWnd) and outbred Wistar rats of the line from which the BB Wistar rats were derived. Most of the observed changes were strain-related (those present in both BBWd and BBWnd but not in control rats) rather than diabetes-related (those in BBWd but neither BBWnd nor control rats) and therefore probably due to the inbreeding process. The BBW strain had significantly lower numbers of white cells and platelets, as well as markedly changed differential white cell counts. Differential counts showed a pattern of lymphopenia, neutrophilia, monocytosis and eosinophilia. It is possible that these white blood cell changes contribute to the increased susceptibility to infection reported for the BBW strain. No significant difference in serum immunoglobulin concentrations was found in any of these three groups of rats. There- fore, hypogammaglobutinemia cannot account for the increased susceptibility to infections, but it is not possible to rule out an abnormality in the distribution of immunoglobulin fractions as an etiological factor.

Cyclosporin prevents diabetes in BB Wistar rats.

Cyclosporin (U.S.A.N. cyclosporine) was used prophylactically to suppress the development of insulin-dependent diabetes mellitus in BB Wistar rats. In this rat strain diabetes spontaneously develops between 60 and 120 days of age and this condition resembles human type 1 diabetes. In 30 (75%) out of 40 control rats glycosuria and hyperglycaemia developed and these rats became insulin-dependent. Their pancreases showed partial to complete destruction of the islets of Langerhans by lymphocytic infiltration. None of the 40 cyclosporin-treated rats became diabetic and their pancreases were histologically normal. Serum cyclosporin was monitored in a subgroup and the dosage was adjusted accordingly; neither hepatotoxicity nor nephrotoxicity was seen in the treated animals.

Proximal motor neuropathy in the BB-Wistar rat.

Proximal motor neuropathy is a well-recognized neuropathic complication in human diabetes mellitus, due to microvasculopathic changes. Spontaneously diabetic BB-Wistar rats maintained at a moderate severity of diabetes developed structural proximal motor neuropathy after long-standing diabetes. This was caused by multiple infarcts in the spinal ventral roots. Endoneurial vessels showed occlusions by platelet aggregates and unstriated fibrin, indicative of a hypercoagulability.

Testicular atrophy in the spontaneously diabetic BB Wistar rat.

Complete gross and microscopic postmortem examinations were performed on 100 BB Wistar diabetic rats, 27 BB Wistar nondiabetic siblings, and 41 Wistar rats, and the incidence of testicular lesions was tabulated. Testicular atrophy was the predominant finding in all three groups of rats, but atrophy occurred at a much younger age in the diabetic rats. There was a strong relationship between the duration of diabetes and the presence of atrophy, which was stronger than the relationship between age and atrophy. The testicular atrophy observed in the diabetic rats was morphologically similar to the senile testicular atrophy in the nondiabetic rats. Histologic findings that were associated with increasing severity of atrophy were multinucleated giant cells in the lumens of seminiferous tubules, increased interstitial connective tissue, Leydig cell hyperplasia, and thickening of the tunica albuginea. Testicular atrophy has also been reported in human diabetics. Therefore, the BB Wistar rat may be a useful model for investigating this aspect of diabetes mellitus.

[Histochemical study of skeletal muscles of the frog, Rana temporaria]. / Etude histochimique des muscles squelettiques de la grenouille, Rana temporaria.

Histochemical profiles of muscles were identified based on staining for myosin ATPase activity. They reveal typical arrangement of muscular fibres with a zoned pattern. Tonic fibres have a unique histochemical profile and are mixed with the most oxydative fast fibres to form toxic zones. Muscles show fast profiles in thigh and tonic or mixed profiles in fore-arm.

The spontaneously diabetic BB Wistar rat. Morphologic and physiologic studies of peripheral nerve.

The spontaneously diabetic "BB" Wistar rat was examined for evidence of peripheral nerve abnormalities by a combined morphologic and physiologic approach. The studies were done on rats kept severely hyperglycemic and frequently ketotic. The peripheral nerves of the lower extremities, including the most distal nerves of the intrinsic foot muscles, revealed only minimal abnormalities by histologic and morphometric examination. Sciatic nerve conduction studies, however, measured over a 2-month period did not show a significant slowing in diabetics compared with age-matched controls, as well as between diabetics and weight-matched controls. In addition, fast anterograde axoplasmic transport studies were correlated with serum glucose results. Rats maintained severely hyperglycemic with or without ketosis had abnormal down flow rats of [3H]leucine compared to controls. Diabetic rats maintained with normal blood glucose levels showed no change in transport rates. These results suggested that persistent hyperglycemia in the "BB" Wistar rat produces significant physiologic but not significant structural abnormalities in the peripheral nerve.