A model of myogenesis in vivo,derived from detailed autoradiographic studies of regenerating skeletal muscle,challenges the concept of quantal mitosis

Summary We have recently shown that myogenesis following severe injury is prolonged compared with minor injury (McGeachie and Grounds 1987). In this previous autoradiographic study 44 mice were injected with tritiated thymidine at various times after muscle injury (0 to 120 h), and samples were taken 9d after injury to determine the percentage of labelled myotube nuclei. In the present study the same experimental data are analysed in detail to reveal how many times labelled muscle precursors divided before fusing to form myotubes.Additional mice were prepared and samples removed 1 h after injection of tritiated thymidine to determine the maximum grain counts of premitotic nuclei. When a labelled premitotic nucleus divides, each of the two daughter nuclei will contain half of the original label. The grain counts of nuclei resulting from sequential divisions of a maximally labelled premitotic nucleus, forms the basis for our detailed analysis which can reveal how many times a muscle precursor has divided after labelling.Nine days after injury the autoradiographic grain counts of labelled myotube nuclei were analysed in detail. The results describe an in vivo model of myogenesis which we use to evaluate quantitatively observations derived from tissue culture studies. The analysis shows that, at the onset of myogenesis in regenerating muscle (30 h after injury), muscle precursors divide only twice before fusing to form myotubes. This observation challenges the concept of quantal mitosis as defined by the tissue culture studies of Quinn et al. (1984, 1985).     

SYNTHESIS OF RNA IN NEURONS OF THE HYPOGLOSSAL NERVE NUCLEUS, AFTER SECTION OF THE AXON, IN MICE

Synthesis of RNA in neurons of the hypoglossal nerve nucleus after axonal section was studied by means of [5-³H]uridine administration and radioautographic counting techniques in mice. The results of the experiments were evaluated by counts of silver grains over the nucleoplasm and cytoplasm of the neurons. RNA synthesis was greater in neurons after axonal section, and this increase was evident from 12 hr after the operation. The greatest increases in the operated side were observed in the 1st, 2nd and 3rd days after operation. In the 7th and 14th days RNA synthesis was still greater in the hypoglossal nucleus of the sectioned nerve but the difference in the control nucleus was not so striking. In the 30th day synthesis of RNA in left and right hypoglossal nuclei was comparable.     

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

Calcitonin gene-related peptide and α-CGRP mRNA expression in cranial motoneurons after hypoglossal nerve injury during postnatal development

Changes in calcitonin gene-related peptide (CGRP) immunoreactivity and α-CGRP mRNA expression were determined in the hypoglossal nucleus after the nerve was crushed or transected in rats at 10, 14 and 21 days postnatal. α-CGRP mRNA expression was determined in normal, noninjured, hypoglossal nuclei at the three ages and after both injuries in 10 and 21 days postnatal rats. Reinnervation and neuronal survival were assayed. Although the three age groups expressed comparable levels of α-CGRP mRNA and its peptide in intact, hypoglossal nuclei, axonal injury produced age-dependent alterations in α-CGRP mRNA and CGRP. In the 21 days postnatal rats, changes in α-CGRP mRNA and peptide mimicked those reported in adult motoneurons after the same injuries. CGRP was elevated until reinnervation after nerve crush, whereas biphasic elevations occurred after nerve transection. In 21 days postnatal rats, increases in α-CGRP mRNA preceded elevations of the peptide but a greater increase resulted initially after nerve transection. An upregulation of α-CGRP mRNA also developed initially after both injuries in 10 days postnatal rats but subsequent elevations of α-CGRP mRNA did not materialize. In contrast, CGRP immunoreactivity did not increase after either injury in 10 days postnatal rats and, in fact decreased. Levels of CGRP immunoreactivity did not differ from normal amounts after either nerve injury in 14 days postnatal rats. Substantial neuronal cell loss occurred after each injury in 10 and 14 days postnatal rats but was not found in 21 days postnatal rats. Tongue reinnervation by surviving motoneurons was established after all injury paradigms except 10 days postnatal transection. The current findings demonstrate an age-dependent correlation between injury-induced expression of CGRP and hypoglossal motoneuron survival.     

Androgen receptors in cranial nerve motor nuclei of male and female rats

This study compared AR proteins in four cranial nerve motor nuclei among male and female rats that were intact, gonadectomized, or gonadectomized and given TP by immunohistochemistry. AR-immunoreactive (ir) neurons were found, in descending order of abundance, in the nucleus ambiguus, hypoglossal nucleus, and the facial and trigeminal motor nuclei of both males and females of intact and gonadectomized plus TP rats. Virtually every neuron of the nucleus ambiguus was AR-ir. In contrast, AR-ir neurons were either restricted to a specific area of the hypoglossal nucleus, or randomly distributed in the facial and trigeminal motor nuclei. The predominant AR-ir site shifted from cell nuclei to the cytoplasm, depending upon the presence or absence of ligand. Sex differences in the amount and staining intensity of AR-ir neurons were discernable in all four motor nuclei of intact rats, and these differences were maintained in gonadectomized plus TP rats, with the exception of the nucleus ambiguus. The immunostaining results were complemented by results from AR binding studies. Cytosolic AR binding values for the hypoglossal and facial motor nuclei of females were only approximately 50% of those of males despite the absence of a sex difference in neuron number. These results indicate that intrinsic sex differences in AR levels and androgenic regulation of AR exist in cranial nerve motor nuclei, and that there are differences in the abundance and distribution pattern of AR responsive neurons in cranial nerve motor nuclei. These results are consistent with the idea that sex differences in AR could account for sex differences observed in nerve regeneration and neuron loss following cranial nerve injury.     

Myogenic cells of regenerating adult chicken muscle can fuse into myotubes after a single cell division in vivo

Autoradiographic studies were carried out on regenerating muscles of adult chickens. Three different muscles of hens were injured, and tritiated thymidine (1 microCi/g) was injected at various times after injury to label replicating muscle precursors. Detailed comparisons of grain counts over premitotic nuclei in samples removed one hour after injection of tritiated thymidine, and of postmitotic myotube nuclei in samples removed 10 days after injury (when labeled precursors had fused to form myotubes), revealed how many times some labeled precursors had divided before fusing into myotubes. DNA synthesis in muscle precursors was initiated 30 h after injury. Grain counts of myotube nuclei indicated that many muscle precursors labeled at the onset of myogenic cell proliferation had divided only once, or twice, before fusing into myotubes. The relationship of these in vivo results to the cell lineage model of myogenesis is discussed.     

Purkinje cell maturation in the light of RNA synthesis

Wistar rats 1- to 90-day-old received an injection of 3H-uridine and were killed 20 min to 44 h later. Autoradiographic examination revealed the highest grain count densities in Purkinje cell nuclei around postnatal day (PD) 6 while the incidence of labelled nuclei stayed at the peak values till PD 15. Silver staining of Purkinje cell nuclei showed that the expression of nucleolar r-RNA coding genes is maximal at PD 15; in some cells it even slightly exceeds adult values. After PD 15, the percentage of labelled Purkinje cell nuclei declined; this was more pronounced in the nucleolar region than outside the nucleolus. The percentage of cells with cytoplasmic labelling culminated on PD 15. The highest grain counts were found in Purkinje cell cytoplasm on PD 6 at 44 h p.i. interval. Reversal in nuclear grain counts at 2 and 6 h p.i. intervals observed between PD 15 and PD 25 suggests faster degradation, or processing and export, of a newly synthesized nuclear RNA in these age groups. Frequency distribution analysis of grain count densities revealed a small group of Purkinje cells with higher incorporation of 3H-uridine both in the nucleolar region and the whole nucleus at PD 15. In situ hybridization of 3H-r-RNA revealed a slight binding excess to DNA of some Purkinje cell nuclei but not in granule cells of 1-month-old rats. These data, together with those published recently by Brodsky et al. (1985), indicate an uneven structural organization and partial overexpression of the genom coding r-RNA synthesis in the population of Purkinje cells.     

Cellular reactions to axotomy in rat superior cervical ganglia includes apoptotic cell death

The cellular response to axonal injury in the superior cervical ganglion was examined by immunofluoresence at intervals from 6 h to 14 days after transection of the internal and external carotid nerves. GAP-43-immunoreactivity (IR) appeared in some neurons in the ganglia 1 day after axotomy, while neurons in control ganglia were GAP-43 negative. In 3 days axotomized ganglia GAP-43-IR structures were increased in number and intensity in nerve fiber bundles, while GAP-43-positive perikarya were restricted to the middle and caudal parts of the ganglia and showed an intensity that was stronger than at 1 day after axotomy. These GAP-43-positive neurons were also galanin positive. In the cranial part of the ganglia, S100-IR in satellite cells was weak at 18 h after axotomy. Peripheral to this area, S100-IR was stronger and co-localized with HSP-72-IR, preferentially located in satellite cells. HSP-72-IR was, however, occasionally observed also in principal neurons at 1 and 3 days after axotomy. In eosin-stained sections, neurons and satellite cells in the cranial part of 1 day axotomized ganglia were reduced in number, and a further loss was noted at 3 days. At 12 h some satellite cells in the cranial part of the ganglia were labelled by the in situ DNA 3'-end labelling method, indicating apoptosis, and at 18 h many cells were labelled. Some neuronal perikarya were also labelled in this region. Labelling was not observed at 1 day or later after axotomy, nor in control ganglia. The results may imply that not only neurons but also satellite cells react to neuronal axonal injury with apoptosis. Neurons in the middle and caudal part of the ganglia survived and showed increased content of GAP-43 and galanin, possibly a sign of regeneration/neuronal plasticity.     

Myogenic cells of regenerating adult chicken muscle can fuse into myotubes after a single cell division in vivo

Autoradiographic studies were carried out on regenerating muscles of adult chickens. Three different muscles of hens were injured, and tritiated thymidine (1 μCi/g) was injected at various times after injury to label replicating muscle precursors. Detailed comparisons of grain counts over premitotic nuclei in samples removed one hour after injection of tritiated thymidine, and of postmitotic myotube nuclei in samples removed 10 days after injury (when labeled precursors had fused to form myotubes), revealed how many times some labeled precursors had divided before fusing into myotubes. DNA synthesis in muscle precursors was initiated 30 h after injury. Grain counts of myotube nuclei indicated that many muscle precursors labeled at the onset of myogenic cell proliferation had divided only once, or twice, before fusing into myotubes. The relationship of these in vivo results to the cell lineage model of myogenesis is discussed.     

KINETICS OF NEUTROPHILIC GRANULOCYTES IN THE BLOOD OF RATS

The kinetics of neutrophilic granulocytes in the blood of rats were investigated using in vivo ³H-TdR labelling and autoradiography. The radioactive precursor was administered by single injection, repeated injections at 5 hr intervals and continuous infusion. The appearance of labelled granulocytes in the blood and in the sputum was recorded up to 120 hr after tracer application. In contrast to results after a single injection of ³H-TdR, complete labelling of the blood granulocyte pool was achieved when the DNA precursor was given by continuous infusion or four repeated injections at 5 hr intervals. In the latter experiments, an exponential replacement of unlabelled blood granulocytes by labelled granulocytes could be demonstrated, the mean intravascular half-life being 5·7 hr. This figure is in good agreement with values obtained by isotopic techniques in other mammalian species.     

Retrograde tracing and toe spreading after experimental autologous nerve transplantation and crush injury of the sciatic nerve: a descriptive methodological study

ABSTRACT: Evaluation of functional and structural recovery after peripheral nerve injury is crucial to determine the therapeutic effect of a nerve repair strategy. In the present study, we examined the relationship between the structural evaluation of regeneration by means of retrograde tracing and the functional evaluation analysis of toe spreading. Two standardized rat sciatic nerve injury models were used to address this relationship. As such, animals received either a 2 cm sciatic nerve defect (neurotmesis) followed by autologous nerve transplantation (ANT animals) or a crush injury with spontaneous recovery (axonotmesis; CI animals). Functional recovery of toe spreading was observed over an observation period of 84 days. In contrast to CI animals, ANT animals did not reach pre-surgical levels of toe spreading. After the observation period, the lipophilic dye DiI was applied to label sensory and motor neurons in dorsal root ganglia (DRG; sensory neurons) and spinal cord (motor neurons), respectively. No statistical difference in motor or sensory neuron counts could be detected between ANT and CI animals. In the present study we could indicate that there was no direct relationship between functional recovery (toe spreading) measured by SSI and the number of labelled (motor and sensory) neurons evaluated by retrograde tracing. The present findings demonstrate that a multimodal approach with a variety of independent evaluation tools is essential to understand and estimate the therapeutic benefit of a nerve repair strategy.     

Initial stages of the myocardial differentiation of the lymph hearts of frog tadpoles. A study by the methods of electron microscopy and electron microscopic autoradiography

Using electron microscope autoradiography, a study was made of the ultrastructure of early stages of muscle differentiation and 3H-thymidine (3H-T) labelled cells in the wall of the developing lymph heart of larvae of Rana temporaria L. The mononucleated postmitotic myoblasts with small bundles of thin and thick myofilaments deprived of Z-bodies were found in the lymph heart wall. No thin or intermediate-sized subsarcolemmal filaments were detected in the cytoplasm of these myoblasts. Myosatellites occurred under the basal lamina of muscle cells at stages 41-42. The primitive muscle-nerve junction was found at stages 44-45. Four hours after a single 3H-T administration only mononuclear cells without myofilaments were labelled. If the fixation was made 72 hours after a single 3H-T administration, the label was found, in addition, on the muscle cell nuclei. These data evidence that at the early stages of muscle differentiation in the developing lymph heart wall DNA synthesis and muscle specific protein synthesis are incompatible.     

Meiotic DNA synthesis during mouse spermatogenesis

The incorporation of radioactivity into various cells in the sequence of spermatogenesis was measured by preparing highly purified spermatozoan nuclei from the cauda epididymidis of mice at daily intervals after injection of (3H)thymidine. The stages of differentiation of these sperm at the time of thymidine administration were calculated from the kinetics of spermatogenesis. The procedure for purification of sperm nuclei included sonication, mechanical shearing, and treatment with trypsin, DNase, Triton X-100, 2M NaC1, and sodium dodecyl sulfate. DNA was isolated from these nuclei by treatment with dithiothreitol and pronase, followed by phenol extraction and ethanol precipitation. The levels of radioactivity in the epididymal sperm head preparations were low (less than 13 dpm/mouse) for 27 days after injection, and then rose dramatically to over 4 times 104 dpm/mouse. Further experiments demonstrated that the 11 dpm of 3H radioactivity contained in sperm heads at 21 or 26 days after injection of (3H)TdR was significantly above background and contamination levels from other cells or other sources. Most of the radioactivity was in the sperm DNA and represented incorporation of tritium from (3H)TdR into the nuclear DNA of meiotic cells at 0.002 percent of the rate of incorporation into S-phase cells. Little, if any, (3H)TdR was incorporation into the DNA of spermatids. The levels of DNA synthesis during the meiotic prophase in the mouse appear to be much lower than those reported for other organisms.     

An Autoradiographic Study of Nucleic Acid and Protein Turnover in the Mammalian Neuraxis

The turnover of nucleic acids and proteins in the central nervous system has been explored by autoradiography following the subarachnoid injection of tagged precursors. Nuclear PNA of neurons and oligodendrocytes becomes radioactive earlier than cytoplasmic PNA after injection of adenine-C¹⁴ and orotic-C¹⁴ acid. By 24 hours following injection, cytoplasmic PNA is radioactive. Radioactivity persists with little decrease for as long as 51 days after an injection of adenine-C¹⁴. The cells of the ependymal lining, choroidal plexus, leptomeninges, blood vessel walls, and Schwann cells also exhibit radioactivity in PNA as judged by the loss of radioactivity following ribonuclease digestion. From the 3rd day on, increasing numbers of the aforementioned cells, with the exception of nerve cells, exhibit ribonuclease-resistant nuclear radioactivity which is abolished by deoxyribonuclease. This radioactivity indicates labelling of nuclear DNA. Following the intrathecal injection of methionine-S³⁵ and glycine-2-H³, nerve cells, oligodendrocytes, cells of ependymal lining, choroidal plexus, leptomeninges, blood vessels, and Schwann cells become radioactive. Nerve cells lose most of their radioactivity within a few hours, first from the cytoplasm and later from the nucleus. Other cell types retain their radioactivity for considerable periods of time. Although astrocytes, microglia, and satellite cells of sensory ganglia do not appear to incorporate labelled precursors into nucleic acids or proteins, reacting phagocytic microglia actively take up labelled amino acids. These results are discussed with particular reference to PNA and protein turnover in nerve cells, oligodendrocytes, and Schwann cells. It is believed that these metabolic activities in neurons are concerned in part with the elaboration of axoplasmic proteins. The nucleoprotein metabolism of oligodendrocytes and Schwann cells may be related to myelin biosynthesis both in the immature and the mature nervous system.     

Investigations on the Incorporation of a Tritiated Ganglioside, GM1, in the Injured and Intact Sciatic Nerves of the Rat

Following injury of their left sciatic nerves by means of a standardized procedure, male rats received intravenous injections of a tritiated ganglioside. GM1, on different days during the process of regeneration. The rats were killed at two different times after the injection and the concentrations of the total radioactivity, nonvolatile radioactivity, and labelled GM1 were estimated in six segments of the crushed and intact sciatic nerves. The segments of the damaged nerves showed higher concentrations of radioactivity and a higher content of GM1 than the corresponding segments of the contralateral nerves. Within the immediate area of the lesion the highest levels were found on the 3rd and 6th days after the injury; the segments distal from the lesion showed the highest levels of activity on days 9 and 12. The nerve segments proximal to the site of the injury showed a low rate of radioactivity incorporation. The higher concentrations of [3H]GM1 in damaged nerves as well as the rate of incorporation as a function of time indicate that exogenous gangliosides may be involved in the processes of regeneration and have a bearing on the latter.     

Proteins of fast axonal transport in the regenerating hypoglossal nerve of the rat

The composition of proteins conveyed by fast axonal transport in growing or regenerating axons is different from that of intact, mature axons. Consistent alterations have been observed in several different types of neurons, but adult peripheral axons (rabbit hypoglossal motoneurons) seemed to be exceptions because during their regeneration there was no increased labelling of a 23 kilodalton (kD) protein associated with the growth state. We examined the composition of fast-transported proteins, labelled by application of [35S]methionine to the hypoglossal nuclei, in intact and regenerating hypoglossal nerves of the rat. Using one- and two-dimensional electrophoresis we detected both increases and decreases in the labelling of specific polypeptides during regeneration. In particular, there was increased labelling of a 23 kD polypeptide. Changes were maximal 7 days after axotomy and subsided thereafter, coincident with reinnervation of the tongue. We conclude that hypoglossal axons show the same changes in transported protein composition which are characteristic of the growth state in other axons. Thus, we have strengthened the correlation between the growth state and changes in synthesis of a set of polypeptides of unknown function.     

Changes in localized arrangement into the hypoglossal nucleus after the neurectomy of unilateral hypoglossal nerve (medial branch) in cats

We studied changes in orofacial behavior and the arrangement of bilateral hypoglossal nuclei after the neurectomy of the medial branch of the unilateral hypoglossal nerve in cats. After recovery from surgery in a head holder, the animals were acclimated to take and chew fish paste (1.8 g) from a spoon and lick milk from a wetted paintbrush. Next we performed a neurectomy in the unilateral hypoglossal nerve after training. We firstly recorded behavior during the taking of fish paste and licking of milk, and then performed a neurectomy in the unilateral hypoglossal nerve. After nerve cutting, the cats’ tongue deviated toward the cut side when they licked food, and bilateral activities of EMGs in the genioglossus muscles became stable in about 1 month. After that, we injected two kinds of fluorescent dye (10% Evans blue, EB, and 3% Fast blue, FB) into the bilateral genioglossus muscles using syringes (0.15 ml in each), respectively. Although each injection of FB and EB into the bilateral genioglossus muscles in normal cats revealed cells positively stained with each dye in the hypoglossal nuclei of each injection site, in cats 1 month after nerve cutting, fluorescent dye was only observed in positive cells in the hypoglossal nucleus of the intact side and the dye injected into the neurectomy side showed a mixture into positive cells of the intact side. The findings suggest that muscles in the neurectomy side may be compensated by regeneration of the peripheral nerves on the intact side.     

STABILITY OF DNA IN PURKINJE CELL NUCLEI OF THE MOUSE : An Autoradiographic Study

Neurons of the mouse were labeled with [³H]thymidine during their prenatal period of proliferation. The ³H activity of the Purkinje cell nuclei was then studied autoradiographically 8, 25, 55, and 90 days after birth. The measured grain number per nucleus decreased by about 14% between the 8th and 25th postnatal days and then remained constant up to 90 days. There was no significant decrease of the ³H activity of the Purkinje cell nuclei after correction of the measured grain number per nucleus for increasing nuclear volume of the growing Purkinje cells and for the influence of [³H]β self-absorption in the material of the sections. Injection of a high dose of [³H]thymidine into young adult mice did not result in ³H labeling of either Purkinje or other neurons in other brain regions. The results agree with the concept of metabolic stability of nuclear DNA. "Metabolic" DNA could not be observed in these experiments.     

EFFECTS OF COLCHICINE ON AXONAL TRANSPORT IN PERIPHERAL NERVES

—Colchicine injected intracisternally markedly inhibited the rapid migration (300-400 mm/day) of labelled proteins in the hypoglossal and vagus nerve of the rabbit. The transport of acetylcholinesterase (EC 3.1.1.7) and choline acetyltransferase (EC 2.3.1.6) previously shown to move with the slow (5-26 mm/day) phase of axoplasmic transport in these nerves, was only partially blocked. In view of this differential effect on axonal flow, we suggest that the neurotubules, on which colchicine acts preferentially, are primarily involved in the rapid (300-400 mm/day) axoplasmic flow. After local injection of colchicine into the nerves both the rapidly migrating labelled proteins and the enzymes (AChE and ChAc) accumulated above the site of injection to the same degree as they accumulate above a nerve ligation. Since this blockage of enzyme transport occurred after concentrations of colchicine much higher than those used for intracisternal injections these findings after local injection may represent more severe effects on axonal transport systems.