.Mammalian skeletal muscle almost invariably regenerates when transected. If the wound is small regeneration may be complete. Cardinal signs of the regenerative process are basophilic, multinucleated myotubes described as early as 1865 by Waldeyer. In mouse muscle the myotubes become prominent about 4 days after transection. Each myotube contains centrally located nuclei which are arranged in linear order. The nuclei increase, markedly, particularly between the fifth and sixth days after transection. Because there is an apparent absence of mitosis within the multinucleated tubes some workers (see Godman, '57) have drawn the inference that nuclei increase amitotically. Lash, Holtzer and Swift ('57) analyzed Feulgen stained regenerative muscle cytophotometrically and found no evidence for cell division within myotubes. They did note a rise in mitotic active among mononucleated cells on the day prior to sudden increases both in the number of tubes and in the number of intratubular nuclei.SUMMARY
Transected mammalian skeletal muscle was treated locally with colchicine at different intervals after initial wounding. Colchicine had no discernible effect on regneration when administered so as to miss the period of greatest cell division among cells of the wound coagulum posited to be myoblasts. When the inhibitor was applied during the period of increased mitotic activity among the cells in question, then muscle regeneration was almost completely curtailed.
Thus there are two points of view concerning the means by which new muscle fibers differentiate during the regenerative process. According to one, muscle regeneration would amount to a distally directed hyperplasia with nuclear increases resulting from some unusual means of multiplication (namely, amotosis). According to the other view, mammalian skeletal muscle regeneration would be the consequence of the fusion of highly proliferative, mononucleated myoblasts of the wound coagulum (for literature and extensive reviews see Godman, '58 and Lash et al '57).
The purpose of this work was to test the latter hypothesis. If regenerative myoblasts are mononucleated cells which multiply mitotically, then a known inhibitor of mitosis (viz., the spindle-poisoning colchicine) should prevent or curtail myotube formation if it applied at the critical two to three day period after transection.
Two groups of experiments are to be reported:
Tissues were fixed on days 2, 3, 4 and 6 following transection. Fixatives were Zenker-formalin or acetic acid-ethanol-formalin. Tissues were handled routinely for paraffin sectioning at 10 u. Staining was with Ehrlich's alum hematoxylin and eosin Y.
Tissues fixed on days 2 and 3 were studied chiefly for mitosis. Mitotic activity is expressed as the percentage of mitotic figures among round cells of the wound coagulum. Signs of regeneration (basophilic multinucleated columns or myotubes) are prominent from day 4 onward.
Four- and 6-day tissues were studied histologically for evidence of muscle regeneration. Muscle was regenerating in each 4- and 6-day specimen whether treated with colchicine or not.
Colchicine-treated two-, three- and 4-day specimens were examined for evidence of nuclear duplications within multinucleated myotubes an injured muscle fibers. Though a given microscopic field might possess many arrested mitotic figures (figs. 3 and 4), cell division was never found within a muscle tube. Occasionally, a dividing cell was in close proximity with a muscle fiber and appeared to be within it. Examination at a magnification of 970 X revealed, always, that these cells were on, rather than within the muscle fiber (see however Pogogeff and Murray, '46; Walker and Bintliff, '60).
It is noted that the decline in mitotic activity from the three-day high took place even though colchicine was administered within the preceding 24-hour period.
Water and saline treated 4- and 6-day tissues presented, without exception, typical pictures of normal mammalian skeletal muscle regeneration (fig. 5). None of the colchicine-treated tissues showed signs of regeneration on days 4 and 6. In the place of numerous and distinct basophilic myotubes, each specimen treated with colchicine on days 2 and 3 showed a wound coagulum made up of numerous and diffusely scattered free cells (cf. figs. 6 and 7). There were many arrested mitotic figures among the latter cells. Close examination of colchicine-treated 4-day specimens revealed an occasional accumulation of nuclei within a common sphere of cytoplasm. These were not inflammatory giant cells, nor were they typical regenerative myotubes. However, the possibility existed that the nuclear accumulations in question were signs of regeneration. For that reason the accumulations were counted in each section of each available 4-day specimen (injected on days 2 and 3), and their numbers were than compared with the typical myotubes found among normal and vehicle treated specimens.
Data in figure 2 illustrate that muscle was severely curtailed upon administration of colchicine on days 2 and 3 after transection. There seemed to be more myotubes per section in normal regenerates than among specimens injected with vehicle. Despite the latter differences, vehicle treated and untreated specimens were indistinguishable from each other histologically.
Colchicine-treated 6-day tissues of the group of experiments being considered were compared with 6-day tissues which had received the alkaloid on days 1 and 2 following transection. The specimens were rated: "A," when they were indistinguishable from normal or vehicle treated regenerates, "B," when regeneration had not taken place and as "C" if the myotubes resembled those of earlier stages of regeneration. Results are presented in table 1.
All cases treated on the first and second days presented normal histological pictures at day 6, and, hence all were rated "A." Treatment on the second and third days led to 6-day regenerates either possessing no signs ("B") of regeneration or resembling sine earlier phase of the process ("C").
Each colchicine-treated specimen was examined for evidence of mitosis within muscle fibers or myotubes. No division, mitotic or amitotic, was found with a multinucleated muscle tube.
It would be a mistake to conclude that the evidence presented is direct proof for the myoblast as a free cell, for the specific action of colchicine could not be ascertained (see also discussions of Eigsti and Dustin. '55). However, Walker and Bintliff ('60) injected tritium labeled thymidine into muscle transected muscle and found, subsequently, that some myotubes nuclei were labeled. Lash (unpublished) noted that a large number of nuclei were labeled when tritiated thymidine was injected coincident with the three-day increase in mitotic activity. He also observed that labeling of myotube nuclei was rare when the isotope was administered at noncritical times.
Studies with isotopes, cytophotometric analsysis (Lash et al '57) and the results obtained in this study favor the view that the myoblast of regenerative mammalian skeletal muscle is mononucleated and that myoblast increases are mitotic. Since experimental evidence does support the latter hypothesis, serious consideration must be given the thesis that regeneration of mammalian muscle is, indeed, a bona fide act of histogenesis.
Fig. 1: Percentages of round cell mitoses in coagulums of colchicine and vehicle treated skeletal muscle wounds.
Fig. 2: EXP refers to nuclear accumulations in 4-day wounds after local injection of colchicine on days 2 and 3 following transection (these were not typical myotubes). LAT indicates the numbers of typical regenerative myotubes encountered in untreated 4-day wounds after injection of vehicle. CON refers to the number of myotubes encountered in untreated 4-day regenerates. The bars indicate maximum as well as minimum numbers of accumulations or myotubes in each 10 u section through the wound coagulum.
Fig. 3: Section through the wound coagulum three days after muscle transection. Specimen was injected with colchicine one and two days after transection. The cells indicated by arrows were seen to contain arrested mitotic figures when examined under oil immersion (note fig. 4). X 1,075:
Fig. 4: Oil immersion photograph of a colchicine arrested mitotic figure within a mononucleated cell. Section was through same region as in figure 3. X 2,425Fig. 5: Myotubes in water-injected 6- day regenerate. X 430.
Fig. 6: Section through the wound coagulum of a 4- day control regenerate. Distilled water was injected on days 2 and 3 following transection. Note the narrow, dark staining myotubes within which are linearly arranged nuclei. X 250:
Fig. 7: Section through wound coagulum in specimen which received in injections of colchicine on days 2 and 3 following transection. Myotubes are not to be found. X 250.
| Treatment | A -- typical 6-day regenerate | B -- absence of regeneration | C -- retarded regeneration* |
|---|---|---|---|
| Colchicine at 24 & 48 hrs. after transection | 7 | 0 | 0 |
| Colchicine at 48 & 72 hrs. after transection | 0 | 8 | 2 |
| Distilled water contralateral to treated wound | 8 | 0 | 0 |
| Normal regenerate | 4 | 0 | 0 |
*typical of day 4 instead of day 6
**Presently at Indiana University, Bloomington