Enzymatic differentiation of arterial and venous segments of the capillary bed during the development of free muscle grafts in the rat

The revascularization of freely grafted muscles in the rat was studied by histochemical reactions that on frozen sections stain the arterial part of the capillary bed blue (alkaline phosphatase [AP] reaction) and the venous part of the capillary bed red (dipeptidyl peptidase IV [DPP IV] reaction). In 112 rats the extensor digitorum longus or soleus muscles were freely grafted and removed at various times up to 93 days following the surgery. In cross section, the capillaries of a normal muscle show a mosaic pattern of staining for the activity of the two enzymes. After grafting, DPP IV activity of capillaries is lost throughout the entire graft within a day or two; but within ischemic muscle, weak and diffuse AP staining persists in capillary remnants for up to 6 days. In the very periphery of the graft AP staining is also preserved in partially damaged capillaries. By 4 days, new AP-positive capillaries can be identified at the periphery of the graft, and in succeeding days AP-positive capillaries are found progressively nearer the center of the graft. At 7 days, the capillary/muscle-fiber ratios are 66% of normal in the periphery of the graft and 44% in the intermediate zone. DPP IV-stained capillaries are not seen until 7 days after grafting. By 60 days, when the grafts have become stabilized, the mosaic pattern of capillary staining has become reestablished. In mature grafts, the number of capillaries per unit area was slightly higher than that in control muscles, but the capillary/muscle-fiber ratio was slightly lower, owing to the smaller than normal cross-sectional areas of the regenerated muscle fibers.     

Functional deficits in skeletal muscle grafts

Individual skeletal muscle fibers degenerate and regenerate with minimal functional deficits. When whole skeletal muscles are grafted in rats or cats by standard grafting techniques, revascularization and reinnervation must occur spontaneously. Under these circumstances, contraction times and maximum velocities of shortening eventually return to control values, but a significant deficit is observed in maximum tetanic tension. Grafts made with anastomosis of nerves or with nerves left intact have smaller deficits in tension development than do standard grafts made without nerve repair. The measurement of contractile properties of single motor units in extensor digitorum longus (EDL) muscles and in EDL grafts in rats indicates that the decreased maximum tetanic tension of whole grafts is due to a 10-20% decrease in the maximum tetanic tension of individual motor units, whereas standard grafts also show a 40-45% decrease in the number of motor units. Compared with control values, the fatigability of 100-mg grafts in rats is decreased, whereas larger 3-g grafts in cats show an increased fatigability. The deficits observed in large grafts can be reduced, but not eliminated, by grafting with neurovascular anastomoses.     

Acetylcholine receptor distribution on regenerating mammalian muscle fibers at sites of mature and developing nerve-muscle junctions

When rat soleus muscles fibers regenerated after notexin-induced damage, AChRs were present at high density on the surface of the new muscle fibers at the sites of the original NMJs, even if the intact motor axons were not present during regeneration. Some AChR molecules which were labelled with R-BgTx before notexin-induced damage persisted for some days at junctional sites after new muscle fibres had regenerated. During muscle fiber degeneration, components of the muscle fiber plasma membrane appeared to remain longer in the junctional region than elsewhere. When muscles on which new "ectopic" NMJs had been forming for at least 2 weeks were damaged, AChR clusters together with sites of high AChE activity were present 2 weeks later on the regenerated muscles in the region of new NMJ formation, even if the "foreign" nerve was not intact during the period of regeneration. If ectopic NMJs had been forming for only 4 days at the time of muscle and nerve damage, neither AChR clusters nor AChE activity were detected on the regenerated muscle fibers.     

The effects of dynamic tension and reduced graft size on muscle regeneration in rabbit free muscle grafts.

In a study of 28 adult New Zealand White rabbits, the influence of tension and size on muscle regeneration in tibialis anterior free muscle grafts (without vascular anastomoses) was examined 6 months after transplantation. Three laboratory models were studied: (1) whole dynamic (WD) graft (allowing ankle excursion and, therefore, variable dynamic physiologic tension), (2) whole static (WS) graft (constant, fixed length and, thus, only isometric tension), and (3) longitudinally sliced (reduced radius) dynamic (SD) model. Bilateral orthotopic grafts of the tibialis anterior muscle were performed in 24 rabbits (eight animals in each of the three different model groups). Controls consisted of normal tibialis anterior muscle from four age-matched rabbits. All tibialis anterior muscle grafts were examined histologically (fiber counts) and functionally (determined by in situ contractile properties under maximal stimulation conditions). The WD grafts demonstrated a significantly higher number of regenerated fibers per muscle cross section (4819 +/- 589) than the WS (2221 +/- 603) or SD (1919 +/- 732) grafts. The amount of tetanic tension in the WD grafts was 35 percent of the control and twice as much as that of the WS grafts (WD 1.0 +/- 0.2 kg versus WS 0.5 +/- 0.4 kg; p less than 0.05). The SD grafts produced approximately one-third as much maximum tetanic tension as the WD grafts (0.3 +/- 0.1 kg versus 1.0 +/- 0.2 kg), demonstrating that the amount of recovery was similar in these two dynamic models, since only the longitudinal middle third of the muscle was grafted in the SD model. Free muscle grafts under dynamic tension, which allows excursion, have shown a greater amount of muscle-fiber regeneration and restoration of function compared with a graft with fixed length. The positive effect of dynamic mechanical tension on small autogenous free muscle grafts (without vascular anastomoses) is clinically significant in the reconstruction of facial and hand neuromuscular deficits when blood vessels are not available for reanastomosis. Future studies using the tibialis anterior WD and SD transplant models will strengthen our understanding of the events of spontaneous revascularization and skeletal muscle regeneration.     

Quantitative differences among EMG activities of muscles innervated by subpopulations of hypoglossal and upper spinal motoneurons during non-REM sleep - REM sleep transitions: a window on neural processes in the sleeping brain

In the rat, a species widely used to study the neural mechanisms of sleep and motor control, lingual electromyographic activity (EMG) is minimal during non-rapid eye movement (non-REM) sleep and then phasic twitches gradually increase after the onset of REM sleep. To better characterize the central neural processes underlying this pattern, we quantified EMG of muscles innervated by distinct subpopulations of hypoglossal motoneurons and nuchal (N) EMG during transitions from non-REM sleep to REM sleep. In 8 chronically instrumented rats, we recorded cortical EEG, EMG at sites near the base of the tongue where genioglossal and intrinsic muscle fibers predominate (GG-I), EMG of the geniohyoid (GH) muscle, and N EMG. Sleep-wake states were identified and EMGs quantified relative to their mean levels in wakefulness in successive 10 s epochs. During non-REM sleep, the average EMG levels differed among the three muscles, with the order being N>GH>GG-I. During REM sleep, due to different magnitudes of phasic twitches, the order was reversed to GG-I>GH>N. GG-I and GH exhibited a gradual increase of twitching that peaked at 70-120 s after the onset of REM sleep and then declined if the REM sleep episode lasted longer. We propose that a common phasic excitatory generator impinges on motoneuron pools that innervate different muscles, but twitching magnitudes are different due to different levels of tonic motoneuronal hyperpolarization. We also propose that REM sleep episodes of average durations are terminated by intense activity of the central generator of phasic events, whereas long REM sleep episodes end as a result of a gradual waning of the tonic disfacilitatory and inhibitory processes.     

Skeletal muscle fiber regeneration following heterotopic autotransplantation in cats.

Whole 3 g extensor digitorum longus (EDL) muscles of cats were autotransplanted. The EDL muscles were either transplanted without denervation prior to transplantation (normal transplants) or denervated 3 to 4 weeks prior to transplantation (pre-denervated transplants). A few peripheral skeletal muscle fibers survived transplantation but most fibers degenerated and then regenerated as the transplant became revascularized. Both normal and pre-denervated muscles regenerated successfully and by 50 days after transplantation fibers which had reinnervated showed high and low myofibrillar ATPase activity. Compared to controls, the smaller mean fiber cross-sectional area of the transplants was due to the large number of small fibers, but some fibers in the transplant were larger than any fibers observed in the controls. Transplants regained 57 percent of the muscle mass of the controls. Contraction and half relaxation times of transplanted muscles were slower than controls, but peak isometric tetanus tension per cm² of muscle was nearly normal. Fifty to 170 days after transplantation, muscles showed low oxidative capacity and fatigued rapidly.     

Morphological aspects of muscle fiber regeneration

Although striated muscle displays remarkable regenerative potential, the three-dimensional cytoarchitecture of the regenerated myofibers is different from that of myofibers formed during fetal development. It has been demonstrated with spaced, serial ultrathin sections that the regenerating myotubes that occur spontaneously (i.e., without secondary trauma) in dystrophic (dy2J) murine muscle and the regenerating fibers found in free whole-muscle transplants of normal, murine extensor digitorum longus muscles branch and recombine, forming a complex syncytium. Multiple motor end-plate regions are observed on the branched syncytia found in dystrophic muscle. Branched fibers persist in long-term grafts and are found with a frequency that indicates that they should be of physiological significance. Although the number of myofibers found in long-term grafts is approximately 68% of that found in control muscle, comparison of the diameter distributions of the regenerated muscle fibers with age-matched control fibers indicates that many of the regenerating fibers fail to achieve normal size. Type IIb fibers appear to be more growth inhibited than type IIa fibers. The size of the motoneuron pool to grafted muscles is smaller than that to control muscles.     

Maturation of spontaneous fetal diaphragmatic activity and fetal response to hypercapnia and hypoxemia

The electromyogram (EMG) of the diaphragm, lateral rectus, and nuchal and hindlimb muscles were studied during spontaneous activity and during hypercapnia or hypoxemia in eight fetal sheep from 0.5 to 0.8 gestation (73-128 days). At the earliest gestational age, diaphragmatic EMG activity was mainly tonic and associated with tonic activity of somatic muscles. The stimulus for the diaphragmatic activity originated centrally. Brief periods of a rapid-eye-movement (REM) state characterized by phasic lateral rectus and diaphragmatic activity and absence of nuchal activity were recognized. Furthermore, from 0.5 to 0.7 gestation onward, activity of all muscles increased. Thereafter increased specificity of activity in relation to the apparent REM and non-rapid-eye-movement (NREM) state occurred. With maturation, phasic diaphragmatic activity increased at the expense of tonic activity. The most striking effect of maturation on apnea was a greater proportion of apnea lasting greater than 1 min, but the total duration of apnea as a percent of a total recording remained unchanged. The quantitative response to hypercapnia during maturation was independent of the pattern of spontaneous diaphragmatic activity. Hypercapnia at 0.5 gestation changed the pattern of diaphragmatic EMG activity from mainly tonic to phasic. Thus the central chemoreceptors and appropriate neuronal pathways are present and functional as early as 0.5 gestation. Hypercapnia at 0.5 gestation caused a shift in diaphragmatic EMG power to lower frequencies similar to that found during control conditions in the older fetus. This might suggest that during maturation there is increased recruitment of phrenic motoneurons. Hypoxemia abolished tonic somatic activity at 0.5 gestation and decreased phasic diaphragmatic activity at more advanced gestational ages. Therefore the central inhibitory mechanisms of hypoxemia are developed by 0.5 gestation.     

Collateral sprouting occurs following end-to-side neurorrhaphy

Recent evidence supports the use of end-to-side neurorrhaphy for the treatment of certain peripheral nerve disorders. However, the mechanism by which nerves regenerate following this procedure is still unclear. To address this question, the authors designed a new end-to-side coaptation model in rats in which the donor nerves were uninjured. The regenerated axons at the coaptation site were observed directly using fluorescent dye as the neural tracer. The sciatic nerve from adult Wistar rats was transplanted between the left and right median nerves. Fifteen rats were divided into three groups. In group I, the donor (right median) nerve was sutured end to side to the divided grafted nerve using a noninjury technique. In group II, the aponeurosis of the spinal muscles was harvested and the sciatic and right median nerves were coapted end to side noninjuriously by wrapping them in the excised aponeurosis. In group III, a perineurial window was created and a partial neurectomy was carried out at the suture site, after which the sciatic and right median nerves were sutured end to side. Sixty days after the operation, nerve regeneration was evaluated by recording action potentials in the grafted nerve, by performing electromyography in the flexor muscles in the forearm, and by histological examination. The grafted nerves were fixed and sectioned, the number of regenerated nerve fibers was counted, and axonal diameters were measured. Fluorescent dye crystal was used, in conjunction with confocal microscopy, to observe the regenerated axons at the co-aptation site. The results showed that nerve regeneration had occurred in the animals, as determined electrophysiologically and histologically. Both the right and left flexor muscles of the forearm contracted simultaneously as a result of indirect electric stimulation of the grafted nerve, which suggests that the regenerated nerve was physiologically connected with the donor nerve. Nerve fiber counts did not show any differences among groups (p > 0.05), but axonal diameters were significantly greater in group III than in the other two groups. Fluorescent dye staining revealed the presence of regenerated nerve fibers beyond the coaptation site. In group III, the regenerating nerves were observed within the whole section of the coaptation site and collateral sprouting was found to occur even at a site distal to the suture. From these results, the authors conclude that in end-to-side neurorrhaphy, nerve regeneration occurs by collateral sprouting from the donor nerve.     

Contractile properties,structure and fiber phenotype of intact and regenerating slow-twitch muscles of mice treated with cyclosporin A

We have studied the contractile properties, structure, fiber-type composition, and myosin heavy chain (MyHC) expression pattern of regenerating and intact soleus muscles of adult CBA/J mice treated with cyclosporin A (CsA) or vehicle solutions (Cremophor, saline). A comparison of muscles after 4-7 weeks drug application with those receiving vehicle showed that the isometric contractile force of intact drug-treated muscles was reduced (tetanus, -21%; twitch, -34%) despite normal mass and muscle cross-sectional area. The frequency of fast-twitch fibers was increased, whereas no innervation deficits, histopathological alterations, or changes in fiber numbers were observed. Regeneration after cryolesion of the contralateral soleus proceeded more slowly in CsA-treated than in vehicle-treated animals. Despite this, when muscle properties reached mature levels (4-7 weeks), muscle mass recovery was better in CsA-treated animals (30% higher weight, 50% more fiber profiles in cross-sections). The force production per unit cross-sectional area was deficient, but not the maximum tension. Twitch time-to-peak and half-relaxation time were shorter than controls correlating with a predominance of fast-twitch fibers (98% Type II fibers versus 16%-18% in control muscles) and fast MyHC isoforms. Partial reversal of this fast phenotype and an increase in muscle force were observed when the animals were left to recover without treatment for 5-8 weeks after CsA application over 7 weeks. The high numbers of fiber profiles in CsA-treated regenerated muscles and increased mass remained unchanged after withdrawal. Thus, CsA treatment has a hyperplastic effect on regenerating muscles, and drug-induced phenotype alterations are much more prominent in regenerated muscles.     

The microscopic morphology of orthotopic free muscle grafts in rabbits

The flexor digitorum superficialis muscle was free grafted (without neurovascular anastomoses) in 122 rabbit forelimbs. Histologic nd histochemical examinations through 6 months after grafting were performed. An early ischemic necrosis of the entire graft, except for a few percent of fibers at the very surface, was consistently seen. Subsequently, there occurred a regeneration of muscle with reconstitution of up to 100 percent of normal numbers of fibers. There was a wide variation in the numbers of fibers regenerated; however, the fiber-free areas were then being replaced by connective tissue. Muscle grafts in 1-month-old rabbits regenerated faster and yielded muscle with evidence of more extensive reinnervation and less connective tissue than 3-month-old animals. In the early postgraft period, minced grafts appeared to be as good as whole ones, but after 1 month, they developed far more connective tissue. Differentiation into fast-twitch and slow-twitch muscle fibers and into high- and low-oxidative fibers began at 2 to 3 weeks after grafting but was not extensive until 3 months. At 6 months, grafts showed areas of normal-appearing muscle interspersed with areas that lacked signs of reinnervation. The earliest sign of regeneration is the appearance of several very elongated nuclei encircling each previously anucleate necrotic muscle fiber. A small amount of basophilic cytoplasm then appears around each new nucleus. As blood vessels grow into the graft, a centripetal wave of phagocytosis is seen, taking 2 to 3 weeks and leaving a bed of immature muscle fibers. We believe this to be the first documentation of regeneration's commencing prior to and thus independently of phagocytosis.     

REGENERATION OF PIGEON FAST AND SLOW MUSCLE FIBER TYPES AFTER PARTIAL EXCISION AND MINCING

The pigeon's metapatagialis muscles, containing fast fibers in two slips and slow fibers in another slip, were excised for a third of their length, minced, and replaced into their previous sites. After regeneration, the pattern of fiber types and their ATPase and oxidative enzymes were examined histochemically. Ultrastructural examination was carried out on the fast fibers. After 4–17 wk the muscles had regenerated into patterns histochemically similar to the controls only within the slip containing fast fibers. The slow slip was much less regenerated, and had a histochemically embryonic composition. Fiber types were characterized and their cross-sectional areas measured, and the degree of atrophy was greatest in the large fast fibers and the slow fibers. Ultrastructural studies revealed a number of alterations of the mitochondria, including dense and light areas in the matrix and an altered pattern of the cristae into parallel tubular or vesicular aggregations. Other changes included dilated sarcoplasmic reticulum, myofibril disorganization, and a compaction of filaments. The slow fibers were thought to be slower in their regeneration rates because of the pattern of multiple innervation's producing a more complex regenerative pattern.     

Effect of foreign innervation on contractile and histochemical properties of the transplanted levator ani muscle of the rat.

Heterotopic transplantation of the levator ani (LA) muscle into the bed of the fast tibialis anterior (TA) or slow soleus (SOL) muscle respectively results in transformation of contractile and histochemical properties of the muscle dependent on the new "foreign" innervation. This transformation is observed after transplantation of minced muscle tissue and of free grafts. The result of transformation is more pronounced in the case of free LA-TA grafts which show progressive shortening of contractile response, whereas the LA-SOL shows slight shortening. The heterotopically transplanted free LA-SOL and the LA-TA grafts become relatively faster than the respective original muscle, suggesting operation of myogenic factors related to the fast LA muscle. Maximal tetanic tension output of the free heterotopic grafts 60 days after transplantation recovers to only about a quarter of the correspondong control muscles. Recovery of speed of contraction in the transplanted LA muscle is similar to that observed after selfreinnervation after crushing the pudendal nerve close to its entry into the muscle. In the heterotopically transplanted muscles the reversal of the originally uniform histochemical fibre pattern to a mixed fibre pattern in respect to ATPase and SDH activity is dependent on the type of innervation. After selfreinnervation of the LA muscle by the pudendal nerve a uniform fibre pattern is maintained with regeneration of the nerve.     

Tissue engineering of peripheral nerves: A comparison of venous and acellular muscle grafts with cultured Schwann cells

Bioengineering is considered to be the laboratory-based alternative to human autografts and allografts. It ought to provide "custom-made organs" cultured from patient's material. Venous grafts and acellular muscle grafts support axonal regeneration only to a certain extent because of the lack of viable Schwann cells in the graft. We created a biologic nerve graft in the rat sciatic nerve model by implanting cultured Schwann cells into veins and acellular gracilis muscles, respectively. Autologous nerve grafts and veins and acellular muscle grafts without Schwann cells served as controls. After 6 and 12 weeks, regeneration was assessed clinically, histologically, and morphometrically. The polymerase chain reaction analvsis showed that the implanted Schwann cells remained within all the grafts. The best regeneration was seen in the control; after 12 weeks the number of axons was increased significantly compared with the other grafts. A good regeneration was noted in the muscle-Schwann cell group, whereas regeneration in both of the venous grafts and the muscle grafts without Schwann cells was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. The venous grafts with Schwann cells showed less fibrous tissue and disorganization than the veins without Schwann cells, but failed to show an excellent regeneration. This might be attributed to the lack of endoneural-tube-like components serving as scaffold for the sprouting axon. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biologic graft with basal lamina tubes as pathways for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

Muscle spindle formation and differentiation in regenerating rat muscle grafts

Muscle spindle development and function are dependent upon sensory innervation. During muscle regeneration, both neural and muscular components of spindles degenerate and it is not known whether reinnervation of a regenerating muscle results in reestablishment of proper neuromuscular relationships within spindles or whether sensory neurons may exert an influence upon differentiation of these spindles. Muscle spindle regeneration was studied in bupivacaine-treated grafts of rat extensor digitorum longus (EDL) muscles. Three types of EDL graft were performed in order to manipulate the extent to which regenerating spindles might be reinnervated: (1) grafts reinnervated following severance of their nerve supply (standard grafts); (2) grafts in which intact nerve sheaths appear to facilitate reinnervation (nerveintact grafts); and (3) grafts in which reinnervation was prevented (nonreinnervated grafts). Complete degeneration of muscle fibers occurred in all grafts prior to regeneration. Initial formation of spindles in regenerating EDL grafts is independent of innervation; intrafusal muscle fibers degenerate and regenerate within spindle capsules that remain intact and viable. The extent of spindle differentiation was evaluated in each type of graft using criteria that included nucleation and ATPase activity, both of which have been shown to be regulated by sensory innervation, as well as the number of muscle fibers/spindle and morphology of spindle capsules.While most spindles contained normal numbers of muscle fibers, most of these fibers were morphologically and histochemically abnormal. Alterations of ATPase activity occurred in all spindles, but were least severe in nerve-intact grafts. While fully differentiated nuclear bag and chain fibers were not observed in regenerated spindles, large, vesicular nuclei, similar to those of normal intrafusal fibers, were present in a small number of spindles in nerve-intact grafts. Sensory nerve terminations were observed only in those spindles that also contained the distinctive nuclei. This study suggests that a specific neurotrophic influence is necessary for regeneration of normal intrafusal muscle fibers and that this influence corresponds to the properly timed sensory neuron-muscle interaction which directs muscle spindle embryogenesis. However, the infrequent occurrence of characteristics unique to intrafusal muscle fibers indicates that reinnervation of regenerating muscle grafts by sensory neurons is inadequate and/or faulty.     

Formation of muscle spindles in regenerated avian muscle grafts

Summary Pigeon muscles lacking muscle spindles were grafted into sites which normally have a muscle containing spindles. The reciprocal transplantations were also made. After two to eight months, the graft of the donor muscle without spindles had regenerated into a muscle containing muscle spindles. The reciprocal grafts, muscles containing spindles transplanted to a site lacking spindle innervation, had neither muscle spindles nor remnants of the spindles. These experiments demonstrate that 1) the innervation is required for formation of the spindle; 2) the original spindles do not survive transplantation; and 3) parts of the original spindle are not required for spindle regeneration.This work was supported in part by NSF grants PCM 77-15960 and PCM 79-16540     

Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts

Summary Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with ³H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later.In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The present experiments provide a direct proof of utilization of donor satellite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.     

Inhomogeneous response of expiratory muscle activity to cold block of the ventral medullary surface.

We assessed the effects of cooling the ventral medullary surface (VMS) on the activity of chest wall and abdominal expiratory muscles in eight anesthetized artificially ventilated dogs after vagotomy and denervation of the carotid sinus nerves. Electromyograms (EMGs) of the triangularis sterni, internal intercostal, abdominal external oblique, abdominal internal oblique, and transversus abdominis muscles were measured with EMG of the diaphragm as an index of inspiratory activity. Bilateral localized cooling (2 x 2 mm) in the thermosensitive intermediate part of the VMS produced temperature-dependent reduction in the EMG of diaphragm and abdominal muscles. The rib cage expiratory EMGs were little affected at 25 degrees C; their amplitudes decreased at lower VMS temperatures (less than 20 degrees C) but by significantly fewer degrees than the diaphragmatic and abdominal expiratory EMGs at a constant VMS temperature. With moderate to severe cooling (less than 20 degrees C) diaphragmatic EMG disappeared, but rib cage expiratory EMGs became tonic and resumed a phasic pattern shortly before the recovery of diaphragmatic EMG during rewarming of the VMS. These results indicate that the effects of cooling the VMS differ between the activity of rib cage and abdominal expiratory muscles. This variability may be due to inhomogeneous inputs from the VMS to expiratory motoneurons or to a different responsiveness of various expiratory motoneurons to the same input either from the VMS or the inspiratory neurons.     

The role of grafted skin in the regeneration of X-irradiated axolotl limbs

The primary aim of these experiments was to follow the cells descended from limb skin through the process of limb regeneration to determine what range of differentiations these cells may assume. Triploid hindlimb or forelimb skin was grafted to the denuded thighs of diploid host axolotls that had previously received 3000 R of X irradiation across both hindlimbs and the intervening pelvic area. The host limbs were then amputated through their grafts and permitted to regenerate. Cartilage, perichondrium, joint connective tissue, general connective tissue, dermis, and epidermis were present in all the regenerated limbs, but only 10% of the regenerates contained muscle. Tabulation of nucleolar numbers showed that the majority of cells in each regenerated tissue originated from the grafted skin. A strong correlation was demonstrated between the forelimb or hindlimb origin of the skin grafts and the number of digits regenerated.     

Innervation of regenerated spindles in muscle grafts of the rat

Features of the nerve supply and the encapsulated fibers of muscle spindles were assessed in grafted and normal extensor digitorum longus (EDL) muscles of rats by analysis of serial 10-microns frozen transverse sections stained for enzymes which delineated motor and sensory endings, oxidative capacity and muscle fiber type. The number of fibers was significantly more variable, and branched fibers were more frequently observed in regenerated spindles than in control spindles. Forty-eight percent of regenerated spindles received sensory innervation. Spindles reinnervated by afferents had a larger periaxial space than did spindles which were not reinnervated by afferents. Regenerated fibers innervated by afferents had small cross-sectional areas, equatorial regions with myofibrils restricted to the periphery of fibers, unpredictable patterns of nonuniform and nonreversible staining along the length of the fiber for 'myofibrillar' adenosine triphosphatase (mATPase) after acid and alkaline preincubation. In contrast, regenerated fibers devoid of sensory innervation resembled extrafusal fibers in that they usually exhibited myofibrils throughout the length of the fiber, no central aggregations of myonuclei, uniform staining for mATPase and a reversal of staining for mATPase after preincubation in an acid or alkaline medium. Approximately thirty percent of encapsulated fibers devoid of sensory innervation stained analogous to a type I extrafusal fiber, a pattern of staining never observed in intrafusal fibers of normal spindles. Groups of encapsulated fibers all exhibiting this pattern of staining reflect that either these fibers may have been innervated by collaterals of skeletomotor axons that originally innervated type I extrafusal fibers or that fibers innervated by only fusimotor neurons express patterns of staining for mATPase similar to extrafusal fibers in the absence of sensory innervation. Sensory innervation may also influence the reestablishment of multiple sites of motor endings on regenerated intrafusal fibers. Those regenerated fibers innervated by afferents had more motor endings than did regenerated fibers devoid of sensory innervation. Differences in size, morphology, and patterns of staining for mATPase and numbers of motor endings between fibers innervated by afferents and fibers devoid of sensory innervation reflect that afferents can influence the differentiation of muscle cells and the reestablishment of motor innervation other than during the late prenatal/early postnatal period when muscle spindles form and differentiate in rats.