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.     

Reinnervation of human skin grafts: a histochemical study

The reinnervation of nine human skin grafts was investigated using histochemical thiocholine methods for the demonstration of cholinesterases. The regenerated cutaneous nerves showed both specific acetylcholinesterase and nonspecific cholinesterase reactions. In the youngest specimens, taken 3 weeks after the grafting, such regenerated nerves were seen both at the subdermal level under the graft and at the margins of the graft. These nerves seemed to orient toward the denervated graft area. The growing nerves were generally distributed in a random fashion. The reinnervation of some hair follicles, erector pili muscles, and sweat glands were observed in well-innervated full-thickness and thick partial-thickness skin grafts. It is suggested that this target-organ control of regenerating nerves occurs as a result of the action of chemotactic factors. A well-innervated graft bed seems to be important for optimum reinnervation of skin grafts. Fibrosis and scarring seem to hamper nerve regeneration.     

A review of free muscle grafting

Free grafting of small muscles is followed by graft necrosis and subsequent reconstitution of graft architecture by the process of muscle regeneration. Large muscles turn instead to scar. Revascularization and reinnervation can occur from the adjacent muscle bed, but reinnervation is more effective when a nerve is implanted or neurorrhaphy is performed. The appearance of a variable amount of connective tissue in a grafted muscle may degrade function. The necessity of "predenervation" remains in question. As we obtain greater experience in the laboratory and in clinical utilization, it is hoped that free muscle grafting will become a more predictable reconstructive procedure.     

Bone regeneration within a coralline hydroxyapatite implant.

The hypothesis that incomplete resorption of osteons in an autogenous cortical bone graft may limit its replacement by new bone regeneration was explored by implanting a hydroxyapatite replica of a coral skeletal structure into bone gaps. This implant contained channels and interconnections similar to those in osteon-evacuated bone grafts. In 6 implanted mandibular defects in dogs, two of which were examined at two, 4, and 6 months, 11 percent, 46 percent, and 88 percent of the implant areas were filled with regenerated bone. The regenerated bone was a woven type at two months, but changed to a lamellar type by 6 months. In two implanted defects examined at 12 months, biodegradation of 29 percent of the implant had occurred. The bone regeneration was physiological, the implant was biocompatible, and the biodegradation began after the bone had regenerated.     

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.     

Regeneration of the arterial wall in microporous,compliant, biodegradable vascular grafts after implantation into the rat abdominal aorta

Summary The ultrastructure of a new type of vascular graft, prepared from a mixture of polyurethane (95 weight %) and poly-L-lactic acid (5 weight %), was examined six weeks after implantation into the abdominal aorta of rats. These microporous, compliant, biodegradable, vascular grafts function as temporary scaffolds for the regeneration of the arterial wall.Smooth muscle cells, covering the grafts, regenerated a neo-media underneath an almost completely regenerated endothelial layer (neo-intima). These smooth muscle cells varied in morphology from normal smooth muscle cells to myofibroblasts. They were surrounded by elastic laminae and collagen fibers.Macrophages, epithelioid cells, multinucleated giant cells, fibroblasts and capillaries were present in the disintegrating graft lattices. The epithelioid cells and multinucleated giant cells engulfed polymer particles of the disintegrating grafts.The regeneration of the endothelial and smooth muscle cells is similar to the natural response of arterial tissue upon injury. The presence of macrophages, epithelioid cells, multinucleated giant cells, fibroblasts and capillaries in the graft lattices resembles the natural response of tissue against foreign body implants. Both of these responses result in the formation of a neo-artery that possesses sufficient strength, compliance and thromboresistance to function as a small caliber arterial substitute.Supported by Grant nr. 82.042 from the Dutch Heart Foundation     

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.     

Ipsilateral and cross-over elongation of the motor nerve by nerve grafting: an experimental study in sheep

In difficult reconstructions, ipsilateral or cross-over nerve grafting is sometimes necessary to achieve reinnervation and motor function. This experimental study in sheep was to answer the question of limitation of elongation of a motor nerve by grafting, the question of the optimal time for suturing the nerve graft to the muscle nerve, and the question of the successful application of this surgical technique in extremities. In 18 sheep, the vastus nerve was elongated by a saphenous nerve graft as long as possible up to 30 cm (step 1). In 10 animals the nerve graft was applied ipsilaterally, and in 8 animals it was used as a cross-over nerve graft to the contralateral limb. The time between nerve grafting and connection of the distal end of the nerve graft to the freshly cut rectus nerve supplying the rectus femoris muscle (step 2) was variable: 0, 3, 6, 9, and 12 months. In all animals, the final experiments (step 3) were performed 6 months after the last operation (step 2). Muscle force measurements in the rectus femoris muscle and quantitative analysis of the number and diameter of myelinated nerve fibers in cross sections of the nerve biopsies at different levels showed that elongation of a motor nerve by nerve grafting is principally not limited. The functional results were rather inhomogeneous and therefore unpredictable (ipsilateral group: maximum tetanic tension = 27 to 172 N; cross-over group: 0 to 227.5 N). Nevertheless, crossover nerve grafting is recommended for selected cases even in extremities. There was no correlation between the time interval between the two operations and the functional or morphologic results, although better functional results were obtained when the distal nerve suture (step 2) was performed some months after nerve grafting (step 1). A clear correlation was found only between the number of regenerated axons in the rectus nerve behind the second suture line and the muscle function.     

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.     

Nerve regeneration after correction of its defect with a vascularized autologous nerve transplant

In chronic experiment performed on 6 dogs, by means of some neurohistological techniques peculiarities of regeneration of nerves have been studied, when their defects are substituted for vascularized and common autoneurotransplants. When the vascularized autoneurotransplants are applied, more favourable conditions are made for the nerve regeneration, than at a free plastisity and amount of regenerated fibers distal to the graft reaches 34-88%, more often making 48-52% of the initial number of fibers in the proximal parts of the nerves in 3 months after the operation. When in the same animals in the contralateral side common autoneurotransplants have been applied, during the same time the amount of fibers regenerated into the peripheral parts of the nerves makes 10-26%, more often 17-20% from the initial level, or in 2-2.5 times less than at substitution of the defects for the vascularized grafts. Absence of an active cellular reaction of the surrounding tissues to the sutural material supramid is used.     

Nerve Cross-Bridging to Enhance Nerve Regeneration in a Rat Model of Delayed Nerve Repair

There are currently no available options to promote nerve regeneration through chronically denervated distal nerve stumps. Here we used a rat model of delayed nerve repair asking of prior insertion of side-to-side cross-bridges between a donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) nerve stump ameliorates poor nerve regeneration. First, numbers of retrogradely-labelled TIB neurons that grew axons into the nerve stump within three months, increased with the size of the perineurial windows opened in the TIB and CP nerves. Equal numbers of donor TIB axons regenerated into CP stumps either side of the cross-bridges, not being affected by target neurotrophic effects, or by removing the perineurium to insert 5-9 cross-bridges. Second, CP nerve stumps were coapted three months after inserting 0-9 cross-bridges and the number of 1) CP neurons that regenerated their axons within three months or 2) CP motor nerves that reinnervated the extensor digitorum longus (EDL) muscle within five months was determined by counting and motor unit number estimation (MUNE), respectively. We found that three but not more cross-bridges promoted the regeneration of axons and reinnervation of EDL muscle by all the CP motoneurons as compared to only 33% regenerating their axons when no cross-bridges were inserted. The same 3-fold increase in sensory nerve regeneration was found. In conclusion, side-to-side cross-bridges ameliorate poor regeneration after delayed nerve repair possibly by sustaining the growth-permissive state of denervated nerve stumps. Such autografts may be used in human repair surgery to improve outcomes after unavoidable delays.     

Reconstruction of ablated rat rectus abdominis by muscle regeneration

Skeletal muscle regeneration is a powerful, naturally occurring process of tissue reconstruction that follows myofiber damage secondary to myotoxic injury that does not normally affect the tissue circulation and scaffold. The ablated tissue, in traumatology and free muscle grafts, is frequently replaced by scars. The final outcome is poor even after in situ myoblast seeding of the harvested muscle. The goal of this study was to identify protocols to reconstruct muscle tissue, even in such adverse environments. The authors applied a step-by-step approach to identify factors favoring the survival of autologous satellite cells and, thus, muscle regeneration. In a rat model of full-thickness rectus abdominis muscle ablation, autologous myoblasts were isolated from the explanted rectus abdominis and seeded in a homologous acellular matrix immediately after wall reconstruction (group 1, five animals). In group 2 (five animals), the ablated rectus abdominis was autografted in situ. In a third group of five rats, Marcaine was injected into both the autograft and the surrounding abdominal wall muscle. Three weeks after surgery, serial cross-sections of the reconstructed abdominal wall were stained with hematoxylin and eosin or embryonic myosin antibody, a well-characterized molecular marker of early myogenesis in development and regeneration. Percentages of the patch area covered by regenerated myofibers were determined by morphometry. When autologous myoblasts were seeded in a homologous acellular matrix, the only myofibers observed to regenerate were those along the border of the patch. Autografting of the middle third of the rectus abdominis muscle similarly resulted in scar formation. The few muscle cells in the graft core were scanty myoblasts that could be detected only by monoclonal embryonic myosin antibody. Although negative for myofiber regeneration, the results in both cases confirmed the mechanical patency of the patches with regard to abdominal organ support. Myofibers were successfully regenerated in the graft by injecting Marcaine into both the autograft and the surrounding muscles. Three weeks after surgery, the patch was paved with young, centrally nucleated myofibers intermixed with young myofibers and myotubes expressing embryonic myosin. The difference in percentage of patch area covered by regenerated myofibers in group 3 (Marcaine injection around the patch, 81.6 +/- 3.0 percent) (mean +/- SD) versus either group 1 (Myoblast-seeded acellular patch, 18.0 +/- 3.0 percent) or group 2 (Autograft, 25.8 +/- 7.0 percent) was statistically significant on independent t test analysis (p < 0.0001). Even an acellular matrix showed some myofiber regeneration after surrounding muscles had been injected with Marcaine. This is the first successful evidence of muscle reconstruction after full-thickness ablation of the middle third of the rectus abdominis. Muscle regeneration seems to be the result of successive waves of migration of angioblasts and then satellite cell-derived myoblasts from the muscles surrounding the patch. The results strongly suggest that vascularization of the scaffold and successive coordinate proliferation of the seeded cells are required for myoblasts to be able to migrate into the patch and differentiate up to myofiber stage.     

Regeneration of the supraoptico-hypophyseal and paraventriculo-hypophyseal tracts in the hypophysectomized rat

Summary The effect of hypophysectomy on the nerve fiber pattern in the median eminence and infundibular stem of the rat has been investigated by a slightly modified Bodian technique. Postoperative changes in the distribution of neurosecretory material and connective tissue and changes in vascularity have also been studied.Extensive regeneration of the fibers of the supraoptico-hypophyseal and paraventriculo-hypophyseal tract could be demonstrated. It is most pronounced at the distal extremity of the infundibular stem but occurs also in the rostral part of the infundibular stem and in the median eminence. Regeneration starts in the second postoperative week and is completed about four weeks later. The nervous regeneration observed in the pituitary area after hypophysectomy is more extensive than is usually encountered after lesions elsewhere in the central nervous system.It could be demonstrated moreover that neurosecretory material accumulates at the same sites in which the terminals of the regenerated nerve fibers can be found. Hypophysectomy also causes an increase in capillary density and connective tissue content of the infundibular stem. Accumulations of neurosecretory material are always found in areas showing a high capillary density and a considerable amount of connective tissue.Factors which might be responsible for the extensive nervous regeneration in the pituitary area are discussed as are the factors determining the pattern of outgrowth of the regenerating nerve fibers. Morphological aspects of storage of posterior pituitary hormones are considered in the light of data in the literature and the results of the present work.Partly supported by a U.S. Public Health grant to Dr. E. Scharrer and a travel grant to the author from the Netherlands Organization for pure scientific Research (ZWO).     

Properties of standard avian slow muscle grafts following long-term regeneration

The tonic anterior latissimus dorsi (ALD) of adult pigeons was orthotopically homografted and evaluated after 11 months of regeneration for histological, histochemical, electromyographic (EMG), and mechanical properties. The resting EMG activity of the grafts was lower in amplitude than that of the controls, but showed the tonic pattern typical for these tonic muscles. The control and grafted muscles had a histochemically homogeneous population of fibers with moderate myofibrillar adenosine triphosphatase activity. Succinic dehydrogenase activity was moderate for the control muscles, but low for the grafts. The regenerated muscles had fewer and smaller fibers and had much larger intersynaptic distances. Both the regenerated and the contralateral control muscles were slow contracting and maintained tetanic tension for prolonged periods with direct electrical stimulation. The relaxation was slower in the grafted muscle than in the control. The grafts produced 40% of the maximum tension of the control muscles, but the rate of tension development was similar between the two groups. The results indicate that the tonic properties were regenerated, but the innervation pattern was altered and the grafted muscles did not have normal mature fibers even after long-term regeneration.     

Spontaneous regeneration of older dystrophic muscle does not reflect its regenerative capacity

Young dystrophic (dy) murine muscle is capable of "spontaneous" regeneration (i.e., regeneration in the absence of external trauma); however, by the time the mice are 8 weeks old, this regeneration ceases. It has been suggested that the cessation of regeneration in dystrophic muscle may be due to exhaustion of the mitotic capability of myosatellite cells during the early stages of the disease. To test this hypothesis, orthotopic transplantation of bupivacaine treated, whole extensor digitorum longus muscles has been performed on 14 to 16-week-old 129 ReJ/++ and 129 ReJ/dydy mice. The grafted dystrophic muscle is able to produce and maintain for 100 days post-transplantation 356 +/- 22 myofibers, a number similar to that found in age-matched dystrophic muscle. The ability of old dystrophic muscle to regenerate subsequent to extreme trauma indicates that the cessation of "spontaneous" regeneration is due to factor(s) other than the exhaustion of mitotic capability of myosatellite cells. Moreover, there is no significant difference in myosatellite cell frequencies between grafted normal and dystrophic muscles (100 days post-transplantation). Myosatellite cell frequencies in grafted muscles are similar to those in age-matched, untraumatized muscles. While grafting of young dystrophic muscle modifies the phenotypic expression of histopathological changes usually associated with murine dystrophy, grafts of older dystrophic muscle show extensive connective-tissue infiltration and significantly fewer myofibers than do grafts of age-matched normal muscle. As early as 14 days post-transplantation, it is possible to distinguish between grafts of old, normal and dystrophic muscles. It is suggested that the connective tissue stroma, present in the dystrophic muscle at the time of transplantation, may survive the grafting procedure.     

The survival of free nonvascularized bone grafts in irradiated areas by wrapping in muscle flaps

In this paper, the concept of vascularizing the bed as opposed to the bone was tested with regard to bone grafting in irradiated areas. Thirteen rabbits underwent cross-transfer of a healthy rib into a bed that received 4500 rads of orthovoltage radiation. Eight of these grafts were wrapped in rotated, nonirradiated latissimus dorsi muscle. At 3 months, these grafts were evaluated radiologically, grossly, and histologically. Seven of eight grafts wrapped in muscle demonstrated evidence of union and survival (88 percent), whereas only one of five of those grafts placed directly into the irradiated bed demonstrated union and survival (20 percent). Statistical analysis showed this to be significant to 97.5 percent, with a lambda 2 value of 5.9.     

Increased phenylalanine incorporation in regenerating skeletal muscle grafts

Skeletal muscle regenerates following grafting, but little is known about protein synthesis and its regulation during regeneration. We determined the sequence of changes in protein synthesis in rat extensor digitorum longus (EDL) muscle by the measurement of phenylalanine (Phe) incorporation into muscle protein at various times after grafting. Compared with control EDL, Phe incorporation in grafts doubled in 1 day, was four- to eight-fold greater from days 2 to 10 after grafting, and then subsided. Tissue mass (wet weight) increased rapidly from days 7 to 20 in EDL grafts. The maximal increase in protein synthesis occurred 7-10 days after grafting, whether or not the nerve was left intact. Autoradiography indicated that incorporated radioactivity was associated with regenerating muscle fibers on day 10. Deficiencies of insulin, pituitary or testicular hormones, or chronic in vivo administration of insulin, growth hormone, testosterone, or tri-iodothyronine did not substantially alter the elevation in incorporation of the Phe into muscle protein 10 days after grafting. The breakdown of EDL protein, measured in vitro simultaneously with protein synthesis, was increased five-fold, and overall protein degradation was elevated six-fold 10 days after grafting. These findings indicate that Phe incorporation is rapidly elevated following grafting of the EDL, and that by days 7-10 reflects synthesis in regenerating muscle fibers. The increase in protein synthesis associated with muscle regeneration at this time appears to be independent of innervation and anabolic hormones.     

Isoenzyme studies of whole muscle grafts and movement of muscle precursor cells

Summary Isoenzymes of glucose-6-phosphate isomerase (GPI: E.C. 5.3.1.9) were used as markers to determine the origin of cells which give rise to new muscle formed in allografts of whole intact muscle. GPI isoenzymes were also employed to see whether host precursor cells, which have been shown to contribute to muscle formation in grafts of minced muscle, can be derived from muscle lying adjacent to grafts.Excellent muscle regeneration was found in allografts of extensor digitorum longus (EDL) muscle examined after 58 days: 12 of 16 grafts contained 80% or more new muscle. Isoenzyme analysis showed that most, and in 2 instances all, new muscle was derived from implanted donor cells; however, there was strong evidence that in 5 grafts some, or all, new muscle must have resulted from host cells moving into the graft. Although hybrid isoenzyme was not detected this was attributed to factors associated with host tolerance which appear to interfere with fusion between host and donor myoblasts.Isografts of minced muscle were placed next to whole EDL muscle allografts to see if cells from allografts moved into adjacent regenerating tissue. Unfortunately, muscle regeneration in minced isografts was poor; only 3 contained 50% or more new muscle and most contained large amounts of fibrous connective tissue. Only a single isoenzyme band was detected in 11 isografts, but in five instances, the presence of a second band showed that cells from EDL allografts were also present. As no hybrid isoenzyme was detected, it is not known whether these cells which had moved into the regenerating minced grafts were muscle precursors, fibroblasts or some other cell types.     

Reaction of intact skeletal muscle on the transplantation of minced muscle tissue

The experiments on rats have revealed that grafting of minced muscle tissue to unaffected gastrochemical muscle of the same animal leads to active regeneration in the graft accompanied by the formation of myogenic elements. In the zone of unaffected muscle contact with the graft the plastic state of muscle tissue was observed: sarcoplasm basophily of muscle fibers, groups and chains of round nuclei in them, the presence of satellite cells, ultrastructural changes, indicative of metabolic activity of the muscle fibers. With the resorption of the graft, these phenomena in the unaffected muscle gradually disappeared.     

Responses to amputation of denervated ambystoma limbs containing aneurogenic limb grafts

The developing neural tubes and associated neural crest cells were removed from stage 30 Ambystoma maculatum embryos to obtain larvae with aneurogenic forelimbs. Forelimbs were allowed to develop to late 3 digit or early 4 digit stages. Limbs amputated through the mid radius-ulna regenerated typically in the aneurogenic condition. Experiments were designed to test whether grafts of aneurogenic limb tissues would rescue denervated host limb stumps into a regeneration response. In Experiment 1, aneurogenic limbs were removed at the body wall and grafted under the dorsal skin of the distal end of amputated forelimbs of control, normally innervated limbs of locally collected Ambystoma maculatum or axolotl (Ambystoma mexicanum) larvae. In Experiment 1, at the time of grafting or 1, 2, 3, 4, 5, 7, or 8 days after grafting, aneurogenic limbs were amputated level with the original host stump. At 7 and 8 days, this amputation included removing the host blastema adjacent to the graft. The host limb was denervated either one day after grafting or on the day of graft amputation. These chimeric limbs only infrequently exhibited delayed blastema formation. Thus, not only did the graft not rescue the host, denervated limb, but the aneurogenic limb tissues themselves could not mount a regeneration response. In Experiment 2, the grafted aneurogenic limb was amputated through its mid-stylopodium at 3, 4, 5, 7, or 8 days after grafting. By 7 and 8 days after grafting, the host limb stump exhibited blastema formation even with the graft extending out from under the dorsal skin. The host limb was denervated at the time of graft amputation. When graft limbs of Experiment 2 were amputated and host limbs were denervated on days 3, 4, or 5, host regeneration did not progress and graft regeneration did not occur. But, when graft limbs were amputated on days 7 or 8 with concomitant denervation of the host limb, regeneration of the host continued and graft regeneration occurred. Thus, regeneration of the graft was correlated with acquisition of nerve-independence by the host limb blastema. In Experiment 3, aneurogenic limbs were grafted with minimal injury to the dorsal skin of neurogenic hosts. When neurogenic host limbs were denervated and the aneurogenic limbs were amputated through the radius/ulna, regeneration of the aneurogenic limb occurred if the neurogenic limb host was not amputated, but did not occur if the neurogenic limb host was amputated. Results of Experiment 3 indicate that the inhibition of aneurogenic graft limb regeneration on a denervated host limb is correlated with substantial injury to the host limb. In Experiment 4, aneurogenic forelimbs were amputated through the mid-radius ulna and pieces of either peripheral nerve, muscle, blood vessel, or cartilage were grafted into the distal limb stump or under the body skin immediately adjacent to the limb at the body wall. In most cases, peripheral nerve inhibited regeneration, blood vessel tissue sometimes inhibited, but other tissues had no effect on regeneration. Taken together, the results suggest: (1) Aneurogenic limb tissues do not produce the neurotrophic factor and do not need it for regeneration, and (2) there is a regeneration-inhibiting factor produced by the nerve-dependent limb stump/blastema after denervation that prevents regeneration of aneurogenic limbs.