Muscle regeneration and the state of the thymus in adult rats under laser irradiation and alloplasty of newborn gastrocnemius muscle and diaphragm

The regeneration of adult rat gastrocnemius muscles has been studied under implantation of new-born rat muscles into area of muscle trauma. Alloplasty was performed using minced gastrocnemius and diaphragm muscles, which differs at birth in animals by degree of differentiation. The rat-recipient area of alloplasty was subjected to He-Ne laser radiation before operation, with the aim of reducing the immune response to allogenic muscle tissue. It has been shown that the number of regenerating myofibers produced from implanted gastrocnemius muscles is more than in alloplants from diaphragms. However, the formation of cartilage, bone, and adipose tissue foci was observed in the alloplastic region throughout the whole regeneration period. After implantation of minced diaphragm muscles, cartilage nodes were observed only in 7-day regenerates. At the end of observation, in the first instance, the area of muscle trauma in adult rat muscles was replaced by adipose tissue, even in the case of initial laser irradiation. After the implantation of diaphragm muscles, the area of trauma was filled with regenerating muscle tissue.     

Plasticity of allogeneic muscle tissue implanted into a traumatized rat muscle on exposure to He-Ne laser light

The combined effect of muscle tissue alloplasty and He-Ne laser light on the regeneration of a traumatized skeletal muscle was studied. In implantation of untreated allogeneic tissue into the region of transverse cutting of the gastrocnemius muscle exposed to laser light in vivo before surgery, the muscle regeneration in recipient rats was successful. Active regeneration of the allogeneic tissue and the muscle tissue of the recipient rat was noted. In implantation of laser-exposed allogeneic tissue into the region of trauma of unexposed gastrocnemius muscle, the muscle regeneration was less successful. The alloplastic implant was replaced by connective tissue and prevented the growth of the muscle tissue of the recipient rat in the defective zone. Implantation of laser-exposed allogeneic muscle tissue into the region of trauma of the muscle x-irradiated to a dose of 20 Gy enhanced destructive processes in it.     

Morphofunctional characteristics of the thymus and muscle regenerates under laser irradiation and alloplasty of adult muscle tissue in the area of trauma

The effect of muscle tissue alloplasty and He-Ne laser radiation on the skeletal muscle regeneration and thymus function was studied. Allogenic muscle tissue was implanted from an adult rat. Without laser irradiation (series 1), initial enhancement of thymus recovery observed on day 7 after alloplasty (a characteristic stress response to operation) was followed by gradual destructive changes in the thymus tissue. On day 30 after alloplasty, connective tissue developed in the implantation area in muscle regenerates, and the muscle tissue accounted for 64 ± 2%. Implantation of unirradiated allografts into the muscles of recipient rats preirradiated with a He-Ne laser (series 2) resulted in a nearly complete destruction of the thymus and suppression of its function; the mitotic index of thymocytes was low. These changes were observed throughout the experiment starting immediately after the operation. In this case, the allogenic transplant retained the ability to develop: the 30-day repairing muscles consisted of 71 ± 2% of muscle tissue. When an allograft preirradiated with a He-Ne laser was implanted into unirradiated rats (series 3), thymus destruction at the beginning of the postoperative period was much less significant than in series 2 but more pronounced than in series 1. Then, thymus recovered more rapidly, the allogenic transplant was resorbed, and the muscle tissue in the regenerates accounted for 62 ± 3%.     

Inducible Expression of Neurotrophic Factors by Mesenchymal Progenitor Cells Derived from Traumatically Injured Human Muscle

Peripheral nerve damage frequently accompanies musculoskeletal trauma and repair of these nerves could be enhanced by the targeted application of neurotrophic factors (NTFs), which are typically expressed by endogenous cells that support nerve regeneration. Injured muscle tissues express NTFs to promote reinnervation as the tissue regenerates, but the source of these factors from within the muscles is not fully understood. We have previously identified a population of mesenchymal progenitor cells (MPCs) in traumatized muscle tissue with properties that support tissue regeneration, and our hypothesis was that MPCs also secrete the NTFs that are associated with muscle tissue reinnervation. We determined that MPCs express genes associated with neurogenic function and measured the protein-level expression of specific NTFs with known functions to support nerve regeneration. We also demonstrated the effectiveness of a neurotrophic induction protocol to enhance the expression of the NTFs, which suggests that the expression of these factors may be modulated by the cellular environment. Finally, neurotrophic induction affected the expression of cell surface markers and proliferation rate of the MPCs. Our findings indicate that traumatized muscle-derived MPCs may be useful as a therapeutic cell type to enhance peripheral nerve regeneration following musculoskeletal injury.     

Effects of autotransplantation of minced muscle tissue and subsequent laser therapy on recovery of the muscle injured by irradiation

A comparative histological investigation of posttraumatic regeneration in irradiated with 30 or 40 Gy and cross-sectioned musculus gastrocnemius of rats after autotransplantation into muscle defect of non-irradiated minced muscle tissue and laser therapy of hind limb in post-operative period was conducted. The obtained results showed that in irradiated with 30 Gy sectioned muscle (control series) the inflammatory reaction, resorption of fibrin in the area of trauma were inhibited and proliferation of muscle tissue from proximal and distal stump was suppressed. The rough connective tissue scar was formed. In experimental series for stimulation of regeneration the method of autografting minced muscle tissue into the defect of irradiated (30 or 40 Gy) cross-sectioned muscle and combination of this method with helium-neon laser rays exposition was used. The more marked recovery was obtained in irradiated with 30 Gy operated muscle after a 10-day treatment of limb with laser rays.     

MALDI imaging mass spectrometry: Discrimination of pathophysiological regions in traumatized skeletal muscle by characteristic peptide signatures

Due to formation of fibrosis and the loss of contractile muscle tissue, severe muscle injuries often result in insufficient healing marked by a significant reduction of muscle force and motor activity. Our previous studies demonstrated that the local transplantation of mesenchymal stromal cells into an injured skeletal muscle of the rat improves the functional outcome of the healing process. Since, due to the lack of sufficient markers, the accurate discrimination of pathophysiological regions in injured skeletal muscle is inadequate, underlying mechanisms of the beneficial effects of mesenchymal stromal cell transplantation on primary trauma and trauma adjacent muscle area remain elusive. For discrimination of these pathophysiological regions, formalin‐fixed injured skeletal muscle tissue was analyzed by MALDI imaging MS. By using two computational evaluation strategies, a supervised approach (ClinProTools) and unsupervised segmentation (SCiLS Lab), characteristic m/z species could be assigned to primary trauma and trauma adjacent muscle regions. Using “bottom‐up” MS for protein identification and validation of results by immunohistochemistry, we could identify two proteins, skeletal muscle alpha actin and carbonic anhydrase III, which discriminate between the secondary damage on adjacent tissue and the primary traumatized muscle area. Our results underscore the high potential of MALDI imaging MS to describe the spatial characteristics of pathophysiological changes in muscle.     

Structural and functional characteristics of muscle allotransplants, formed under different conditions of laser irradiation

It is known that a low-energy laser radiation can cause reflex suppression of immunity. The present experiments were designed to determine the plastic activity of allogenic muscle tissue in different conditions of a previous action of laser rays. The cross homotransplantation of gastrocnemius muscles was carried out between intact rats, or between rats in which 14 days before transplantation each hind leg was subjected to low-energy He-Ne laser radiation in dose of 7.5-9 J/cm2 (10 procedures, the duration of each exposure was equal to 5 min), or between intact and radiated rats. It was shown that the donor muscle tissue survived longer when a nonradiated muscle was transplanted into the radiated muscle bed. The axons grew into the donor muscle tissue. More allogenic muscle tissue was involved in contractile reaction when stimulation was carried out via the nerve. Laser radiation of a homotransplant alone, or that of a homotransplant and a muscle bed in the recipient was less effective. So, He-Ne laser radiation of the area of a planned allotransplantation decreased the transplant immunity response and favoured a longer development of allogenic muscle tissue. The viability of donor muscle tissue therewith increased, if the muscle allograft had not been subjected to a previous laser radiation.     

Repair mechanisms involved in muscle regeneration following partial excision of the rat gastrocnemius muscle

The sequential cytological events of the regeneration process, after partial excision of the gastrocnemius muscle in the rat, were followed by light and electron microscopy. During the first 2 days after injury leukocytes and macrophages infiltrate into the traumatized area. Myogenic regeneration is then characterized by mainly two repair mechanisms. Mononucleated cells, that populate the excised area, most probably fuse together to give rise to newly formed multinucleated myotubes that further develop to striated myofibers. Another mechanism involves the repair of injured muscle fibers by the possible fusion of mononucleated cells with their necrotic cut ends. Consequently, by addition of nuclei and new muscular material, sarcoplasmic outgrowths from the injured fibers are formed. It is concluded that mainly two repair mechanisms are involved in the regeneration process following partial excision of a muscle: addition of new muscle fibers in a process similar to that of embryonic myogenesis and also meristic growth from the injured fibers.     

Nuclear proliferation (“nucleosis”) in damaged skeletal muscle A critical review

Summary The formation of myotubes and their role in regeneration of skeletal muscle are discussed. It is pointed out that some recent observations and theories contradict each other. Furthermore, where early regeneration of transplanted skeletal muscle is described, no attention is given to the wanting nerve supply, thus assuming muscle regeneration in the absence of nerve supply.Strangely enough, those who studied traumatized muscle paid no attention to the fact that myotubes occur not only in traumatized skeletal muscle, but also in denervated skeletal muscle, in dystrophies, especially the myotonic type, and, in abortive form, in injured heart muscle. Thus, no statement is available whether the various workers would consider the myotubes in denervated or dystrophic muscle to be different in morphology, origin and fate from those in traumatized muscle and, if considered different, give the reasons for it. In my opinion, the trauma proper and the catabolic and reparative processes in traumatized tissue should be expected to damage the nerves in and around the injured tissue (denervation in situ), which should contribute to the atrophy of muscle fibers with the ensuing nuclear proliferation (nucleosis). If myotubes in denervated and in traumatized muscle are basically identical, their morphogenesis from granulation tissue or from dissociated muscle fibers cannot be valid.This work was supported by grants from the Muscle Dystrophy Association of Canada and the Medical Research Council of Canada. The assistance of Dr. J. C. Lee is gratefully acknowledged.     

Differentiation and regeneration potential of mesenchymal progenitor cells derived from traumatized muscle tissue

Mesenchymal stem cell (MSC) therapy is a promising approach to promote tissue regeneration by either differentiating the MSCs into the desired cell type or by using their trophic functions to promote endogenous tissue repair. These strategies of regenerative medicine are limited by the availability of MSCs at the point of clinical care. Our laboratory has recently identified multipotent mesenchymal progenitor cells (MPCs) in traumatically injured muscle tissue, and the objective of this study was to compare these cells to a typical population of bone marrow derived MSCs. Our hypothesis was that the MPCs exhibit multilineage differentiation and expression of trophic properties that make functionally them equivalent to bone marrow derived MSCs for tissue regeneration therapies. Quantitative evaluation of their proliferation, metabolic activity, expression of characteristic cell-surface markers and baseline gene expression profile demonstrate substantial similarity between the two cell types. The MPCs were capable of differentiation into osteoblasts, adipocytes and chondrocytes, but they appeared to demonstrate limited lineage commitment compared to the bone marrow derived MSCs. The MPCs also exhibited trophic (i.e. immunoregulatory and pro-angiogenic) properties that were comparable to those of MSCs. These results suggest that the traumatized muscle derived MPCs may not be a direct substitute for bone marrow derived MSCs. However, because of their availability and abundance, particularly following orthopaedic injuries when traumatized muscle is available to harvest autologous cells, MPCs are a promising cell source for regenerative medicine therapies designed to take advantage of their trophic properties.     

Upregulation of uncoupling protein homologues in skeletal muscle but not adipose tissue in posttraumatic insulin resistance

Metabolic alterations after surgical stress include peripheral insulin resistance and increased utilization of fat as a fuel substrate. An up-regulation of skeletal muscle uncoupling proteins (UCPs) has been associated with physiologic states of insulin resistance and enhanced fat metabolism in rodents. We examined whether posttraumatic insulin resistance induced the UCPs in gastrocnemius and soleus muscle and white adipose tissue in an experimental model of surgical trauma. Insulin sensitivity was significantly reduced in isolated soleus muscles but unchanged in adipocytes after trauma. In traumatized rats, mRNA and protein contents of UCP2 and UCP3 and were significantly increased in both muscle types. UCP2 protein content in adipose tissue was unaltered by surgical stress. Circulating NEFAs and glycerol were reduced after surgical trauma. We hypothesize that the changes in UCP2 and UCP3 gene and protein expression are involved in the regulation of substrate utilization in posttraumatic insulin resistance.     

Mechanism of formation of intramedullary cavities and their role in the regeneration of the spinal cord

The spinal cord preparations of 38 dogs and 20 rabbits have been studied with the aim to investigate the influence of the cerebrospinal fluid on the spinal cord nervous tissue. The spinal cord preparations of 8 patients having trauma of the vertebral column with interruption of the spinal cord have also been studied. As demonstrate histological investigations, the cerebral tissue of the pieces, put into the flask with liquor, in the subarachnoidal space of the canine spinal cord, in diastasis between the ends of the cut spinal cord during 6 h up to 7 days, swells, becomes edematous. Cavities occupying about 30% of the area in the slices studied appear in it. At hemisection of the rabbit spinal cord without closure of the defect in the meninx vasculosa with the glue MK-6, the area of the cavity formation varies from 24 up to 35%, comparing the whole area of the preparation, while in rabbits with hemisection and successive gluing of the defect in the meninx vasculosa the area of the nervous tissue destruction makes 13-18%. It has been proved that the scar forming in the traumatized segment of the spinal cord does not present a continuous formation, but contains a large amount of cavities that prevent regeneration of nerve fibers. The experimental data concerning lysing effect of the cerebrospinal fluid on the traumatized nervous tissue are confirmed by the results obtained at investigating the preparations of the spinal cord of the patients died as the cause of the spinal cord trauma.     

Regenerative Activity of Muscle Allografts and the State of Thymus under Conditions of Long-Term Laser Treatment of Adrenal Glands and Transplantation Site Prior to Surgery

Both gastrocnemius muscles were cross-allografted between the intact rats and the rats whose adrenal glands and right shins were exposed to He–Ne laser radiation before the surgery (632.8 nm, 2.5–3.0 mW/cm², 10 sessions over 14 days at a cumulative dose of 15–18 J/cm² per rat). By the time of grafting, the activity of adrenal glands decreased, while the activity of thymus increased. As a result, the allogenic muscle tissue was resorbed more actively in the irradiated rats, especially in the right, irradiated shin. At the same time, reinnervation of the grafts containing a certain amount of survived allogenic muscle tissue was more active in the right shin, compared to the left, unirradiated one. Survival of the donor muscle tissue and its improved reinnervation were observed in a higher proportion of grafts in the intact rats. The allografts from the right (preirradiated) shin of animals with irradiated adrenal glands proved to be more viable, compared to the left (unirradiated) shin.     

Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma

Although microdialysis is widely used to sample endogenous and exogenous substances in vivo, interpretation of the results obtained by this technique remains controversial. The goal of the present study was to examine recent criticism of microdialysis in the specific case of dopamine (DA) measurements in the brain extracellular microenvironment. The apparent steady-state basal extracellular concentration and extraction fraction of DA were determined in anesthetized rat striatum by the concentration difference (no-net-flux) microdialysis technique. A rate constant for extracellular clearance of DA calculated from the extraction fraction was smaller than the previously determined estimate by fast-scan cyclic voltammetry for cellular uptake of DA. Because the relatively small size of the voltammetric microsensor produces little tissue damage, the discrepancy between the uptake rate constants may be a consequence of trauma from microdialysis probe implantation. The trauma layer has previously been identified by histology and proposed to distort measurements of extracellular DA levels by the no-net-flux method. To address this issue, an existing quantitative mathematical model for microdialysis was modified to incorporate a traumatized tissue layer interposed between the probe and surrounding normal tissue. The tissue layers are hypothesized to differ in their rates of neurotransmitter release and uptake. A post-implantation traumatized layer with reduced uptake and no release can reconcile the discrepancy between DA uptake measured by microdialysis and voltammetry. The model predicts that this trauma layer would cause the DA extraction fraction obtained from microdialysis in vivo calibration techniques, such as no-net-flux, to differ from the DA relative recovery and lead to an underestimation of the DA extracellular concentration in the surrounding normal tissue.     

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 x-ray irradiated gastrocnemius muscle in old rats after stimulation

In the present study the implantation of nonexposed muscle tissue to the site of injury in irradiated musculus gastrocnemius and following laser therapy were applied in order to stimulate this muscle's posttraumatic regeneration in old rats. It was shown that a far larger amount of functionally active muscle tissue was formed at the site of injury compared with rats received laser therapy alone or the ones which were only implanted nonexposed minced muscle tissue. The muscle tissue consisted of muscle fibers which originated from the grafted pieces of nonexposed skeletal muscle and the ones produced by myofibers of muscle stumps recovered after irradiation. The connective tissue developed more evenly. The formation of adipose tissue was not observed at the site of injury. Moreover, the skin wound healing and the hair growth were stimulated as well.     

A satellite cell mitogen from crushed adult muscle

Single fiber-satellite cell units from skeletal muscle of adult rats were used to study the regulation of satellite cell proliferation. The satellite cells remained quiescent during culture in serum-containing medium but could be induced to enter the cell cycle by exposure to a saline extract of crushed adult muscle. The activity in the extract has a molecular weight greater than 30K and is heat and trypsin sensitive. The mitogenic activity does not result from transferrin. Little or no activity was obtained from crushed extracts of heterologous tissues. Proliferation of myogenic cells from rat embryos was also stimulated by the muscle mitogen but growth of muscle fibroblasts was not enhanced. The time response of satellite cell proliferation after exposure to the muscle mitogen showed that the cells enter DNA synthesis after a lag period of 18 hr and proliferate with a generation time of 12 hr. This confirms that satellite cells in adult muscle are in G0, or an extended G1. The mitogen is also effective in stimulating muscle growth and myoblast fusion in vivo when injected into 1-week-old rat pups. These experiments suggest that muscle regeneration is initiated by the release of an endogenous mitogen from traumatized muscle.     

Sources of myoblast development in skeletal muscle regeneration

The regeneration of skeletal muscle tissue was studied upon pharmacological denervation, trauma and combined damage of skeletal muscles in mice. It is suggested that the formation of myoblasts proceeds not only via development of the cells-satellites but also by separation of nucleo-sarcoplasmic territories of the muscle fibres. The ratio of two forms of development of the cells is determined by the experimental conditions which are discussed.     

Transplantation of devitalized muscle scaffolds is insufficient for appreciable de novo muscle fiber regeneration after volumetric muscle loss injury

Volumetric muscle loss (VML) is a traumatic and functionally debilitating muscle injury with limited treatment options. Developmental regenerative therapies for the repair of VML typically comprise an ECM scaffold. In this study, we tested if the complete reliance on host cell migration to a devitalized muscle scaffold without myogenic cells is sufficient for de novo muscle fiber regeneration. Devitalized (muscle ECM with no living cells) and, as a positive control, vital minced muscle grafts were transplanted to a VML defect in the tibialis anterior muscle of Lewis rats. Eight weeks post-injury, devitalized grafts did not appreciably promote de novo muscle fiber regeneration within the defect area, and instead remodeled into a fibrotic tissue mass. In contrast, transplantation of vital minced muscle grafts promoted de novo muscle fiber regeneration. Notably, pax7+ cells were absent in remote regions of the defect site repaired with devitalized scaffolds. At 2 weeks post-injury, the devitalized grafts were unable to promote an anti-inflammatory phenotype, while vital grafts appeared to progress to a pro-regenerative inflammatory response. The putative macrophage phenotypes observed in vivo were supported in vitro, in which soluble factors released from vital grafts promoted an M2-like macrophage polarization, whereas devitalized grafts failed to do so. These observations indicate that although the remaining muscle mass serves as a source of myogenic cells in close proximity to the defect site, a devitalized scaffold without myogenic cells is inadequate to appreciably promote de novo muscle fiber regeneration throughout the VML defect.