Enhanced liver regeneration following acute hepatectomy by low-level laser therapy.

OBJECTIVE: The aim of the present study was to investigate the effect of low-level laser therapy (LLLT) on liver regeneration following hepatectomy. BACKGROUND: LLLT has been found to modulate various biological processes. MATERIALS AND METHODS: Twelve mature male rats were used. The liver was exposed, and 70% of it was excised. The rats were assigned randomly to two groups: control, non-laser treated, and experimental, laser-treated (diode [Ga-Al-As] laser 804 nm) group. For determination of newly formed blood vessels and proliferating cells, 5-Bromo-2'deoxyuridine (BrdU) was injected intraperitoneally. The rats were sacrificed 2 d post hepatectomy, and histological sections from each liver were processed for analysis of new blood-vessel formation using BrdU immunostaining kit. Mesenchymal stem cells (MSCs) were assessed using c-kit immunostaining. BrdU-labeled cells were counted as for estimation of newly formed hepatic cells. RESULTS: It was found that the number of proliferating cells (BrdU positive cells) per area in the regenerating regions of the livers were significantly (p < 0.01) 2.6-fold higher in the laser-treated rats than in the control non-laser-treated rats. The density of the newly formed blood vessels and c-kit immunopositive cells in the regenerating area of the laser-treated livers was significantly (p < 0.01) 3.3- and 2.3-fold respectively higher than the control non-laser treated livers. CONCLUSION: It is concluded that LLLT following acute hepatectomy most probably stimulates a significant enhancement of liver regeneration conducive to both the formation of new hepatocytes and MSCs and angiogenesis in the regenerating liver.

Correlation between rate of bony ingrowth to stainless steel, pure titanium, and titanium alloy implants in vivo and formation of hydroxyapatite on their surfaces in vitro.

The rate of bony ingrowth to identical metal implants made of either pure titanium (cpTi), titanium alloy (Ti-6Al-4V), or stainless steel 316L (SS) inserted to the medullar canal of the femur in rats was investigated. The kinetics of spontaneous deposition of hydroxyapatite (HA) globules on the aforementioned metals in vitro during incubation in simulated body fluid (SBF) was also studied. It was found that the rate of increased bonding strength between the cpTi implants and the host bone was the highest, whereas around the SS implants it was the slowest. At 10 days postimplant insertion, the shear strength of the cpTi implants was 2.2- and 4-fold significantly higher than for the Ti-6Al-4V and the SS implants, respectively. Spontaneous formation of the HA globules on the cpTi and Ti-6Al-4V implants that were incubated in the SBF was observed as early as 6 and 10 days after incubation in SBF, respectively, whereas on SS implants, deposition of HA was evident only after 2 weeks of in vitro incubation in SBF. It is concluded that the chemical surface characteristics and the biocompatibility of the implants probably play a key role in the process of bone growth next to them, during the formation of bone in vivo. The rate of bony ingrowth to various metal implants alloys inserted into the medullar canal of rats correlates well with the induction of apatite formation on them during incubation in vitro with SBF.

Ga-As (808 nm) laser irradiation enhances ATP production in human neuronal cells in culture.

OBJECTIVE: The aim of the present study was to investigate whether Ga-As laser irradiation can enhance adenosine triphosphate (ATP) production in normal human neural progenitor (NHNP) cells in culture. METHODS: NHNP were grown in tissue culture and were treated by Ga-As laser (808 nm, 50 mW/cm(2), 0.05 J/cm(2)), and ATP was determined at 10 min after laser application. RESULTS: The quantity of ATP in laser-treated cells was 7513 +/- 970 units, which was significantly higher (p < 0.05) than the non-treated cells, which comprised 3808 +/- 539 ATP units. CONCLUSION: Laser application to NHNP cells significantly increases ATP production in these cells. These findings may explain the beneficial effects of low-level laser therapy (LLLT) in stroked rats. Tissue culture of NHNP cells might offer a good model to study the mechanisms associated with promotion of ATP production in the nervous system by LLLT.

Low-level laser irradiation promotes proliferation and differentiation of human osteoblasts in vitro.

OBJECTIVES: The aim of the present study was to investigate the effect of low-level laser irradiation on proliferation and differentiation of a human osteoblast cell line. BACKGROUND DATA: It was previously found that low-level laser therapy (LLLT) enhances bone repair in experimental models. MATERIALS AND METHODS: Cultured osteoblast cells were irradiated using He-Ne laser irradiation (632 nm; 10 mW power output). On the second and third day after seeding the osteoblasts were exposed to laser irradiation. The effect of irradiation on osteoblast proliferation was quantified by cell count and colorimetric MTT (dimethylthiazol tetrazolium bromide) assay 24 and 48 h after second irradiation. RESULTS: A significant 31-58% increase in cell survival (MTT assay) and higher cell count in the once-irradiated as compared to nonirradiated cells was monitored. Differentiation and maturation of the cells was followed by osteogenic markers: alkaline phosphatase (ALP), osteopontin (OP), and bone sialoprotein (BSP). A two-fold enhancement of ALP activity and expression of OP and BSP was much higher in the irradiated cells as compared to non-irradiated osteoblasts. CONCLUSION: We conclude that LLLT promotes proliferation and maturation of human osteoblasts in vitro. These results may have clinical implications.

Promotion of angiogenesis by low energy laser irradiation.

The effect of low energy laser (He-Ne) irradiation (LELI) on the process of angiogenesis in the infarcted rat heart and in the chick chorioallantoic membrane (CAM), as well as the proliferation of endothelial cells in tissue culture, was investigated. Formation of new blood vessels in the infarcted rat heart was monitored by counting proliferating endothelial cells in blood vessels. In the CAM model, defined areas were laser-irradiated or nonirradiated and blood vessel density was recorded in each site in the CAM at various time intervals. Laser irradiation caused a 3.1-fold significant increase in newly formed blood vessels 6 days post infarction, as compared with nonirradiated rats. In the CAM model, a slight inhibition of angiogenesis up to 2 days post irradiation and a significant enhancement of angiogenesis in the laser-irradiated foci as compared with control nonirradiated spots were evident. The LELI caused a 1.8-fold significant increase in the rate of proliferation in endothelial cells in culture over nonirradiated cells. It is concluded that LELI can promote the proliferation of endothelial cells in culture, which may partially explain the augmentation of angiogenesis in the CAM model and in the infarcted heart. These results may have clinical significance by offering therapeutic options to ameliorate angiogenesis in ischemic conditions.

Impact of low level laser irradiation on infarct size in the rat following myocardial infarction.

Low energy level irradiation (LLLI) has been found to modulate biological processes. The effect of LLLI on the development of acute myocardial infarction (MI) was investigated following chronic ligation of the left anterior descending (LAD) coronary artery in laboratory rats. The hearts of 22 rats were laser irradiated (LI) using a diode laser (804 nm, 38 mW power output) through the intercostal muscles in the chest following MI and on day 3 post MI. In the control non laser irradiated (NLI) group (19 rats) MI was induced experimentally and laser irradiation was not applied. All rats were sacrificed 21 days post MI. Size, thickness and relative circumferential length of the infarct, as well as other parameters, were determined from histological sections stained with Masson's trichrome and hearts stained with triphenyl tetrazolium chloride (TTC) using histomorphometric methods. The infarct size (expressed as percent of total left ventricle area) of the LI rats was 10.1+/-5.8, which was significantly lower (65%; P<0.01) than the infarct size of NLI rats which was 28.7+/-9.6. Correlatively, the ratio of circumferential length of the infarcted area was significantly lower (2-fold; P<0.01) in the LI rats as compared to the NLI rats. LLLI of the infarcted area in the myocardium of experimentally induced MI rats, at the correct energy, duration and timing, markedly reduces the loss of myocardial tissue. This phenomenon may have an important beneficial effect on patients after acute MI or ischemic heart disease.

Detailed endocardial mapping accurately predicts the transmural extent of myocardial infarction.

OBJECTIVES: This study delineates between infarcts varying in transmurality by using endocardial electrophysiologic information obtained during catheter-based mapping. BACKGROUND: The degree of infarct transmurality extent has previously been linked to patient prognosis and may have significant impact on therapeutic strategies. Catheter-based endocardial mapping may accurately delineate between infarcts differing in the transmural extent of necrotic tissue. METHODS: Electromechanical mapping was performed in 13 dogs four weeks after left anterior descending coronary artery ligation, enabling three-dimensional reconstruction of the left ventricular chamber. A concomitant reduction in bipolar electrogram amplitude (BEA) and local shortening indicated the infarcted region. In addition, impedance, unipolar electrogram amplitude (UEA) and slew rate (SR) were quantified. Subsequently, the hearts were excised, stained with 2,3,5-triphenyltetrazolium chloride and sliced transversely. The mean transmurality of the necrotic tissue in each slice was determined, and infarcts were divided into <30%, 31% to 60% and 61% to 100% transmurality subtypes to be correlated with the corresponding electrical data. RESULTS: From the three-dimensional reconstructions, a total of 263 endocardial points were entered for correlation with the degree of transmurality (4.6 +/- 2.4 points from each section). All four indices delineated infarcted tissue. However, BEA (1.9 +/- 0.7 mV, 1.4 +/- 0.7 mV, 0.8 +/- 0.4 mV in the three groups respectively, p < 0.05 between each group) proved superior to SR, which could not differentiate between the second (31% to 60%) and third (61% to 100%) transmurality subgroups, and to UEA and impedance, which could not differentiate between the first (<30%) and second transmurality subgroups. CONCLUSIONS: The degree of infarct transmurality extent can be derived from the electrical properties of the endocardium obtained via detailed catheter-based mapping in this animal model.

Long-term effect of low energy laser irradiation on infarction and reperfusion injury in the rat heart.

Low-energy laser irradiation (LELI) has been found to modulate biological processes. The present study investigated the effect of LELI on infarct size after chronic myocardial infarction (MI) and ischemia-reperfusion injury in rats. The left anterior descending (LAD) coronary artery was ligated in 83 rats to create MI or ischemia-reperfusion injury. The hearts of the laser-irradiated (LI) rats received irradiation after LAD coronary artery occlusion and 3 days post-MI. At 14, 21, and 45 days post-LAD coronary artery permanent occlusion, infarct sizes (percentage of left ventricular volume) in the non-laser-irradiated (NLI) rats were 52 +/- 12 (SD), 47 +/- 11, and 34 +/- 7%, respectively, whereas in the LI rats they were significantly lower, being 20 +/- 8, 15 +/- 6, and 10 +/- 4%, respectively. Left ventricular dilatation (LVD) in the chronic infarcted rats was significantly reduced (50-60%) in LI compared with NLI rats. LVD in the ischemia-reperfusion-injured LI rats was significantly reduced to a value that did not differ from intact normal noninfarcted rats. Laser irradiation caused a significant 2.2-fold elevation in the content of inducible heat shock proteins (specifically HSP70i) and 3.1-fold elevation in newly formed blood vessels in the heart compared with NLI rats. It is concluded that LELI caused a profound reduction in infarct size and LVD in the rat heart after chronic MI and caused complete reduction of LVD in ischemic-reperfused heart. This phenomenon may be partially explained by the cardioprotective effect of the HSP70i and enhanced angiogenesis in the myocardium after laser irradiation.

Attenuation of infarct size in rats and dogs after myocardial infarction by low-energy laser irradiation.

BACKGROUND AND OBJECTIVE: The aim of the present study was to investigate the possibility that low-energy laser irradiation attenuates infarct size formation after induction of chronic myocardial infarction (MI) in small and large experimental animals. STUDY DESIGN/MATERIALS AND METHODS: Laser irradiation was applied to the infarcted area of rats and dogs at various power densities (2.5 to 20 mW/cm(2)) after occlusion of the coronary artery. RESULTS: In infarcted laser-irradiated rats that received laser irradiation immediately and 3 days after MI at energy densities of 2.5, 6, and 20 mW/cm(2), there was a 14%, 62% (significant; P < 0.05), and 2.8% reduction of infarct size (14 days after MI) relative to non--laser-irradiated rats, respectively. In dogs, a 49% (significant; P < 0.01) reduction of infarct size was achieved. CONCLUSION: The results of the present study indicate that delivery of low-energy laser irradiation to infarcted myocardium in rats and dogs has a profound effect on the infarct size after MI.

Skeletal muscle cell activation by low-energy laser irradiation: a role for the MAPK/ERK pathway.

Low-energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum-starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum-starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI- and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N-terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3-sec irradiation induced ERK1/2 phosphorylation, a 12-sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis alpha (TNF-alpha) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.

Low-energy laser irradiation reduces formation of scar tissue after myocardial infarction in rats and dogs.

BACKGROUND: Low-energy laser irradiation (LELI) has been found to attenuate various biological processes in tissue culture and experimental animal models. The aim of the present study was to investigate the effect of LELI on the formation of scar tissue in experimentally induced chronic infarct in rats and dogs. METHODS AND RESULTS: Myocardial infarction (MI) was induced in 50 dogs and 26 rats by ligation of the left anterior descending coronary artery. After induction of MI, the laser-irradiated (LI) group received laser irradiation (infrared laser, 803-nm wavelength) epicardially. Control MI-induced non-laser irradiated (NLI) dogs were sham-operated, and laser was not applied. All dogs were euthanized at 5 to 6 weeks after MI. Infarct size was determined by TTC staining and histology. The laser treatment (P:<0.05) lowered mortality significantly, from 30% to 6.5%, after induction of MI. The infarct size in the LI dogs was reduced significantly (P:<0.0001) (52%) compared with NLI dogs. Histological observation of the infarct revealed a typical scar tissue in NLI dogs and cellularity in most of the LI dogs. Only 14+/-3% of the mitochondria in the cardiomyocytes in the ischemic zone (4 hours after MI) of LI MI-induced rats were severely damaged, compared with 36+/-1% in NLI rats. Accordingly, ATP content in that zone was 7.6-fold (significantly) higher in LI than in NLI rats. CONCLUSIONS: Our observations indicate that epicardial LELI of rat and dog hearts after chronic MI caused a marked reduction in infarct size, probably due to a cardioprotective effect of the LELI.

Three-dimensional endocardial impedance mapping: a new approach for myocardial infarction assessment.

Precise identification of infarcted myocardial tissue is of importance in diagnostic and interventional cardiology. A three-dimensional, catheter-based endocardial electromechanical mapping technique was used to assess the ability of local endocardial impedance in delineating the exact location, size, and border of canine myocardial infarction. Electromechanical mapping of the left ventricle was performed in a control group (n = 10) and 4 wk after left anterior descending coronary artery ligation (n = 10). Impedance, bipolar electrogram amplitude, and endocardial local shortening (LS) were quantified. The infarcted area was compared with the corresponding regions in controls, revealing a significant reduction in impedance values [infarcted vs. controls: 168.8 +/- 11. 7 and 240.7 +/- 22.3 Omega, respectively (means +/- SE), P < 0.05] bipolar electrogram amplitude (1.8 +/- 0.2 mV, 4.4 +/- 0.7 mV, P < 0. 05), and LS (-2.36 +/- 1.6%, 11.9 +/- 0.9%, P < 0.05). The accuracy of the impedance maps in delineating the location and extent of the infarcted region was demonstrated by the high correlation with the infarct area (Pearson's correlation coefficient = 0.942) and the accurate identification of the infarct borders in pathology. By accurately defining myocardial infarction and its borders, endocardial impedance mapping may become a clinically useful tool in differentiating healthy from necrotic myocardial tissue.

Recovery from sarafotoxin-b induced cardiopathological effects in mice following low energy laser irradiation.

OBJECTIVE: Low energy laser irradiation has been shown to accelerate various biological processes, including regeneration of injured tissues. In the present work we studied the effect of low energy laser irradiation on ischemic mice hearts, following administration of sarafotoxin-b, a powerful vasoconstrictor peptide of the endothelin/sarafotoxin family. METHODS: Immediately after injection of the toxin and two days later, hearts were exposed to low energy laser irradiation. Eight days after the injection the mice were sacrificed and their hearts were processed for light and electron microscopy. RESULTS: Sarafotoxin-b induced an approximate 2-fold increase in the relative cavity volume of the left ventricle. Low energy laser irradiation of the sarafotoxin-injected mice caused a significant decrease (62%) in the left ventricular dilatation. Electron microscopy of the sarafotoxin-injected mice hearts revealed that the extent of formation of large vacuoles in the cytoplasm of the cardiomyocytes as well as disorganization of the myofibrils were much higher than in the laser irradiated mice. CONCLUSIONS: Our study indicates that low energy laser irradiation enhanced recovery of the damaged cardiomyocytes from the ischemia induced by sarafotoxin-b.

Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro.

Low-energy laser (He-Ne) irradiation was found to promote skeletal muscle regeneration in vivo. In this study, its effect on the proliferation and differentiation of satellite cells in vitro was evaluated. Primary rat satellite cells were irradiated for various time periods immediately after preparation, and thymidine incorporation was determined after 2 days in culture. Laser irradiation affected thymidine incorporation in a bell-shaped manner, with a peak at 3 s of irradiation. Three seconds of irradiation caused an induction of cell-cycle regulatory proteins: cyclin D1, cyclin E and cyclin A in an established line of mouse satellite cells, pmi28, and proliferating cell nuclear antigen (PCNA) in primary rat satellite cells. The induction of cyclins by laser irradiation was compatible with their induction by serum refeeding of the cells. Laser irradiation effect on cell proliferation was dependent on the rat's age. At 3 weeks of age, thymidine incorporation in the irradiated cells was more than twofold higher than that in the controls, while at 6 weeks of age this difference had almost disappeared. Myosin heavy chain (MHC) protein levels were twofold lower in the irradiated than in the control cells, whereas the proliferation of the irradiated cells was twofold higher. Fusion percentage was lower in the irradiated compared to non-irradiated cells. In light of these data, the promoting effect of laser irradiation on skeletal muscle regeneration in vivo may be due to its effect on the activation of early cell-cycle regulatory genes in satellite cells, leading to increased proliferation and to a delay in cell differentiation.

Enhancement of bone apposition to stainless steel cortical screws by surface modification using heat treatment: an experimental study.

OBJECTIVE: The purpose of the present study was to test whether the surface of stainless steel cortical screws modified by an oxidation process (heat treatment) resulted in enhancement of bone apposition as a consequence of better bone apposition to the metal surface. DESIGN: Control and heat-treated commercial cortical screws (stainless steel 316L) were inserted alternately into the tibiae of eight goats with a fixed insertion torque. Fluorochrome bone label was given during the six-week experimental period, after which the goats were killed and the extraction torque force measured. The screws and the adjacent bone were processed for histology. MAIN OUTCOME MEASURE: It was hypothesized that the heat-treated transcortical metal screws would have a greater extraction torque than untreated control screws. RESULTS: The extraction torque of the heat-treated screws was 0.59 +/- 0.06 newton-meters, which was significantly (p < 0.0001) higher (1.7-fold) than that of the control screws (0.35 +/- 0.02 newton-meters). Histomorphometric measurements demonstrated a 65 percent, significant (p < 0.05), increase in the area of fluorescence (indication of new bone deposition) adjacent to the heat-treated implant versus the control screws. CONCLUSIONS: Heat treatment of the cortical screws prior to insertion significantly increases fixation strength to the host bone in a large animal model. The clinical applicability will be to achieve bone apposition similar to that seen with titanium implants but with a stiff low-cost material.

Histology and enzymatic activity in the postnatal development of limb muscles in rodents.

The present work examines how increases in spontaneous motor capabilities during postnatal development are reflected in enzymatic activity and the histology of hindlimb muscles of the dormouse (Eliomys melanurus), the jird (Meriones tristrami), the vole (Microtus socialis), and the spiny mouse (Acomys cahirinus). The precocial neonate of the spiny mouse had the most advanced developmental state of young myofibers with striations as early as 1 week after delivery. At the same age, the altricial neonate vole had less developed muscles compared to the spiny mouse, but was more mature compared to other altricial species. The dormouse was the least developed, with numerous myoblasts and few myotubes at 1 week after delivery. These differences in myogenic development were conspicuous throughout postnatal development. Similar differences between the species were also evident at the biochemical level, as measured in the kinetics of activity of the enzyme creatine-phosphokinase immediately after delivery. On postnatal day 7, the creatine-phosphokinase level in the spiny mouse was fourfold higher than in the dormouse or vole. The enzymatic activity of acid phosphatase decreased during the first week postdelivery in the spiny mouse while peaking in the first, second, and third week in the jird, vole, and dormouse, respectively. These results support the notion that precocial species undergo certain developmental stages in utero, whereas, the same stages commence in altricials only postnatally. For the tested altricial species, the results illustrate that limb muscles in the vole, which displays more basic gaits, mature before limb muscles of the jird and dormouse, which display more specialized gaits.

Promotion of bone repair in the cortical bone of the tibia in rats by low energy laser (He-Ne) irradiation.

The effect of low energy laser (He-Ne) irradiation on bone repair in the cortical part of the tibia of the rat was investigated using biochemical and radioactive labeling methods. A fixed round hole was created in the lateral aspect of the tibia and the newly formed tissue was collected from the gap in the cortical bone. Alkaline phosphatase (ALP) and calcium progressively accumulated at the site of injury, peaking at 9 and 13 days postinjury, respectively. Direct irradiation of the hole injury with He-Ne laser on days 5 and 6 postinjury altered osteoblastic activity at the injured site as reflected by alkaline phosphatase activity. The laser irradiation also caused a significant increase ( approximately 2-fold) in calcium accumulation at the site of injury for 9-18 days postinjury. The rate of calcium deposition, measured by radioactive calcium, was significantly higher (approximately 2-fold) in the laser-irradiated rats as compared with controls. It is concluded that the process of bone repair in a hole created in the rat tibia is markedly enhanced by direct He-Ne laser irradiation of the injured site at the optimal energy level and time postinjury.

Effect of cadmium on bone repair in young rats.

The effect of cadmium (Cd) in drinking water on repair of bone at a site of hole injury to the tibia of young rats was followed using quantitative methods. The rats (3-4 wk old) were given 20 ppm and 200 ppm Cd for 5 wk and compared to a control group. A slight reduction (about 10%) in body weight and water and food consumption was observed in cadmium-exposed rats as compared to control rats. Clinical chemistry tests in the blood and histology of kidney, liver, and bone did not indicate changes related to Cd toxicity. A significant reduction (43%) in alkaline phosphatase (ALP) and tartarate-resistant acid phosphatase (TRAP) (46%) enzymatic activity was observed at 4 and 7 d postinjury respectively, in the site of injury in the rats receiving 200 ppm Cd in drinking water as compared to control rats. Calcium accumulation in the newly formed repair tissue at the site of injury was also significantly reduced (53%) at 13 d postinjury in the Cd-treated (200 ppm) rats as compared to control rats. It is concluded that Cd probably exhibits an effect on the bone repair process as reflected by reduction in ALP activity (osteoblastic cells) and mineralization at the site of injury in the tibia of young rats.

Regeneration in denervated toad (Bufo viridis) gastrocnemius muscle and the promotion of the process by low energy laser irradiation.

BACKGROUND: It is known that while denervated skeletal muscles have the ability to regenerate, maturation of regenerated myofibres does not take place under these conditions. Denervation also causes elevation of "invasive" and satellite cells, but the role of these cells in the regeneration process after injury to the denervated muscle is still unknown. Low energy lasers have recently been found to modulate and accelerate physiological processes in cells. The aim of the present study was to compare regeneration in denervated and innervated amphibian muscles and to investigate whether this process in denervated muscles can be stimulated by low energy laser irradiation prior to injury in these muscles. METHODS: Denervated gastrocnemius muscles of toads were irradiated with He-Ne laser (6.0 mW, 31.2 J/cm2) 7 days postdenervation (control muscle received red light irradiation at the same wavelength). Nine days after denervation cold injury was performed on the site of irradiation of both groups of muscles. At 14 days postinjury all muscles were removed and processed for histology and histomorphometric analysis of mononucleated cells, myotubes, and young myofibres in the regenerated zone. RESULTS: The volume fraction (percent of total injured zone) of the various histological structures in the injured zones 14 days after cold injury in the denervated (9 days prior to injury) muscles did not differ from innervated injured muscles at the same time interval postinjury. The mononucleated cells and myotubes in the laser irradiated muscles comprised 49 +/- 4% and 6 +/- 1% of the injured area, respectively, which was significantly lower than their volume fraction (67 +/- 2% and 11 +/- 2%, respectively) in the control muscles. The young myofibres populated 34 +/- 4% of the total injured area in the denervated and laser irradiated muscles which was significantly higher than their volume fraction (12 +/- 2%) in control denervated muscles. CONCLUSIONS: It is concluded that initial stages of regeneration can also take place in skeletal denervated and injured muscles of amphibians. The kinetics of the regeneration process are identical in denervated and innervated muscles. The process of regeneration in denervated muscles can be markedly enhanced if the muscle is irradiated by low energy laser prior to injury, probably by activation (stimulation of proliferation and/or differentiation) cells in the muscles that are "recruited" and participate in the process of regeneration.