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Low-energy laser irradiation promotes the survival and cell cycle entry of skeletal muscle satellite cells
Abstract
Low energy laser irradiation (LELI) has been shown to promote skeletal muscle cell activation and proliferation in primary cultures of satellite cells as well as in myogenic cell lines. Here, we have extended these studies to isolated myofibers. These constitute the minimum viable functional unit of the skeletal muscle, thus providing a close model of in vivo regeneration of muscle tissue. We show that LELI stimulates cell cycle entry and the accumulation of satellite cells around isolated single fibers grown under serum-free conditions and that these effects act synergistically with the addition of serum. Moreover, for the first time we show that LELI promotes the survival of fibers and their adjacent cells, as well as cultured myogenic cells, under serum-free conditions that normally lead to apoptosis. In both systems, expression of the anti-apoptotic protein Bcl-2 was markedly increased, whereas expression of the pro-apoptotic protein BAX was reduced. In culture, these changes were accompanied by a reduction in the expression of p53 and the cyclin-dependent kinase inhibitor p21, reflecting the small decrease in viable cells 24 hours after irradiation. These findings implicate regulation of these factors as part of the protective role of LELI against apoptosis. Taken together, our findings are of critical importance in attempts to improve muscle regeneration following injury.
... PBM therapy, as a non-drug and non-invasive physical therapy method, has been clinically used for the treatment of a variety of diseases such as Alzheimer's disease and hair loss [21][22][23]. PBM therapy is reported to trigger a series of signal pathways and be involved in a variety of biological processes, such as promoting cell survival and proliferation [24], increasing ATP synthesis [25], facilitating wound healing, and regulating neuronal functions [26]. Furthermore, recent studies reveal that PBM therapy promotes skeletal muscle cells proliferation [24,27]. ...
... PBM therapy is reported to trigger a series of signal pathways and be involved in a variety of biological processes, such as promoting cell survival and proliferation [24], increasing ATP synthesis [25], facilitating wound healing, and regulating neuronal functions [26]. Furthermore, recent studies reveal that PBM therapy promotes skeletal muscle cells proliferation [24,27]. However, the effect of PBM therapy on cancer cachexia-induced muscle wasting has not been explored. ...
... PBM therapy has been used in clinical to regulate neuronal functions, promote hair growth, reduce pain and inflammation under pathological conditions [38,39]. In addition, PBM therapy with a He-Ne laser (632.8 nm, 4.5 mW; 1.8 mm beam diameter) has been shown in recent studies to activate quiescent satellite cells (myogenic stem cells) and promote the regeneration of skeletal muscle [24,27]. Further research results suggested that PBM therapy promoted protein translation through the PI3K/AKT and Ras/Raf/ERK pathways to drive quiescent skeletal muscle satellite cells into the cell cycle and promote their proliferation, without side-effects [27]. ...
Cancer cachexia-associated muscle wasting as a multifactorial wasting syndrome, is an important factor affecting the long-term survival rate of tumor patients. Photobiomodulation therapy (PBMT) has emerged as a promising tool to cure and prevent many diseases. However, the effect of PBMT on skeletal muscle atrophy during cancer progression has not been fully demonstrated yet. Here, we found PBMT alleviated the atrophy of myotube diameter induced by cancer cells in vitro, and prevented cancer-associated muscle atrophy in mice bearing tumor. Mechanistically, the alleviation of muscle wasting by PBMT was found to be involved in inhibiting E3 ubiquitin ligases MAFbx and MuRF-1. In addition, transcriptomic analysis using RNA-seq and GSEA revealed that PI3K/AKT pathway might be involved in PBMT-prevented muscle cachexia. Next, we showed the protective effect of PBMT against muscle cachexia was totally blocked by AKT inhibitor in vitro and in vivo. Moreover, PBMT-activated AKT promoted FoxO3a phosphorylation and thus inhibiting the nucleus entry of FoxO3a. Lastly, in cisplatin-treated muscle cachexia model, PBMT had also been shown to ameliorate muscle atrophy through enhancing PI3K/AKT pathway to suppress MAFbx and MuRF-1 expression. These novel findings revealed that PBMT could be a promising therapeutic approach in treating muscle cachexia induced by cancer.
Graphical Abstract
Schematic representation of the signaling pathway for PBM therapy ameliorates cancer cachexia-associated muscle wasting. In cancer cachexia mice, myostatin and activin released by tumor cells act on the corresponding receptor ActRIIB by vascular transportation. Subsequent Smad2/3 are phosphorylated to reduce AKT activity and suppress FoxO3a phosphorylation. Dephosphorylated FoxO3a are translocated into the nucleus and induce the transcription of target genes (MAFbx and MuRF-1) which regulate the ubiquitin–proteasome systems, resulting in muscle wasting. After receiving PBM therapy, PI3K-AKT signaling is activated, and then stimulates protein synthesis by activating mTOR. mTOR activates the P70S6K, leading to protein synthesis. AKT also phosphorylates and inhibits the nucleus entry of FoxO3a, thereby alleviating muscle wasting.
... PBM therapy, as a non-drug and non-invasive physical therapy method, has been clinically used for the treatment of a variety of diseases such as Alzheimer's disease and hair loss [17][18][19]. PBM therapy is reported to trigger a series of signal pathways and be involved in a variety of biological processes, such as promoting cell survival and proliferation [20], increasing ATP synthesis [21], facilitating wound healing, and regulating neuronal functions [22]. Furthermore, recent studies reveal that PBM therapy promotes skeletal muscle cells proliferation [20,23]. ...
... PBM therapy is reported to trigger a series of signal pathways and be involved in a variety of biological processes, such as promoting cell survival and proliferation [20], increasing ATP synthesis [21], facilitating wound healing, and regulating neuronal functions [22]. Furthermore, recent studies reveal that PBM therapy promotes skeletal muscle cells proliferation [20,23]. However, the effect of PBM therapy on cancer cachexiainduced muscle wasting has not been explored. ...
... PBM therapy has been used in clinical to regulate neuronal functions, promote hair growth, reduce pain and in ammation under pathological conditions [38,39]. In addition, PBM therapy with a He-Ne laser (632.8 nm, 4.5 mW; 1.8 mm beam diameter) has been shown in recent studies to activate quiescent satellite cells (myogenic stem cells) and promote the regeneration of skeletal muscle [20,23]. Further research results suggested that PBM therapy promoted protein translation through the PI3K/AKT and Ras/Raf/ERK pathways to drive quiescent skeletal muscle satellite cells into the cell cycle and promote their proliferation, without side-effects [23]. ...
Cancer cachexia-associated muscle wasting as a multifactorial wasting syndrome, is an important factor affecting the long-term survival rate of tumor patients. Photobiomodulation therapy (PBMT) has emerged as a promising tool to cure and prevent many diseases. However, the effect of PBMT on skeletal muscle atrophy during cancer progression has not been fully demonstrated yet. Here, we found PBMT alleviated the atrophy of myotube diameter induced by cancer cells in vitro , and prevented cancer-associated muscle atrophy in mice bearing tumor. Mechanistically, the alleviation of muscle wasting by PBMT was found to be involved in inhibiting E3 ubiquitin ligases MAFbx and MuRF-1. In addition, transcriptomic analysis using RNA-seq and GSEA revealed that PI3K/AKT pathway might be involved in PBMT-prevented muscle cachexia. Next, we showed the protective effect of PBMT against muscle cachexia was totally blocked by AKT inhibitor in vitro and in vivo . Moreover, PBMT-activated AKT promoted FoxO3a phosphorylation and thus inhibiting the nucleus entry of FoxO3a. Lastly, in cisplatin-treated muscle cachexia model, PBMT had also been shown to ameliorate muscle atrophy through enhancing PI3K/AKT pathway to suppress MAFbx and MuRF-1 expression. These novel findings revealed that PBMT could be a promising therapeutic approach in treating muscle cachexia induced by cancer.
... Silent satellite cells beneath the basement membrane of muscle fibers play an essential role in muscle regeneration. LIL can be waking up these satellite cells and entering the proliferative phase [17][18][19]. ...
... This is to be expected because the role of ChABC in repairing muscle is not to create a new muscle fiber but to connect the muscle fibers to create a functional syncytium [23,25]. Unlike ChABC, the main role of photobiomodulation with LIL is to create new muscle fibers by stimulating silent satellite cells [19,34]. ...
... According to these explanations, the effect of ChABC on functional variables (manometric findings) and the effect of LIL on tissue variables is justifiable. An abundance of mitochondria as photoreceptors in the muscle tissue is a logical assumption for the laser's effect on muscle repair by angiogenesis [18] and stimulating satellite cells to produce new myofibrils [19,34]. Inconsistent with these statements, our study results also show an increase in ki67 (proliferation factor) and VEGF mRNA expressions in the laser group compared with sphincterotomized animals. ...
Background
Photobiomodulation with low-intensity laser (LIL) and chondroitinase ABC (ChABC) can repair damaged muscle tissue, so the aim of this study was to investigate the effect of co-administration of these two factors on anal sphincter repair in rabbits.
Methods
Male rabbits were studied in 5 groups (n = 7): Control (intact), sphincterotomy, laser, ChABC and laser + ChABC. 90 days after intervention were evaluated resting and maximum squeeze pressures, number of motor units, collagen amount, markers of muscle regeneration and angiogenesis.
Results
Resting pressure in the Laser + ChABC group was higher than the sphincterotomy, laser and ChABC groups ( p < 0.0001). Maximum squeeze pressure in the all study groups was higher than sphincterotomy group ( p < 0.0001). In the laser + ChABC and ChABC groups, motor unit numbers were more than the sphincterotomy group ( p < 0.0001). Collagen content was significantly decreased in the laser ( p < 0.0001) and laser + ChABC groups. ACTA1 ( p = 0.001) and MHC ( p < 0.0001) gene expression in the Laser + ChABC group were more than the laser or ChABC alone. VEGFA ( p = 0.009) and Ki67 mRNA expression ( p = 0.01) in the Laser + ChABC group were more than the laser group, But vimentin mRNA expression ( p < 0.0001) was less than the laser group.
Conclusion
Co-administration of ChABCs and photobiomodulation with LIL appears to improve the tissue structure and function of the anal sphincter in rabbits more than when used alone.
... EMG findings in the laser group also confirmed muscle contractility and presence by recording the action potentials of motor units. The role of LLL therapy in muscle regeneration is underscored by its stimulation of satellite cell proliferation and differentiation, 34 as well as increases in myofibrils, 35 antiapoptotic properties, 34 anti-inflammatory properties 21 and angiogenesis. 19 Our results further confirm LLL's efficacy in muscle repair by showing increased muscle mass in the laser group compared to the control group. ...
... EMG findings in the laser group also confirmed muscle contractility and presence by recording the action potentials of motor units. The role of LLL therapy in muscle regeneration is underscored by its stimulation of satellite cell proliferation and differentiation, 34 as well as increases in myofibrils, 35 antiapoptotic properties, 34 anti-inflammatory properties 21 and angiogenesis. 19 Our results further confirm LLL's efficacy in muscle repair by showing increased muscle mass in the laser group compared to the control group. ...
... Red and near-infrared light are the main light sources for PBMT. Several studies have shown that red light has the ability to promote the proliferation of both neural stem cells and skeletal muscle satellite cells [10,11]. However, the information about the modulation of the heart by red light is still limited [12,13]. ...
... Red light in the visible range has shown pro-proliferative functions on different types of cells [10,11,15]. However, the regulatory effects of LED-Red on CM proliferation are still unknown. ...
Photobiomodulation (PBM) has emerged as an alternative therapy involved in modulating a variety of biological effects. In this study, we verified whether PBM can affect cardiac physiological activity in mice through noninvasive irradiation using light-emitting diodes at a wavelength of 630 nm (LED-Red). We found that the PBM involved in regulating the repair of injured myocardium is wavelength-limited. LED-Red caused cardiomyocytes (CMs) that had exited the cell cycle to divide and proliferate again, and the cell proliferation ratio increased significantly with the accumulation of intracellular photopower. In addition, LED-Red promoted myocardial revascularization and myocardial regeneration, reduced the area of fibrosis in mice with myocardial infarction (MI), and thus improved cardiac contractile function. In regard to the mechanism, miRNA sequencing analysis showed that low-power LED-Red irradiation could induce differential changes in miRNAs in CMs. Among them, miR-136-5p was identified as a cardiac photo-sensitive miRNA and was obviously inhibited after stimulation, which produced a proliferation-promoting effect on CMs. Subsequent luciferase reporter assays confirmed the involvement of Ino80 as a binding target of miR-136-5p in the regulatory process of CM proliferation. Similarly, LED-Red irradiation elevated intracellular Ino80 expression. After knockdown of Ino80, the proliferation-promoting effect of LED-Red on CMs was inhibited. Collectively, this study demonstrates that LED-Red can promote CM proliferation by inhibiting cardiac photo-sensitive miRNA- miR-136-5p expression through targeting Ino80. The findings provided a new potential strategy for the treatment of ischemic cardiomyopathy (ICD).
... As células satélite, uma população de células-tronco musculares esqueléticas, são geralmente reconhecidas como a principal e, possivelmente, a única fonte de regeneração muscular pós-natal (Frontera & Ochala, 2015). Estudos anteriores revelaram o potencial da PBMT em promover a proliferação de células satélites e inibir sua apoptose in vitro (Bendov et al., 1999;Shefer et al., 2001;Shefer et al., 2002). Ben-Dov et al., (1999) irradiado com comparação ao grupo que não recebeu irradiação, relatando um papel protetor da PBMT contra a apoptose de células satélites (Shefer et al., 2002). ...
... Estudos anteriores revelaram o potencial da PBMT em promover a proliferação de células satélites e inibir sua apoptose in vitro (Bendov et al., 1999;Shefer et al., 2001;Shefer et al., 2002). Ben-Dov et al., (1999) irradiado com comparação ao grupo que não recebeu irradiação, relatando um papel protetor da PBMT contra a apoptose de células satélites (Shefer et al., 2002). ...
A atrofia do tecido muscular estriado esquelético é um processo complexo causado por um desequilíbrio entre a degradação e síntese de proteínas miofibrilares, levando à redução da força muscular e, consequentemente, influencia o comportamento emocional, a saúde mental e a qualidade de vida dos indivíduos. Desta forma, a atenuação da atrofia e estímulo para formação de um novo tecido muscular é um desafio para reabilitação. Esforços consideráveis têm sido dedicados ao estabelecimento de novos tratamentos, entretanto, ainda faltam dados experimentais e clínicos confiáveis para sua aplicação clínica. Dentre as recursos terapêuticos disponíveis, a terapia por fotobiomodulação (PBMT, do inglês photobiomodulation therapy) apresenta um grande potencial, visto que é frequentemente utilizada como estratégia terapêutica promissoras para a reabilitação do tecido muscular estriado esquelético. Dentro deste contexto, o objetivo deste estudo é proporcionar, através de uma revisão narrativa, uma compreensão das evidências atuais disponíveis sobre a importância da PBMT no tratamento de atrofia do músculo esquelético. As buscas foram realizadas nas bases de dados bibliográficas do PubMed/MEDLINE, Biblioteca virtual da saúde (BVS), Web of Science e SciELO. As evidências encontradas neste estudo direcionam que a PBMT pode ser proposta como uma intervenção terapêutica eficaz no tratamento da atrofia muscular, devido ao potencial de estimular fatores regulatórios miogênicos que promovem a ativação e proliferação de células satélites e consequente aumento da formação de fibras musculares, assim como atenuar e apoptose de celular as vias de proteólise da fibra muscular.
... Cell apoptosis stands as one of the pathophysiological mechanisms in normal brain aging and neurodegenerative diseases. The phenomenon of PBM anti-apoptotic properties was initially observed in skeletal muscle satellite cells by Shefer et al. [102]. The study highlighted that laser irradiation at a wavelength of 632.8 nm could diminish the levels of p53, p21, and Bax, while concurrently elevating Bcl-2 levels 24 h post-irradiation, thus safeguarding skeletal muscle satellite cells from apoptosis. ...
Neurodegenerative diseases involve the progressive dysfunction and loss of neurons in the central nervous system and thus present a significant challenge due to the absence of effective therapies for halting or reversing their progression. Based on the characteristics of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), which have prolonged incubation periods and protracted courses, exploring non-invasive physical therapy methods is essential for alleviating such diseases and ensuring that patients have an improved quality of life. Photobiomodulation (PBM) uses red and infrared light for therapeutic benefits and functions by stimulating, healing, regenerating, and protecting organizations at risk of injury, degradation, or death. Over the last two decades, PBM has gained widespread recognition as a non-invasive physical therapy method, showing efficacy in pain relief, anti-inflammatory responses, and tissue regeneration. Its application has expanded into the fields of neurology and psychiatry, where extensive research has been conducted. This paper presents a review and evaluation of studies investigating PBM in neurodegenerative diseases, with a specific emphasis on recent applications in AD and PD treatment for both animal and human subjects. Molecular mechanisms related to neuron damage and cognitive impairment are scrutinized, offering valuable insights into PBM’s potential as a non-invasive therapeutic strategy.
... 26,27 It was revealed that laser radiation inhibited nuclear factor kappa B (NF-κB), the key inflammatory transcription factor, and the related signaling pathways like inflammatory cytokines. 28,29 LLLT also reduced oxidative stress by inhibiting the production of reactive oxygen species (ROS) and inducible form of nitric oxide synthase (iNOS) expression in oxidatively-stressed cells. 24,[29][30][31] Moreover, low-level laser radiation enhanced the production and release of various small molecules and growth factors, including TGF-β, brain-derived neurotrophic factor (BDNF), platelet-derived growth factor, and glial-derived neurotrophic factor. ...
Introduction: Multiple sclerosis (MS) is an autoimmune disease. Inflammatory cells, cytokines, and chemokines play a major role in the pathogenesis of the disease. Low-level laser therapy (LLLT) as a photobiostimulation approach could affect a wide range of cellular responses. LLLT inhibits the inflammatory signaling pathway, improves cell viability, inhibits apoptosis, modulates immune responses, and induces the production of growth factors.
Methods: In this review, we discuss the effect of LLLT on cellular responses and its application in the treatment of MS. Such keywords as “low-level laser therapy”, “photobiomodulation” and “multiple sclerosis” were used to find studies related to laser therapy in MS in Google scholar, PubMed, and Medline databases.
Results: LLLT reduced the inflammatory immune cells and mediators. It also enhanced the regeneration of neurons.
Conclusion: Investigations showed that besides current treatment strategies, LLLT could be a promising therapeutic approach for the treatment of MS.
... In addition to aiding cellular respiration and the inflammatory response, PBM is also beneficial in stimulating stem cells and progenitor cells. 34 In vivo and in vitro animal injury models have shown an increase in satellite cell activation around myofibrils treated with PBM, 35,36 leading to a rise in new myofibril formation. 37,38 Irradiated muscle tissues promote a notable maturation of young myofibrils compared with those not irradiated. ...
Context:
Contusion and soft tissue injuries are common in sports. Photobiomodultion, light and laser therapy, is an effective aid to increase healing rates and improve function after various injury mechanisms. However, it is unclear how well photobiomodulation improves function after a contusion soft tissue injury. This study aimed to determine the effects of a pulsed red and blue photobiomodulation light patch on muscle function following a human thigh contusion injury.
Design:
Single-blinded randomized control trial design.
Methods:
We enrolled 46 healthy participants. Participants completed 5 visits on consecutive days. On the first visit, participants completed a baseline isokinetic quadriceps strength testing protocol at 60°/s and 180°/s. On the second visit, participants were struck in the rectus femoris of the anterior thigh with a tennis ball from a serving machine. Immediately following, participants were treated for 30 minutes with an active or placebo photobiomodulation patch (CareWear light patch system, CareWear Corp). Following the treatment, participants completed the same isokinetic quadriceps strength testing protocol. Participants completed the treatment and isokinetic quadriceps strength test during the following daily visits. We normalized the data by calculating the percent change from baseline. We used a mixed model analysis of covariance, with sex as a covariate, to determine the difference between treatment groups throughout the acute recovery process.
Results:
We found the active photobiomodulation treatment significantly increased over the placebo group, quadriceps peak torque during the 180°/s test (P = .030), and average power during both the 60°/s (P = .041) and 180°/s (P ≤ .001) assessments. The mean peak torque and average power of 180°/s, at day 4, exceeded the baseline levels by 8.9% and 16.8%, respectively.
Conclusions:
The red and blue photobiomodulation light patch improved muscle strength and power during the acute healing phase of a human thigh contusion injury model.
... Impaired cerebral blood flow (CBF) is another chief cause of brain damage in neurodegeneration, but the dissociation of nitrous oxide induced by transcranial NIR light can improve blood circulation and oxygenation (Figures 1 and 2), as NO is a powerful vasodilator [61]. Furthermore, many studies have suggested that the enhanced mitochondrial function brought about by PBM treatment reduces apoptosis [62,63], while the activation of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neuronal growth factor (NGF) by PBM augment neurogenesis [64]. ...
Alzheimer’s disease (AD) is a neurodegenerative disease and the world’s primary cause of dementia, a condition characterized by significant progressive declines in memory and intellectual capacities. While dementia is the main symptom of Alzheimer’s, the disease presents with many other debilitating symptoms, and currently, there is no known treatment exists to stop its irreversible progression or cure the disease. Photobiomodulation has emerged as a very promising treatment for improving brain function, using light in the range from red to the near-infrared spectrum depending on the application, tissue penetration, and density of the target area. The goal of this comprehensive review is to discuss the most recent achievements in and mechanisms of AD pathogenesis with respect to neurodegeneration. It also provides an overview of the mechanisms of photobiomodulation associated with AD pathology and the benefits of transcranial near-infrared light treatment as a potential therapeutic solution. This review also discusses the older reports and hypotheses associated with the development of AD, as well as some other approved AD drugs.
... The ability of PBMT to stimulate stem cells is highly relevant because the specialized satellite cells present in skeletal muscles function as stem or progenitor cells. 4 These mononucleated cells are located between the sarcolemma and the basement membrane, and are responsible for the well-known ability of muscles to grow and proliferate in response to exercise training. ...
... Moreover, it has been accounted for that LED treatment two times per day (670 nm) altogether diminished the quantity of striatal and cortical neurons going through apoptosis incited by exposure to rotenone and MPP+ [138]. LED pre-treatment with 670 nm light at fluences of 4 [137] and 30 J/cm2 [24] brought about the significant rescue of primary neurons from apoptosis induced by various neurotoxins. Advantageous impacts of PBM at frequencies of ~ 810 nm on mitochondrial structure and MMP breakdown have been displayed in different in vitro neurotoxicity models [71,138,112]. ...
Photobiomodulation (PBM) portrays the utilization of red or near infrared light to stimulate, heal, recover, and protect tissue that has either been harmed, is degenerating, or, else likely is in risk of dying. The brain experiences various issues that can be ordered into three general groupings: traumatic (stroke, traumatic brain injury, and global ischemia), degenerative diseases (dementia, Alzheimer's and Parkinson's), and psychiatric (depression, anxiety, post-traumatic stress disorder). There is some proof that this multitude of apparently different circumstances can be advantageously impacted by applying light to the head. There is even the likelihood that PBM could be utilized for cognitive enhancement in normal healthy individuals. In this transcranial PBM (tPBM) application, near infrared (NIR) light is frequently applied to the forehead in view of the better entrance (no hair, longer wavelength). A few workers have utilized lasers, yet as of late the presentation of modest light emitting diode (LED) arrays has permitted the improvement of light radiating head helmets or "brain caps". This review will cover the mechanisms of action of photobiomodulation to the brain and sum up some of the key pre-clinical studies and clinical trials that have been embraced for different brain disorders.
... Aimbre et al. [38] noted that LLLT reduced neutrophil influx and the expression of IL-1β micro-ribonucleic acid. LLLT can inhibit nuclear factor kappa B (NF-κB), the key inflammatory transcription factor, and the related signaling pathways [39,40]. LLLT can also suppress other important mediators of inflammation, namely, macrophage-associated inflammatory proteins and pro-inflammatory cytokines [41]. ...
Little is known about alternative treatment options for rhinosinusitis (RS). We aimed to evaluate the efficacy of low-level laser therapy (LLLT) for RS in experimentally induced rabbit models of RS. A total of 18 rabbits were divided into four groups: a negative control group (n = 3), an RS group without treatment (n = 5, positive control group), an RS group with natural recovery (n = 5, natural recovery group), and an RS group with laser irradiation (n = 5, laser-treated group). Computed tomography and histopathological staining were performed for each group. mRNA and protein expression levels of local cytokines (IFN-γ, IL-17, and IL-5) were also measured. Tissue inflammation revealed a significant improvement in the laser-treated group compared with the RS and natural recovery groups (p < 0.01). In addition, sinus opacification in the CT scans and cytokine expression was reduced in the laser-treated group, though without statistical significance. LLLT could be an effective option for the management of RS concerning radiological, histological, and molecular parameters.
... Most PBM studies have shown that laser irradiation, especially in the red and NIR regions of the spectrum, can improve cell viability and proliferation, as documented in various cell types including fibroblasts, endothelial cells, skeletal cells, keratinocytes, myoblasts, and stem cells [37][38][39]. For example, 650 nm GaAlAs laser could increase the proliferation, differentiation, and secretion of the growth factors of the adipose-derived MSCs [40,41] while PBM at 660 nm (5 mW, 6, 10, 12 J/cm 2 ) could significantly increase the proliferation and viability of the bone marrow MSCs (BMSCs) [42]. ...
Photo biomodulation (PBM) as a non-invasive and safe treatment has been demonstrated the anti-inflammatory potential in a variety of cell types, including stem cells. However, further investigations using different laser parameters combined with more accurate methods such as quantitative measurement of inflammatory gene expression at the mRNA level are still necessary. The aim of this study was to evaluate the effect of 532 nm green laser on cell proliferation as well as expression of inflammatory genes in human adipose-derived mesenchymal stem cells (hADMSCs) using RNA sequencing (RNA-seq) technique and confirmatory RT-PCR. hADMSCs were cultured in DMEM low glocuse medium with 10% fetal bovine serum until the fourth passage. Cultured cells were divided in two groups: control group (no laser irradiation) and laser group, irradiated with 532 nm laser at 44 m J/cm² with an output power of 50 mW and a density of 6 mW/cm², every other day, 7 s each time. The cell viability was assessed using MTT assay 24 h after each irradiation on days 3, 5, and 7 after cell seeding, followed by performing RNA-seq and RT-PCR. The MTT assay showed that PBM increased cell proliferation on day 5 after irradiation compared to day 3 and decreased on day 7 compared to day 5. In addition, gene expression analysis in hADMSCs using RNA-seq revealed down-regulation of inflammatory genes including CSF2, CXCL2, 3, 5, 6, 8, and CCL2, 7. These results indicate that 532 nm PBM with the parameters used in this study has a time-dependent effect on hADMSCs proliferation as well as anti-inflammatory potential.
Graphical abstract
... LLLT has shown beneficial effect on muscle cells by modulation of matrix metalloproteinase activity and gene expression. A study concluded that LLLT activates skeletal muscle satellite cells by enhancing their proliferation, inhibiting differentiation and regulating protein synthesis [22]. ...
Patients treated for head and neck cancer commonly suffer from oral mucositis, ulceration, bacterial infection causing pain, discomfort, impairment of speech and deglutition, leading to dehydration, poor nutrition, and mortality. Low level laser therapy (LLLT) is a promising state-of-the-art method which can be used effectively to reduce these side effects. It works on the principle of photo-bio stimulation. LLLT induces a biological response through energy transfer within the cells. It has anti-inflammatory effects, stimulates the immune system, increases of blood flow, upsurges cellular metabolism, and boosts rapid wound healing.
... Low Level Laser (LLL): summary-reduced ROS production in an hormetic fashion in C2C12 cells C2C12 muscle cells can be made to contract via direct electrical stimulation, affecting numerous biochemical and morphological analyses. Amongst these changes are the production of ROS and subsequent mitochondrial dysfunctions, leading to muscular fatigue from a decrease in ATP production (Pan et al. 2006;Mesquita-Ferrari et al. 2015;Shefer et al. 2002;Wehrle et al. 1994). Xu et al. (2003) proposed that LLL treatment may be useful in preventing muscular fatigue since cytochromes in the respiratory chain can absorb laser irradiation at specific wavelengths, which then enhances electron transfer and reduces free radical formation leading to greater ATP production (Assis et al. 2013). ...
Sarcopenia is a significant public health and medical concern confronting the elderly. Considerable research is being directed to identify ways in which the onset and severity of sarcopenia may be delayed/minimized. This paper provides a detailed identification and assessment of hormetic dose responses in animal model muscle stem cells, with particular emphasis on cell proliferation, differentiation, and enhancing resilience to inflammatory stresses and how this information may be useful in preventing sarcopenia. Hormetic dose responses were observed following administration of a broad range of agents, including dietary supplements (e.g., resveratrol), pharmaceuticals (e.g., dexamethasone), endogenous ligands (e.g., tumor necrosis factor α), environmental contaminants (e.g., cadmium) and physical agents (e.g., low level laser). The paper assesses both putative mechanisms of hormetic responses in muscle stem cells, and potential therapeutic implications and application(s) of hormetic frameworks for slowing muscle loss and reduced functionality during the aging process.
... These cells are usually not actively proliferating and thus not proceeding through the cell cycle. Progression of satellite cells through the cell cycle is tightly controlled by negative regulators, such as the highly expressed cyclin-dependent kinase inhibitor p21 [2,3]. Physical contact with the ECM, also promotes maintenance of satellite cells in a state of quiescence or G0 state [4e7]. ...
Satellite cells are generally quiescent in vivo. Once activated, progression through the cell cycle begins. Immortalised myoblasts from a single cell line are fairly homogenous in culture, but primary human myoblasts (PHMs) demonstrate heterogeneity. This phenomenon is poorly understood however may impact on PHM expansion. This study aimed to evaluate cell cycle transition from growth to synthesis phases of the cell cycle (G1 to S phase) and total mRNA relevant to this transition in PHM clones derived from 2 donor biopsies. Proportions of cells transitioning from G1 to S phase were evaluated at 2-hourly intervals for 24 h (n = 3 for each) and total mRNA quantified. Both PHM clones revealed an exponential transition from G1 to S phase over time, with a significantly slower rate for PHMs from S9.1 compared to S6.3, which had a higher proportion of PHMs in S phase for most time-points (p < 0.05). After 24 h the proportion of PHMs in S phase was ∼13% (S6.3) compared to ∼22% (S9.1). Gene transcription increased as cells progressed from G1 to S phase. Although total RNA increased with similar linearity in both clones, S6.3 PHMs had consistently (10 out of 12 time points) significantly higher concentrations. Validating the 2-hourly assessment over 24 h, a 4-hourly assessment from 8 to 32 h revealed similar differences but included the beginning of a plateau. This study demonstrates that PHMs from different donors differ in both cell cycle progression and overall transcriptome revealing new aspects in the heterogeneity of isolated satellite cells in vitro.
... The non-destructive energy exerted by PBMT stimulates a series of photobiological reactions reported to be beneficial for therapeutic purposes by the principle of biomodulation, i.e., using the native raw materials produced within our body to produce favorable changes in tissues [18]. Several cellular signaling pathways were triggered by PBMT application to promote cell growth, survival, proliferation, collagen synthesis, and differentiation [19][20][21][22]. In vitro, it has shown to be favorable on bone fracture repair by increasing mitochondrial activity thus ATP synthesis, DNA/RNA synthesis in osteoblasts, cell proliferation, cell viability and the expression of alkaline phosphatase (ALP) [23][24][25][26]. ...
The purpose of this study is to examine the prospective therapeutic effects of photobiomodulation on the healing of bone defects in diabetic mellitus (DM) using rat models to provide basic knowledge of photobiomodulation therapy (PBMT) during bone defect repair. For in vitro study, an Alizzarin red stain assay was used to evaluate the effect of PBMT on osteogenic differentiation. For in vivo study, micro-computed tomography (microCT) scan, H&E and IHC stain analysis were used to investigate the effect of PBMT on the healing of the experimental calvarial defect (3 mm in diameter) of a diabetic rat model. For in vitro study, the high glucose groups showed lower osteogenic differentiation in both irradiated and non-irradiated with PBMT when compared to the control groups. With the PBMT, all groups (control, osmotic control and high glucose) showed higher osteogenic differentiation when compared to the non-irradiated groups. For in vivo study, the hyperglycemic group showed significantly lower bone regeneration when compared to the control group. With the PBMT, the volume of bone regeneration was increasing and back to the similar level of the control group. The treatment of PBMT in 660 nm could improve the bone defect healing on a diabetic rat calvarial defect model.
... Using D-galactose-induced aging mice, Salehpour and collaborators (Salehpour et al., 2017) noted that red (660 nm) and near-infrared (810 nm) laser for 6 weeks attenuated the impairment in spatial and episodic-like memories. One hypothesis to explain these effects may be related to the capacity of PBM to regulate neuronal functions (Eells et al., 2003;Rojas et al., 2008;Freitas and Hamblin, 2016), including cell proliferation (Shefer et al., 2002;Gao et al., 2006), DNA and protein synthesis (Feng et al., 2012), and oxidative energy metabolism (Wang et al., 2017b). ...
Aging is often accompanied by exacerbated activation of cell death-related signaling pathways and decreased energy metabolism. We hypothesized that transcranial near-infrared laser may increase intracellular signaling pathways beneficial to aging brains, such as those that regulate brain cell proliferation, apoptosis, and energy metabolism. To test this hypothesis, we investigated the expression and activation of intracellular signaling proteins in the cerebral cortex and hippocampus of aged rats (20 months old) treated with the transcranial near-infrared laser for 58 consecutive days. As compared to sham controls, transcranial laser treatment increased intracellular signaling proteins related to cell proliferation and cell survival, such as signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB), also known as Akt that is linked to glucose metabolism. In addition, ERK is linked to memory, while ERK and JNK signaling pathways regulate glucose metabolism. Specifically, the laser treatment caused the activation of STAT3, ERK, and JNK signaling proteins in the cerebral cortex. In the hippocampus, the laser treatment increased the expression of p70S6K and STAT3 and the activation of Akt. Taken together, the data support the hypothesis that transcranial laser photobiomodulation improves intracellular signaling pathways linked to cell survival, memory, and glucose metabolism in the brain of aged rats.
... Skeletal muscle regeneration starts after chemical and/ or mechanical injury [15,16], and leads to the release of cytokines, prostaglandins, nitric oxide, growth factors, and the consequent activation of satellite cells [17,60]. Some studies showed that PBMT treatment could stimulate those processes, for instance increasing the expression of MRFs transcripts, such as MyoD and MyoG, which are known to play essential roles on reparative myogenesis [52,61,62]. ...
Envenoming caused by snakebites is a very important neglected tropical disease worldwide. The myotoxic phospholipases present in the bothropic venom disrupt the sarcolemma and compromise the mechanisms of energy production, leading to myonecrosis. Photobiomodulation therapy (PBMT) has been used as an effective tool to treat diverse cases of injuries, such as snake venom-induced myonecrosis. Based on that, the aim of this study was to analyze the effects of PBMT through low-level laser irradiation (904 nm) on the muscle regeneration after the myonecrosis induced by Bothrops jararacussu snake venom (Bjssu) injection, focusing on myogenic regulatory factors expression, such as Pax7, MyoD, and Myogenin (MyoG). Male Swiss mice (Mus musculus), 6–8-week-old, weighing 22 ± 3 g were used. Single sub-lethal Bjssu dose or saline was injected into the right mice gastrocnemius muscle. At 3, 24, 48, and 72 h after injections, mice were submitted to PBMT treatment. When finished the periods of 48 and 72 h, mice were euthanized and the right gastrocnemius were collected for analyses. We observed extensive inflammatory infiltrate in all the groups submitted to Bjssu injections. PBMT was able to reduce the myonecrotic area at 48 and 72 h after envenomation. There was a significant increase of MyoG mRNA expression at 72 h after venom injection. The data suggest that beyond the protective effect promoted by PBMT against Bjssu-induced myonecrosis, the low-level laser irradiation was able to stimulate the satellite cells, thus enhancing the muscle repair by improving myogenic differentiation.
... PBM is commonly used for skin regeneration, reducing wound inflammation, and proliferation of injured tissue. PBM stimulates the proliferation of mesenchymal cells, 26 fibroblasts, 27 osteoblasts, 28 endothelial cells, and stem cells. 29 Although many studies have demonstrated that PBM has a positive effect on thyroid cells, the results are inconsistent. ...
Objective:
This study aimed to investigate whether photobiomodulation (PBM) restores normal thyroid follicular cells affected by ionizing radiation, and to determine the mechanism of PBM on thyroid function.
Background:
Despite diverse applications of PBM to medical therapy, there has been no evidence of its involvement with thyroid function.
Methods:
A light emission diode (850 nm) array was used at 2, 5, and 10 J/cm2 for in vitro analysis in human thyroid N-Thy-3.1 cells, and at 120 J/cm2 for in vivo analysis in C58BL6 mice. Cell survival and proliferation were evaluated through clonogenic and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assays. Cell cycle was measured using flow cytometry. Cell cycle markers, such as p53, retinoblastoma (Rb), and E2F1, were investigated by western blot analysis. In vitro levels of cyclic adenosine monophosphate (cAMP) and thyroglobulin (TG) and in vivo levels of cAMP, TG, thyroid-stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) were measured using enzyme-linked immunosorbent assay.
Results:
A lethal dose for N-Thy-3.1 cells was 6 Gy. PBM at 2 J/cm2 was the most effective for causing cell cycle arrest by ionizing radiation. PBM regulated p53, Rb, and cAMP expression levels in vitro. PBM restored proliferation by regulating Rb and p53 in ionizing radiated thyroid follicular tissues. PBM also recovered cAMP, TG, and thyroid function marker expression (TSH, T3, and T4) by ionizing radiation in vivo.
Conclusions:
PBM restored ionizing radiation-induced thyroid follicular cell dysfunction by increasing cAMP proliferation and expression. PBM is effective for ionizing radiation-induced hypothyroidism by complementing cell proliferation and cAMP, presenting a novel method for clinical application.
... This article is protected by copyright. All rights reserved proliferation [12,13], accelerating wound healing [14,15] and regulating neuronal functions [16,17]. Zhang and colleagues reported that PBMT could protect megakaryocytes from mitochondrial injury and apoptosis, bolster megakaryocytes maturation, and then ameliorate thrombocytopenia [18,19]. ...
Background
Chemotherapy‐induced thrombocytopenia (CIT) can increase the risk of bleeding, which may delay or prevent the administration of anticancer treatment schedules. Photobiomodulation therapy (PBMT), a non‐invasive physical treatment, has been proposed to improve thrombocytopenia; however, its underlying regulatory mechanism is not fully understood.
Objective
To further investigate the mechanism of thrombopoietin (TPO) in megakaryocytopoiesis and thrombopoiesis.
Methods
Multiple approaches such as western blotting, cell transfection, flow cytometry and animal studies were utilized to explore the effect and mechanism of PBMT on thrombopoiesis.
Results
PBMT prevented a severe drop in platelet count by increasing platelet production, and then ameliorated CIT. Mechanistically, PBMT significantly upregulated hepatic TPO expression in a thrombocytopenic mouse model, which promoted megakaryocytopoiesis and thrombopoiesis. The levels of TPO mRNA and protein increased by PBMT via the Src/ERK/STAT3 signaling pathway in hepatic cells. Furthermore, the generation of the reactive oxygen species was responsible for PBMT‐induced activation of Src and its downstream target effects.
Conclusions
Our research suggests that PBMT is a promising therapeutic strategy for the treatment of CIT.
... A possible significance has been suggested based on increasing knowledge of low-intensity light therapy (LILT) or photobiomodulation (PBM) therapy, that uses low intensities of light with wavelengths in the visible (VIS), red, and near-IR (NIR) (Tafur et al., 2010;Kim et al., 2017). These modalities have been shown to influence a wide variety of cellular functions, including gene expression, growth and proliferation, survival, and differentiation (Shefer et al., 2002;Zhang et al., 2003). These functions are primarily mediated by raising the levels of adenosine triphosphate (ATP), a process in which cytochrome c oxidase appears to be the primary photo acceptor and transducer of photo signals in these regions of the light spectrum (Karu, 2010;Poyton and Ball, 2011). ...
Once regarded solely as the energy source of the cell, nowadays mitochondria are recognized to perform multiple essential functions in addition to energy production. Since the discovery of pathogenic mitochondrial DNA defects in the 1980s, research advances have revealed an increasing number of common human diseases, which share an underlying pathogenesis involving mitochondrial dysfunction. A major factor in this dysfunction is reactive oxygen species (ROS), which influence the mitochondrial-nuclear crosstalk and the link with the epigenome, an influence that provides explanations for pathogenic mechanisms. Regarding these mechanisms, we should take into account that mitochondria produce the majority of ultra-weak photon emission (UPE), an aspect that is often ignored – this type of emission may serve as assay for ROS, thus providing new opportunities for a non-invasive diagnosis of mitochondrial dysfunction. In this article, we overviewed three relevant areas of mitochondria-related research over the period 1960–2020: (a) respiration and energy production, (b) respiration-related production of free radicals and other ROS species, and (c) ultra-weak photon emission in relation to ROS and stress. First, we have outlined how these research areas initially developed independently of each other – following that, our review aims to show their stepwise integration during later stages of development. It is suggested that a further stimulation of research on UPE may have the potential to enhance the progress of modern mitochondrial research and its integration in medicine.
... One possible explanation for the susceptibility of injured skeletal muscle to treatment with LLLT, demonstrated by the reconstitution of muscle fibers, may be related to its mitochondrial content and the presence of satellite cells. These cells, stimulated by LLLT [34], are located above the muscle fiber but beneath the sarcolemma, and are involved in skeletal muscle regeneration and participate in its growth, providing nuclei and cytoplasmic mass to the muscle fiber, which argues that LLLT promotes recovery of muscle atrophy in association with satellite cell proliferation and angiogenesis [35,36]. ...
The purpose of the present work was to study the effect of low-level laser therapy (LLLT): helium-neon (He-Ne) and gallium arsenide (Ga-As) laser on the histomorphology of muscle and mitochondria in experimental myopathy in rats. Thirty Suquía strain female rats were distributed in groups: (A) control (intact), (B) injured, (C) injured and treated with He-Ne laser, (D) injured and treated with Ga-As laser, (E) irradiated with He-Ne laser on the non-injured muscle, and (F) irradiated with Ga-As laser on the non-injured muscle. Myopathy was induced by injecting 0.05 mg/rat/day of adrenaline in the left gastrocnemius muscle at the same point on five consecutive days, in groups B, C, and D. LLLT was applied with 9.5 J cm−2 daily for seven consecutive days in groups C, D, E, and F. The muscles were examined with optic and electronic microscopy. The inflammation was classified as absent, mild, and intense and the degree of mitochondrial alteration was graded I, II, III, and IV. Categorical data were statistically analyzed by Chi-square and the Fisher-Irwin Bilateral test, setting significant difference at p < 0.05. The damage found in muscle and mitochondria histomorphology in animals with induced myopathy (B) was intense or severe inflammation with grade III or IV of mitochondrial alteration. They underwent significant regression (p < 0.001) compared with the groups treated with He-Ne (C) and Ga-As (D) laser, in which mild or moderate inflammation was seen and mitochondrial alteration grades I and II, recovering normal myofibrillar architecture. No differences were found between the effects caused by the two lasers, or between groups A, E, and F. Group A was found to be different from B, C, and D (p < 0.001). LLLT in experimental myopathy caused significant muscular and mitochondrial morphologic recovery.
... There is evidence that biostimulation by low-level laser therapy (LLLT) increases cell proliferation, collagen synthesis and activation of growth factors [34,35]. The LLLT applied during the first hours after injury optimizes repair process of muscle tissue by increasing proliferation of satellite cells, mobility of myoblasts and growth of young myofibrils concomitantly with the formation of new blood vessels at the irradiated area [36][37][38][39]. The benefits of photobiostimulation promoted by low energy laser irradiation to minimize muscle inflammation, myofibers losses caused by venom myotoxins and improved regeneration have been already experimentally demonstrated [16][17][18][19]. ...
Myotoxins present in Bothrops venom disrupt the sarcolemma of muscle fibers leading to the release of sarcoplasmic proteins and loss of muscle homeostasis. Myonecrosis and tissue anoxia induced by vascularization impairment can lead to amputation or motor functional deficit. The objective of this study was to investigate the dynamic behavior of motor function in mice subjected to injection of Bothrops jararacussu venom (Bjssu) and exposed to low-level laser therapy (LLLT). Male Swiss mice received Bjssu injection (830 μg/kg) into the medial portion of the right gastrocnemius muscle. Three hours later the injected region was irradiated with diode semiconductor Gallium Arsenide (GaAs- 904 nm, 4 J/cm²) laser following by irradiation at 24, 48 and 72 hours. Saline injection (0.9% NaCl) was used as control. Gait analysis was performed 24 hours before Bjssu injection and at every period post-Bjssu using CatWalk method. Data from spatiotemporal parameters Stand, Maximum Intensity, Swing, Swing Speed, Stride Length and Step Cycle were considered. The period of 3 hours post venom-induced injury was considered critical for all parameters evaluated in the right hindlimb. Differences (p<0.05) were concentrated in venom and venom + placebo laser groups during the 3 hours post-injury period, in which the values of stand of most animals were null. After this period, the gait characteristics were re-established for all parameters. The venom + laser group kept the values at 3 hours post-Bjssu equal to that at 24 hours before Bjssu injection indicating that the GaAs laser therapy improved spatially and temporally gait parameters at the critical injury period caused by Bjssu. This is the first study to analyze with cutting edge technology the gait functional deficits caused by snake envenoming and gait gains produced by GaAs laser irradiation. In this sense, the study fills a gap on the field of motor function after laser treatment following snake envenoming.
This chapter describes our animal and human studies using red to near-infrared lasers and LEDs, a noninvasive and relatively inexpensive intervention, which we are investigating for neuroprotection and for the augmentation of cognitive brain functions. The first part deals with animal studies on the prevention of neurodegeneration and behavioral deficits by photobiomodulation. We describe light parameters that prevent optic neuropathy in vivo. These include fractionated exposure to light wavelengths that correspond to the absorption spectrum of the mitochondrial respiratory enzyme cytochrome oxidase. We show that such light stimulates cell respiration and upregulates cytochrome oxidase activity and the antioxidant enzyme superoxide dismutase. The human studies in the second part of the chapter demonstrate that photobiomodulation may be translated from animals to humans as a noninvasive, safe, nonpharmacological, and cost-effective method for augmentation of brain functions. The translation to human studies shows that transcranial infrared laser stimulation of the forehead causes cognitive enhancement associated with cerebrovascular oxygenation of the prefrontal cortex. Noninvasive methods such as near-infrared spectroscopy, magnetic resonance imaging, and electroencephalography were able to monitor metabolic, hemodynamic, and electrophysiological effects on human brains in real time. Combining photobiomodulation with such neurophysiological monitoring allows for calibration of the laser dose for individuals based on their own particular cerebrovascular response. This approach might also permit identifying potential candidates for photobiomodulation before clinical presentation of neurodegenerative effects. This research may have a broad impact, not only relevant for cognitive enhancement in healthy people but also potential for the noninvasive treatment of neurodegenerative and neurocognitive disorders.
Background and purpose:
Physical and therapeutic strategies to maintain and rehabilitate skeletal muscle mass, strength, and postural balance are clinically relevant to improve the health, well-being, and quality of life of older adults. The purpose of this study was to investigate the effects of photobiomodulation (PBM)/laser therapy combined with a resistance training (RT) program on quadriceps hypertrophy and strength, and postural balance in older women.
Methods:
In a randomized, triple-blinded, placebo-controlled design, twenty-two older women (age 66.6 ± 5.2 years) were engaged in a supervised 10-wk RT program (2 times per week) involving unilateral leg extension exercise, in which each leg of the same participant was randomly assigned to receive active (λ = 808 nm, optical output = 100 mW, total energy = 42 J) or placebo laser PBM immediately before the RT sessions. Maximal dynamic strength by unilateral knee extension 1-repetition maximum (1RM), muscle hypertrophy by vastus lateralis muscle thickness, and postural balance by one-legged stance test on a force platform were assessed before and after the training program.
Results:
Significance statistical analysis revealed a similar improvement (time P = .003) from pre- to posttraining for muscle hypertrophy and strength, and postural balance between active and placebo laser conditions. However, clinical interpretation for muscle hypertrophy showed a moderate effect (effect size [ES] = 0.58) for the active laser and a small effect (ES = 0.38) for the placebo laser. Clinical difference was not noticed between conditions for other analyzed variables.
Conclusions:
These findings indicate that RT alone can be clinically important for counteracting the deleterious effects of aging on muscle size, strength, and balance, and that applying laser PBM therapy before the RT sessions may further improve gains in muscle hypertrophy.
Potent neuroprotective effects of photobiomodulation with 670 nm red light (RL) have been demonstrated in several models of retinal disease. RL improves mitochondrial metabolism, reduces retinal inflammation and oxidative cell stress, showing its ability to enhance visual function. However, the current knowledge is limited to the main hypothesis that the respiratory chain complex IV, cytochrome c oxidase, serves as the primary target of RL. Here, we demonstrate a comprehensive cellular, molecular, and functional characterization of neuroprotective effects of 670 nm RL and 810 nm near-infrared light (NIRL) on blue light damaged murine primary photoreceptors. We show that respiratory chain complexes I and II are additional PBM targets, besides complex IV, leading to enhanced mitochondrial energy metabolism. Accordingly, our study identified mitochondria related RL-and NIRL-triggered defense mechanisms promoting photoreceptor neuroprotection. The observed improvement of mitochondrial and extramitochondrial respiration in both inner and outer segments is linked with reduced oxidative stress including its cellular consequences and reduced mitochondria-induced apoptosis. Analysis of regulatory mechanisms using gene expression analysis identified upregulation α-crystallins that indicate enhanced production of proteins with protective functions that point to the rescued mitochondrial function. The results support the hypothesis that energy metabolism is a major target for retinal light therapy.
Abstract Background Anal sphincter injury leads to fecal incontinence. Based on the regenerative capability of laser and human adipose-derived stem cells (hADSCs), this study was designed to assess the effects of co-application of these therapies on anal sphincter recovery after injury. Design Male rabbits were assigned to equal groups (n = 7) including control, sphincterotomy, sphincterotomy treated with laser (660 nm, 90 s, immediately after sphincterotomy, daily, 14 days), hADSCs (2 × 106 hADSCs injected into injured area of the sphincter immediately after sphincterotomy), and laser + hADSCs. Ninety days after sphincterotomy, manometry and electromyography were performed, sphincter collagen content was evaluated, and Ki67, myosin heavy chain (MHC), skeletal muscle alpha-actin (ACTA1), vascular endothelial growth factor A (VEGFA), and vimentin mRNA gene expression were assessed. Results The laser + hADSCs group had a higher resting pressure compared with the sphincterotomy (p
Peripheral neuropathy (PN) is a serious complication of diabetes mellitus (DM) and is known to be resistant to conventional treatment. Photobiomodulation (PBM) is demonstrated to be effective in treating PN and in protecting nerve fiber damage. To better understand the mechanisms underlying the regenerative effects of PBM on diabetic neuropathy, we conducted a study in an in vitro model of diabetes induced by glucose neurotoxicity. Neuro 2A cells (1 × 10⁴ cells/ well; N2A) were cultured in Minimum Essential Medium (MEM) supplemented with high glucose concentrations (100 mM) for 48 h and after the incubation period were submitted to either one or three consecutive applications of PBM, once a day (low-level InGaAlP, continuous wave mode, 660 nm, 30 mW, 1.6 J/cm², 15 s, per well). Cell viability was measured by MTT method, neurotoxicity by LDH release, neurite outgrowth was evaluated through morphometric analysis, and AKT/ERK protein expression levels were assessed by western blotting. Results demonstrate that PBM increased N2A viability as well as induced neurogenesis observed by the increase in neurite outgrowth being this effect modulated by AKT activation. Data obtained herein reinforce the regenerative potential of PBM in the treatment of PN and strongly suggests that phototherapy should be considered adjuvant in the treatment of diabetes.
Satellite cells, a population of skeletal muscular stem cells, are generally recognized as the main and, possibly, the sole source of postnatal muscle regeneration. Previous studies have revealed the potential of low-level laser (LLL) irradiation in promoting satellite cell proliferation, which, thereby, boosts the recovery of skeletal muscle from atrophy. The purpose of this study is to investigate the beneficial effect of LLL on disuse-induced atrophy. The optimal irradiation condition of LLL (808 nm) enhancing the proliferation of Pax7+ve cells, isolated from tibialis anterior (TA) muscle, was examined and applied on TA muscle of disuse-induced atrophy model of the rats accordingly. Healthy rats were used as the control. On one hand, transiently, LLL was able to postpone the progression of atrophy for 1 week through a reduction of apoptosis in Pax7−veMyoD+ve (myocyte) population. Simultaneously, a significant enhancement was observed in Pax7+veMyoD+ve population; however, most of the increased cells underwent apoptosis since the second week, which suggested an impaired maturation of the population. On the other hand, in normal control rats with LLL irradiation, a significant increase in Pax7+veMyoD+ve cells and a significant decrease of apoptosis were observed. As a result, a strengthened muscle contraction was observed. Our data showed the capability of LLL in postponing the progression of disuse-induced atrophy for the first time. Furthermore, the result of normal rats with LLL irradiation showed the effectiveness of LLL to strengthen muscle contraction in healthy control.
Purpose: The aim of the present study was to investigate the effectiveness of photobiomodulation therapy (PBMT) on muscle recovery based on inflammation (interleukin-10 [IL-10]; tumor necrosis factor-α [TNFα]), muscle damage markers (creatine kinase [CK]; lactate dehydrogenase [LDH]), delay onset muscle soreness (DOMS), and countermovement jump performance (CMJ) after two sprint interval training (SIT) sessions compared with a placebo condition (part-I), as well as to compare the effectiveness of PBMT with active recovery (AR) and cold-water immersion (CWI) (part-II).
Methods: Part-I was conducted as a double-blind, randomized and placebo-controlled study and part-II as a parallel-group study. Thirty-six men participated in the studies (12 participants in part-I and 36 participants in part-II). Volunteers performed two SITs interspaced by 24-h (SIT1 and SIT2) to mimic the effect of accumulating 2 consecutive days of SIT. In part-I, only after SIT2, PBMT [Total energy: 600J (300J per leg in 5 spots); wavelength: 660–850 nm] or placebo interventions were performed, while in part-II PBMT (part-I data), AR (15-min; 50% of the maximal aerobic power), or CWI (10-min; 10°C) were carried out, also after SIT2. Blood samples were collected before (i.e., baseline), and 0.5, 1, 24, 48, and 72-h after SIT2, while CMJ and DOMS were measured before, 24, 48, and 72-h after SIT2.
Results: In part-I, there were no interactions between PBMT and placebo conditions for any blood markers (P ≥ 0.313), DOMS (P = 0.052), and CMJ (P = 0.295). However, an effect of time was found with increases in LDH, CK, and IL-10 (P ≤ 0.043) as well as a decrease in DOMS at 72-h compared with 24-h (P = 0.012). In part-II, there were no interactions between the PBMT, AR, and CWI groups for any markers at the same moments (P ≥ 0.189) and for the peak and integral values (P ≥ 0.193), for DOMS (P = 0.314) and CMJ (P = 0.264). However, an effect of time was found with an increase in CK and IL-10 (P = 0.003), while DOMS decreased at 48 and 72-h compared with 24-h (P = 0.001).
Conclusion: In summary, PBMT had no effect on inflammation, muscle damage, CMJ performance, or DOMS after two consecutive sprint interval training sessions compared to placebo, CWI, and AR strategies.
This study aimed to investigate the effects of low-level laser therapy (LLLT) on muscle repair in rats with chronic alcohol intake. Thirty male Wistar rats were distributed into three groups: injured tibialis anterior (TA) muscle without treatment (IC); chronic ingestion of alcohol plus injured TA muscle (AI); and chronic ingestion of alcohol plus injured TA plus LLLT (AIL). Each group was divided into two different subgroups, and rats were sacrificed on days 3 and 7 post-injury. Morphological features in the injured areas were similar with or without alcohol intake (IC and AI); however, LLLT promoted a decrease in the number of inflammatory cells and destroyed zones, as well as improved tissue organization (AIL). In general, alcohol intake caused a decrease in myogenic regulatory factors (MyoD and myogenin) and vascular endothelial growth factor in the AI group. Moreover, LLLT promoted recovery of these factors to the same level as in animals without alcohol intake (IC and AIL). LLLT was able to increase the expression of myogenic and vascular growth factors and stimulate skeletal muscle regeneration in rats with chronic alcohol intake.
Low-level laser irradiation (LLLI) has been used as a non-invasive method to improve muscular regeneration capability. However, the molecular mechanisms by which LLLI exerts these effects remain largely unknown. Here, we described global gene expression profiling analysis in C2C12 myoblasts after LLLI that identified 514 differentially expressed genes (DEG). Gene ontology and pathway analysis of the DEG revealed transcripts among categories related to cell cycle, ribosome biogenesis, response to stress, cell migration, and cell proliferation. We further intersected the DEG in C2C12 myoblasts after LLLI with publicly available transcriptomes data from myogenic differentiation studies (myoblasts vs myotube) to identify transcripts with potential effects on myogenesis. This analysis revealed 42 DEG between myoblasts and myotube that intersect with altered genes in myoblasts after LLLI. Next, we performed a hierarchical cluster analysis with this set of shared transcripts that showed that LLLI myoblasts have a myotube-like profile, clustering away from the myoblast profile. The myotube-like transcriptional profile of LLLI myoblasts was further confirmed globally considering all the transcripts detected in C2C12 myoblasts after LLLI, by bi-dimensional clustering with myotubes transcriptional profiles, and by the comparison with 154 gene sets derived from previous published in vitro omics data. In conclusion, we demonstrate for the first time that LLLI regulates a set of mRNAs that control myoblast proliferation and differentiation into myotubes. Importantly, this set of mRNAs revealed a myotube-like transcriptional profile in LLLI myoblasts and provide new insights to the understanding of the molecular mechanisms underlying the effects of LLLI on skeletal muscle cells.
Background:
This study aimed at estimating the effectiveness of two commonly used modalities-extracorporeal shock wave therapy (ESWT) and low-level laser therapy (LLLT)-each combined with usual care (exercises and orthotic supports) in comparison to only usual care to relieve pain in patients with plantar fasciitis (PF).
Methods:
Participants with PF were randomly allocated into 3 groups: ESWT (n = 25), LLLT (n = 24), and control (n = 17). All participants received a home exercise program with orthotic support. The ESWT group received 2000 shock waves with 0.02 mJ/mm2 for 3 sessions, once a week; LLLT group received gallium-aluminum-arsenide laser with 850 nm wavelength for 10 sessions, 3 times a week. Pain was measured by Foot Function Index-pain subscale (FFI-p) and Numerical Rating Scale for pain (NRS-p). The scores were recorded at baseline, third week, and third month after the treatment. Analysis was performed using repeated measures ANOVA.
Results:
There was a significant improvement in pain over the 3 months in all groups on both FFI-p ( P < .001) and NRS-p ( P < .001). In NRS-p, LLLT group had significantly lower pain than ESWT ( P = .002) at the third week and control ( P = .043) and ESWT ( P = .003) at third month. In FFI-p total score, ESWT group had higher pain than LLLT ( P = .003) and control ( P = .035) groups at third week and LLLT ( P = .010) group at third month.
Conclusion:
When LLLT and ESWT were combined with usual care, LLLT was found to be more effective than ESWT in reducing pain in PF at short-term follow-up.
Level of evidence:
Level II, comparative study.
Brain photobiomodulation (PBM) therapy using red to near-infrared (NIR) light is an innovative treatment for a wide range of neurological and psychological conditions. Red/NIR light is able to stimulate complex IV of the mitochondrial respiratory chain (cytochrome c oxidase) and increase ATP synthesis. Moreover, light absorption by ion channels results in release of Ca²⁺ and leads to activation of transcription factors and gene expression. Brain PBM therapy enhances the metabolic capacity of neurons and stimulates anti-inflammatory, anti-apoptotic, and antioxidant responses, as well as neurogenesis and synaptogenesis. Its therapeutic role in disorders such as dementia and Parkinson’s disease, as well as to treat stroke, brain trauma, and depression has gained increasing interest. In the transcranial PBM approach, delivering a sufficient dose to achieve optimal stimulation is challenging due to exponential attenuation of light penetration in tissue. Alternative approaches such as intracranial and intranasal light delivery methods have been suggested to overcome this limitation. This article reviews the state-of-the-art preclinical and clinical evidence regarding the efficacy of brain PBM therapy.
The pathogenesis of myonecrosis caused by myotoxins from bothropic venom is associated with local extracellular matrix (ECM) disintegration, hemorrhage, and inflammation. Search for alternative methods associated with serum therapy is mandatory to neutralize the fast development of local damage following snakebites. The experimental use of photobiomodulation therapy (PBMT) in murine models has shown promising results relative to structural and functional recovery from bothropic snakebite-induced myonecrosis. This study pioneered in using Raman and Fourier transform infrared (FTIR) spectroscopies to characterize biochemical alterations in the gastrocnemius that had been injected with Bothrops jararacussu venom and exposed to local PBMT. Results show that vibrational spectra from lyophilized and diluted venom (1307 cm ⁻¹) was also found in the envenomed gastrocnemius indicating venom presence in the unirradiated muscle 48 h post-injection; but any longer visible after PBMT at this time exposure or 72 h post-injection regardless irradiated or not. Raman and FTIR analyses indicated that the bands with higher area and intensity were 1657 and 1547 cm⁻¹ and 1667 and 1452 cm⁻¹, respectively; all are assignments for proteins, especially collagen, and are higher in the PBMT-exposed gastrocnemius. The infrared spectra suggest that laser treatment was able to change protein in tissue and that such change indicates collagen as the main target. We hypothesize that the findings reflect remodeling of ECM with key participation of collagen and faster tissue recovery for an anabolic condition.
Photobiomodulation (PBM) therapy contributes to pain relief, wound healing, and tissue regeneration. The pulsed wave (PW) mode has been reported to be more effective than the continuous wave (CW) mode when applying PBM to many biological systems. However, the reason for the higher effectiveness of PW-PBM is poorly understood. Herein, we suggest using delayed luminescence (DL) as a reporter of mitochondrial activity after PBM treatment. DL originates mainly from mitochondrial electron transport chain systems, which produce reactive oxygen species (ROS) and adenosine triphosphate (ATP). The decay time of DL depends on the pulse frequencies of applied light, which correlate with the biological responses of human dental pulp stem cells (hDPSCs). Using a low-power light whose wavelength is 810 nm and energy density is 38 mJ/cm², we find that a 300-Hz pulse frequency prolonged the DL pattern and enhanced alkaline phosphatase activity. In addition, we analyze mitochondrial morphological changes and their volume density and find evidence supporting mitochondrial physiological changes from PBM treatment. Our data suggest a new methodology for determining the effectiveness of PBM and the specific pulse frequency dependency of PBM in the differentiation of hDPSCs.
The goal of this study is to estimate the effects of low-level laser irradiation (LLLI) on the superovulatory response according to the number of corpora lutea (CL), follicles (F) and the embryo yield. In recent years, while searching for new, more efficient and organic methods to improve superovulatory response and embryo yield with respect to the conventional methods, low-level laser irradiation (LLLI) is a more sensitive and less costly technology that can be used to improve animal reproduction, namely, artificial insemination and the embryo production system. The dairy-cow donors were treated for superovulation with Pluset
• Background:
In 1967 a few years after the first working laser was invented, Endre Mester in Semmelweis University Budapest, Hungary wanted to find out if laser might cause cancer. He took some mice, shaved the hair off their backs, divided them into two groups and gave a laser treatment with a low powered ruby laser to one group. They did not get cancer and to his surprise the hair on the treated group grew back more quickly than the untreated group. That was how "laser biostimulation" was discovered.
o Effect of laser on hair Growth of mice (in Hungarian). Mester, E. Szende, B. and Tota, J.G. (1967). Kiserl Orvostud 19. 628-631
• Purpose of the work:
The effects of pulsed monochromatic light, with fixed pulsations and wavelengths, on the healing of pressure ulcers were evaluated in this prospective, randomized, controlled study.
• Approach and methodology:
A placebo-controlled, double-blind study using low energy photon therapy (LLLT) was performed in ten patients with bedsore on the back. Treatment was given three times a week for 10 weeks, using monochromatic (red) optical sources; diode 660nm (GaAl-660). The patients who were randomized to placebo treatment received sham therapy from an identical-appearing light source from the same delivery system.
• Results:
Ten patients with bedsore were randomized to receive LLLT or placebo therapy. At the conclusion of the study, the percentage of the initial ulcer area remaining unhealed in the LLLT and placebo groups was 24.4% and 84.7%, respectively (P = 0.0008). The decrease in ulcer area (compared to baseline) observed in the LLLT and placebo groups was 193.0 mm2 and 14.7 mm2, respectively (P = 0.0002).
One patient dropped out of the study, complaining of lack of treatment efficacy; he was found to be randomized to the placebo group. There were no adverse effects.
• Conclusions:
In this placebo-controlled, double-blind study LLLT was an effective modality for the treatment of bedsore which were resistant to conventional medical management.
The results are encouraging as pulsed monochromatic light increased healing rate and shortened healing time. This will positively affect the quality of life in elderly patients with pressure ulcers.
Keywords:
Bedsore Healing, Soft Tissue Healing, Decubitus Ulcer Healing, Low Level Laser ,Wound Healing.
Low-intensity laser irradiation exerts effects on various biological objects and is currently exploited in various branches of medicine. From a practical point of view, irradiation in the near-infrared range seems most attractive since it has the highest penetration characteristics for human tissues. In the present work, we studied the effects of 835 nm infrared low-intensity laser irradiation on two melanoma cell lines, Mel IL and MeWo, in culture. The data that we obtained indicate that low-intensity infrared irradiation impacts the growth rate of Mel IL and MeWo melanoma cell lines and that the response dynamics of the two cell lines are similar, in spite of certain quantitative differences. Stimulation of cell growth occurs within a relatively narrow range of low doses (about 0.17 J/cm²). With significantly higher doses, deceleration of growth occurs instead. Рге-treatment of cells with low-dose radiation, however, protects them from the negative influence of higher doses. The protective action of low doses develops gradually, within about 10–30 min, and persists for at least 3 h. Рге-irradiation of cells also widens the range of stimulatory action with low-dose radiation. A theoretical model that explains the dynamics of cellular responses to various irradiation doses has been proposed.
Multinucleated myofibers, the functional contractile units of adult skeletal muscle, harbor mononuclear Pax7+ myogenic progenitors on their surface between the myofiber basal lamina and plasmalemma. These progenitors, known as satellite cells, are the primary myogenic stem cells in adult muscle. This chapter describes our laboratory protocols for isolating, culturing, and immunostaining intact myofibers from mouse skeletal muscle as a means for studying satellite cell dynamics. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are plated in dishes coated with PureCol collagen (formerly known as Vitrogen) and maintained in a mitogen-poor medium (± supplemental growth factors). Employing such conditions, satellite cells remain at the surface of the parent myofiber while synchronously undergoing a limited number of proliferative cycles and rapidly differentiate. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. These EDL myofibers are routinely plated individually as adherent myofibers in wells coated with Matrigel and maintained in a mitogen-rich medium, conditions in which satellite cells migrate away from the parent myofiber, proliferate extensively, and generate numerous differentiating progeny. Alternatively, these EDL myofibers can be plated as non-adherent myofibers in uncoated wells and maintained in a mitogen-poor medium (± supplemental growth factors), conditions that retain satellite cell progeny at the myofiber niche similar to the FDB myofiber cultures. However, the adherent myofiber format is our preferred choice for monitoring satellite cells in freshly isolated (Time 0) myofibers. We conclude this chapter by promoting the Nestin-GFP transgenic mouse as an efficient tool for direct analysis of satellite cells in isolated myofibers. While satellite cells have been often detected by their expression of the Pax7 protein or the Myf5nLacZ knockin reporter (approaches that are also detailed herein), the Nestin-GFP reporter distinctively permits quantification of satellite cells in live myofibers, which enables linking initial Time 0 numbers and subsequent performance upon culturing. We additionally point out to the implementation of the Nestin-GFP transgene for monitoring other selective cell lineages as illustrated by GFP expression in capillaries, endothelial tubes and neuronal cells. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular, can also be isolated and analyzed using protocols described herein. Collectively, this chapter provides essential tools for studying satellite cells in their native position and their interplay with the parent myofiber.
低出力レーザー照射（Low-power laser irradiation: LLI）は細胞増殖を含め，様々な生物学的過程に影響を及ぼすことが報告されている．われわれは808 nm・60 mWのLLIがヒト由来脳腫瘍細胞（glioblastoma A-172）の細胞増殖を抑制することを報告した．そこで，その効果に用いるレーザーの波長による違いがあるのかどうかを明らかにする目的で，同一の実験系において異なる波長の405 nm LLIを用いて，A-172への照射効果を検討した．細胞の継代培養には一般的な培養法を用いて，35 mm ペトリ皿に1.2×104 cells/mlの細胞濃度で, また96 ウェル・プレートに0.3×104 cells/mlで均一に播き，一晩培養した．405 nmの半導体レーザー発生装置（27 mW）を用いて，その出力をペトリ皿の中央部または各ウェルへ誘導した（照射面積：7.07 mm2）．非照射群と照射群（20, 40, 60分連続照射）に分け， 培養開始時，照射直後，24時間後，48時間後の細胞増殖を位相差顕微鏡で観察しながらデジタルカメラで撮影し，細胞計数を行った．またMTT法を用いて，照射後48時間後の生細胞数を吸光光度法により測定した．定点撮影法によるすべての計数結果から，非照射群と比較して，照射群は3群とも細胞増殖率の有意な低下を示し（p
Hamstring injuries are among the most frequent in sports. The high relapse rate is a challenge for sports medicine and has a great impact on athletes and sport teams. The treatment goal is to provide the athlete the same functional level as before the injury. Thus, functional rehabilitation is very important to the success of the treatment. Currently, several physical therapy modalities are used, according to the stage of the lesion, such as cryotherapy, laser therapy, therapeutic ultrasound, therapeutic exercise, and manual therapy. However, the evidence of the effectiveness of these modalities in muscle injuries is not fully established due to the little scientific research on the topic. This article presents an overview of the physiotherapy approach in the rehabilitation of hamstring muscle injuries.
119071, Москва У молодых и взрослых крыс проводили лазеротерапию обеих икроножных мышц, аутотранспланти-рованных in situ в целом или измельченном состоянии. Исследовали также тимус и динамику массы тела как показатель гомеостаза. Установлено, что эффективность импульсного инфракрасного лазерно-го излучения (890 нм) в отношении регенерации мышц (количество мышечной ткани, реиннервация) определялась степенью повреждения мышц. Однако лазеротерапия улучшала регенерацию мышц даже после крайне тяжелой мышечной травмы, вызванной полным разрушением ее целостности и потерей половины мышечной массы. В регенератах содержалось сравнительно небольшое количество мышеч-ной ткани, но она была функционально активна. У молодых крыс положительный эффект был выше, чем у взрослых. Лазерное излучение влияло на общее состояние организма. У всех животных при обоих видах травмы к моменту завершения лазеротерапии (10 сут) в той или иной степени наблюдалось сни-жение массы тела, а на 30-е сутки — постепенное увеличение. Как у молодых, так и у взрослых крыс между опытом и контролем на фоне практически одинаковой массы тимуса и количества коркового вещества отмечались различия в пролиферативной активности лимфоидных клеток. Это указывает на незавершенность восстановительных процессов в тимусе.
As lesões dos isquiotibiais estão entre as mais frequentes do esporte. A alta taxa de recidivas representa um desafio para a medicina esportiva e apresenta grande impacto para atletas e clubes esportivos. O objetivo do tratamento é proporcionar ao atleta o mesmo nível funcional anterior à lesão. Dessa forma, a reabilitação funcional é muito importante para o sucesso do tratamento. Atualmente, usam‐se várias modalidades fisioterápicas de acordo com o estágio da lesão: crioterapia, laserterapia, ultrassom terapêutico, terapia manual e cinesioterapia. Entretanto, as evidências da eficácia dessas modalidades nas lesões musculares ainda não estão completamente estabelecidas, devido à baixa investigação científica sobre o tema. O presente artigo apresenta uma revisão sobre a abordagem fisioterápica na reabilitação das lesões musculares de isquiotibiais.
Cells from multicellular organisms self-destroy when no longer needed or when damaged. They do this by activating genetically controlled machineries that lead to apoptosis. Skeletal muscles in adult animals are fully differentiated syncytial cells. Apoptosis has been described in developing and, recently, in adult skeletal muscle. The cellular and molecular aspects of myoblast and myofibre apoptosis and their role in disease are analysed in this review. Alterations in the pathways that regulate myoblasts proliferation/differentiation lead to induction of apoptosis during myogenesis both in vivo and in vitro. In adult muscle myofibres apoptosis seems to start from segmental areas of myofibres often producing loss of a single myonucleus. The bcl2/bax system is active in muscle when apoptosis occurs. On the other hand conflicting results are reported on the role played by FasL/Fas system. These findings are confirmed by in vitro results on myotubes and on their susceptibility to apoptosis. Though apoptosis has been shown to occur in the skeletal muscle, the role played in diseases and the pattern followed in myogenic cells are far from being clear.
During the terminal stage of skeletal myogenesis, myoblasts stop replicating, fuse to form multinucleate fibers, and express the genes that encode the proteins that convey contractile capacity. Because of this dramatic shift in proliferative state, morphology, and gene expression, it has been possible to readily identify and quantitate terminally differentiating myoblasts. In contrast, it is not clear whether the proliferating cells that give rise to postmitotic myoblasts are equally distinct in their phenotype and in fact whether distinct stages in skeletal myogenesis precede the onset of terminal differentiation. To address these questions, monoclonal antibodies and immunofluorescence microscopy were used to determine that replicating myoblasts from newborn rats do express a muscle-specific phenotype. To identify replicating cells, incorporation of 5-bromo-2'-deoxyuridine (BrdUrd) into DNA was assayed by using anti-BrdUrd antibody. The developmentally regulated, muscle-specific, integral membrane protein H36 and the intermediate-filament protein desmin were scored as markers of the myogenic phenotype. The percentage of BrdUrd+ (i.e., proliferative) cells among H36+ and desmin+ myoblasts was equal to the percentage of BrdUrd+ cells in the entire population, indicating that the expression of H36 and desmin is uniformly characteristic of replicating myoblasts. Inhibition of protein synthesis before and during growth in BrdUrd did not alter the frequency of desmin and H36 immunofluorescence in BrdUrd+ cells. Thus, desmin and H36 were present in the replicating myoblasts prior to the onset of growth in BrdUrd. These results were confirmed using H36+ cells selected by flow cytometry: these purified H36+ myoblasts replicate, express desmin, and differentiate. Similar results were obtained with mouse myoblasts. Desmin expression in these mammalian cells differs from that in chicken embryo myoblasts: only a small proportion of replicating chicken embryo myoblasts express desmin. That replicating mammalian myoblasts have a muscle-specific phenotype serves to define a distinct stage in myogenic development and a specific cell in the myogenic lineage. Further, it implies that there is a regulatory event activated during myogenesis that precedes terminal differentiation and that is required for expression of those genes whose products distinguish the replicating myoblast.
Little is known about the regulation of apoptosis in fibroblasts although several model systems including serum deprivation and treatment with staurosporine or topoisomerase inhibitors have been used to induce apoptosis in vitro. To validate a reproducible in vitro model for the study of apoptosis in fibroblasts, we cultured density-inhibited monolayer cultures of Balb/c 3T3 fibroblasts in Dulbecco's modified essential medium plus 15% fetal calf serum and then withdrew serum. Time-lapse video microscopy demonstrated that within minutes of serum withdrawal, cells lost substrate attachment and floated to the top of the liquid growth medium. There was a time-dependent increase in the number of non-adherent cells. Some of these cells regained attachment and spread momentarily, but they eventually rounded up and lost attachment permanently. In contrast to serum-containing cultures in which similar morphological changes were followed by mitosis, in serum-free cultures repeated attempts at mitosis were followed by permanent attachment loss and presumably cell death. To assess whether all the non-adherent cells were in fact dead, the percentages of cells that continued to proliferate upon return to serum-supplemented conditions was computed. After various periods of serum starvation a decreasing proportion (approx. 75% at 30 minutes; < 2% at 24 hours) of the non-adherent cells could be rescued by addition of serum. Transmission electron microscopy of cells 3 hours after serum withdrawal showed that the majority (approximately 60%) of non-adherent cells exhibited marked intranuclear chromatin condensation but maintained integrity of cell and nuclear membranes and cell organelles, morphological changes consistent with those of apoptotic cell death. Scanning electron microscopy of cultures 3 hours following serum withdrawal showed rounded cells with marked surface blebbing. Fluorescence and confocal microscopy revealed increased intensity of nuclear staining with DAPI while actin filaments became indistinct or collapsed around the nucleus. After cycloheximide treatment to inhibit protein synthesis, there was no reduction of apoptosis. Gel electrophoresis of DNA from both control and 3 hour-serum-deprived cells showed intact DNA with no oligonucleosomal length fragmentation. After serum withdrawal, intracellular calcium was reduced by about 32% over 5 minutes as measured by fura2 ratio fluorimetry in single cells. Serum-starved cells showed a time-dependent shrinkage in mean cell diameter compared to trypsinized, adherent control cells (at 0 hours, mean diameter = 18.0 microns--viable; at 4 hours, mean diameter = 15.5 microns--apoptotic). Flow cytometric analysis showed increased propidium iodide staining and reduced fluorescein diacetate uptake over 3 hours, changes that were contemporaneous with the reduction of cell diameter.(ABSTRACT TRUNCATED AT 400 WORDS)
Conventional methods for isolating myogenic (satellite) cells are inadequate when only small quantities of muscle, the tissue in which satellite cells reside, are available. We have developed a tissue culture system that reliably permits isolation of intact, living, single muscle fibers with associated satellite cells from predominantly fast and slow muscles of rat and mouse; maintenance of the isolated fibers in vitro; dissociation, proliferation, and differentiation of satellite cells from each fiber; and removal of the fiber from culture for analysis.
Treatment of L929 cells with tumor necrosis factor alpha (TNFalpha) activates a programmed cell death pathway resulting in apoptosis. We investigated the intracellular signaling pathways activated in L929 cells by TNFalpha. TNFalpha robustly activates Jun kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family. In addition, p42(MAPK) is activated, but a 10-fold greater concentration of TNFalpha was required for substantial MAPK activation than was needed for maximal JNK stimulation. Simultaneous treatment of L929 cells with fibroblast growth factor (FGF-2) significantly reduced the apoptotic response to TNFalpha. FGF-2 substantially activated the Raf/MEK/MAPK (where MEK is mitogen-activated protein kinase kinase) pathway but did not affect TNFalpha activation of JNK. These results indicate that although JNK may play an important role in transmitting the TNFalpha signal from the cell surface to the nucleus, activation of the JNK pathway is not sufficient to induce apoptosis. Expression of dominant-negative Asn-17 Ras in L929 cells diminished the FGF-2 stimulation of p42(MAPK) and eliminated the protective effect of FGF-2. Asn-17 Ras expression did not affect JNK activity and had no effect on TNFalpha activation of JNK. Pharmacological inhibition of MEK-1 activity by incubation of cells with the compound PD 098059 blocked p42(MAPK) activation and FGF-2 protection against apoptosis. Interestingly, activated Val-12 Ras expression substantially enhanced TNFalpha-mediated apoptosis in L929 cells, but Val-12 Ras did not constitutively activate MAPK in L929 cells and FGF-2 partially protected Val-12 Ras-expressing cells from TNFalpha-mediated apoptosis. Our data indicate that activation of the MAPK pathway mediates an FGF-2 protective effect against apoptosis and highlights the important role that integration of multiple intracellular signaling pathways plays in the regulation of cell growth and death.
c-Jun, a signal-transducing transcription factor of the AP-1 family, normally implicated in cell cycle progression, differentiation and cell transformation, recently has also been linked to apoptosis. To explore further the functional roles of c-Jun, a conditional allele was generated by fusion of c-Jun with the hormone-binding domain of the human estrogen receptor (ER). Here we demonstrate that increased c-Jun activity is sufficient to trigger apoptotic cell death in NIH 3T3 fibroblasts. c-Jun-induced apoptosis is evident at high serum levels, but is enhanced further in factor-deprived fibroblasts. Furthermore, apoptosis by c-Jun is not accompanied by an increase in DNA synthesis. Constitutive overexpression of the apoptosis inhibitor protein Bcl-2 delays the c-Jun-mediated cell death. The regions of c-Jun necessary for apoptosis induction include the amino-terminal transactivation and the carboxy-terminal leucine zipper domain, suggesting that c-Jun may activate cell death by acting as a transcriptional regulator. We further show that alpha-fodrin, a substrate of the interleukin 1beta-converting enzyme (ICE) and CED-3 family of cysteine proteases, becomes proteolytically cleaved in cells undergoing cell death by increased c-Jun activity. Moreover, cell-permeable irreversible peptide inhibitors of the ICE/CED-3 family of cysteine proteases prevented the cell death.
Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax -/- neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.
We and others have recently shown that loss of the mitochondrial membrane potential (Deltapsi) precedes apoptosis and chemical-hypoxia-induced necrosis and is prevented by Bcl-2. In this report, we examine the biochemical mechanism used by Bcl-2 to prevent Deltapsi loss, as determined with mitochondria isolated from a cell line overexpressing human Bcl-2 or from livers of Bcl-2 transgenic mice. Although Bcl-2 had no effect on the respiration rate of isolated mitochondria, it prevented both Deltapsi loss and the permeability transition (PT) induced by various reagents, including Ca2+, H2O2, and tert-butyl hydroperoxide. Even under conditions that did not allow PT, Bcl-2 maintained Deltapsi, suggesting that the functional target of Bcl-2 is regulation of Deltapsi but not PT. Bcl-2 also maintained Deltapsi in the presence of the protonophore SF6847, which induces proton influx, suggesting that Bcl-2 regulates ion transport to maintain Deltapsi. Although treatment with SF6847 in the absence of Ca2+ caused massive H+ influx in control mitochondria, the presence of Bcl-2 induced H+ efflux after transient H+ influx. In this case, Bcl-2 did not enhance K+ efflux. Furthermore, Bcl-2 enhanced H+ efflux but not K+ flux after treatment of mitochondria with Ca2+ or tert-butyl hydroperoxide. These results suggest that Bcl-2 maintains Deltapsi by enhancing H+ efflux in the presence of Deltapsi-loss-inducing stimuli.
We investigated the potential of cultured myoblasts to generate skeletal muscle in an ectopic site. Myoblasts from a clonal cell line or from expanded primary cultures were injected under the skin of the lumbar region of adult syngenic Balb/c mice. One to 7 weeks after injection, distinct muscles, of greater mass in mice injected with clonal myoblasts (6-78 mg, n=37) than in mice injected with primary myoblasts (1-7 mg, n=26), had formed between the subcutaneous panniculus carnosus muscle and the trunk muscles of host animals. These ectopic muscles exhibited spontaneous and/or electrically-evoked contractions after the second week and, when stimulated directly in vitro, isometric contractile properties similar to those of normal muscles. Histological, electron microscopical and tissue culture examination of these muscles revealed their largely mature morphology and phenotype. The fibres, most of which were branched, were contiguous, aligned and capillarised, exhibited normal sarcormeric protein banding patterns, and expressed muscle-specific proteins, including desmin, dystrophin, and isoforms of developmental and adult myosin heavy chain. Enveloping each fibre was a basal lamina, beneath which lay quiescent satellite cells, which could be stimulated to produce new muscle in culture. Presence of endplates (revealed by alpha-bungarotoxin and neurofilament staining), and the eventual loss of expression of neural cell adhesion molecule and extrasynaptic acetylcholine receptors, indicated that some fibres were innervated. That these muscle fibres were of implanted-cell origin was supported by the finding of Y-chromosome and a lack of dystrophin in ectopic muscles formed after subcutaneous injection of, respectively, male myoblasts into female mice and dystrophin-deficient (mdx) myoblasts into normal C57Bl/10 muscle. Our results demonstrate that an organised, functional muscle can be generated de novo from a disorganised mass of myoblasts implanted in an extramuscular subcutaneous site, whereby the host contributes significantly in providing support tissues and innervation. Our observations are also consistent with the idea that myogenic cells behave like tissue-specific stem cells, generating new muscle precursor (satellite) cells as well as mature muscle. Subcutaneous implantation of myoblasts may have a range of useful applications, from the study of myogenesis to the delivery of gene products.
The antimitotic nucleoside cytosine arabinoside (araC) causes apoptosis in postmitotic neurons for which two mechanisms have been suggested: (1) araC directly inhibits a trophic factor-maintained signaling pathway required for survival, effectively mimicking trophic factor withdrawal; and (2) araC induces apoptosis by a p53-dependent mechanism distinct from trophic factor withdrawal. In rat sympathetic neurons, we found that araC treatment for 12 hr induced approximately 25% apoptosis without affecting NGF-maintained signaling; there was neither reduction in the activity of mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) or protein kinase B/Akt, a kinase implicated in NGF-mediated survival, nor was there c-Jun N-terminal kinase (JNK) activation or c-Jun N-terminal phosphorylation, events implicated in apoptosis induced by NGF withdrawal. However, araC treatment, but not NGF-withdrawal, elevated expression of p53 protein before and during apoptosis. Additionally, araC-induced apoptosis was suppressed in sympathetic neurons from p53 null mice. Although MAPK/ERK activity is not necessary for NGF-induced survival, it protected against toxicity by araC, because inhibition of the MAPK pathway by PD98059 resulted in a significant increase in the rate of apoptosis induced by araC in the presence of NGF. Consistent with this finding, ciliary neurotrophic factor, which does not cause sustained activation of MAPK/ERK, did not protect against araC toxicity. Our data show that, in contrast to NGF deprivation, araC induces apoptosis via a p53-dependent, JNK-independent mechanism, against which MAPK/ERK plays a substantial protective role. Thus, NGF can suppress apoptotic mechanisms in addition to those caused by its own deprivation.
The effect of serum starvation on the expression and phosphorylation of PKC-alpha and p53 in Chinese hamster V79 cells was investigated. Serum starvation led to growth arrest, rounding up of cells and the appearance of new PKC-alpha and p53 bands on Western blots. Prolonged incubation (> or = 48 hr) in serum-deprived medium led to cell detachment and death. Moving cells to fresh medium containing 10% serum before, but not after, cell detachment reversed the changes observed in PKC-alpha and p53, and also prevented later cell detachment. Radiolabelling studies showed that the higher-molecular-weight PKC-alpha and p53 bands result from increased phosphorylation, while a lower-molecular-weight PKC-alpha band reflects newly synthesized protein. Immunocomplex kinase assays have shown that the increased phosphorylation of PKC-alpha is associated with its increased activity. To study the relationship between PKC-alpha, p53 and cell death, cells were treated either with TPA, to down-regulate PKC or with staurosporine, to inhibit PKC activity. Staurosporine, a potent PKC inhibitor and inducer of programmed cell death, caused the appearance of new PKC-alpha and p53 bands similar to those induced by serum starvation. If serum starvation was preceded by prolonged (48 hr) TPA treatment to down-regulate PKC-alpha, cell detachment and death did not take place within the same time frame. Intracellular fractionation of cells demonstrated that increased expression of PKC-alpha and the appearance of the associated higher and lower molecular-weight bands occurred in the nucleus. These data highlight the association of PKC-alpha and p53 with cellular events leading to cell death.
Three members of the family of protease-activated receptors (PARs), PARs-1, -3 and -4, have been identified as thrombin receptors. PAR-1 is expressed by primary myoblast cultures, and expression is repressed once myoblasts fuse to form myotubes. The current study was undertaken to investigate the hypothesis that thrombin inhibits myoblast fusion. Primary rodent myoblast cultures were deprived of serum to promote myoblast fusion and then cultured in the presence or absence of thrombin. Thrombin inhibited myoblast fusion, but another notable effect was observed; 50% of control cells were apoptotic within 24 h of serum deprivation, whereas less than 15% of thrombin-treated cells showed signs of apoptosis. Proteolysis was required for the effect of thrombin, but no other serine protease tested mimicked the action of thrombin. Neither a PAR-1- nor a PAR-4-activating peptide inhibited apoptosis or fusion, and myoblast cultures were negative for PAR-3 expression. Myoblasts exposed to thrombin for 1 h and then changed to medium without thrombin accumulated apoptosis inhibitory activity in their medium over the subsequent 20 h. Thus the protective action of thrombin appears to be effected through cleavage of an unidentified thrombin receptor, leading to secretion of a downstream apoptosis inhibitory factor. These results demonstrate that thrombin functions as a survival factor for myoblasts and is likely to play an important role in muscle development and repair.
During the last few years, detection of apoptosis has evolved from a predominantly morphological basis to the use of ever more specific techniques. The methods widely used to visualize DNA fragmentation in tissue sections are now supplemented by a variety of specific antisera against components of the cell death pathways. Essential requirements for apoptosis detection techniques include high sensitivity for apoptotic cells, the ability to differentiate between apoptotic and necrotic cell death and other forms of DNA damage, and the distinction between different stages of the cell death process. In this overview, we will focus on recent technical advances in apoptosis detection covering improvements of in situ DNA fragmentation techniques, as well as pointing out some of the new tools available for the detection of apoptotic cells in tissue.
This paper reviews studies on the basic principles of biostimulation of wound healing by various low-energy lasers. It looks at the mechanism of action of biostimulation as well as the lasers effect on cell proliferation, collagen synthesis, and would healing.
Mitochondrial physiology is disrupted in either apoptosis or necrosis. Here, we report that a wide variety of apoptotic and necrotic stimuli induce progressive mitochondrial swelling and outer mitochondrial membrane rupture. Discontinuity of the outer mitochondrial membrane results in cytochrome c redistribution from the intermembrane space to the cytosol followed by subsequent inner mitochondrial membrane depolarization. The mitochondrial membrane protein Bcl-xL can inhibit these changes in cells treated with apoptotic stimuli. In addition, Bcl-xL-expressing cells adapt to growth factor withdrawal or staurosporine treatment by maintaining a decreased mitochondrial membrane potential. Bcl-xL expression also prevents mitochondrial swelling in response to agents that inhibit oxidative phosphorylation. These data suggest that Bcl-xL promotes cell survival by regulating the electrical and osmotic homeostasis of mitochondria.
Myoblast transplantation (MT) is a potential approach for gene transfer into skeletal muscle, the efficiency of which depends upon the number of copies of donor genome incorporated into the host tissue. We have developed a system for quantitative studies of MT that measures amounts of donor-derived genome in host muscles and estimates the contributions of donor cell survival and proliferation in vivo.
[14C]thymidine-labeled, male myoblasts were transplanted into female muscles, providing two donor cell markers, Y chromosome and [14C]. The markers were measured in muscle extracts by slot blotting and scintillation counting, respectively.
In each extract, the amount of Y chromosome was used to quantify donor-derived genome, whereas the radiolabel provided an estimate of cell survival. Furthermore, the different modes of inheritance of the markers meant that proliferation of surviving donor cells was detected as a change in marker ratio.
This system provides a method for assessing potential improvements of MT.
Apoptosis plays an important role during neuronal development, and defects in apoptosis may underlie various neurodegenerative
disorders. To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of mitogen-activated
protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC-12 pheochromocytoma
cells. NGF withdrawal led to sustained activation of the JNK and p38 enzymes and inhibition of ERKs. The effects of dominant-interfering
or constitutively activated forms of various components of the JNK-p38 and ERK signaling pathways demonstrated that activation
of JNK and p38 and concurrent inhibition of ERK are critical for induction of apoptosis in these cells. Therefore, the dynamic
balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether
a cell survives or undergoes apoptosis.
The effect of low-energy laser (He-Ne) irradiation on the process of skeletal muscle regeneration after cold injury to the gastrocnemius muscle of the toad (Bufo viridis) was studied using quantitative histological and morphometric methods. The injured zones in the experimental toads were subjected to five direct He-Ne laser (632.8 nm wavelength) irradiations (6.0 mW for 2.3 min) every alternate day starting on the fourth day postinjury. Muscles that were injured as above, and subjected to redlight irradiation, served as a control group. Morphometric analysis was performed on histological sections of injured areas at 9, 14, and 30 days postinjury. At 9 days postinjury, mononucleated cells populated 69.3% ± 16.8% of the total area of injury. Thereafter, their volume fraction (percent of total injured zone) decreased gradually but more rapidly in the laserirradiated muscle than in the control. The volume fraction of the myotubes in the laser-irradiated muscles at 9 days of muscle regeneration was significantly higher (7.0% ± 2.2%) than in the control muscle (1.2% ± 0.4%). Young myofibers in the laser-irradiated muscles populated 15.5% ± 7.9% and 65.0% ± 9.5% of the injured area at 9 and 14 days of muscle regeneration, respectively, while in control muscles these structures were not evident at 9 days and made up only 5.3% ± 2.9% of the traumatized area at 14 days postinjury. The volume fraction of the young myofibers further increased by 30 days of muscle regeneration making up 75.7% ± 13.2% of the traumatized area, while in the laser-irradiated muscles most of the injured zone was filled with mature muscle fibers. It is concluded that He-Ne laser irradiation during the regeneration process markedly promotes muscle maturation in the injured zone following cold injury to the toad gastrocnemius muscle. © 1993 Wiley-Liss, Inc.
Myoblast transplantation has been proposed as a potential therapy for Duchenne muscular dystrophy (DMD). A Y-chromosome-specific probe was used to track the fate of donor male myoblasts injected into dystrophic muscles of female mdx mice (which are an animal model for DMD). In situ analysis with the Y-probe showed extremely poor survival of isolated normal male (C57B1/10Sn) donor myoblasts after injection into injured or uninjured muscles of dystrophic (mdx) and normal (C57B1/10Sn) female host mice. A decrease in the numbers of donor (male) myoblasts was seen from 2 days and was marked by 7 days after injection: few or no donor myoblasts were detected in host muscles examined at 3–12 months. There was limited movement of the injected donor myoblasts and fusion into host myofibers was rare. The results of this study strongly suggest that the failure of clinical trials of myoblast transplantation therapy in boys with DMD may have been due to rapid and massive death of the donor myoblasts soon after myoblast injection. © 1996 John Wiley & Sons, Inc.
Compression injury of a central nerve results in its degeneration with irreversible loss of function due to the inability of the mammalian central nervous system (CNS) to regenerate. In contrast, the CNS of lower vertebrates has a high capacity to regenerate. Recently, low energy laser irradiation was shown to attenuate degeneration in injured CNS nerves. The optic nerves of rats were subjected to moderate crush, calibrated so that some electrophysiological activity was preserved. The nerves were then subjected to low energy laser irradiation (10.5 mW, 2 min daily) for various periods. The electrical activity of the nerves, distal to the site of injury, was determined by measuring the compound action potential at the termination of the experiment. Two weeks of irradiation begun immediately after injury and continued daily thereafter, resulted in a compound action potential which was significantly higher (mean +/- S.E.M. 1856 +/- 535 microV) than that of non-irradiated injured nerves (351 +/- 120 microV). The effect was temporary and subsided within a week. This two-week irradiation was slightly more effective than a treatment lasting one week (1406 +/- 225 microV) and was significantly more effective than 4 days of irradiation (960 +/- 133 microV). The number of treatments is therefore important. The time at which the treatment commences relative to the injury is also critical. Irradiation initiated two hours after the crush was about half as effective as immediate irradiation (810 +/- 42 microV). No apparent effect was evident when the laser was applied for the first time 5 h, or longer, after the crush.(ABSTRACT TRUNCATED AT 250 WORDS)
MitochondriaLaserTransmembrane proton gradientMembrane potentialATP synthesisOxygen uptake
Normal adult mouse tibialis anterior muscles were perfused continuously with 3H-thymidine for nine days. Quantitation of the satellite cell population in these muscles reveals that not only is the frequency of satellite cell nuclei low, but that those present are mitotically quiescent.
The effect of low-energy laser (He-Ne) irradiation on the rate of skeletal muscle regeneration after partial excision of the rat gastrocnemius muscle was studied using quantitative histological morphometric methods. The injured zones of the experimental rats were subjected to direct He-Ne laser (632.8 nm wavelength) irradiation (6.0 mW for 2.3 min) immediately following injury and once daily thereafter for 5 days. Muscles that were injured as above and subjected to red or room light irradiation served as a control group. The volume fraction (percent of total volume of injured zone) of the mononucleated cells in the injured zone decreased gradually with time after injury, but more rapidly in the laser irradiated muscles than in the control. At 3 days post-injury the myotubes in the laser-irradiated muscles populated a significantly higher percentage (13.9 +/- 1.1%) of the injured area than in the control muscles (7.8 +/- 1.0%). The volume fraction of the young myofibers in the laser irradiated muscles exceeded 30.6 +/- 2.2% and 49.6 +/- 5.6% at 8 and 11 days post-injury, respectively, while in control muscles these structures comprised only 9.6 +/- 1.0% and 27.2 +/- 3.8% of the injured zone at 8 and 11 days after injury, respectively. It is concluded that He-Ne laser irradiation during the regeneration process promotes muscle maturation in the injured zone following partial excision of the rat gastrocnemius muscle.
The vast majority of cortical thymocytes die during T cell development while those that survive this selective process accumulate in the medulla. bcl-2, an inner mitochondrial membrane protein, has been shown to inhibit apoptosis in certain cell lines. In the thymus, bcl-2 is regionally localized to the mature T cells of the medulla. To assess the role of bcl-2 in the programmed death of thymocytes, we generated transgenic mice that redirected bcl-2 expression to cortical thymocytes. bcl-2 protected immature CD4+8+ thymocytes from glucocorticoid, radiation, and anti-CD3-induced apoptosis. Moreover, bcl-2 altered T cell maturation, resulting in increased percentages of CD3hi and CD4-8+ thymocytes. Despite this, clonal deletion of T cells that recognize endogenous superantigens still occurred. This transgenic model indicates that multiple death pathways operate within the thymus that can be distinguished by their dependence on bcl-2.
Early death is the fate of most developing T lymphocytes. Because bcl-2 can promote cell survival, we tested its impact in mice expressing an E mu-bcl-2 transgene within the T lymphoid compartment. The T cells showed remarkably sustained viability and some spontaneous differentiation in vitro. They also resisted killing by lymphotoxic agents. Although total T cell numbers and the rate of thymic involution were unaltered, the response to immunization was enhanced, consistent with reduced death of activated T cells. No T cells reactive with self-superantigens appeared in the lymph nodes, but an excess was found in the thymus. These observations, together with previous findings on B cells, suggest that modulated bcl-2 expression is a determinant of life and death in normal lymphocytes.
We studied influences of low-intensity argon laser irradiation at various wavelengths on mitochondrial respiration in vitro. Isolated guinea pig liver mitochondria were suspended in an isotonic buffer solution (pH 7.4, 37 degrees C). The mitochondrial suspension was introduced into a constant temperature reaction chamber in which an irradiation fiber, a thermocouple, an oxygen electrode, and a stirrer were installed. Under respiratory conditions of state 4, state 3, and uncoupled respiration, mitochondrial oxygen consumption was measured during low-intensity argon laser irradiations at 351nm, 458 nm, and 514.5 nm. The 351 nm and the 458 nm irradiations at 200 mW inhibited uncoupled respiration by 19% and 11%, respectively, and the irradiation at 351 nm inhibited state 3 respiration as well by 10%. In contrast, the 514.5 nm irradiation enhanced both state 3 and uncoupled respiration by 6-7%. Temperature reference experiments indicated that the thermal effect alone could not account for the effects of laser irradiation on mitochondrial oxygen consumption. These results suggest that the 351 nm and the 458 nm laser irradiation may injure the mitochondrial inner membrane, while the 514.5 nm laser irradiation may slightly promote the rate of ATP synthesis.
The effect of low-energy laser (He-Ne) irradiation on the process of neoformation of blood capillaries during regeneration in the toad (Bufo viridis) gastrocnemius muscle was studied using histomorphometric methods. The injured zones of the experimental toads were subjected to four direct He-Ne laser irradiations (632.8 nm wavelength; 6.0 mW for 2.3 min) every alternate day, commencing on the second day after injury. Muscles that were injured as above and subjected to red light irradiation served as control. The volume density (cm3/cm3) of the capillaries in the injured zone at 9 days after injury was significantly (P < 0.01) higher (0.09 +/- 0.006) than in the control muscles (0.048 +/- 0.007). At 14 days after injury, the volume density in the injured zone of the control muscles further increased, while the value in the laser-irradiated muscles remained unchanged. The surface density (m2/cm3) of the capillaries in the injured zone was 2.3-fold higher in the laser-irradiated muscles than in the control muscles at 9 days after injury. The surface density further increased in the control muscles between 9 and 14 days after injury, while in the laser-irradiated muscles there was a decrease in this value during the above period. The surface-to-volume ratios of the capillaries in the injured zone of control and laser-irradiated muscles indicate a straighter, rather than a convoluted appearance between 9 and 14 days after injury. It is concluded that He-Ne laser irradiation during skeletal muscle regeneration in the toad markedly promotes the process of neoformation of blood vessels in the injured zone.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell transplantation has potential benefits for tissue replacement in the the enhancement of tissue regeneration and as cell-mediated gene therapy for systemic diseases. The transplantation of myoblasts into skeletal muscle also allows gene transfer into cells of the host since myoblasts fuse with host fibers thereby forming hybrid myofibers. The success of myoblast transplantation can be determined by a variety of measures, such as the percentage of myoblasts that fuse, the number of hybrid myofibers formed, or the level of transgene expression. Each measure is a reflection of the fate of the transplanted cells. In order to compare different measures of transplantation efficacy, we followed the fate of transplanted myoblasts expressing the marker enzyme beta-galactosidase (beta-gal) in two different assays. Two weeks after transplantation, the number of hybrid myofibers was determined histochemically, whereas transgene (beta-gal) expression was measured biochemically. To control for variabilities of transplantation among different animals, we obtained both measurements from each muscle by using alternate cryosections in the two assays. Within each individual muscle, both hybrid fiber number and beta-gal expression were maximal at the site of implantation and diminished in parallel with distance from the site. However, for determining the success of transplantation among groups of muscles, these two measures of efficacy yielded discordant results: the transplants with the highest number of hybrid fibers were not the transplants with the greatest beta-gal activity. Such discrepancies are likely due to regional variations at the transplantation site that arise when cells are introduced into a solid tissue. These results demonstrate the importance of multiple measures of cell fate and transplantation efficacy for studies of cell transplantation and for the application of such studies to cell therapy and cell-mediated gene therapy.
The transmission of extracellular signals into their intracellular targets is mediated by a network of interacting proteins that regulate a large number of cellular processes. Cumulative efforts from many laboratories over the past decade have allowed the elucidation of one such signaling mechanism, which involves activations of several membranal signaling molecules followed by a sequential stimulation of several cytoplasmic protein kinases collectively known as mitogen-activated protein kinase (MAPK) signaling cascade. Up to six tiers in this cascade contribute to the amplification and specificity of the transmitted signals that eventually activate several regulatory molecules in the cytoplasm and in the nucleus to initiate cellular processes such as proliferation, differentiation, and development. Moreover, because many oncogenes have been shown to encode proteins that transmit mitogenic signals upstream of this cascade, the MAPK pathway provides a simple unifying explanation for the mechanism of action of most, if not all, nonnuclear oncogenes. The pattern of MAPK cascade is not restricted to growth factor signaling and it is now known that signaling pathways initiated by phorbol esters, ionophors, heat shock, and ligands for seven transmembrane receptors use distinct MAPK cascades with little or no cross-reactivity between them. In this review we emphasize primarily the first MAPK cascade to be discovered that uses the MEK and ERK isoforms and describe their involvement in different cellular processes.
We have studied the effect of adding extra satellite cells or soluble factors from crushed muscle on regeneration of minced fragments from rat tibialis muscle. The muscle mince was wrapped in an artificial epimysium to prevent adhesions and cell immigration from adjacent muscles. Regeneration was quantitatively assessed by electrophoretic determination of the muscle-specific form of creatine kinase. Control minces exhibited three periods of change in creatine kinase activity during a 7-week regeneration period. Activity fell rapidly during the first week, then rose gradually from 1-3 weeks and increased more rapidly from 3-7 weeks. To augment the original complement of myogenic cells, satellite cells were isolated from the contralateral muscle, purified by density gradient centrifugation, and expanded in culture for 3 days before adding to the muscle mince. The added cells resulted in a 3-fold enhancement of creatine kinase activity throughout the regeneration period. Soluble muscle extract incorporated into a collagen matrix also stimulated regeneration when added to muscle mince. The extract accelerated the rate of creatine kinase increase during the 1-3 week period beyond that observed in the control or cell augmented mince, suggesting that factors in the extract may facilitate revascularization or reinnervation. The specific activity of creatine kinase was increased in regenerates augmented with both cells and extract, indicating that the effects enhance primarily myogenic processes.
The bax gene promoter region contains four motifs with homology to consensus p53-binding sites. In cotransfection assays using p53-deficient tumor cell lines, wild-type but not mutant p53 expression plasmids transactivated a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase. In addition, wild-type p53 transactivated reporter gene constructs containing a heterologous minimal promoter and a 39-bp region from the bax gene promoter in which the p53-binding site consensus sequences reside. Introduction of mutations into the consensus p53-binding site sequences abolished p53 responsiveness of reporter gene plasmids. Wild-type but not mutant p53 protein bound to oligonucleotides corresponding to this region of the bax promoter, based on gel retardation assays. Taken together, the results suggest that bax is a p53 primary-response gene, presumably involved in a p53-regulated