Primary myogenic cells see the light: improved survival of transplanted myogenic cells following low energy laser irradiation

BACKGROUND AND OBJECTIVES: There is a substantial need for finding new avenues to promote muscle recovery when acute skeletal muscle loss extends beyond the natural capacity of the muscle to recover. Maintenance and regeneration of skeletal muscles depend mainly on resident stem cells known as satellite cells. Nevertheless, there are situations in which a significant loss of muscle tissue exhausts the satellite cell pool. For such cases, cell therapy and tissue engineering are becoming promising alternatives. Thus far, attempts to supplement damaged host muscles with donor satellite cells by means of myoblast transplantation therapy were mostly unsuccessful due to massive and rapid loss of donor cells within few hours after transplantation. This study aims at following the effects of low-energy-laser irradiation on the fate of implanted myoblasts. STUDY DESIGN: Primary myogenic cells, harvested from male rat skeletal muscles, were irradiated with low energy laser, seeded on a biodegradable scaffold and expanded in vitro. The scaffold containing cells was transplanted into partially excised muscles of host female rats. Donor cells were identified in the host muscle tissue, using Y-chromosome in situ hybridization. RESULTS: In this study, we show that laser irradiated donor primary myogenic cells not only survive, but also fuse with host myoblasts to form a host-donor syncytium. CONCLUSIONS: Our data show that the use of low energy laser irradiation (LELI), a non-surgical tool, is a promising means to enhance both the survival and functionality of transplanted primary myogenic cells.     

Kinetics of plasma heat shock protein HSP-70 release in coronary artery surgery: on-pump versus off-pump

AIM: Heat shock protein HSP-70 is known as protective chaperone molecule synthetized in response following ischemia and stress agents. It is detected in the myocardium and endothelium as well as in the circulation. Damaged as well as viable but exposed to stress cells contribute to the release of HSP-70 into the circulation. The aim of the study was to investigate if cardiopulmonary bypss (CPB) leads to more circulating HSP-70, on the basis of comparison dynamics of plasma concentration HSP-70 in 8 men undergoing procedures with the use of CPB (coronary artery bypass grafting, CABG group) and 8 men undergoing off-pump surgery (OPCAB group). METHODS: Blood samples were taken preoperatively, twice intraoperatively, immediately after surgical procedure (1 h) and 24-hours thereafter. The concentration of plasma HSP-70 was measured by means of immunoassay. The derived results were compared statistically with the frequency of incidence postoperative atrial fibrillation (AF). RESULTS: In CABG group was observed continuous gradual increase of plasma HSP-70 concentration during the operation with the peak 1 h after surgery (P<0.01), in striking contrast to OPCAB group, in which was detected small, but non statistically significant increase of HSP-70 1 h after operation. Significantly more of circulating HSP-70 it was detected in CABG group during the operation and 1 h after surgery (CABG vs OPCAB, respectively P<0.015 and P<0.028). In both groups among patients witch AF it was found higher postoperative values of circulating HSP-70 compared with the non-AF group (P=0.0415). CONCLUSIONS: The use of CPB leads to significant more release of HSP-70 into the circulation. According to our findings high plasma concentration of HSP-70 may be the measure of operative cellular stress, ischemia or injury and may be related with greater onset of postoperative AF. High circulating HSP-70 levels is connected with higher incidence of postoperative AF after open heart surgery.     

Attenuation of the process of muscle regeneration in the toad gastrocnemius muscle by low energy laser irradiation

The effect of number, frequency, and timing of HeNe (6.0 mW; 31.2J/cm²) and Ga-As-diode (average power at 2.82 Hz-0.005 mW) laser irradiations on the process of muscle regeneration at 14 days following cold injury to the toad gastrocnemius muscle was investigated using histomorphometric methods. The volume fraction (percent of total injured area) of mononucleated cells, myotubes and degenerated fibers was 10 ± 1%, 0%, and 4 ± 1%, respectively, in the HeNe laser irradiated muscles (5 irradiations every alternate day, beginning on the 4th day after injury), whereas in the control nonirradiated muscles, these values were significantly higher comprising 57 ± 2% (P < 0.01), 11 ± 1% and 10 ± 2% (P < 0.05), respectively. The volume fraction of young myofibers in injured areas that were subjected to the same laser irradiation regime was 8.6-fold significantly higher (P < 0.01) than their volume fraction in control muscles. The histomorphometric results were the same for injured zones of muscles that were laser irradiated only once, on the 9th day postinjury, and for those that received five consecutive irradiations every alternate day. Muscle regeneration was equally promoted by single Ga-As-diode laser or HeNe irradiation. Multiple irradiations of Ga-As-diode laser caused some pathological changes in the newly formed muscular structures. It is concluded that the process of skeletal muscle regeneration is markedly promoted by low energy laser irradiations, but that the effect depends on the number, timing, and frequency of irradiations and the type of laser used. © 1994 Wiley-Liss, inc.     

Irradiation at 780 nm increases proliferation rate of osteoblasts independently of dexamethasone presence

BACKGROUND AND OBJECTIVES: We have previously shown that phototherapy increases cell growth and impairs protein secretion of fibroblasts. Our objective was to study the effect of phototherapy on osteoblast-like cells in culture treated with dexamethasone. STUDY DESIGN/MATERIALS AND METHODS: Rat calvaria osteoblast-like cells were previously treated or not with dexamethasone and then, they were irradiated or not with a GaAlAs diode laser (wavelength of 780 nm, 10 mW, 3 J/cm2). Adhesion, proliferation, and osteonectin synthesis were analyzed. RESULTS: Phototherapy increased the proliferation rate of cells independently of dexamethasone presence. Adhesion and osteonectin synthesis were not significantly influenced by laser and/or dexamethasone. CONCLUSIONS: Based on the conditions of this study we concluded that phototherapy acts as a proliferative stimulus on osteoblast-like cells, even under the influence of dexamethasone. Thus, we suggest that phototherapy can be of importance as co-adjuvant in bone clinical manipulation in order to accelerate bone regeneration.     

HSP-25 and HSP-90 stabilize Na,K-ATPase in cytoskeletal fractions of ischemic rat renal cortex

BACKGROUND: We recently designed an in vitro system based on differential Triton-extractability of Na,K-ATPase from the cytoskeletal protein fraction isolated from rat renal cortex after renal ischemia. In the present study, we hypothesized that heat shock protein (HSP)-70, HSP-25 and HSP-90 work synergistically to stabilize the cytoskeletal anchorage of Na,K-ATPase. METHODS: Cellular proteins were fractionated by differential centrifugation into cytoskeletal pellets (I-PEL) obtained early (exhibiting abnormally high Triton extractability of Na,K-ATPase) and non-cytoskeletal supernatants (R-SUP) obtained late (exhibiting high abundance of HSP) after renal ischemia. For assessment of the role of HSP-70, HSP-25 and HSP-90 upon in vitro re-compartmentalization, I-PEL was either incubated in R-SUP with/without HSP antibodies, or in buffer with/without HSPs at different titers and combinations. Effects were evaluated by changes of Triton extractability of Na,K-ATPase after co-incubation. RESULTS: R-SUP was shown to contain significant amounts of HSP-70, HSP-25 and HSP-90. Incubation of I-PEL in R-SUP reduced Triton extractability of Na,K-ATPase. Addition of antibodies against each HSP significantly abolished these effects of R-SUP. Incubation of I-PEL with purified HSP-70, HSP-25 or HSP-90 each partly reproduced the effects of R-SUP, whereas the combination of all three HSP demonstrated a strong and more than additive effect on the cytoskeletal stabilization of Na,K-ATPase. CONCLUSIONS: The molecular mechanisms responsible for postischemic re-compartmentalization of Na,K-ATPase in rat renal cortex likely involves interactions between HSP-70, HSP-25 and HSP-90, stress proteins known to be induced in the ischemic kidney.     

Heat shock protein-70 repairs proximal tubule structure after renal ischemia

BACKGROUND: Recent studies have suggested a role of heat shock protein (HSP)-70 in cytoskeletal repair during cellular recovery from renal ischemia. The aim of this study was to test the hypothesis that HSP-70 interacts in vitro with cytoskeletal elements obtained from rat renal cortex during early reflow after renal ischemia. METHODS: Cellular proteins were fractionated into cytoskeletal pellets and noncytoskeletal supernatants by Triton X-100 extraction of rat renal cortex obtained after 15 minutes or 18 hours of reflow after 45 minutes of renal ischemia, or from controls. Aliquots of isolated pellets were coincubated with aliquots of isolated supernatants in different combinations. A repeat Triton extraction was performed, and differential distribution of Na, K-ATPase or HSP-70 was assessed by Western blots and densitometric analysis. RESULTS: Coincubation of cytoskeletal pellets obtained during early reflow after renal ischemia (exhibiting severe injury of the cytoskeletal anchorage of Na,K-ATPase) and noncytoskeletal supernatant obtained during later reflow (showing high HSP expression) resulted in specific translocation of HSP-70 from the supernatant into the pellet, functionally associated with dose-dependent stabilization of Na,K-ATPase within this cytoskeletal fraction. These effects could be reproduced by incubation with purified HSP-70 and were abolished by the addition of anti-HSP-70 antibodies. CONCLUSION: These data support the hypothesis that HSP-70 interacts with cytoskeletal elements during the restoration of proximal tubule cell structure and polarity after renal ischemia. This experimental approach represents a new in vitro assay to study further the role of HSP in cellular repair.     

Expression of desmin in normal and laser (HeNe) irradiated regenerating skeletal muscles in the toad (Bufo viridis) and the white rat

The expression of desmin was investigated using immunohistochemical methods in normal and low-energy laser (HeNe) irradiated regenerating rat and toad gastrocnemius muscles following partial excision in the former and cold injury in the latter. During the initial stages of regeneration, presumptive myoblasts immunoreacted to desmin antibodies while other mononucleated cells remained unstained. At a later stage the cytoplasm of the myotubes was intensely stained with anti-desmin. At all time intervals there were more mature myogenic structures in the injured zones of the laser irradiated rat or toad muscles than non-irradiated muscles as indicated by the positive immunoreactivity to desmin. It is concluded that desmin immunostaining provides additional information on the role of histological structures during the regeneration process. The process of skeletal muscle regeneration follwing injury is markedly promoted by low-energy direct laser irradiation during the regeneration process. The results also indicate that the rate of differentiation of myoblasts from undifferentiated stem cells is enhanced by the laser irradiation.     

Usefulness and clinical application of phototherapy: preface and comments

The following articles describe topics of phototherapy including low reactive laser therapy by diode laser device, semiconductor as a medium consisting of aluminum, gallium and arsenic, near infrared light irradiator using halogen lamp, and xenon light by high-intensity electrical stimulation of xenon gas. In addition, the applications of phototherapy in the clinical medicine such as rehabilitation, orthopedics and pain clinic are described. Phototherapy is a useful and safe method for pain relief.     

缺血后处理对大鼠骨骼肌缺血再灌注损伤的影响

目的 探讨缺血后处理对大鼠骨骼肌缺血再灌注损伤的影响以及应用缺血后处理的时机.方法 将32只大鼠随机分成四组,采用切断患肢全部皮肤、肌肉和神经,保留患肢股动静脉的动物模型,通过夹闭和开放股动静脉造成骨骼肌缺血和再灌注损伤.采用测定骨骼肌缺血4 h.再灌注1 h后血清丙二醛(MDA)、骨骼肌髓过氧化物酶(MPO),再灌注6 h后骨骼肌的死亡程度来观察缺血后处理对大鼠骨骼肌缺血再灌注损伤的影响,以及再灌注5 min后应用缺血后处理是否对骨骼肌缺血再灌注损伤有保护作用.结果 对骨骼肌缺血4 h再灌注6 h的损伤,再灌注开始后即刻应用30 s缺血、30 s再通,三次循环的缺血后处理对骨骼肌的缺血再灌注损伤即有保护作用,不仅减少了骨骼肌再灌注区域中性粒细胞浸润(MPO)和血清氧自由基水平(MDA)水平,而且减少了骨骼肌的死亡程度;再灌注5 min后应用缺血后处理并没有降低骨骼肌缺血再灌注区域的MPO和血清MDA水平,也没有降低骨骼肌缺血再灌注后的死亡程度,与直接缺血再灌注组相同,对骨骼肌缺血再灌注损伤并没有保护作用.结论 骨骼肌缺血后再灌注开始前立刻应用缺血后处理对大鼠骨骼肌缺血再灌注损伤有一定的保护效果,可以减少骨骼肌缺血再灌注损伤后的死亡程度;缺血后处理应用时机非常重要,再灌注5 min后应用缺血后处理则失去对骨骼肌缺血再灌注损伤的保护作用.     

Cultured epithelial cells response to phototherapy with low intensity laser

BACKGROUND AND OBJECTIVES: Little is known about the intracellular response of epithelial cells to phototherapy. The aim of this in vitro study was to analyze the effect of phototherapy with low-energy lasers with different wavelengths and powers on cultured epithelial cell growth under different nutritional conditions. STUDY DESIGN/MATERIALS AND METHODS: Epithelial cell cultures (Vero cell line) grown in nutritional deficit in culture medium supplemented with 2% fetal bovine serum (FBS) were irradiated with low-energy laser from one to three times with a GaAlAs laser (660 nm) and InGaAlP (780 nm), 40 and 70 mW, respectively, with 3 or 5 J/cm2. Cell growth was indirectly assessed by measuring the cell mitochondrial activity. RESULTS: Nonirradiated cell cultures grown in nutritional regular medium supplemented with 10% FBS produced higher cell growth than all cultures grown in nutritional deficit irradiated or not. The overall cell growth of cultures grown under nutritionally deficit conditions was significantly improved especially when irradiated with 780 nm for three times. CONCLUSIONS: Phototherapy with the laser parameters tested increases epithelial cell growth rate for cells stressed by growth under nutritionally deficient states. This cell growth improvement is directly proportional to the number of irradiations; however, was not enough to reach the full cell growth potential rate of Vero epithelial cell line observed when growing under nutritional regular condition.     

蝮蛇抗栓酶对骨骼肌缺血再灌注损伤保护的实验研究

目的探讨骨骼肌缺血再灌注损伤前后微循环变化及蝮蛇抗栓酶(Svate)的保护作用.方法 24只新西兰大白兔随机分成实验组和对照组,以气囊止血带造成兔左下肢缺血再灌注损伤模型.实验组分别在缺血前及再灌注前给予Svate静注,对照组给予等量生理盐水.用活体微循环显微镜观察兔缺血前及再灌注后骨骼肌微循环的变化并作血液流变学检测.结果骨骼肌缺血再灌注后对照组:全血粘度增高、血流缓慢、红细胞严重聚集、纤维蛋白原增加,大量白细胞附壁和白色微栓形成,无复流现象严重,微血管有渗出、渗血,骨骼肌组织损伤较重.随再灌注时间的推移,微循环障碍逐渐加重;实验组经Svate保护,血液流变学状况、微循环障碍明显好转,骨骼肌组织损伤较轻.结论①骨骼肌缺血再灌注后造成严重的微循环障碍和肌组织损伤;②Svate通过疏通微循环对骨骼肌缺血再灌注损伤起保护作用.     

肌细胞生成素在分化培养人体肌卫星细胞表达的研究

目的 研究肌细胞生成素(myogenin)在人体肌卫星细胞分化培养中的表达规律，探索肌细胞生成素在肌卫星细胞分化中的作用。方法 取6例正常成人的骨骼肌，消化、分离肌卫星细胞，肌卫星细胞生长培养后进行分化培养。在分化培养过程中，观察肌卫星细胞形态，计算其融合率，应用半定量RT—PCR技术检测细胞总RNA中肌细胞生成素mRNA的表达量。结果 在肌卫星细胞分化培养过程中，肌细胞生成素mRNA的表达增加，其规律与肌卫星细胞分化、融合相一致。结论 肌细胞生成素参与人体肌卫星细胞分化过程。     

Ozone oxidative preconditioning is mediated by A1 adenosine receptors in a rat model of liver ischemia/ reperfusion.

The liver is damaged by sustained ischemia in liver transplantation, and the reperfusion after ischemia results in further functional impairment. Ozone oxidative preconditioning (OzoneOP) protected the liver against ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the role of A(1) adenosine receptor on the protective actions conferred by OzoneOP in hepatic I/R. By using a specific agonist and antagonist of the A(1) subtype receptor (2-chloro N6 cyclopentyladenosine, CCPA and 8-cyclopentyl-1,3-dipropylxanthine, DPCPX respectively), we studied the role of A(1) receptor in the protective effects of OzoneOP on the liver damage, nitiric oxide (NO) generation, adenosine deaminase activity and preservation of the cellular redox balance. Immunohistochemical analysis of nuclear factor-kappa B (NF-kappaB), tumor necrosis factor alpha (TNF-alpha) and heat shock protein-70 (HSP-70) was performed. OzoneOP prevented and/or ameliorated ischemic damage. CCPA showed a similar effect to OzoneOP + I/R group. A(1)AR antagonist DPCPX blocked the protective effect of OzoneOP. OzoneOP largely reduced the intensity of the p65 expression, diminished TNF-alpha production, and promoted a reduction in HSP-70 immunoreactivity. In summary, OzoneOP exerted protective effects against liver I/R injury through activation of A(1) adenosine receptors (A(1)AR). Adenosine and (.)NO produced by OzoneOP may play a role in the pathways of cellular signalling which promote preservation of the cellular redox balance, mitochondrial function, glutathione pools as well as the regulation of NF-kappaB and HSP-70.     

Selective and non-selective cox inhibitors up-regulate heat shock protein-70 expression and protect human monocytes and macrophages from the cytotoxic effect of hydrogen peroxide

Heat shock protein 70 (HSP-70) is a molecular chaperone that protects cells against stress stimuli. The expression of HSP-70 is normally up regulated in response to stress. However, in patients with severe sepsis, HSP-70 expression is suppressed. We hypothesized that inhibitors of cyclooxygenase (COX) could up regulate the expression of HSP-70 in human macrophages and monocytes, which in turn will improve the resistance of these cells to stress. Human monocytes and macrophages were untreated or treated with aspirin, ibuprofen or NS-398, a selective COX-2 inhibitor, for 1.5 to 24 hrs. A parallel group was exposed to a 30-min heat shock at 42°C. Western blotting and real-time polymerase chain reaction quantified, respectively, time dependent changes in HSP-70 protein expression and mRNA production. To evaluate whether elevated HSP-70 expression protects the cells from stress, cell viability was determined after exposure of cells to cytotoxic concentrations of hydrogen peroxide. Changes in cell viability were monitored using propidium iodide and fluorescent microscopy. Treatment of the monocytes or macrophage cultures with either aspirin, ibuprofen or NS-398 caused a statistically significant increase (p < 0.05; n = 4) of about 2.5 fold in HSP-70 mRNA expression that peaked at 1.5 hrs. Heat shock caused a 25-fold increase in HSP-70 mRNA expression at 1.5 hrs. The COX inhibitors also triggered a statistically significant increase (p < 0.05; n = 8) of about 2-fold in HSP-70 expression within 1.5 hrs of treatment, while the heat shock triggered a 3-5 fold increase in both cell populations. The protein levels remained elevated for 24 hrs in all treated groups. Significantly, pretreatment of the cells with either the COX inhibitors or the heat shock protected the cells from the cytotoxic effect of hydrogen peroxide. These findings suggest that both selective and non-selective COX inhibitors may be as effective as a febrile response in augmenting the body’s resistance to stress.     

Effects of phototherapy on newborn rat testicles

Phototherapy is the most widespread treatment for lowering bilirubin concentration in neonates. In the routine, phototherapy has some side effects including skin eruption, fluid loss, abdominal distention, mild hemolysis and mild thrombocytopenia. The aim of the study was to investigate the possible mutagenic and gametocidal side effects of 72 h continuous phototherapy on the rat testicle. We observed decreases in spermatogonia numbers per tubule (S/T values), tubular fertilization index (TFI) and sperm sertoli cell index (SSCI), which are the most reliable methods in estimating future fertility potential, due to sensitivity to phototherapy. The differences between study and control groups for S/T, TFI and SSCI values were statistically significant (p = 0.008, p = 0.02 and p = 0.004, respectively). There were significant differences in seminiferous tubule diameters between the control and study groups (p < 0.005), but no significant difference in DNA index values between the control (0.66 +/- 0.12) and study (0.59 +/- 0.05) groups (p > 0.05). As a conclusion, phototherapy seems to have some side effects on the newborn rat testicle. Further studies with larger groups, designed for investigation of the effects of phototherapy on seminiferous tubules, may give more beneficial results.     

Acute allograft rejection occurs independently of inducible heat shock protein-70

Dendritic cells (DCs) are key mediators of the innate response to transplantation. Yet, the substances that activate these cells during acute allograft rejection remain elusive. Previous work has suggested that heat shock protein (HSP)-70 is associated with acute allograft rejection. Hence, the goal of this study was to determine whether HSP-70 activates DCs and plays a critical role in acute allograft rejection in an experimental model that is dependent on innate MyD88 signaling. Our in vitro data indicate that HSP-70 does not activate DCs. In vivo transplant studies demonstrate that HSP-70 levels are not increased during acute allograft rejection and that an absence of the inducible form of HSP-70 neither delays acute allograft rejection, impairs DCs maturation, nor alters Th1 immune responses during acute allograft rejection. In conclusion, our results indicate that HSP-70 in our experimental models does not play an essential role in acute allograft rejection.     

Low-level laser therapy combined to functional exercise on treatment of fibromyalgia: a double-blind randomized clinical trial

This study aims to investigate the effects of low-level laser therapy (LLLT) combined to a functional exercise program on treatment of FM. A double-blind and placebo-controlled randomized clinical trial composed of 22 women divided into two groups: placebo group (functional exercise program associated with placebo phototherapy n?=?11) and laser group (same exercise program associated with active phototherapy; n?=?11). Each session lasted from 40 to 60 min and was performed three times a week for 8 weeks. Phototherapy (808 nm, 100 mW, 4 J, and 142.85 J/cm² per point) was bilaterally applied to different points of the quadriceps (8), hamstrings (6), and triceps sural muscles (3) immediately after each exercise session. Pre- and post-intervention evaluations regarding pain (sites, intensity, and threshold), functional performance (balance, functional tests), muscle performance (flexibility and isokinetic variables), depression, and quality of life were conducted. A reduction in pain and improvement in functional and muscular performance, depression, and quality of life were observed in both groups (p?<?0.05); however, with no significant differences between them (p?>?0.05). In conclusion, the benefic effects of functional exercise were not improved by combination with LLLT.     

Maintenance Phototherapy for the Treatment of Early-stage Mycosis Fungoides

Mycosis fungoides (MF) is the most common type of cutaneous T cell lymphoma. Phototherapy is a first-line treatment option of early stages MF. The present study aimed at assessing the efficacy of phototherapy in Tunisian patients with MF treated with phototherapy and evaluate the efficacy of maintenance phase.     

Protective effect of anisodamine on reperfusion injury of skeletal muscles in rabbit

Anisodamine is an alkaloid isolated from a Chinese plant, which was subsequently synthesized. Its chemical structure is similar to atropine. It inhibits cholinergic nerve function, improves microcirculation, and was reported to have a protective effect on reperfusion injury in various organs. We used anisodamine in a rabbit model with ischemia and reperfusion injury of hind limb muscles. We evaluated its effect on skeletal muscle cells, using transmission electron microscopy, and analyzed lipid peroxidation by measuring malondialdehyde and lactate dehydrogenase blood concentrations. We found that malondialdehyde and lactate dehydrogenase concentrations after 1 hour of reperfusion were lower in animals treated with anisodamine than in controls. Damage to membrane structures and myofilaments in muscle cells was less severe after anisodamine treatment. Our findings indicate that anisodamine protects skeletal muscles with ischemia and reperfusion injury.