Nivaldo Antonio Parizotto

Dose-dependency of Low-energy HeNe Laser Effect in Regeneration of Skeletal Muscle in Mice

Abstract. We evaluated the effect on mice skeletal muscle regeneration of different doses (2.6, 8.4, and 25 J/cm2) of HeNe laser (λ 632.8 nm; power, 2.6 mW; spot size, 0.007 cm2) applied directly to intact skin of injured muscle. Muscle injury was induced in both right and left Tibialis anterior (TA) muscles by ACL myotoxin (5 mg/kg). Right TA muscles were irradiated daily for 5 days while contralateral muscles received a sham treatment. Only the 2.6 J/cm2 dose resulted in changes such as increased mitochondrial density and muscle fibre in the TA muscles as compared to sham groups (3280±704 µm2 versus 2110±657 µm2, p=0.02). We concluded that the HeNe effect on mouse muscle regeneration is dose-specific: only 2.6 J/cm2 increased muscle fibre area and mitochondrial density.

The effect of therapeutic ultrasound on repair of the achilles tendon (tendo calcaneus) of the rat

The purpose of this study was to evaluate the effects of therapeutic ultrasound (US) on the healing process in the Achilles tendon (Tendo calcaneus) of Wistar rats after tenotomy. Sonication was performed at a frequency of 1 MHz, an intensity of 0.5 W/cm(2) (SATA), for 5 min, over a period of 14 consecutive days in two modalities (n = 15); in both continuous and pulsed modes. The control group was divided into tenotomized, mock-sonicated and nontenotomized tendons (n = 15). On the 15th postoperative day, the tendons were removed and analyzed by using the polarized light microscopy, with the purpose of detecting and measuring the organization of collagen fibers through birefringence. The results showed a high birefringence for the tendons treated using the pulsed mode (p < 0.001), revealing the best organization and aggregation of collagen bundles. Sonication in the continuous mode induced a decrease (p = 0.047) in the ability to quicken the healing process. These findings suggest that US therapy is beneficial in the early healing process of tendons when the pulsed mode is used.

Androgenic-Anabolic Steroids Associated with Mechanical Loading Inhibit Matrix Metallopeptidase Activity and Affect the Remodeling of the Achilles Tendon in Rats:

Background The indiscriminate use of anabolic-androgenic steroids has been shown to induce pathologic changes in the Achilles tendon in several situations. Purpose To study tendon remodeling in rats treated with anabolic-androgenic steroids combined with an exercise program. Study Design Controlled laboratory study. Methods Wistar rats were grouped as follows: sedentary (group I), injected with anabolic-androgenic steroids only (group II), trained only (group III), and trained and injected with anabolic-androgenic steroids (group IV). The trained groups performed jumps in water: 4 series of 10 jumps each, with an overload of 50% to 70% of the animals body weight and a 30-second rest interval between series, for 6 weeks. Anabolic-androgenic steroids (5 mg/kg) were injected subcutaneously. Activity of matrix metallopeptidases, a marker for tendon remodeling, was analyzed in tissue extracts by zymography on gelatin–sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Results Morphological analyses of tendons showed that in group II, the most external layer that covers the tendon was thicker with aggregation of the collagen fibers, suggesting an increase in collagen synthesis. In group IV, an inflammatory infiltrate and fibrosis in tendons as well as a pronounced increase of the serum corticosterone level were observed. This training protocol upregulated matrix metallopeptidase activity, whereas anabolic-androgenic steroid treatment strongly inhibited this activity. The appearance of lytic bands with molecular masses of approximately 62 and 58 kDa suggests the activation of matrix metallopeptidase–2. Conclusion Anabolic-androgenic steroid treatment can impair tissue remodeling in the tendons of animals undergoing physical exercise by downregulating matrix metallopeptidase activity, thus increasing the potential for tendon injury. Clinical Relevance Since the AAS abuse is so widespread, a better comprehension of the pathological effects induced by these drugs may be helpful for the development of new forms of therapy of AAS-induced lesions.

Low-level laser (light) therapy (LLLT) on muscle tissue: performance, fatigue and repair benefited by the power of light

Abstract The use of low level laser (light) therapy (LLLT) has recently expanded to cover areas of medicine that were not previously thought of as the usual applications such as wound healing and inflammatory orthopedic conditions. One of these novel application areas is LLLT for muscle fatigue and muscle injury. Since it is becoming agreed that mitochondria are the principal photoacceptors present inside cells, and it is known that muscle cells are exceptionally rich in mitochondria, this suggests that LLLT should be highly beneficial in muscle injuries. The ability of LLLT to stimulate stem cells and progenitor cells means that muscle satellite cells may respond well to LLLT and help muscle repair. Furthermore the ability of LLLT to reduce inflammation and lessen oxidative stress is also beneficial in cases of muscle fatigue and injury. This review covers the literature relating to LLLT and muscles in both preclinical animal experiments and human clinical studies. Athletes, people with injured muscles, and patients with Duchenne muscular dystrophy may all benefit. Zusammenfassung Die Anwendung der Low-Level-Laser (Licht)-Therapie (LLLT) hat sich in letzter Zeit um Bereiche der Medizin erweitert die über die üblichen Anwendungen wie Wundheilung und Behandlung von entzündlichen orthopädischen Leiden hinausgehen. Einen solchen neuen Bereich stellt der Einsatz der LLLT bei Muskelermüdung und Muskelverletzung dar. Seit es Konsens ist, dass die Mitochondrien die wichtigsten Photoakzeptoren innerhalb einer Zelle darstellen, und es bekannt ist, dass Muskelzellen besonders reich an Mitochondrien sind, hat sich die Idee entwickelt, dass die LLLT auch bei Muskelverletzungen nutzbringend eingesetzt werden könnte. Da es möglich ist, Stammzellen und Vorläuferzellen mittels LLLT zu stimulieren, liegt die Vermutung nahe, dass Muskel-Satellitenzellen auf die LLLT gut ansprechen und diese Therapie genutzt werden könnte, um Muskeln zu reparieren. Darüber hinaus ist die Fähigkeit der LLLT, Entzündungen zu reduzieren und oxidativen Stress zu verringern auch in Fällen von Muskelermüdung und -verletzungen vorteilhaft. Der vorliegende Review-Artikel gibt einen Überblick über die aktuelle Literatur zum Thema LLLT und Muskeln und umfasst sowohl vorklinische Tierversuche als auch klinische Studien am Menschen. Von den Forschungsergebnissen können Sportler, Menschen mit Muskelverletzungen und Patienten, die an einer Muskeldystrophie vom Typ Duchenne erkrankt sind, gleichermaßen profitieren.

Low-Level Laser Therapy (808 nm) Reduces Inflammatory Response and Oxidative Stress in Rat Tibialis Anterior Muscle After Cryolesion

Muscle regeneration is a complex phenomenon, involving coordinated activation of several cellular responses. During this process, oxidative stress and consequent tissue damage occur with a severity that may depend on the intensity and duration of the inflammatory response. Among the therapeutic approaches to attenuate inflammation and increase tissue repair, low‐level laser therapy (LLLT) may be a safe and effective clinical procedure. The aim of this study was to evaluate the effects of LLLT on oxidative/nitrative stress and inflammatory mediators produced during a cryolesion of the tibialis anterior (TA) muscle in rats.

ELECTRICAL STIMULATION IMPAIRS EARLY FUNCTIONAL RECOVERY AND ACCENTUATES SKELETAL MUSCLE ATROPHY AFTER SCIATIC NERVE CRUSH INJURY IN RATS

Neuromuscular recovery after peripheral nerve lesion depends on the regeneration of severed axons that re‐establish their functional connection with the denervated muscle. The aim of this study was to determine the effects of electrical stimulation (ES) on the neuromuscular recovery after nerve crush injury in rats. Electrical stimulation was carried out on the tibialis anterior (TA) muscle after sciatic nerve crush injury in a rat model. Six ES sessions were administered every other day starting from day 3 postinjury until the end of the experiment (day 14). The sciatic functional index was calculated. Muscle excitability, neural cell adhesion molecule (N‐CAM) expression, and muscle fiber cross‐sectional area (CSA) were accessed from TA muscle. Regenerated sciatic nerves were analyzed by light and confocal microscopy. Both treated (crush+ES) and untreated (crush) groups had their muscle weight and CSA decreased compared with the normal group (P < 0.05). Electrical stimulation accentuated muscle fiber atrophy more in the crush+ES than in the crush group (P < 0.05). N‐CAM expression increased in both crush and crush+ES groups compared with the normal group (P < 0.05). Regenerated nerves revealed no difference between the crush and crush+ES groups. Nevertheless, functional recovery at day 14 post‐injury was significantly lower in crush+ES group compared with the crush group. In addition, the crush+ES group had chronaxie values significantly higher on days 7 and 13 compared with the crush group, which indicates a decrease in muscle excitability in the crush+ES animals. The results of this study do not support a benefit of the tested protocol of ES during the period of motor nerve recovery following injury. Muscle Nerve, 2010

Low level laser therapy increases angiogenesis in a model of ischemic skin flap in rats mediated by VEGF, HIF-1α and MMP-2

It is known that low level laser therapy is able to improve skin flap viability by increasing angiogenesis. However, the mechanism for new blood vessel formation is not completely understood. Here, we investigated the effects of 660 nm and 780 nm lasers at fluences of 30 and 40 J/cm(2) on three important mediators activated during angiogenesis. Sixty male Wistar rats were used and randomly divided into five groups with twelve animals each. Groups were distributed as follows: skin flap surgery non-irradiated group as a control; skin flap surgery irradiated with 660 nm laser at a fluence of 30 or 40 J/cm(2) and skin flap surgery irradiated with 780 nm laser at a fluence of 30 or 40 J/cm(2). The random skin flap was performed measuring 10×4 cm, with a plastic sheet interposed between the flap and the donor site. Laser irradiation was performed on 24 points covering the flap and surrounding skin immediately after the surgery and for 7 consecutive days thereafter. Tissues were collected, and the number of vessels, angiogenesis markers (vascular endothelial growth factor, VEGF and hypoxia inducible factor, HIF-1α) and a tissue remodeling marker (matrix metalloproteinase, MMP-2) were analyzed. LLLT increased an angiogenesis, HIF-1α and VEGF expression and decrease MMP-2 activity. These phenomena were dependent on the fluences, and wavelengths used. In this study we showed that LLLT may improve the healing of skin flaps by enhancing the amount of new vessels formed in the tissue. Both 660 nm and 780 nm lasers were able to modulate VEGF secretion, MMP-2 activity and HIF-1α expression in a dose dependent manner.

Effects of 660 and 780 nm low‐level laser therapy on neuromuscular recovery after crush injury in rat sciatic nerve

Post‐traumatic nerve repair is still a challenge for rehabilitation. It is particularly important to develop clinical protocols to enhance nerve regeneration. The present study investigated the effects of 660 and 780 nm low‐level laser therapy (LLLT) using different energy densities (10, 60, and 120 J/cm2) on neuromuscular and functional recovery as well as on matrix metalloproteinase (MMP) activity after crush injury in rat sciatic nerve.

Low-Level Laser Therapy Induces Differential Expression of Osteogenic Genes During Bone Repair in Rats

OBJECTIVES The aim of this study was to measure the temporal pattern of the expression of osteogenic genes after low-level laser therapy during the process of bone healing. We used quantitative real-time polymerase chain reaction (qPCR) along with histology to assess gene expression following laser irradiation on created bone defects in tibias of rats. MATERIAL AND METHODS The animals were randomly distributed into two groups: control or laser-irradiated group. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation started 24 h post-surgery and was performed for 3, 6, and 12 sessions, with an interval of 48 h. A 830 nm laser, 50 J/cm(2), 30 mW, was used. On days 7, 13, and 25 post-injury, rats were sacrificed individually by carbon dioxide asphyxia. The tibias were removed for analysis. RESULTS The histological results revealed intense new bone formation surrounded by highly vascularized connective tissue presenting slight osteogenic activity, with primary bone deposition in the group exposed to laser in the intermediary (13 days) and late stages of repair (25 days). The quantitative real-time PCR showed that laser irradiation produced an upregulation of BMP-4 at day 13 post-surgery and an upregulation of BMP4, ALP, and Runx 2 at day 25 after surgery. CONCLUSION Our results indicate that laser therapy improves bone repair in rats as depicted by differential histopathological and osteogenic genes expression, mainly at the late stages of recovery.

Low level laser therapy (830 nm) improves bone repair in osteoporotic rats: Similar outcomes at two different dosages

BACKGROUND AND OBJECTIVE The goal of this study was to investigate the effects of low level laser therapy (LLLT) in osteoporotic rats by means of subjective histopathological analysis, deposition of collagen at the site of fracture, biomechanical properties and immunohistochemistry for COX-2, Cbfa-1 and VEGF. MATERIAL AND METHODS A total of 30 female Wistar rats (12weeks-old, ±250g) were submitted to ovariectomy (OVX). Eight weeks after the OVX, a tibial bone defect was created in all animals and they were randomly divided into 3 groups (n=10): control bone defect group (CG): bone defects without any treatment; laser 60J/cm(2) group (L60): animals irradiated with LLLT, at 60J/cm(2) and laser 120J/cm(2) group (L120): animals irradiated with LLLT, at 120J/cm(2). RESULTS In the laser treated groups, at both fluences, a higher amount of newly formed bone was evidenced as well as granulation tissue compared to control. Picrosirius analysis demonstrated that irradiated animals presented a higher deposition of collagen fibers and a better organization of these fibers when compared to other groups, mainly at 120J/cm(2). COX-2, Cbfa-1 or VEGF immunoreactivity was detected in a similar manner either 60J/cm(2) or 120J/cm(2) fluences. However, no differences were shown in the biomechanical analysis. CONCLUSION Taken together, our results support the notion that LLLT improves bone repair in the tibia of osteoporotic rats as a result of stimulation of the newly formed bone, fibrovascularization and angiogenesis.