Thirty-five male Wistar rats of 150 days of age and average weight of 0.486g SD=0.05 were used, provided from the Central bioterium, of the São Paulo State University (UNESP), Botucatu-SP campus (Brazil), and maintained in the Faculty of Science and Technology – FCT/UNESP, Presidente Prudente Campûs, SP, Brazil. The animals were kept in plastic boxes at a controlled temperature (22 SD=2°C) and 12-h light/dark cycle with free access to water and food (standard laboratory chow).

All procedures were previously approved by the ethics committee for animal use from FCT/UNESP, Presidente Prudente Campûs, protocol 01/2013.

The animals were randomly allocated into five groups of seven animals each:

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  Control Group (C): animals remained in the bioterium and were euthanized paired with the experimental groups.

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  Muscle Injury Control Group (IC): animals submitted to muscle injury, remained in the bioterium and were euthanized paired with the experimental groups.

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  Muscle Injury treated with LLLT (ILT): animals submitted to muscle injury, received laser application daily for seven days.

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  Muscle Injury treated with PRP (IP): animals submitted to muscle injury, received PRP application immediately after the injury.

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  Muscle Injury treated with LLLT and PRP (ILTP): animals submitted to muscle injury, application of both protocols mentioned above.

The animals of the IP and ILTP groups underwent cardiac puncture for preparation of PRP and were then submitted to the muscle injury protocol. The other groups were submitted to the injury protocol immediately after confirmation of anesthesia. The C group was not submitted to any procedure.

After the lesion protocol, the PRP was immediately applied with the animals still under anesthesia, as well as the first LLLT session. The details of each protocol are described below.

Blood collection was performed through cardiac puncture in the animals of the IP and ILTP groups. The animals were submitted to anesthesia by intraperitoneal administration of ketamine (70mg/kg) and xylazine (15mg/kg),7 and after confirmation of anesthesia a cardiac puncture was performed using a 0.2mL disposable syringe containing sodium citrate at 10%, 4mL of blood was obtained from each animal. Immediately after the puncture, saline solution was injected to restore blood volume.

The collected blood was centrifuged at 200g for 15min, splitting the sample into three parts: red bottom fraction, composed primarily of red blood cells; intermediate yellow-straw fraction (buffy coat), with the serum component; and the top fraction, composed of the blood plasma.

The top fractions were pipetted, including the buffy coat and the pipetted contents were centrifuged again at 500g for 10min. Next, 0.2mL of the bottom content PRP was pipetted.16

Blood and PRP samples were analyzed in the laboratory of the Veterinary Hospital of the Universidade do Oeste Paulista (UNOESTE) by means of an automatic blood cell analyzer (pocH 100iy Diff, Sysmex®). The analysis was performed on two blood samples and three PRP samples, for confirmation of platelets.17

In the IP and ILTP groups the muscle injury was performed immediately after cardiac puncture, avoiding the application of new doses of anesthesia. In the IC and ILT groups, the animals received an intraperitoneal injection of xylazine and ketamine, as described above. After confirmation of anesthesia, each animal was placed on the damage inductor equipment, in a supine position, with the hip in slight flexion, knee extension, and ankle in plantar flexion, the right leg attached to the machine with adhesive tape (duct tape). After positioning in the equipment, two electrodes were placed on the paw of the animal, on the calcaneal tendon and popliteal fossa, respectively. Electrical stimulation was applied suddenly to the positioned animal until full contraction of the lower limb in plantarflexion. Immediately afterwards, the equipment was fired, which promoted abrupt dorsiflexion of the lower limb of the animal while it was stimulated; the electrical current was stopped immediately after dorsiflexion. The dorsiflexion stimulation caused by the equipment and interruption of the current, in total, took an average of 2s to complete. This procedure was repeated until totaling 10 series, with a 30s interval between applications. In each series 2.25J was released, totaling 22.5J of energy applied to the muscle injury. This protocol was adapted from Pachioni et al.18

In animals of the IP and ILTP groups, 100μL of PRP was injected using a sterile syringe. The syringe with the needle was placed on the injured limb in the distal third of the tibia to be applied in the belly of the soleus muscle. The application of PRP was performed within 6h of the muscle injury protocol and withdrawal of blood.11

Diode laser equipment was used (Coherent, Laser Cube), previously calibrated, with a wavelength of 637nm (visible red), output power of 25mW, beam 1mm in diameter, 3.18W/cm2 power density, and 10s of application, totalizing an energy per point of 0.25J.

The laser was applied to a single point, perpendicular to the muscle lesioned region, for 10s. The LLLT started on the day of injury and was conducted for seven consecutive days with 24h intervals between applications. This protocol was adapted from Iyomasa et al.19 and Luo et al.6 The dose was 31.85J/cm2, and the total dosage after seven applications was 222.95J/cm2.

Euthanasia was performed 24h after the final laser therapy session paired with the other groups, totaling seven days between muscle injury and euthanasia. The animals were euthanized with an overdose of xylazine and ketamine, following ethical principles in animal research. The soleus muscle was removed surgically. For all analyses the muscle belly was used since it is the most damaged location as seen in the Pachioni et al.18 study.

The samples were submitted to the freezing method for non-fixed tissues and stored at −75°C in an ultra-low temperature freezer, Coldlab CL580-80V. Cuts measuring 5μm in thickness were made in a cryostat microtome at −20°C, placed on slides and stained with hematoxylin-eosin (HE) for analysis of the structure of the muscles, and Picrosirius red for collagen analysis.20,21

The images of the HE slides were performed using an optical microscope Nikon Eclipse 50i, attached to an Infinity 1 camera. Next, the images were analyzed using the NIS-Elements D3 software. The qualitative analysis was based on the morphology of the muscle fibers, based on features such as the shape (polygonal, rounded, angled), connective tissue (endomysium and the perimysium), and inflammatory infiltrate fibers in a state of necrosis and phagocytosis. The semi-quantitative analysis of morphological characteristics was performed by the frequency of occurrence in each animal, and classified per intensity: absent (0), mild (1), moderate (2) and intense (3). This method was adapted from De Souza et al.22

The Picrosirius stained slides were photographed by a polarized light microscope Leica DM 4000B coupled to a DFC500 camera, Leica, belonging to the Faculdade de Odontologia, UNESP, Araçatuba Campûs, using a 10× objective. After, using ImageJ software,21 the mean gray value representing the collagen intensity in the image was measured.

For the semi-quantitative analysis of morphological characteristics, a frequency table was set up with the groups and the intensity of the variables.

For collagen analysis, the Shapiro–Wilk test was performed to verify data normality. After confirmation of normal distribution, the analysis of variance ANOVA One-Way test was performed with Tukey post hoc. All analyses were performed using SPSS software v.22; the significance level was 5%.

The C group presented normal morphology, with polygonal fibers, peripheral nuclei and no presence of inflammatory infiltrate (Fig. 2). The endomysium and perimysium showed no alterations (Fig. 2). There were angular and rounded cells in some animals (Table 1).

Figure 2.

Images of histological slides stained with HE, magnification 200×. Arrow, polymorphic cell; arrow head, rounded fibers; cross, angled fibers; star, inflammatory infiltrate; circle, phagocytosis; square, necrosis.

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The IC group presented intense polymorphic fibers in all animals, fibers in the process of phagocytosis, as well angular and rounded fibers with a moderate to intense presence in all animals (Table 1). The epimysium and perimysium showed a great extent of structural disarrangement followed by intense inflammatory infiltrate in most animals (Fig. 2).

The ILT group presented intense polymorphic fibers in all animals; rounded and angular fibers had similar occurrences with the majority being mild or moderate (Table 1). Inflammatory infiltrate, as well as intense phagocytose fibers, appeared in 60% of animals. Moreover, this feature looked better than in the IC group, presenting less extensive inflammatory process and better distribution on the muscle (Fig. 2).

The IP group presented an intense presence of polymorphic fibers in 80% of the animals and mild presence in the other 20%. Intense inflammatory infiltrate was present in 20% of the animals with moderate in 60% and mild in 20%.

Table 1 demonstrates that in the group with both treatments (ILTP), intense polymorphic fibers were found in 75% of the animals and moderate in 25%. Rounded and angular fibers presented mild (75% and 100%) or absent frequency (25%). In addition, the ILTP group presented lesser amount of inflammatory cells and smaller injured area resulting in a lower structural disorder compared to the other groups (Fig. 2).

The Picrosirius stained slides were analyzed by mean gray values that represents the sum of collagen deposition density, divided for the area of image.

The ILT (2.99 SD=1.13) showed significantly more collagen compared to the C (1.88 SD=0.41, p=0.012) and IP (2.04 SD=0.44, p=0.018) (Fig. 3).

Figure 3.

Values of means and standard deviation of the mean gray values of collagen. *, statistical difference compared to C (p<0.05); +, statistical difference compared to IP (p<0.05).

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Samples that represent the distribution and organization of the collagen are presented in Fig. 4. Organization and even distribution of collagen is observed across the entire image in the control group (C), different from that found in the groups that suffered muscle injury (IC, ILT, IP, and ILTP), where the collagen is increased and concentrated at the injury sites (Fig. 4).

Figure 4.

Images of histological slides stained with Picrosirius red and photographed under polarized light. Magnification 100×.

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