Effect of low-level laser therapy on the expression of inflammatory mediators and on neutrophils and macrophages in acute joint inflammation

Sixty animals were randomly distributed into four groups of 15 animals each. The first group (control) did not receive any kind of intervention; the second group (injury), received induction but did not receive any treatment; the third group (LLLT 50 mW) was treated with LLLT at 50 mW; and rats of the fourth group (LLLT 100 mW) were treated with LLLT at 100 mW. All the groups were evaluated 24 hours post injury (five animals per group, at each experimental time point).

The animals were anesthetized with an intramuscular injection of a 7% ketamine solution (Cetamin; Syntec, Cotia, SP, Brazil) and 0.3% xylene solution (Xilazin; Syntec) at a proportion of 2:1 (0.2 ml/100 g). The induction of OA was then performed following previously published methods [27, 28]. Specifically, 200 μl injections were administered in the right knee of the hind leg of each animal with a 4% papain solution dissolved in 10 ml saline solution, to which 10 ml cysteine solution (0.03 M) was added. This solution was used as the activator to produce cartilage injury. The animals were then immediately submitted to the administration of LLLT.

An arsenide and aluminum gallium-type diode laser with a wavelength of 808 nm from Photon Laser III DMC (Sao Carlos, SP, Brazil) was used. The optical power was calibrated using a Newport multifunction optical meter (Model 1835C; Newport Corporation, Irvine, CA, USA). The dose and parameters are summarized in Table 1.

Laser irradiation was given transcutaneously at two points: medial and lateral. Laser irradiation was performed immediately after the papain-cysteine injection, on the right knee in groups at power output of 50 and 100 mW. The control and injury groups received no treatment and served as the negative and positive control groups, respectively, for the comparative histomorphometric analysis. Animals were immobilized by means of grip and were irradiated at an angle of 90° at the surface of the tissue.

After receiving the treatment, on the day of euthanasia a procedure for obtaining the washed articular synovial fluid was performed. The articular cavity was washed with 1 ml physiologic serum into the intracapsular knee space, the material was immediately centrifuged at 1,500 rpm/5 minutes, as described previously [29], and the supernatant was stored at -80°C for analysis of inflammatory mediators.

Articular synovium was washed by injecting 200 μl phosphate-buffered saline (PBS) in the joint cavity, because much material (washed) was recovered (usually around 20 to 30 μl). The recovered lavage was centrifuged at 1,500 rpm for 5 minutes, and the cell pellet was suspended in 200 μl PBS. Total cells were then counted in a hemocytometer (Neubauer chamber, 10 μl; diluting solution, Turk's solution 1:5). The remaining articular material was washed thoroughly and used for the differential cell count (neutrophils, eosinophils, macrophages, and lymphocytes). For this counting, the washed joint was processed in a cytocentrifuge (Eppendorf, Hamburg, Germany) at 450 rpm for 6 minutes, and the slides containing the cell pellet were stained with Diff-Quick. As shown in previous studies, 300 cells were counted on each slide [29, 30].

The amount of IL-1β and TNFα in washed articular synovium was quantified using the enzyme-linked immunosorbent assay, as per the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA.). For this purpose, 96-well plates were coated with 100 ml monoclonal antibody for each cytokine: anti-IL-1β and TNFα diluted in sodium carbonate buffer (0.1 M, pH 9.6). The plates were incubated (4°C) for 18 hours. For blocking, the plates were washed four times with PBS containing 0.05% Tween 20 and then filled with 300 μl/well of blocking solution (3% gelatin in PBS containing 0.05% Tween 20 (Sigma, St. Louis, MO, USA) at 37°C for 3 hours before being subjected to a new cycle of washes. Next, 100 ml properly diluted samples or standards of recombinant cytokines were added to the plate and incubated for 18 hours at 4°C. After washing, 100 μl respective biotinylated antibodies specific for the detection of each cytokine was added and left for 1 hour at room temperature. After washing the plates, the volume of 100 μl streptavidin-peroxidase was added and left for 1 hour at room temperature (22°C) followed by further washes. The reaction was visualized by adding 100 μl/well solution of 3,3',5,5'-tetramethylbenzidine and stopped by adding 50 μl/well sulfuric acid (2 N). The reading was performed in a Spectrum Max Plus 384 spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA, USA) at a wavelength of 450 nm with correction at 570 nm. The sample concentrations were calculated from standard curves obtained with recombinant cytokines. The limit of detection for IL-1β and TNFα was 1.95 pg/ml, while that for IL-6 was 3.13 to 300 pg/ml [19].

One microgram of total RNA was used for cDNA synthesis and real-time polymerase chain reaction (PCR) gene expression analysis. Initially, contaminating DNA was removed using DNase I (Invitrogen Life Technologies, Carlsbad, CA, USA) at a concentration of 1 unit/μg RNA in the presence of 20 mM Tris-HCl, pH 8.4, containing 2 mM MgCl₂ for 15 minutes at 37°C, followed by incubation at 95°C for 5 minutes for enzyme inactivation. Reverse transcription was then carried out in a 200 μl reaction mixture in the presence of 50 mM Tris-HCl, pH 8.3, 3 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM dNTPs, and 50 ng random primers with 200 units of Moloney murine leukemia virus-reverse transcriptase (Invitrogen). The reaction conditions were as follows: 20°C for 10 minutes, 42°C for 45 minutes, and 95°C for 5 minutes.

The reaction product was amplified by real-time PCR on the 7000 Sequence Detection kit using the SYBRGreen core reaction system (ABI Prism, Applied Biosystems, Foster City, California, USA). The thermal cycling conditions were as follows: 50°C for 2 minutes, then 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Experiments were performed in triplicate for each data point.

IL-10 and IL-1β mRNA abundance was quantified as a relative value compared with an internal reference, β-actin, whose abundance did not change between the varying experimental conditions. Primers used for real^-time PCR are as follows: IL-6 [GenBank:E02522], forward primer 5'-TCCTACCCCAACTTCCAATGCTC-3' and reverse primer 5'-TTGGATGGTCTTGGTCCTTAGCC-3'; and IL-1 [GenBank:M98820], forward primer 5'-CACCTCTCAAGCAGAGCACAG-3' and reverse primer 5'-GGGTTCCATGGTGAAGTCAAC-3' [20].

At the end of each treatment, animals of each group were identified, weighed, and subsequently euthanized by inhalation of carbon dioxide. This method confers rapid loss of consciousness in response to hypoxia attributed to depression of vital centers and is performed in a carbon dioxide chamber. The tibio-femoral articulation of the right hind leg of each animal was separated for analysis of the cartilaginous tissue of the knee. The material was immediately fixed using a 10% buffered formaldehyde solution and submitted to histological procedures.

The material was decalcified with ethylenediamine tetraacetic acid and submitted to the classic histological method for embedment in paraffin, dehydration in increasing concentrations of alcohol, clearing with xylol in order to allow the penetration of paraffin, impregnation in paraffin baths and insertion in molds, cross-sectional cuts to a thickness of 5 μm, and finally mounting on a synthetic balsam. Sections were then stained with hematoxylin and eosin to perform histopathological analysis.

The data were tabulated using Microsoft Excel 2007 software and initially assessed for normality using the Shapiro-Wilk test. As normal distribution was observed, analysis of variance with Tukey's post-hoc test was used for comparisons between 7, 14, and 21 days within each group as well as between the control, injury, 50 mW LLLT, and 100 mW LLLT groups. All data are expressed as mean and standard deviation values. The GraphPad Prism 5 software program (GraphPad Software, San Diego, CA, USA) was used, with significant difference from the null hypothesis considered when P < 0.05.

The data presented show the total count of viable cells in washed joint fluid that were enumerated in the Neubauer chamber. The control group had fewer cells (6.8 ± 0.5) than the injury group (72.8 ± 5.3) whereas the 50 mW LLLT group (32.3 ± 0.8) and the 100 mW LLLT group (45.8 ± 2.3) had intermediate values. In the differential analysis for neutrophils, the groups treated with 50 mW LLLT (18.88 ± 5.3) and 100 mW LLLT (19.88 ± 3.2) showed a reduction in the absolute number of neutrophils compared with the injury group (48.7 ± 7.3); however, only the 50 mW group showed no statistical difference (P > 0.05) when compared with the control group (4.1 ± 0.9). This clearly demonstrates that LLLT reduced the number of neutrophils to approximately the level observed in the control group (Figure 1A).

In Figure 1B the effect of LLLT on the differential count of macrophages can be seen. By statistical analysis (analysis of variance) we found statistical difference for the injury group and 100 mW LLLT group compared to control group (P < 0.05)., The 50 mW LLLT group has significant difference compared to injury group and 100 mW LLLT group (P < 0.05). No difference was observed between 50 mW LLLT group and control group (P > 0.05).

In Figure 2A, we can observe that there is a significant difference between the control group and the group challenged with joint injury (P < 0.05) in expression of IL-1β. Treatment with 50 mW LLLT reduced the expression of IL-1β to values similar to the control group, and significantly lower than the injury group (P < 0.05). In the 100 mW LLLT group, despite not having shown a statistically significant difference (P > 0.05) compared with the control group, there was a reduction in IL-1β expression in this treatment group.

Figure 2B represents the effect of LLLT on IL-6 mRNA expression. In these experiments, IL-6 expression was significantly increased after papain injection when compared with animals from the control groups. Only treatment with 50 mW LLLT was markedly efficient in reducing papain-induced IL-6 mRNA expression.

Figure 3 shows the results of analysis of protein expression of TNFα in the articular synovial lavage. We can observe that the injury inflammation induced by papain led to a statistically significant increased expression of TNFα compared with the control group and the 50 mW LLLT group (P < 0.05). However, the 100 mW LLLT group showed a significantly lower expression of TNFα compared with the injury group (P < 0.001), similar to values in the control group.

Upon histological examination, the control group had general features that were consistent with synovial joint normality. The joint spaces did not have inflammatory exudate, and the synovial membranes had thickened intima and subintima showing typical characteristics. The surfaces of coated articular hyaline cartilage were homogeneous, and there was active endochondral ossification of the epiphysis. The control group also had epiphyseal bone marrow with a normal pattern that was filled with red bone marrow. The articular meniscus consisted of thick collagen fibers and chondrocytes, and ossification was present (Figure 4A, B).

In the injury group (induction of the inflammatory process without any treatment) the synovial joint showed acute inflammatory process. The joint spaces had fibrinous and hyaline material that adhered to the surface of the synovial membrane, and some areas had an acute inflammatory infiltrate. The synovial membrane had an intima with normal thickness, but the subintimal layer showed signs of acute inflammatory infiltrate and dilated blood vessels. Similar to that in the control group, the surfaces coated with articular hyaline cartilage were homogeneous and the epiphyseal bone marrow had the normal fill of red bone marrow. Moreover, the epiphysis of the bone marrow presents antagonistic in the process of cellular degeneration (Figure 4C, D).

The group treated with 50 mW LLLT showed synovial joints with few morphological changes, such as high amounts of fibroblast cells and presence of discrete inflammatory cells (a few neutrophils) in the underlying connective tissue of the anterior cruciate ligament (that is, the joint capsule; Figure 4E, F).

The group treated with 100 mW LLLT showed signs of an acute inflammatory process in the synovial joint. The articular spaces were filled with acute inflammatory infiltrate (exudate) and the presence of red blood cells and hyaline material. Apart from this, the synovial membrane had an intima of normal thickness. The layer subintima showed signs of acute inflammatory infiltration and dilated blood vessels. Surfaces coated with articular hyaline cartilage were homogeneous, and the epiphyseal bone marrow had a normal pattern and was filled with red bone marrow. The articular meniscus consisted of thick collagen fibers and chondrocytes, as shown in Figure 4G, H.