2.1.

Experimental Groups

This study was approved and conducted in accordance with the Animal Care Committee guidelines of the Federal University of São Carlos (CEP 040/2010). Animals were maintained at 19°C to 23°C on a 12:12 h light-dark cycle in the Animal Experimentation Laboratory of the Federal University of São Carlos. Rats were housed in plastic cages and had free access to water and standard food.

Eighty male Wistar rats (weighing $300\pm 20\mathrm{g}$, 12 to 13 weeks) were randomly distributed into four groups ($n=20$): intact control group (IG), injured control group (CG), injured laser treated group at $10\mathrm{J}/{\mathrm{cm}}^2$ (L10), and injured laser treated group at $50\mathrm{J}/{\mathrm{cm}}^2$ (L50). The animals were distributed into two subgroups, with different periods of sacrifice (5- and 8-weeks postsurgery).

2.2.

Anterior Cruciate Ligament Transection (ACLT)

The animals were submitted to general anesthesia induced by intraperitoneal injection of xilazin (Syntec^®, R. Soluções Lar, Cotia, São Paulo, Brasil, $20\mathrm{mg}/\mathrm{kg}$, IP) and ketamin (Agener^®, Av. do Café, São Paulo, Brasil at $40\mathrm{mg}/\mathrm{kg}$, IP) and subjected to the ACLT of the left hind paw. The left knee was shaved, then sterilized and draped in sterile fashion. A medial arthrotomy was performed. Then, the patella was dislocated and the anterior cruciate ligament was isolated and transected. The ACLT was confirmed with Lachman testing by the surgeon and an observer.²¹ After being irrigated with sterile saline solution, the wounds were closed in layers and antiseptically treated. Rats were given appropriate postoperative care and allowed free activities in individual cages.

2.3.

Treatments

Laser treatment started 2 weeks after the surgery and it was performed for 15 and 30 sessions for each subgroup, using the following protocol: five consecutive days of irradiation with an interval of 2 days, for 3 and 6 weeks, respectively. The LLLT was applied at two points (on the medial and lateral sides of the joint), using the punctual contact technique. A low-energy Ga-Al-As laser (Theralaser, DMC^® São Carlos, São Paulo, Brazil) was used at 830 nm, continuous wave diode, with a $0.028\text{-}{\mathrm{cm}}^2$ beam diameter, a power output of 30 mW, fluence at $10\mathrm{J}/{\mathrm{cm}}^2$ (irradiation time of 10 s, energy per point 0.3 J), and fluence at $50\mathrm{J}/{\mathrm{cm}}^2$ (irradiation time of 47 s, energy per point 1.4 J). On the respective days, animals were euthanized individually by carbon dioxide asphyxia. The knee joints were removed for analysis.

2.4.

Histological Analysis

After harvesting, the specimens were fixated in 4% formaldehyde for 2 days, followed by decalcified in 4% ethylenediaminetetraacetic acid . The specimens were divided into two pieces, using a blade, at the mean point between both condyles, perpendicular to the articular surface. Samples were embedded in paraffin blocks and histological sections were done. Therefore, thin sections (6 μm) were prepared in the sagittal plane, starting from the medial margin of the joint using a micrometer (Leica RM—2145, Germany). Laminas were stained with hematoxylin and eosin (HE, Merck, Germany), Safranin-O (Merck, Germany), and Picrossirius-Red (Merck, Germany). Moreover, three sections were obtained for the imunohistoquemical analysis.

2.5.

Histological Descriptive Analysis

Histopathological alterations in the articular cartilage were evaluated by two blinded observers. For descriptive analysis, the samples were stained with HE to evaluate cartilage structure, amount of cells and cellular organization. The specimens were examined using a light microscopy ($100\times$) (Leica Microsystems AG, Wetzlar, Germany).

2.6.

Semi-Quantitative Analysis

The severity of the OA lesion was graded on a scale using the Mankin scoring system (Table 1).^22,23 Samples were stained with Safranin- O and histological evaluation system was performed along all of the extension. At least three sections of each specimen were examined using light microscopy ($100\times$) (Leica Microsystems AG, Wetzlar, Germany). The mean Mankin score of the two observers was calculated.

Table 1

Mankin score for the histological grading of cartilage degeneration.

2.7.

Morphometric Analysis

The morphometric study was carried out using one randomized slide stained with HE. The cartilage thickness and number of chondrocytes in each area were quantitatively scored using the computer-based image analysis Axiovision 3.1 Image Analysis (Carl Zeiss, Oberkochen, Germany). To count the number of condrocytes, three areas of $80,000{\mu \text{m}}^2$, at the anterior, central, and posterior region of each slide were chosen. Within each area, cells were marked and the chondrocytes average was calculated. Thickness was also measured in three regions, one central and two laterals (300-mm left and right from the first region), from subcondral bone to articular surface.²⁴

2.8.

Picrossirius Red Analysis

Histological sections stained by the Picrosirius-polarization method were viewed under polarized light (Carl Zeiss, Oberkochen, Germany) to assess the collagen organization in the cartilage tissue.^25–28 Similarly to the morphometric analysis, three areas of 80,000 μm² (anterior, central, and posterior region) of each slide were chosen to quantify the amount of collagen using the software ImageJ (Version 1.45, National Institutes of Health, Bethesda, Maryland). In each field, an indirect evaluation of the total collagen fibers organization based on the birefringence of the collagen fiber bundles after staining with Picrosirius was performed. Two experienced observers (PO and AR) performed the scoring in a blinded manner.²⁹

2.9.

Immunohistochemistry

The TNF-$\alpha$, IL-$1\beta$, MMP 13, and Col-1 immunoexpression were determined using the streptavidin-biotin-peroxidase method. Sections at 3 μm were deparaffinized in three changes of xylene and rehydrated in a graded series of ethanol to distilled water. For antigen retrieval, slides were placed in 0.01 M citrate-buffer pH 6.0 and heated in a microwave for three cycles of 5 min each at 850 W. Endogenous peroxidases were quenched by incubation in 3% ${\mathrm{H}}_2{\mathrm{O}}_2$ for 20 min at room temperature. Sections were incubated overnight at 4°C with primary antibody: TNF-$\alpha$ (polyclonal rabitt anti-rat, ab6671, Abcam, Cambrige, MA, UK), interleukin-1 (IL-$1\beta$) (polyclonal rabitt anti-rat, sc-7884, Sta Cruz biotechnology, California, USA), metalloprotein 13 (MMP 13) (polyclonal rabitt anti-rat, ab75606, abcam, Cambrige, MA, UK) and Collagen type 1 (Col-1) (anti-Col 1A monoclonal primary antibody, Sta Cruz Biotechnology, USA). Subsequently, sections were incubated. Which tissue was used as positive control for each antibody tested in immunohistochemistry technique with biotinylated secondary antibody (LSA B, Dak ocytomation) for 30 min, washed in phosphate-buffered saline, and incubated with streptavidin peroxidase conjugate (LSA B, Dakocytomation) for 30 min. Finally, the reaction was developed using 3,3′-Dianobenzidine tetrahydrocloride (Sigma, St. Louis, Missouri) for 5 min. Slides were briefly counterstained in hematoxylin and rehydrated, and cover slips added. Negative and positive controls were to run simultaneously. Positive controls were represented by mammary tissue. Negative controls were made by eliminating the primary antibody as established in previous studies conducted by our group.³⁰ The TNF-$\alpha$, IL-$1\beta$, MMP 13, and Col-1 immunoexpressions were evaluated both qualitatively (presence of the immunomarkers) and quantitatively in predetermined fields using a light microscope ((Leica Microsystems AG, Wetzlar, Germany).

2.10.

Immunohistochemical Semiquantitative Analysis

Sections stained using immunohistochemistry were analyzed for the percentages of immunopositive cells in control and experimental animals. A total of 1000 cells were evaluated in 3 to 5 fields at $400\times$ magnification. These values were used as labeling indices.

2.11.

Statistical Analysis

The normality of all variables’ distribution was verified using the Shapiro–Wilk W test. For the variable that exhibited normal distribution, comparisons among the groups were made using one-way analysis of variance (ANOVA) with pos hoc Tukey’s test. For the variable that exhibited nonnormal distribution, Kruskal Wallis test was used. STATISTICA version 7.0 was used to carry out the statistics analysis. Values of $p<0.05$ were considered statistically significant.

3.1.

General Findings

Neither postoperative complications nor behavioral changes were observed. The rats rapidly returned to their normal diet and showed no weight loss during the experimentation. None of the animals died during the experiment and no infection in the surgical site was observed.

3.2.

Histological Analysis

3.2.1.

Descriptive analysis

Histopathological analysis revealed that after 5 weeks, cartilage tissue of IG presented a normal structure, without signs of fibrillation. Moreover, in the superficial region, the chondrocytes were displayed in a parallel arrangement and in the intermediate region they were organized in columns [Fig. 1(a)]. The CG presented signs of fibrillation, irregularities in the articular surface and chondrocytes displayed in a disorganized disposition [Fig. 1(c)]. In the laser treated groups, at both fluencies, the articular surface showed initial signs of fibrillation and condrocytes organized in a normal orientation, resembling the intact animals [Figs. 1(e) and 1(g)].

Fig. 1

Representative photomicrographs the experimental groups. Organization of chondrocytes (thin arrow →), fibrillation and irregularities (thick arrow ), cartilage joint (hash), subchondral bone (asterisk). (a) IG 5 weeks; (b) IG 8 weeks; (c) CG 5 weeks; (d) CG 8 weeks; (e) L10 5 weeks; (f) L10 8 weeks; (g) L50 5 weeks; (h) L50 8 weeks. Scale bar, 100 μm.

After 8 weeks, the histological findings revealed that IG presented normal cartilage structure [Fig. 1(b)]. In the control animals, the degenerative process progressed, presenting intense fibrillation, surface irregularities and hipercellularity, with chondrocytes displayed in a disorganized way [Fig. 1(d)]. The L10 presented a better tissue organization compared to CG, with a moderate amount of cells, with slight fibrillation, and irregularities [Fig. 1(f)]. Cartilage structure of the L50 was more disorganized compared to L10, with a moderate presence of chondrocytes and fibrillation in articular surface [Fig. 1(h)].

3.2.2.

Semi-quantitative analysis

The semi-quantitative analysis demonstrated that, after 5 weeks, IG presented significantly lower scores in comparison to CG and L50. After 8 weeks, Mankin scores observed in intact animals were significantly lower compared to CG, L10 and L50 (Fig. 2). No other difference was observed.

Fig. 2

Mankin score. IG: intact control group; CG: injured control group; L10: injured laser treated group at $10\mathrm{J}/{\mathrm{cm}}^2$; L50: injured laser treated group at $50\mathrm{J}/{\mathrm{cm}}^2$. * $p\le 0,05$.

3.3.

Morphometric Analysis

3.3.1.

Cellularity

Five weeks postsurgery, similar results in the cellularity analysis were found for all groups. In the second period evaluated, the number of condrocytes in the CG was higher compared to IG, L10 and L50 ($p=0.0052$, 0.00017, 0.00015, respectively) [Fig. 3(a)].

Fig. 3

Results of quantitative analysis. (a) Cellularity and (b) Thickness. IG: intact control group; CG: injured control group; L10: injured laser treated group at $10\mathrm{J}/{\mathrm{cm}}^2$; L50: injured laser treated group at $50\mathrm{J}/{\mathrm{cm}}^2$. * $p\le 0,05$.

3.3.3.

Collagen fibers

No difference was observed in collagen fiber evaluation comparing the results found in the experimental groups at the two periods analyzed (Fig. 4).

Fig. 4

Results of the collagen fiber evaluation. IG: intact control group; CG: injured control group; L10: injured laser treated group at $10\mathrm{J}/{\mathrm{cm}}^2$; L50: injured laser treated group at $50\mathrm{J}/{\mathrm{cm}}^2$.

3.4.

Immunohistochemistry

Figures 5Fig. 6Fig. 7–8 showed the qualitative immunoexpression for IL-$1\beta$, TNF-$\alpha$, MMP-13, and Col-1, respectively. It is possible to observe that all the immunoreactivities were observed in the nucleus of chondrocytes.

Fig. 5

Representatives sections of IL-$1\beta$ immunohistochemistry. (a) IG 5 weeks; (b) IG 8 weeks; (c) CG 5 weeks; (d) CG 8 weeks; (e) L10 5 weeks; (f) L10 8 weeks; (g) L50 5 weeks; (f) L50 8 weeks. Scale bar, 100 μm.

Fig. 6

Representatives sections of tumor necrosis factor (TNF)-$\alpha$ immunohistochemistry. (a) IG 5 weeks; (b) IG 8 weeks; (c) CG 5 weeks; (d) CG 8 weeks; (e) L10 5 weeks; (f) L10 8 weeks; (g) L50 5 weeks; (h) L50 8 weeks. Scale bar, 100 μm.

Fig. 7

Representatives sections of metalloprotein (MMP)-13 immunohistochemistry. (a) IG 5 weeks; (b) IG 8 weeks; (c) CG 5 weeks; (d) CG 8 weeks; (e) L10 5 weeks; (f) L10 8 weeks; (g) L50 5 weeks; (h) L50 8 weeks. Scale bar, 100 μm.

Fig. 8

Representatives sections of collagen (Col)-1 immunohistochemistry. (a) IG 5 weeks; (b) IG 8 weeks; (c) CG 5 weeks; (d) CG 8 weeks; (e) L10 5 weeks; (f) L10 8 weeks; (g) L50 5 weeks; (h) L50 8 weeks. Scale bar, 100 μm.

Figure 9 reveals the results quantitative of IL-$1\beta$, TNF-$\alpha$, MMP-13, and Col-1 immunoexpression. Results indicated that a lower IL-$1\beta$ expression was observed in the IG compared to the other groups at both experimental periods analyzed (5- and 8-weeks postsurgery) [Fig. 9(a)]. Similar findings of IL-$1\beta$ expression were demonstrated in the other experimental injured groups (treated and control groups).

Fig. 9

(a) Results of the TNF-$\alpha$ expression. (b) Results of the IL-$1\beta$ expression. (c) Results of the MMP-13 expression. IG: intact control group; CG: injured control group; L10: injured laser treated group at $10\mathrm{J}/{\mathrm{cm}}^2$; L50: injured laser treated group at $50\mathrm{J}/{\mathrm{cm}}^2$. * $p\le 0,05$.

Quantitative analysis of TNF-$\alpha$ showed that no difference was observed between the experimental groups after 5 weeks. Eight weeks postsurgery, the expression of TNF-$\alpha$ was higher in the CG, L10, and L50 compared to IG. No other difference was detected [Fig. 9(b)].

Similar results were observed in the MMP-13 immunoexpression evaluation. At the first experimental period, no difference was observed. After 8 weeks, the expression of MMP-13 was higher in the treated and nontreated OA groups compared to IG [Fig. 9(c)].

The analysis of Col-1 showed that 5-weeks postsurgery, a significant increase of this protein was found in the L10 compared to the other groups. In the second experimental period, intact animals showed a lower expression of col-1 compared to the other groups. Moreover, CG and L10 showed a significantly higher expression compared to L50 [Fig. 9(d)].