The effects of photobiomodulation therapy on Staphylococcus aureus infected surgical wounds in diabetic rats . A microbiological , histopathological , and biomechanical study 1

PURPOSE: To evaluate the effects of photobiomodulation therapy (PBMT) at 685 nm on diabetic wound healing in rats suffering from bacterial infection induced by Staphylococcus aureus (S. aureus). METHODS: Thirty streptozotocin-induced diabetic rats were allocated into two groups: control and PBMT. A 4-cm full-thickness linear-incision was made on the dorsal midline and was contaminated with S. aureus. The wounds in the PBMT group were irradiated daily for 5 consecutive days, starting 3 days after the induction and always in the mornings. RESULTS: The result revealed that PBMT resulted in a significant decrease in S. aureus CFU in the PBMT group in comparison to the control group (P<0.05). The length of wounds, in the 2nd and 3rd weeks, in the PBMT group were significantly shorter compared to the control group (P<0.05). PBMT caused a significant increase in the histological parameters in comparison to the control group (P<0.05). Moreover, PBMT significantly increased the breaking strength of the surgical scars produced in the skin of the PBMT group when compared to the control group (P<0.05). CONCLUSION: Photobiomodulation therapy may be useful in the management of wound infection through a significant bacterial growth inhibition and an acceleration of wound healing process.


Introduction
The healing process of wounds has attracted the attention of researchers since the beginning of its identification, especially with regard to the factors that delay or prevent it.The most important repair failures in skin wounds are those that occur in the diabetes mellitus (DM) or infections 1 .DM associated with suppression of the inflammatory reaction, decreases the migration of fibroblasts, reduced angiogenesis, changes in the proliferation of endothelial cells, and fibroblasts.On the other hand, it increases the apoptosis of them while decreasing collagen production 2 .
Wound infections are among the most common problems in diabetic people.Infection is one factor that changes the physiological progress of the wound healing 3,4 .The bacteria inhibit the angiogenesis and the proteolytic enzymes that may affect the extracellular matrix, preventing the wound contraction 5 .
Staphylococcus aureus (S. aureus), a potentially pathogenic bacterium, is a bacterium that frequently colonizes the skin.
However, if there is a break in the skin from a wound, or if there is a suppression of systemic defense, then colonizing S. aureus can cause an infection.Infections with S. aureus have been a challenge for many years.The confined population and the widespread use of antibiotics have led to the development of antibiotic-resistant strains of S. aureus 6 .
The antibacterial role of PBMT has been investigated, utilizing an approach known as photodynamic antimicrobial therapy or lethal photosensitization 15 .In this treatment, the light emitted by a PBMT activates a specific photosensitizer triggering biochemical changes capable of killing bacteria 16,17 .The specific photosensitizer links itself to a target microorganism and is activated by the light at a specific wavelength causing the release of reactive oxygen species.Increased levels of free radicals may cause cell death by exhausting the molecules of adenosine triphosphate and lipid peroxidation 18,19 .In addition to its antibacterial effects, the PBMT has also been shown to have impression on several other factors including toxin secretion by bacteria 20 , cytokines 21 , and synthesis and remodeling of collagen and angiogenesis 22 in wounds.PBMT seems promising for the treatment of diabetic wounds where healing is impaired and the treatment is further complicated by infection [23][24][25][26] .Therefore, the study was carried out to evaluate the bio-stimulatory effects of PBMT at 685 nm and 3 J/cm 2 , by in vivo microbiological, histological, and biomechanical analysis on diabetic wound healing in rats suffering from bacterial infection induced by S. aureus.

Methods
The experimental procedures with the animals used in the present study were approved by the Ethics Committee of the Islamic Azad University, Faculty of Veterinary Medicine, Tehran, Iran, under the reference code 031-4/2015.
Thirty adult male Wistar rats, weighing 200-250g, were obtained from a local colony.Subjects were randomly classified into two groups i.e. the control group and the PBMT group.Each group consisted of fifteen rats, and out of these 15, five were used for breaking strength measurement, five for bacterial load determination, and five for histopathological analysis.The rats were housed in individual plastic cages bedded with soft pad and maintained at 25±2°C, 50-60% humidity, and in a 12h/12h light/ dark cycle.The animals were fed a standard pelleted rodent diet and water ad libitum.

Experimental induction of diabetes in rats
Diabetes mellitus was induced by a single injection of streptozotocin (200 mg/kg, i.p.) in the volume of 0.2 ml, to the overnight fasting rats.Streptozotocin was dissolved in normal saline.Hyperglycemia was measured by a commercial glucometer (One Touch Sure Step, Lifescan, Milpitas, CA).After 14 days, rats with blood glucose level of >300 mg/dL 27 were considered as diabetic models.

Experimental protocol
On the day of the experiment, all rats were weighed and anesthetized using an intramuscular injection with a combination of ketamine 50 mg/kg and xylazine 10 mg/kg.The lumbar region of the rats was shaved and scrubbed with povidone-iodine.Under sterile conditions, a 4 cm full-thickness linear incision was made by a surgery blade on the dorsal midline.The wound area was contaminated with one aliquot of 50 μL of a saline solution containing approximately 5×10 7 CFU/mL of S. aureus, and afterwards, the infected skin wound was closed with 4-0 nylon strands using simple separate sutures at a distance of 1 cm from each other.Bacteria were allowed to grow in wounds for 48 h.Infection signs were observed on the 2nd post-wounding day, suggesting development of infection in the wounds.All surgical procedures were performed by a surgeon, and the animals were allowed to move freely in their individual cages.

Laser irradiation
A 685 nm InGaAlP (15 mW, 3 J/cm 2 , spot of 0.028 cm2, Teralaser; DMC ® São Carlos-SP, Brazil) was used in the present study.The mean power of the laser equipment was measured with a power measuring device before the study.The infected wounds were irradiated daily for five consecutive days, starting on the 3rd day post-induction, and always in the mornings.The animals were immobilized by the assistant, and the contact technique was used with perpendicular application divided into five points of the wound, for 5 min.The control group animals underwent sham irradiation with the turned off equipment.

Macroscopic analysis
All rats were examined daily for general appearance during which they proceeded to macroscopic evaluation of the wound, and the data were recorded on separate sheets.For the macroscopic analyses, the length of the wounds was measured by one person using a caliper on the days 0, 3, 5, 8, 14 and 21 postsurgery.The sutures were removed 14 days post operation.

Euthanasia and tissue sampling
At the end of the trial, euthanasia was performed using an overdose of anesthetic drugs, and then, skin samples were harvested.

Bacteriological analysis
For the purpose of bacterial load determination, a 1 cm × 2 cm sample tissue, including the wound was homogenized in 1ml phosphate-buffered saline (3500 rpm, 5 min), and was then centrifuged (10.000×g) for 10 min.The supernatant was collected and plated on TSA agar plates.After incubation in the dark at 37°C for 48h, the number of colonies was counted.Measuring the bacterial load was done by multiplying the number of colonies with dilution factor and volume of supernatant obtained during the tissue homogenization and was presented as log CFU/ml 28 .

Histopathological analysis
The skin specimens for histopathological analysis were processed following the standard methods (fixed in 10% formalin for 24h, embedded in paraffin, cut sections of 5 μm thickness, and stained by hematoxylin and eosin).The histological sections were evaluated by one pathologist who was blinded to the experiment as suggested by Gál et al. 29 by the following scale: 0, 1, 2, and 3 (Table 1).

Breaking strength analysis
For the purpose of measuring the breaking strength of scars, a strip of skin, 7 cm long and with the same widths of wound diameter was carefully removed by a scalpel in the manner that the wound was located at the middle of the strip.Then, the segment was pulled at both ends by two clamps until being broken using a machine for biomechanical assays (Zwick/Roell, Ulm, Germany).

Statistical analysis
A Kruskal-Wallis or Student's t-test was performed to compare the two mean scores.A value of P < 0.05 was considered the significance level.

Results
Data analysis shows that inhibition of bacterial growth was achieved by PBMT.In detail, mean bacterial numbers in PBMT group (0.51 × 10 1 ± 0.2 × 10 1 CFU/ml) were significantly lower (P < 0.05) than in the control group (8.4 × 10 7 ± 1.8 × 10 7 CFU/ml).Figure 1 illustrates wound lengths throughout this study.The length of wounds in the PBMT group were significantly shorter compared to those of the control group (P < 0.05).There was no significant difference in the mean scores for the decrease in the lengths of wounds between the PBMT and the control groups on the 3rd, 5th and 8th days.However, the differences between the two groups on the 14th and 21st days were significant (P < 0.05).Histological results appear in Table 2.According to the results, PBMT caused a significant increase in both the number of macrophages and the new blood vessels (P < 0.05).The rats in the PBMT group exhibited significantly elevated fibroblast numbers as well as elevated collagen deposition in comparison to the control group (P < 0.05).The light microscopy results are shown in Figures 3 and 4.   The breaking strength of scars in the rats' skin is presented in the Figure 5. PBMT significantly increased the breaking strength of surgical scars produced in the skin of PBMT group when compared to the control group (P < 0.05).

Discussion
The healing process of wounds can be partitioned into four stages (i.e.hemostasis, inflammation, proliferation, and remodeling) each of which can be affected by many factors.The highest prevalence of repair failures often occurs in the early stages.
Infection is an important factor that prolongs the inflammation stage.S. aureus Infections are more frequent in diabetic patients and potentially increase their morbidity and mortality.
Infection in diabetes is caused by the hyperglycemia that favors neutrophil dysfunction, antioxidant depression, angiopathies, and neuropathy 30 .In order to treat bacterial infections and prevent their progression, various treatment strategies such as antibiotics have been employed.However, concerns about the reduction of drug penetration in infected areas, drug resistance, and their side effects have led to the increased use of physical agents such as PBMT.
Several studies demonstrated the positive results of the use of PBMT for the eradication of microorganism.Kaya et al. 31 demonstrated that the use of PBMT at 808 nm, caused bacterial death in experimental chronic osteomyelitis induced by S. aureus.
Other researchers investigated the effects of blue light (470 nm) on S. aureus in an in vitro study and suggested that PBMT with a low dose may be an affordable and practical alternative to other treatment types 32 .The findings of the present study are aligned with the previous studies which showed that the use of PBMT is clinically effective in killing bacteria.Our results showed that laser irradiation resulted in a significant decrease in S. aureus CFU in the PBMT group in comparison to the control group.PBMT has beneficial effects on morphological changes in diabetic wound healing.A previous study 33 reported that PBMT enhanced wound healing in diabetic mice, as evidenced by wound closure and the histopathological evaluation of wounds of treated and untreated animals.Kana et al. 34 found that maximum enhancement of skin wound healing when utilizing a laser was reached at a daily dose of 4 J/cm 2 .Kawalec et al. 35 also witnessed the positive effect of 980 nm laser radiation at 18 J/cm 2 on wound healing in diabetic mice.In the present study, the PBMT group showed an accelerated process of inflammation and proliferation that is in agreement with previous studies 36,37 .Furthermore, in the current study, the PBMT wounds showed an increase in the amount of collagen fibers and a more evident remodeling of connective tissue, and this corresponded with the results of the study where the PBMT at 685 nm with a dose 2.5 J/cm 2 was utilized for skin wound treatment 38 .
The beneficial effects of the PBMT on tensile strength in the experimental diabetic and nondiabetic wounds have been examined.Lyons et al. 39 showed a substantial improvement in skin tensile strength and a significant improvement in the total collagen content.Stadler et al. 40 , using an incisional model, showed a beneficial effect of PBMT (830 nm) at 5 J/cm 2 on the skin wound tensile strength tests in diabetic mice; they evaluated on 11th and the 23rd post-treatment days.This study provides preliminary data which suggest that PBMT is capable of enhancing wound tensile strength.It was observed that the control group showed significant reduction in the breaking strength of scars produced in the skin when compared to the PBMT group.According to our results, it is confirmed that the healing of diabetic wounds in the PBMT group were accelerated by PBMT, although the wounds were infected by the same bacteria as the wounds of the control group.Therefore, the authors emphasize the need for further study to evaluate the possible effects of the PBMT on infectious wounds.Hopefully this study will stimulate further research in this scope.

Conclusions
Diabetic rat showed that the use of photobiomodulation therapy may be useful in the management of wound infection through a significant bacterial growth inhibition and an acceleration of wound healing process.Moreover, interventions that shorten The effects of photobiomodulation therapy on Staphylococcus aureus infected surgical wounds in diabetic rats.

FIGURE 1 -
FIGURE 1 -The wound lengths (cm) in the two groups during different days.

Figure 2
Figure 2 demonstrates improvement in the PBMT group in macroscopic study.

FIGURE 2 -
FIGURE 2 -Improvement process of wound healing in the two groups.

FIGURE 3 -
FIGURE 3 -Representative histological images of the wound (skin) area as seen by hematoxylin and eosin staining from control group on 21 day following surgery.The epithelial gap (arrows).The connective tissue of the superficial dermis is formed by a loose connective containing active fibroblasts (*).(×40).

FIGURE 4 -
FIGURE 4 -Representative histological images of the wound (skin) area as seen by hematoxylin and eosin staining from PBMT group on 21 day following surgery.The epithelial gap in treatment group (×40).Compare the gap with the Figure 3.

FIGURE 5 -
FIGURE 5 -The effect of photobiomodulation therapy (PBMT) on breaking strength at 21 days after induction of infectious wound in diabetic rat.PBMT was performed daily from 3 to 8 days after wounding.*Statistically significant difference (p0.05) in comparison with the control.

TABLE 1 -
Explanation of scale used in evaluation of histological sections.