2.1.

Bacterial Strains

A multidrug resistant strain of S. aureus was used to infect wounds. It was a clinical isolate obtained from Gazi University, Department of Microbiology, Ankara, Turkey. A single colony was used to inoculate tryptic soy broth and cultured overnight at 37°C. Bacterial suspension was then centrifuged, the supernatant was discarded and the pellet was dissolved in phosphate-buffered saline (PBS) to approximately ${10}^6$ to ${10}^7\mathrm{CFU}/\mathrm{ml}$. This suspension was used to infect wounds on animals.

2.2.

Photosensitizer and Light Source

ICG (Pulsion Medical Systems AG, Munich, Germany) solution was freshly prepared in PBS before each experiment and kept in the dark to protect it from photobleaching. All the experiments were also performed in the dark. An 808-nm diode laser was used as the light source. It is a continuous-mode laser with a maximum output power of 2 W. Laser light was delivered to the target tissue with a $1000\text{-}\mu \mathrm{m}$ optical fiber that was coupled to the original fiber of the laser. To illuminate an area of $1{\mathrm{cm}}^2$, a collimator was attached to the end of the $1000\text{-}\mu \mathrm{m}$ optical fiber. The distance between the tip of optical fiber and the target tissue was fixed and the power of the laser was controlled before each experiment with a power meter (Newport 1918-C, California).

2.3.

Animals and Abrasion Wound Model

Randomly selected Wistar albino female rats, 2- to 3-months old, weighing 170 to 220 g were used. They were obtained from Vivarium, Center for Life Sciences and Technologies Research at Bogazici University. All experiments were approved by Institutional Ethics Committee for the Local Use of Animals in Experiments of Bogazici University.

Animals were anesthetized by intraperitoneal (i.p.) injection of ketamine and xylazine mixture ($90\mathrm{mg}/\mathrm{kg}$ ketamine, $10\mathrm{mg}/\mathrm{kg}$ xylazine) before wound creation and laser application. The dorsal skin of the animals was shaved by an electric razor, and then the skin was cleaned by 70% (v/v) alcohol. To create abrasion wounds, 21-gauge needles were used to scratch an area of approximately $1{\mathrm{cm}}^2$ on the upper layer of the epidermis. After creating the wounds, $50\mu \mathrm{l}$ of bacterial suspension was added to the scratched area of the wound with the help of the tip of a pipette. There was a 30 min of waiting period for diffusion of bacteria into the wound.

2.4.

In Vivo Experiments

In this study, four groups were formed to investigate and compare the effect of ICG-PDT application. Experimental group comprises “PDT-applied wounds,” which received both laser light application and ICG. As positive controls, “laser-applied wounds,” which only received laser light and “ICG-applied wounds,” which only received ICG were created. In the “control group,” wounds received neither laser nor ICG as a negative control. Three wounds were created on each animal; one of them was assigned for negative control and the other two were assigned for PDT or positive controls.

In the PDT groups, ICG solution was added to the wounds after inoculation of bacteria. Immediately after the addition of ICG solution, irradiation of the wound by laser was started. First, $10\mu \mathrm{l}$ of ICG solution was added and an additional $10\mu \mathrm{l}$ of ICG was added to the wound at 3-min intervals until the total volume of $50\mu \mathrm{l}$ ($5\times 10\mu \mathrm{l}$) was reached during laser application. Laser irradiation lasted for 15 min in each application. The output power was 500 mW, therefore, the laser energy dose transferred to the wound was $450\mathrm{J}/{\mathrm{cm}}^2$.

In the laser groups, wounds were irradiated with $450\mathrm{J}/{\mathrm{cm}}^2$ of laser energy without any ICG after the inoculation of bacteria.

In an ICG group, specific ICG concentrations were added to the wounds without any laser irradiation and the ICG administration was the same as in the PDT groups.

Following these applications, the wounds were removed using sterile scissors and forceps. Tissue samples were cut at the boundaries of the wounds and these samples were put in 5 ml of PBS. After the weights of the samples were calculated, they were compressed in a buffer solution to release viable bacteria from the tissue by using a sterile pestle. Viable bacteria in these solutions were calculated using a serial dilution method. The aliquots were serially diluted in PBS solution by $1/10$ dilution factor and spread on tryptic soy agar plates and incubated overnight at 37°C. Colonies counted on these plates were then multiplied by a dilution factor to calculate the amount of bacteria within the corresponding tissue sample. Then colony-forming units (CFU) per gram were calculated for each wound depending on the weight of the tissue sample extracted from the animals:

$\mathrm{CFU}/\text{gram}=\text{colonies}\text{counted}\text{on}\text{plates}\times \text{dilution}\text{factor}/\text{weight}\text{of}\text{tissue}\text{sample}$.

2.5.

Wound Healing and Histological Analysis

In order to observe the wound healing period, 2-day, 4-day, 7-day and 11-day groups were formed with five animals per group with a single wound per animal. The optimum combination of ICG concentration and energy dose were used to treat the infected wound on each animal and then these animals were followed for 2, 4, 7, and 11 days. On the treatment day, the initial size of the wounds was precisely measured by Vernier calipers and the size of the wounds was measured every day until they were sacrificed. Wounds were removed after sacrificing for further histological analysis. Due to ethical considerations, animals with untreated wounds were not allowed to live during the healing process, and were sacrificed immediately after the first day. They were just used to compare the immediate response of the experimental group and positive controls in terms of bactericidal and/or thermal effects. Besides the PDT-treated wounds, the wounds of five animals were treated with an antibacterial cream with 2% mupirocin to form a control group for comparing the antibacterial effect of the conventional treatment with the effect of ICG-PDT during the healing process. These animals were followed for 14 days after infection and treatment and how quickly they were healed after treatment was assessed by measuring the wound sizes with Vernier calipers.

Removed tissue samples were fixed in 10% PBS-formalin solution for 2 to 3 days. After fixation, samples were processed in Tissue Processor (Leica TP 1020). These samples were embedded in paraffin blocks and sectioned to $6\text{-}\mu \mathrm{m}$ thickness by microtome (Leica RM 2255). These sections were stained with hematoxylin–eosin. Stained slides were assessed under a light microscope (Nikon Eclipse 80i, Japan) to observe the epithelial lining, re-epithelialization, inflammation, and collagen formation.

2.6.

Temperature Measurements

Temperature change was monitored by a $20\text{-}\mu \mathrm{m}$ K-type thermocouple, which has a response time of 0.1 s and measures changes of 0.1°C. The tip of the thermocouple was inserted into the wounds created during measurements. First, the temperature of the wound was measured before laser irradiation. Then the temperature increase was measured in the presence and absence of ICG immediately after illumination.

2.7.

Statistical Analysis

All the viable cell count data were normalized by dividing it with its corresponding data in the control wound. These normalized data were analyzed with one-way analysis of variance and then two-tailed Student $t$ test for statistical significance. $p$ values lower than 0.05 were considered as significantly different.

3.1.

Antibacterial Effect of PDT on Infected Abrasion Wounds

The laser energy dose ($450\mathrm{J}/{\mathrm{cm}}^2$) was applied together with 500, 1000, and $2000\mu \mathrm{g}/\mathrm{ml}$ of ICG on infected abrasion wounds. As shown in Fig. 1, a significant reduction in cell viability was observed in the PDT groups. The reduction was around 90% and corresponds to 1 to 2 logarithmic decrease in CFU/gram. In the laser group, viable cell count after irradiation was nearly the same as in the control group. In ICG groups, a decrease was observed in the bacterial cell count when compared with the control group. However, the data in this group were not significantly different from the data in the control and laser groups. As expected, PDT groups were significantly different from all other control groups. But PDT groups were not significantly different from each other, showing that a decrease in cell viability did not depend on the ICG concentrations applied in this study.

Fig. 1

Bacterial cell viability on abrasion wounds after laser, indocyanine green (ICG), and photodynamic therapy (PDT) applications. Laser output power was 500 mWatt, irradiation time was 15 min, ICG concentrations used were 500, 1000, and $2000\mu \mathrm{g}/\mathrm{ml}$. Asterisk represents the statistical difference with respect to control ($p<0.05$). $n\ge 8$ number of wounds in each group.

3.2.

Wound Healing

Figure 2 shows the percentage reduction in the size of the PDT and 2% mupirocin-treated wounds during 14 days. PDT parameters chosen for the healing period were $450\mathrm{J}/{\mathrm{cm}}^2$ of energy dose and $500\mu \mathrm{g}/\mathrm{ml}$ of ICG. In the first 2 days, the area of PDT-treated wounds decreased by nearly 40%. Then the healing process slowed down for 1 to 2 days, and then accelerated again. After the fifth day, the size of the wounds decreased more than 50%. At the 11th day, wounds were barely visible and their sizes approached zero.

Fig. 2

The percentage reduction in size of PDT-treated wounds and 2% mupirocin-treated wounds. $n=5$ number of wounds/animals in each group.

The area of 2% mupirocin-treated wounds decreased only 20% in the first 6 days. Then the healing process of these wounds increased and nearly reached to the healing process of the PDT-treated wounds, however, then it slowed down again and approximately 75% of the wound healing was observed on the 11th day. 2% mupirocin-treated wounds were still visible after 14 days. The healing process of the PDT-treated wounds was much faster than the 2% mupirocin-treated wounds, as clearly seen in Fig. 2.

3.3.

Histological Analysis

As shown in Fig. 3(a), a disrupted epithelial lining can be clearly observed on the newly opened wound. The integrity of the epidermis was destroyed because of the scratches with the needles. In order to assess whether any thermal damage occurred due to laser irradiation, tissue samples were removed after PDT application. Figure 3(b) shows this tissue sample in which the epithelial lining was disrupted as in the tissue sample shown in Fig. 3(a). Even though a high concentration of ICG was used, no thermal destruction was observed in the tissue. In Fig. 3(c), the wound sample removed at day 2 was shown. Tissue was covered with a thick scab. Beneath the scab, the epithelial lining, which became thicker, was observed. The integrity of the epidermis was recovered but was not uniform. The numbers of the fibroblasts increased and were concentrated at the edge of the wound. At day 4, it was observed that the scab of the wound almost completely disappeared, but some remnants were still present. The epithelial lining became thicker, therefore, the integrity of the epidermis was provided. Fibroblast cells at the edge of the wound were still high in number [Fig. 3(d)]. At day 7, the scab on the wound disappeared completely. The epithelial lining started to become thinner than it was at day 4. The integrity of the epidermis was still preserved and the number of fibroblast cells decreased. The wound healing process was almost completed [Fig. 3(e)]. At day 11, the scar of the wound was nearly invisible. The wound healing process was completed in 11 days as shown in Fig. 3(f). The epithelial lining reached its normal thickness, the integrity of the epidermis was uniform and fewer fibroblasts were observed.

Fig. 3

Histological image of (a) wound that was newly opened and had not yet received ICG or laser; (b) wound that was immediately removed after PDT application; (c) PDT-treated wound that was removed at second day after application; (d) PDT-treated wound that was removed at fourth day after application; (e) PDT-treated wound that was removed at seventh day after application; (f) PDT-treated wound that was removed at 11th day after application. Hematoxylin–eosin staining; original magnification (mag): (a, b, c, d, e, f) $100\times$.

Figure 4 shows the wound morphologies during the healing process. The sample in Fig. 4(a) is a newly opened wound and the sample in Fig. 4(b) is a PDT-treated sample. The PDT-treated sample had a scab on it at the second day [Fig. 4(c)]. The scab on the wound diminished at the fourth day [Fig. 4(d)]. The scab totally disappeared, there was only a small redness, and the wound size remarkably decreased at seven days [Fig. 4(e)]. There were not any scabs or redness, and only small scars were in the place of the wound at the 11th day [Fig. 4(f)].

Fig. 4

Wound appearance of (a) a sample that was newly opened and had not yet received ICG or laser; (b) sample that was immediately removed after PDT application; (c) PDT-treated sample that was removed at second day after application; (d) PDT-treated sample that was removed at fourth day after application; (e) PDT-treated sample that was removed at seventh day after application; (f) PDT-treated sample that was removed at 11th day after application.

3.4.

Temperature Measurements

The temperature of the wound before laser irradiation was measured as $22.54\pm 0.29°\mathrm{C}$. In the absence of ICG, $450\mathrm{J}/{\mathrm{cm}}^2$ of energy dose caused only a 1.32°C temperature change after 15-min illumination. In the presence of ICG, the temperature change was 7.68°C after same duration of illumination (Table 1). ICG caused more than a 6°C change. During laser illumination, the maximum temperature reached was 39.53°C. It was still below the critical point of 45°C at which hyperthermia begins.⁴⁶

Table 1

Temperature change after the laser application in the presence of ICG or without ICG.