Quantitative and qualitative evaluation of recovery process of a 1064 nm laser on laser-induced skin injury: in vivo experimental research

The 1064 nm laser was used to irradiate the skin of miniature pigs. The output range of laser power is approximately 12 W–955 W, and the radiation duration t, by using the equation $Q=Pt,$ and $H=\frac{4Q}{\pi {{d}^{2}}}$, can be used to compute the energy Q and laser irradiation dose H. According to the above method, the minimum laser irradiation is 7.64 J cm⁻², and the maximum output radiation dose is 608.28 J cm⁻². The relationship between laser output current and output power is determined using the linear fitting method. The linear fitting equation in these experiments has a linear correlation coefficient R²  >  0.99. Hence, the output power of the laser used in this experiment has a good linear relationship with the electric current. The light path of laser irradiation on the skin of live miniature pigs is shown in figure 1. The laser power meter is used to measure the laser power through an aperture of 1 cm in diameter.

Zoom In Zoom Out Reset image size

The 5  ×  10 grid arrays were set as the laser irradiation area, as shown in figure 2. The injury area was shaved before laser irradiation. Each grid is an irradiation point position, and the 5 grids in each column are the same laser irradiation dose. The laser irradiation spot diameter was approoximately 1 cm, the irradiation time was 0.5 s, The dose range was 29.4– 708.2 J cm⁻². And the laser power was irradiated successively from small to large.

Zoom In Zoom Out Reset image size

Six pigs with white hair were chosen. The weight of each pig was approximately 20–25 kg. Conventional miniature pigs were anesthetized by intramuscular injection of 3% sodium pentobarbital solution by 1 ml kg⁻¹ injection dose. A laser irradiation area of approximately 15 cm by 30 cm was delimited on both sides of the spine of miniature pigs, and 5  ×  10 grid arrays were set. Animal experiments with pigs were performed in accordance with the Beijing Institute of Radiation Medicine Experiment Animal Center-approved animal protocols. This study was approved by the Beijing Institute of Radiation Medicine Experiment Animal Center-approved Animal Protocols. All animal experiments with pigs were performed in accordance with the guidelines of the IACUC-DWZX-2019-502.

The laser irradiation power was irradiated successively from small to large. During the irradiation process, the changes in skin lesions such as erythema, white coagulation spots and brown burned dots were observed and recorded in real time. The erythema reaction was observed at low dose, and erythema color and duration were recorded immediately after irradiation. A small white central coagulation spot appeared when the radiation dose increased. When the dose increased again, the area of the white coagulation spot increased, and the central part of the skin showed a hazel burned dot. The irradiation dose and irradiation time of each type of damage spot (erythema, white spot and burned dot) were observed and recorded.

After irradiation with an IR laser at 0, 3, 7, 14, 21 and 28 d, the healing areas of skin wounds were observed and token photographs for measurement after being re-anesthetized. The animals were anesthetized by same way, and skin tissue was removed from the wound prior to animal euthanasia. The skin tissue was stored in 4% neutral formalin solution, and H&E staining was performed for the pathological sections.

One pig was sacrificed to acquire the samples at the one time point. And five samples were acquired at the same irradiation dose. After paraffin sections were made from the damaged spot tissue, H&E staining was performed. We observed the histopathological changes of the skin under the light microscope, including the epidermal, dermis and subcutaneous tissue layer of the skin, as well as the structural changes in hair follicles.

With the increase in the radiation power density, the acute damage caused by the laser to the skin will lead to burning and even gasification, producing all white blisters with white spots in the middle of erythema. Erythema is the lightest reaction and is reversible. The incidence rate of the erythema reaction is determined by observing the erythema visible to the naked eye (vascular congestion) immediately after irradiation. At least two specialist observed and evaluated the erythema; then, we recorded the occurrence points and irradiation points of erythema to calculate the incidence rate of the erythema reaction. After irradiation, the white spots present were observed and recorded; this information was obtained to calculate the incidence of the white spot reaction. The incidence of the burned dot reaction was calculated by the same method.

Matlab software was used to analyze the data results, which were expressed as $\overline{x}\pm s$. The fitting equation was obtained by using the linear fitting method; the linear correlation coefficient of R²  >  0.98 was highly linear. The damage threshold ED₅₀, which is that the incidence of erythema was 50% of the corresponding laser dose, and the 50% confidence interval were computed.

The laser dose was observed immediately after irradiation from smallest to largest; as the dose increased, the skin damage was observed in the following order: faint erythema, slight erythema, small white spot in the center of erythema, and small burned dot in the center of erythema. The faint erythema disappeared after a few seconds, the mild erythema disappeared after a few seconds, the erythema disappeared after 1–2 min, and the small central white spot and the small burned dot around the burned dot disappeared 30 min after irradiation with the 1064 nm laser, as shown in figure 3.

Zoom In Zoom Out Reset image size

Three days after the laser irradiation of miniature pigs, there was no seepage on the damaged spot and wound surface, and some wounds had a thin scab. After 7 d, all the damaged spots and wounds had scabbed. After 14 d, the shrinkage of the lesion wound surface decreased. After 21 d, the scab formation was still obvious at the highest dose, no signs of scab removal were observed, and the healing was slow. After 28 d, the lesion wound had healed further, but the scab was still obvious at the highest dose and healed slowly, as shown in figure 4.

Zoom In Zoom Out Reset image size

The following skin injury reactions were observed: when the laser irradiation dose was 29.4 J cm⁻², the incidence of erythema in the pig skin was only 13.3%; when the laser irradiation doses were 39.1 and 48.8 J cm⁻², the incidence of erythema in pig skin was 25.0% and 45.7%, respectively. When the laser irradiation dose reached 77.9 J cm⁻², the incidence of erythema in pig skin reached 100%, but white coagulation lesions were not observed. The laser irradiation dose and incidence of erythema in pig skin is shown in table 1.

Figure 5 shows that there was a good linear relationship between erythema reaction incidence and irradiation dose in the range of 29.4–77.9 J cm⁻².

Zoom In Zoom Out Reset image size

The following erythema and leukoplakia responses were observed: when the laser irradiation dose was 126.4 J cm⁻², the incidences of erythema and leukoplakia were 14.3% and 85.7% (n  =  35); when the laser irradiation dose was 174.8 J cm⁻², the incidences of erythema and leukoplakia were 56.5% and 43.5% (n  =  23); when the laser irradiation dose was 223.3 J cm⁻², the incidences of erythema and leukoplakia were 20.0% and 80.0% (n  =  35); and when the laser irradiation dose was higher than 417.3 J cm⁻², the incidence of porcine skin burn spots was 100.0%. The damage threshold was ED₅₀  =  47.4 J cm⁻². Depending on the reaction of erythema, white spot, and burnt spots, we can separate the irradiation dose as low, moderate, and high dose, that is, the low dose is low than 126.4 J cm⁻², the moderate dose is large than 126.4 J cm⁻² and low than 223.3 J cm⁻², and the high dose is large than 223.3 J cm⁻². The results demonstrated that as the dose increases, skin injury plaques progress from mild to severe. There is a good linear relationship between the incidence of erythema reaction and the dose within a certain dose range.

Morphological observation of normal skin from miniature pigs showed that the epidermal layer cells were orderly arranged, closely connected with the dermis and clearly demarcated. The collagen fibers of the dermis were arranged in dense and clear structures, and the normal number of sebaceous glands, hair follicles and other skin accessories were seen with clear structures. (The row 'normal' in figures 6–8).

Zoom In Zoom Out Reset image size

Zoom In Zoom Out Reset image size

Zoom In Zoom Out Reset image size

Six hours after irradiation with the 1064 nm laser for 0.5 s, tissue damage showed a trend to increase gradually with the increased laser irradiation dose. In the groups receiving the 126.4 J cm⁻² and 223.3 J cm⁻² doses, the local epidermal cell polarity changed, vacuoles appeared around the epidermal nucleus, cracks appeared between the epidermis and dermis, and the structures around the epidermal superficial glands were loose. In the group receiving the 320.3 J cm⁻² dose, the cell polarity of the whole epidermis was changed, the epidermis and dermis were separated, and collagen degeneration was observed. In the group receiving the 514.3 J cm⁻² dose, the epidermal tissue detached from the dermal tissue, and the local epidermal cells appeared necrotic.

Three days after irradiation with the 1064 nm laser, in the groups receiving the 126.4 J cm⁻² and 223.3 J cm⁻² doses, the local epidermal tissue gradually thickened, and local epidermal basal cells and epidermal superficial gland cells began to proliferate. It can be seen that the proliferated epidermis began to cover the wound surface or that the new epidermis grew and extended to the wound area; however, there were still cracks between the epidermis and the dermis, and the epidermal superficial gland structure was loose. In the groups receiving the 320.3 J cm⁻² dose, the new epidermis extended to the wound area, the epidermal superficial gland cells began to proliferate, and the local blood vessels in the epidermis were still congested. In the group receiving the 514.3 J cm⁻² dose, large areas of epidermal necrosis and exfoliation could be seen. The newly formed epidermis extended to the wound surface and grew, and some superficial dermal gland cells started to proliferate in the wound.

Seven days after irradiation with the 1064 nm laser, in the group receiving the 126.4 J cm⁻² dose, hyperplasia of the epidermal deep cells and epidermal superficial glands were active. The new epidermis extended to the wound surface and grew. Bleeding could be seen under the local epidermis. In the groups receiving the 223.3 J cm⁻² and 320.3 J cm⁻² doses, collagen fibrosis and necrosis could be seen locally in the superficial dermis, hyperplasia and activity of glands was observed in the superficial dermis, late granulation tissue could still be seen locally, and new epidermis could be seen extending to the wound surface under the necrotic and exfoliated epidermis in large areas. In the group receiving the 514.3 J cm⁻² dose, the new epidermis extended to the wound surface under the necrotic and exfoliated epidermis, and the epidermal gland cells in the epidermis began to proliferate.

Fourteen days after irradiation with the 1064 nm laser, in the group receiving the 126.4 J cm⁻² dose, the local epidermis layer was thicker, and the new epidermis had not completely covered the wound surface. In the groups receiving the 223.3 J cm⁻² and 320.3 J cm⁻² doses, the new epidermis extended to the wound surface, the remaining granulation tissue in the superficial dermis was gradually replaced by the new collagen fibers, and the new epidermis did not completely cover the wound surface. In the group receiving the 514.3 J cm⁻² dose, the new epidermis was thick and extended to the wound surface, but it did not completely cover the wound surface. A small amount of late granulation tissue and dysplasia of glands was seen in the superficial dermis.

Twenty-one days after irradiation with the 1064 nm laser, in the group receiving the 126.4 J cm⁻² dose, the local epidermis layer was thicker, and the epidermis and dermis returned to the normal tissue structure. In the group receiving the 223.3 J cm⁻² dose, there were patchy inflammatory cells infiltrating into the epidermis and fewer collagen fibers in the superficial dermis. In the groups receiving the 320.3 J cm⁻² and 514.3 J cm⁻² doses, the new epidermis under the necrotic epidermis was thick and extended to the wound surface. The epidermis in the superficial dermis shows dysplasia of glands, and the new epidermis had not completely covered the wound surface.

Twenty-eight days after irradiation with the 1064 nm laser, in the group receiving the 26.4 J cm⁻² dose, the epidermis and dermis returned to normal tissue structure. In the group receiving the 223.3 J cm⁻² dose, the newly formed epidermis layer was still slightly thicker, the proliferation of cells under the epidermis was active, the collagen fiber content in the superficial dermis began to increase, and inflammatory cell infiltration foci could still be seen between the epidermis and the superficial dermis. In the group receiving the 320.3 J cm⁻² dose, the new epidermis was thick and thick, and the new granulation tissue could be seen in the superficial epidermis. The collagen fiber content in the local superficial epidermis was low, and the old epidermal tissue could still be clearly exfoliated. In the group receiving the 514.3 J cm⁻² dose, the epidermal layer was thicker than that of the exfoliated epidermis, and the epidermal cells were not hyperplastic.

Three days after irradiation with the 1064 nm laser, there was a tendency of tissue repair in different in each group, but this tendency decrease with the increase in laser irradiation intensity. Thus, 7–14 d after laser irradiation, the wound surface of each dose group did not return to normal tissue structure, and the recovery trend was slower in the group receiving the 223.3 J cm⁻² irradiation dose; 21–28 d after laser irradiation, the group receiving the 126.4 J cm⁻² basically recovered to normal tissue structure, while the other irradiated groups did not recover to normal tissue structure. In particular, in the group receiving the 514.3 J cm⁻² high intensity dose, the recovery trend was slower in the high-intensity group, which was consistent with the general observation trend.