Dynamic panoramic presentation of skin function after fractional CO2 laser treatment

Haoran Guo; Xiangyu Zhang; Hui Li; Chuhan Fu; Ling Jiang; Yibo Hu; Jinhua Huang; Jing Chen; Qinghai Zeng

doi:10.1016/j.isci.2023.107559

. 2023 Aug 9;26(9):107559. doi: 10.1016/j.isci.2023.107559

Dynamic panoramic presentation of skin function after fractional CO₂ laser treatment

Haoran Guo ^1,³, Xiangyu Zhang ^2,³, Hui Li ¹, Chuhan Fu ¹, Ling Jiang ¹, Yibo Hu ¹, Jinhua Huang ¹, Jing Chen ¹, Qinghai Zeng ^1,^4,^∗

PMCID: PMC10462835 PMID: 37649701

Summary

Fractional CO₂ laser, as a typical ablative laser, has been used to assist in the treatment of many skin diseases, such as photoaging, atrophic scar, hypertrophic scar, superficial pigmentation, vitiligo, and so on. However, the dynamic changes in skin function after fractional CO₂ laser treatment are still unclear. This study explored the changes in local skin function and possible regulatory mechanisms after fractional CO₂ laser treatment for 1, 3, 5, and 7 days through transcriptome high-throughput sequencing. The results showed that fractional CO₂ laser tended to transform the “lesions” into “normal skin”, regulate the skin barrier, coordinate the rearrangement of collagen, enhance the local microvascular circulation, activate the immune system to secrete a large number of cytokines, and act as an auxiliary tool to assist drug transport. In conclusion, according to the basic principle of destruction before reconstruction, fractional CO₂ laser plays a key role of balancer in skin reconstruction.

Subject areas: Surgery, Surgical procedure, Human physiology

Graphical abstract

Highlights

•
Fractional CO₂ laser plays a key role of balancer in skin reconstruction
•
Fractional CO₂ laser mobilizes the skin barrier regulation and defense system
•
Fractional CO₂ laser normalizes collagen arrangement
•
Fractional CO₂ laser promotes drug metabolism

Surgery; Surgical procedure; Human physiology

Introduction

Fractional CO₂ laser is a technology based on fractional photothermolysis with a wavelength of 10600 nm¹. It can be absorbed by various water-bearing structures in the skin tissue.² The earliest application of CO₂ laser can be traced back to 1964. These early CO₂ lasers released energy well absorbed by water and skin. This property enabled its use as a surgical knife due to its highly concentrated energy beam.³ Subsequently, the CO₂ laser became popular in the 1980s and 1990s as a traditional ablative laser and was commonly used for wrinkle removal.⁴

The wrinkle removal was promoted by selective targeting of water under the skin tissue. The treatment enhanced the removal of the full layer of epidermis and part of the dermis that initiated the wound repair mechanism.² The CO₂ laser treatment also stimulated tissue remodeling by generating new collagen fibers and improving skin texture, thereby removing wrinkles.⁵ This and other skin rejuvenation applications made CO₂ laser a gold standard in dermatology.⁶ However, its wide use is limited due to delayed erythema, persistent pigmentation, and difficult wound healing,⁷ especially for the skin around the non-facial areas, such as the anterior chest, palmar, plantar, and neck.⁸ It was observed that prolonged course of tissue repair may be related to the small number of migrated stem cells in the parts with fewer hair follicles.⁹ A nonablative laser was introduced to improve treatment outcomes and the adverse reactions after treatment in the same period. However, due to the recurrent symptoms after treatment and low patient satisfaction, the use of nonablative laser was limited.¹⁰ In 2004, Mainstein used fractional mode to treat photoaging for the first time, which opened the upsurge of fractional CO₂ laser use.¹¹

Fractional CO₂ laser has changed the previous CO₂ laser treatment model, which overcomes the significant side effects in the full-layer skin rejuvenation due to traditional CO₂ laser,¹² and at the same time improves collagen fiber synthesis due to its substantial stimulatory effect.¹³ When applied to the skin, these beams initiate uniform three-dimensional cylindrical thermal damage zones of the same size and arrangement and are termed microscopic thermal zone (MTZ).¹⁴ This MTZ is a property of fractional CO₂ laser and precisely damages the targeted area while sparing the surrounding skin tissue,⁴ which allows the formation of healthy skin islands leading to the formation of a stem cell repair bank in post-wound repair treatment process and can rapidly migrate to the MTZ region to complete the regenerative repair of the epidermis.¹⁵ It is generally believed that MTZ accounts for less than 40% of the whole governance area, which ensures that the skin can be regenerated within 24–48 h. Compared with the traditional CO₂ laser, in fractional CO₂ lasers, reduced exposure leads to fewer adverse reactions and promotes wound healing activity.¹⁶

At present, fractional CO₂ laser is being used to treat wrinkles, photoaging, atrophic scar, hypertrophic scar, vitiligo, and superficial pigmentation.¹⁷ These fractional CO₂ lasers also assist in drug delivery.¹⁸ However, the dynamic changes near the lesion area are not well understood. Therefore, this study tracked the dynamic changes in the skin tissue repair process after a single fractional CO₂ laser treatment using skin magnifier and HE staining. Also, through high throughput sequencing of the transcriptome, the changes in the gene expression profile of Cavia porcellus back skin after the 1, 3, 5, and 7 days of fractional CO₂ laser treatment were investigated. In addition, the study also explores the dynamic changes in skin functions, especially in the skin barrier, collagen arrangement, drug metabolism, immune system and skin defense system.

Results

Dynamic changes of gene expression profile after fractional CO₂ laser treatment

The quality control analysis of the transcriptome data of the control group and the fractional CO₂ laser group was presented in the form of PCA (Figure 1A). On the 1st day after fractional CO₂ laser treatment, 1300 DEGs showed significant changes (Log2Foldchange >1 or < −1, p < 0.05). A total of 567 genes were upregulated and 733 genes were downregulated. On the 3rd day, 672 DEGs were identified with 291 genes upregulated and 381 genes downregulated. While on the 5th and 7th days, the number of DEGs significantly reduced. There were 471 DEGs on the 5th day, with 264 genes upregulated and 207 genes downregulated. While only 466 DEGs were identified on 7th day with 402 genes upregulated genes and 64 genes downregulated (Figures 1B and 1C). Interestingly, we found that upregulated DEGs doubled on the 7th day compared with the 5th day, which was different from the trend of continuous downregulation (Figure 1C). Therefore, we further analyzed the upregulated DEGs for the 7th day and performed enrichment analysis using GO and KEGG databases. GO identified biological processes regulating adaptive immunity, including regulation of cell adaptation mediated by integrin, positive thymic T cell selection, and leukocyte heating or rolling. In contrast, the cellular component is involved in the synthesis of various protein structures, whereas the plasma membrane of organelles tertiary granule membrane, phagocytic cup, and mast cell granule were upregulated. The GO molecular function identified an increased interaction of various cytokines (Figure S1A). Similar results were obtained with KEGG analysis-cytokine receptor interaction and chemokine signaling pathways were significantly upregulated. Several other signaling pathways orchestrating adaptive immunity and microbial infection, such as Th17 cell differentiation, Th1 and Th2 cell differentiation and Staphylococcus aureus infection were also significantly activated on the 7th day (Figure S1B).

We also investigated the dynamic changes occurring in these DEGs at different time points. We sorted them from large to small according to the log2Foldchange and extracted the top 15 upregulated genes and the 15 genes that were highly downregulated every day to draw a time sequence heatmap (Figure 1D). The top 30 DEGs that change every day are separately mapped in Figure 1E.

Dynamic changes of functions and signaling pathways in skin tissue after fractional CO₂ laser treatment

It can be seen from Figure 1 that after fractional CO₂ laser treatment, several genes were significantly affected compared with the control group on the 1st, 3rd, 5th, and 7th days. To determine the treatment-driven changes in skin tissue, we extracted DEGs for GO and KEGG enrichment analysis. For GO enrichment analysis, according to Enrichment Score, we sort the biological process, cellular component, and mobile function modules and extracted the top five functional items on the 1st, 3rd, 5th and 7th days (p < 0.05). As shown in Figure 2A, the GO biological process mainly identified immunity-related signatures, including the activation of the adaptive immune system. For example, genes related to T cell-mediated immunity, positive thymic T cell selection and positive regulation of immunoglobulin production altered significantly throughout the period, with the highest activation on the 7th day. Cellular components identified laminin-5 complex, keratin filament, cornified envelope, and active filament bundle genes related to keratinization and cytoskeleton formation. The functional enrichment identified processes related to energy generation and chemokine interaction in the molecular function that were significantly enriched.

Dynamic functions and signaling pathways changes in skin tissue after fractional CO₂ laser treatment

(A) The GO and KEGG enrichment analysis of differentially expressed genes (DEGs, Log2Foldchange >1 or < −1, p < 0.05).

(B) The GESA-GO and GSEA-KEGG enrichment analysis of all genes. This experiment has at least 3 biological replicates.

For KEGG enrichment analysis, the top 20 signaling pathways were presented. We identified pathways related to microbial infection – S. aureus infection and Leishmaniasis, and adaptive immunity pathways-Th17 cell differentiation and Antigen processing and presentation significantly upregulated. These pathways also showed a gradually increasing trend over time, which shows that even with a normal skin appearance, the internal cellular components alter significantly. Further analyses using GESA-GO and GSEA-KEGG enrichment on all genes in descending order according to Log2Foldchange (Figure 2B) revealed several observations. According to the GSEA-GO results, we identified pathways related to muscle tissue structure significantly decreasing during this period. These include pathways such as skeletal muscle contraction, myosin filament, M band, etc. It may be due to heat conduction produced by laser on the subcutaneous muscle tissues of C. porcellus. We also found that inflammatory and immune response pathways, such as neutrophil chemotaxis, innate immune response, and inflammatory response, significantly upregulated. Interestingly, the immune response, especially the adaptive immune response, was more evident on the 7th day. At the same time, GESA-KEGG results also reflected signaling pathways related to inflammation-TNF signaling pathway, IL-17 signaling pathway, and Cytokine-cytokine receptor interaction significantly activated. Moreover, pathways related to pathogen infection were also active during this period.

The potential effect of fractional CO₂ laser on skin barrier regulation

The energy released by fractional CO₂ laser can be efficiently absorbed by skin tissue rich in water, and a conical ablation zone is formed in skin tissue. This zone consists of a vaporized tissue, peripheral thermal coagulation zone and peripheral thermal effect zone.¹⁹ Like previous studies, immediately after a single fractional CO₂ laser treatment to the skin, the MTZ area was vaporized by the high-power laser beam, and the surrounding tissues formed a thermal coagulation zone. After one day, the epidermis was not completely repaired, and the conical crater in the MTZ was filled with necrotic skin tissue. During this, the skin erythema was the most serious. On the 3rd day, the skin formed a thick scab, skin erythema gradually subsided, and the infiltration of inflammatory cells decreased gradually. Until the 7th day, the skin returned to normal structure (Figures 3A and 3B; Figure S2A). Next, the signaling pathways from GESA-KEGG related to skin barrier repair was presented. We found that tight junction representing the tightness of the skin barrier,²⁰ showed persistent down-regulation on the 1st, 3rd, 5th, and 7th days after a single fractional CO₂ laser. Meanwhile, Focal adhesion, ECM-receptor interaction, cell adhesion molecules (CAMs) and adherens junction showed a downward trend on the 1st day followed by a significant upward trend on the 5th and 7th days. In contrast, the VEGF signaling pathway related to angiogenesis²¹ has been continuously upregulated during this period (Figure 3C). At the same time, we collected all the genes related to these pathways, sorted them from large to small according to the log2Foldchange of day 1, and reserved the top 15 and the bottom 15 genes to draw a heatmap (Figure 3D). Here, we found that Tnc, Spp1, Col4a6, and other genes changed regularly with time.

Fractional CO₂ laser normalizes collagen arrangement

As a kind of ablative laser, fractional CO₂ laser strongly stimulates the synthesis of new collagen fibers and the remodels them by starting the wound repair mechanism. The present research shows that fractional CO₂ laser is not only used to treat skin diseases with collagen reduction such as photoaging and atrophic scars, but also to treat hypertrophic scars and keloids.²² Consistent with previous studies, fractional CO₂ laser treatment severely damaged the collagen structure on the 1st day after treatment in this sequencing results. It is evident from functional GSEA-GO analysis that collagen binding (NES = −1.696968553, p < 0.01), collagen trimer (NES = −2.153327627, p < 0.001), collagen containing extracellular matrix (NES = −1.795670124, p < 0.001) showed significant down-regulation (Figure 4A). This is the damage caused by the laser thermal effect and the collagen decomposition caused by increased MMP secretion (Figure 1E). Over time, the skin structure gradually recovered. On the 7th day, the collagen binding (NES = 1.820103817, p < 0.01), collagen trimer (NES = 1.974690406, p < 0.001), collagen fiber organization (NES = 1.924559352, p < 0.001), collagen containing extracellular matrix (NES = 2.06612039, p < 0.001) was upregulated compared with control group. The collagen catabolic process was still highly activated (NES = 2.06612039, p < 0.001) (Figure 4B). It was consistent with previous research results that collagen decomposition increased in a short time after fractional CO₂ laser treatment. With the prolongation of time, the body started the wound repair mechanism promoting the increase of collagen.²³ At the same time, we collected all the genes related to the collagen metabolism pathway for ssGSEA analysis and calculated the statistical difference through the limma package. The result is like Figures 4A and 4B, but the interesting thing is that on the 7th day, two opposite functional items, positive regulation of collagen biosynthetic process (p < 0.001) and negative regulation of collagen biosynthetic process (p < 0.05), were both upregulated (Figure 4C). At the same time, we also collected several classical signaling pathways regulating collagen metabolism. We found that Jak-STAT and PI3K-Akt signaling pathway were significantly upregulated on the 5th and 7th days after fractional CO₂ laser treatment. In contrast, the TGF-beta signaling pathway showed a downward trend on the 3rd, 5th, and 7th days. WNT signaling pathway showed no noticeable change (Figure S2B). It seems that fractional CO₂ laser did not blindly promote collagen regeneration but preferentially altered collagen content and distribution, suggesting that fractional CO₂ laser activates collagen remodeling. However, this does not simply promote the increase of collagen but shows the normalization of collagen distribution.

Fractional CO₂ laser promotes drug metabolism

At present, fractional CO₂ laser, as a new technical means, is gradually being used as an auxiliary treatment to assist drug delivery in vitiligo, psoriasis, and other skin diseases characterized by relatively deep lesions.²⁴^,²⁵ In this study, it was found that after a single fractional CO₂ laser treatment, drug metabolism-other enzymes were significantly upregulated throughout the period (p < 0.05) and drug metabolism-cytochrome P450 was significantly downregulated on the 1st day (NES = −1.559063327, p < 0.05) and upregulated on the 5th (NES = 1.524121117, p < 0.05) and 7th (NES = 1.739642649, p < 0.01) days (Figure 5A). At the same time, we extracted the genes related to these two pathways to map the heatmap of Top30 DEGs (Figure 5B) and mapping the PPI network (Figure 5C) online through STRING, which formed two modules centered on Upp1 and LOC100715394 (the encoded protein belongs to the GST superfamily), respectively. Previous studies have shown that it is best to use fractional CO₂ laser within 24 h when assisting drug delivery to treat skin diseases because the epidermal regeneration has not been completed at this time, and the drug can be directly delivered to the focus.²⁶ However, the ssGSEA results of drug metabolism in this study showed that drug transport was still significantly upregulated on the 3rd (p < 0.01), 5th (p < 0.001) and 7th (p < 0.01) days and drug transmembrane transport were highly active on the 5th (p < 0.05) and 7th (p < 0.001) days (Figure 5D).

Fractional CO₂ laser affects the skin defense system

After treatment with fractional CO₂ laser, an open wound is formed on the skin. Therefore, the expert consensus of fractional laser recommends that preventive antiviral and anti-microbial infections should be carried out one day before surgery to five days after treatment. In addition, active infections (mainly herpes virus infections) have recently been accepted as contraindications for fractional CO₂ laser treatment.²⁷ Similarly, according to the GSEA-KEGG enrichment analysis of Figure 6A, the pathways related to virus and microbial infection were significantly upregulated from the 1st day to the 7th day. Pathways related to innate immunity, such as Nod-like receptor signaling pathway (p < 0.05) and Toll-like receptor signaling pathway (p < 0.05), are activated throughout this period. Moreover, the related adaptive immunity pathways were obviously upregulated and even stronger on the 7th day after treatment (Figure 6B). However, there are no obvious infection-related symptoms in the C. porcellus’ back skin. Preventive use of antiviral and antibiotic treatment may be beneficial to the body’s inflammatory repair and prevent infection.

Fractional CO₂ laser mobilizes the skin defense system

(A) The GSEA-KEGG enrichment analysis of microbial and virus infection-related pathways.

(B) After fractional CO₂ laser treatment, the immune system was activated and on the 7th day, the adaptive immune-related pathway was more significantly upregulated. This experiment has at least 3 biological replicates.

Discussion

Fractional CO₂ laser has been widely used to treat photoaging, wrinkles, atrophic scars, hypertrophic scars, vitiligo, and superficial pigmentation.¹⁷ However, due to its limited knowledge of the mechanism of action in treating different diseases and the relatively long recovery time after surgery, many clinicians were deterred from a fractional CO₂ laser. In this study, we used C. porcellus as animal models and explore the dynamic changes of skin after fractional CO₂ laser treatment through high throughput sequencing of transcriptome, revealing the potential role of fractional CO₂ laser in skin barrier regulation, collagen arrangement, drug metabolism, immune system and skin defense system, and providing a basis for clinical use.

At the molecular level, we found that after a single fractional CO₂ laser irradiation, several DEGs showed a downward trend over time. However, the number of DEGs upregulated significantly increased on the 7th day, and the GO functional are related to inflammation, pathogen infection, immunity, etc. Moreover, the KEGG signaling pathways of them were significantly upregulated. Even though the skin tissue on the back of C. porcellus had recovered to normal structure on the 7th day after treatment, the adaptive immune system-related genes were highly upregulated. At the organizational level, the skin barrier was in a down-regulation trend after the fractional CO₂ laser treatment. However, with the extended time, many signaling pathways related to the skin barrier were significantly enhanced compared with the untreated group. The changes of collagen metabolism like in previous studies.²⁸ Collagen structure is significantly downregulated on the 1st day after treatment and upregulated on the 7th day after treatment. However, both positive regulation of the collagen biosynthetic process and negative regulation of the collagen biosynthetic process were upregulated on the 7th day. In terms of use, the fractional CO₂ laser is often used as an auxiliary tool for drug delivery within the 24 h after treatment. However, it was found that drug metabolism-other enzymes and Drug metabolism-cytochrome P450 were still significantly upregulated on the 5th and 7th days of treatment in this study. Next, many signaling pathways related to microbial infection, viral infection and immunity were activated after treatment, even on the 7th day, highlighting that the skin tissues are still at potential risk of infection.

The energy released by a fractional CO₂ laser is absorbed by the water-rich skin tissue and converted into heat.¹ Immediately after treatment, the skin will show signs of thermal burns, and the tissue will gradually recover to a stable state as time goes on. According to our sequencing results, the total number of DEGs in the skin after a single fractional CO₂ laser treatment reached 1300 on the 1st day, 672 on the 3rd day, and only more than 400 on the 5th and 7th days. The changes in these DEGs also reflect the process of gradual restoration and stability of skin tissue. However, different from what we have known, the number of genes upregulated on the 7th day increased significantly. Through GO and KEGG enrichment analysis, many functional modules and signaling pathways related to adaptive immunity were significantly upregulated, such as TNF, IL-17 signaling pathway and cytokine-cytokine receptor interaction. Current research shows that various cytokines involved in these pathways promote wound healing and tissue repair, etc. For example, TNF-ɑ, by mediating VEGFA and ERK1/2 signaling pathways, upregulated the activity of brain microvascular endothelial cells and promoted wound healing.²⁹ The IL-17 signaling pathway regulates skin repair by safeguarding the metabolic adaptation of wound epithelial cells.³⁰ The GO and KEGG enrichment analysis on the 1st, 3rd, 5th, and 7th days shown that the enrichment scores of many functional items and signaling pathways on the 7th day were higher than those on the 1st day, suggesting that these subsequently upregulated functions and pathways might be involved in long-term skin repair and reconstruction. So, we speculate that many cytokines still promote the repair and remodeling of damaged skin in various ways on the 7th day or even longer after the fractional CO₂ laser treatment.

The skin forms an effective barrier between organisms and the environment, preventing pathogen invasion and resisting chemical and physical attacks, while the physical barrier of skin is mainly composed of stratum corneum and tight junction.³¹ We found that even on the 7th day after fractional CO₂ laser treatment, the eschar in MTZ had completely fallen off, and the epidermis had been reconstructed, However, the tight junction was still in a downregulated state. It suggests that even though the basic structure of the skin has been repaired, the barrier function of the skin is still not perfect. From the perspective of postoperative care, this indicates that our skin is still very fragile at that time. We, therefore, need to extend the use of products to repair skin barriers and strengthen the awareness of moisturizing and sun protection. From the perspective of clinical drug use, it is suggested that fractional CO₂ laser can increase the percutaneous absorption of topical applied medicine. It is still practical to daub therapeutic drugs on the 7th day after treatment. At the same time, we also found that the signaling pathways related to skin barrier, such as focal adhesion, ECM receiver interaction, cell adhesion motors (CAMs) and adherens junction,³² showed an overall upward trend on the 5th and 7th days after treatment. Therefore, we believe that the effect of topical drugs on the wound is better from the moment of treatment to the 3rd day.

The energy released by a fractional CO₂ laser is converted into heat and stimulates the generation and remodeling of new collagen, which is the primary mechanism of fractional CO₂ laser to achieve the skin "rejuvenation" effect. The microvascular destruction caused by laser heat causes tissue ischemia to induce the secretion of collagenase and matrix metalloproteinase (MMP), leading to the dissolution of old collagen, and then the skin tissue starts the damage repair mechanism.³³ Type III collagen, as the main component of granulation tissue during wound healing,³⁴ increased significantly after thermal injury. Studies showed that the mRNA level of procollagen type Ⅰ and III peaked on the 21st day after treatment and remained for 6 months. With the progress of the healing, type I collagen is gradually deposited. In contrast, the number of components of type III collagen is reduced to a certain level until they are like healthy skin.³⁵^,³⁶^,³⁷ This indicates that the treatment effect of fractional CO₂ laser is maintained for a longer time. Too frequent laser irradiation maybe is not recommended. In our study, we found that on the 1st day after treatment, the tissue rich in collagen structure decreased significantly. On the 7th day after treatment, the collagen trimer, collagen fiber organization, collagen containing extracellular matrix and other pathways were significantly upregulated compared with the control group (Figures 4A and 4B). However, at the same time, we found that both positive regulation of collagen biosynthetic process and negative regulation of collagen biosynthetic process were obviously upregulated on the 7th day (Figure 4C) and some classical signaling pathways that stimulate collagen synthesis, such as Jak-STAT and PI3K-Akt signaling pathway were significantly upregulated on the 5th and 7th days. In contrast, the TGF-beta signaling pathway showed a downward trend (Figure S2B). The TGF-beta signaling pathway is a classical signaling pathway that stimulates collagen synthesis and plays an essential role in treating hypertrophic scars such as keloids.³⁸ Some study found that fractional CO₂ laser degrades large and wide collagen bundles in keloids in a short time, disturbs the original disordered collagen arrangement, makes skin reconstruction highly orderly, reduce fibroblast proliferation and inhibits TGF-b1 secretion, which increases collagen synthesis.³⁹ Fractional CO₂ laser activates collagen remodeling, not simply promoting the increase of collagen but showing the normalization of collagen distribution, which shows the transformation process of “scar to skin”.

The stratum corneum is the main barrier of drug delivery, and fractional laser can achieve controllable destruction and ablation. Laser-assisted drug penetration may increase local drug concentration and penetration depth through the ablation of the skin barrier, optical breakdown of optical mechanical wave and photothermal effect.⁴⁰ Current research shows that fractional CO₂ laser, as a new technical means, is also gradually being used as an auxiliary treatment means to assist drug delivery in treating vitiligo and other skin diseases.⁴¹ For example, a prospective, single-blinked study reported in 2022 found that fractional CO₂ laser combined with 5-fluorouracil had a more significant effect on re-pigmentation than 5-fluorouracil alone, and the patient’s compliance was higher.⁴² However, there is no specific time range for the external use of drugs after fractional CO₂ laser, but it is believed that the best effect can be achieved within 24 h after treatment, according to the change in tissue structure. Because at this time, the epidermis is vaporized to form a pore to transport drugs. A similar result was found in an animal experiment that when the small molecule compound sodium fluoride (with fluorescence) was applied within 6 h after a fractional CO₂ laser exposure, the fluorescence intensity (FI) increased. The FI was the highest when applied within 30 min after laser treatment, while, after 24 h of laser exposure, its FI was like that of non-laser treated skin.²⁶ While in our sequencing results, we found that although the skin tissue basically completed the epidermal regeneration within three days (the sampling point was the 3rd day), the drug metabolism other enzymes and Drug metabolism cytochrome P450 were still significantly up regulated later. And even drug transport and drug transmembrane transport signaling pathways are obviously active on the 5th and 7th days. Human cytochrome P450, as membrane-bound hemoproteins, plays an essential role in drug detoxification, cell metabolism and body balance. In humans, almost 80% of oxidative metabolism and about 50% of the total elimination of common clinical drugs can be attributed to one or more different cytochrome P450.⁴³ It suggests that within 7 days after fractional CO₂ laser treatment, the skin still has a strong ability to absorb and decompose drugs. A histological experiment of an earlier optical coherence tomography study also suggested that although the channels caused by fractional CO₂ laser will re-epithelialize in about two days, dermal remodeling often requires at least three weeks.⁴⁴ Due to the dermal tissue not yet completely recovered, the body will promote the permeability of local microcirculation to repair the damaged structure, enhancing drug percutaneous absorption capacity. Therefore, we speculate that although the epidermis reconstruction has been completed, extending the use time of local drugs may bring better adjuvant treatment effect. In addition, the accelerated degradation of drugs by cytochrome P450 is also beneficial to reduce the occurrence of adverse drug reactions. Through this study, we have learned that fractional CO₂ laser can not only provide a direct contact channel for drug delivery, but also can increase the drug metabolism ability of the skin which is beneficial to improve the efficacy of topical medication or accelerate the degradation of drugs and reduce side effects. Of course, other factors may still play an important role, including the depth and density of pores, the scope of the surrounding tissue coagulation zone, the time of local application, and the physical and chemical properties of local drugs.⁴⁵

Although the current fractional laser expert consensus has proposed the preventive use of antibiotics and antiviral drugs before and after fractional CO₂ laser treatment,²⁷ many patients still have doubts about using such drugs when receiving treatment. In this study, we found that after a single fractional CO₂ laser treatment on the back skin of C. porcellus, many signaling pathways related to innate immunity, adaptive immunity, microbial and viral infection were significantly upregulated. These pathways were still significantly activated on the 7th day, even some of them were more significantly upregulated on the 7th day than the 1st day. It suggests that it is very necessary for us to use anti-inflammatory, anti-microbial and anti-virus drugs in fractional CO₂ laser to prevent hyperpigmentation after excessive inflammation and infection caused by pathogens. On the other hand, it also suggests that cytokines released by appropriate inflammatory reaction may participate in the long-term process of subsequent skin structure reconstruction.

In conclusion, the gene expression profile of fractional CO₂ laser-treated skin will undergo dynamic changes over time. Skin barrier, collagen arrangement, drug metabolism, immune system and skin defense system all present obvious characteristics of timing change after fractional CO₂ laser treatment. Depending on the principle of the thermal effect of “first destruction” and “then reconstruction”, fractional CO₂ laser can competently assist in the treatment of a variety of skin diseases.

Limitations of the study

There are still many shortcomings in this study, such as the short observation time for animal efficacy, unclear long-term effects, and species differences between C. porcellus and human skin tissue. These shortcomings limit the conclusion. No clinical experiments have been conducted and there is a lack of dynamic observation evidence for the treatment of different diseases. And this study only explored the effects of fractional CO₂ laser therapy, without assisting other lasers or drugs. The detailed mechanism exploration will be explored in the follow-up study.

Ethics approval

This research has been approved by the Experimental Animal Ethics Committee of Central South University (CSU-2022-0597), and the research data in this paper is allowed to be published.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins

Hematoxylin	Servicebio	G1004
Differentiation solution	Servicebio	G1039
Anti-blue solution	Servicebio	G1040
Eosin	Servicebio	G1001

Deposited data

RNA-seq	This paper	GEO accession numbers: GSE234691 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE234691)

Software and algorithms

R	N/A	R 4.2.1, https://www.r-project.org/
STRING	N/A	Version: 11.5, https://cn.string-db.org/
GraphPad Prism	N/A	Version: 9.0, https://www.graphpad.com/

Experimental models: Organisms/strains

Cavia porcellus	Hunan SJA Laboratory Animal Co., Ltd	Four-week-old, white, female

Other

Fractional CO₂ laser	Third Xiangya Hospital	Kinglaser, KL type, JiLin, China
CBS skin analysis	Third Xiangya Hospital	CBS-802, Taiwan, China
Inverted microscope	Third Xiangya Hospital	Olympus, Japan

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Resource availability

Lead contact

Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request.

Corresponding author: Qinghai Zeng (zengqinghai@csu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Experimental model and study participant details

Animals and treatment

Four-week-old white female Cavia porcellus were acquired from the Hunan SJA Laboratory Animal Co., Ltd (Hunan, China). The study was reviewed and approved by the Experimental Animal Ethics Committee of Central South University (CSU-2022-0597). A total of 12 Cavia porcellus were used in the experiment, and were divided into 4 time points for testing and sampling (3 Cavia porcellus treated each time). The detailed grouping description is as follows: number the experimental animals from 1 to 12 according to their body weight and divide the numbers by 4. Animals with a remainder of 1 will be in Group A (Day 1), those with a remainder of 2 will be in Group B (Day 3), those with a remainder of 3 will be in Group C (Day 5), and those with a remainder of 4 will be in Group D (Day 7). After adapting to the environment for four days, the back skin of Cavia porcellus was depilated one day before a single fractional CO₂ laser treatment. After anesthesia, the right side was irradiated with a single fractional CO₂ laser (Kinglaser, KL type, JiLin, China). The energy of the laser is 140 mJ, the size of the spot is 2 × 2 cm², and the spacing between the beams is 0.6 mm, while the left of back skin was not irradiated. Post irradiation, the skin on both sides of the back was incubated with sterile ice towels to cool down. On day 1, 3, 5 and 7 after treatment, a skin magnifier (CBS-802, CBS skin analysis, Taiwan, China) was used to record the change of skin, which was equipped with a 5-megapixel lens and can enlarge the skin image to 50 x and allows us to clearly observe the subtle changes in the skin.

Method details

High-throughput transcriptome sequencing

On day 1, 3, 5 and 7 after treatment, the back skin was collected for high-throughput transcriptome sequencing (repeat with more than three in each group). Fresh skin tissues were immediately placed in sterile cryopreservation tubes and rapidly frozen in liquid nitrogen to prevent RNA degradation. Subsequently, liquid nitrogen was used to cause skin tissue fragmentation. The total RNA was extracted from the tissue using the Trizol reagent and was subjected to general transcriptome sequencing and data analysis on the Illumina platform (Shanghai Ouyi Company).

Hematoxylin-eosin staining

The paraffin embedding was dewaxed, sectioned, and hydrated using a mixture of xylene and ethanol. The sections were stained with hematoxylin dye (G1004, Servicebio) for 5 min. The sections were washed under running water, and the slices were treated with a differentiation solution and an anti-blue solution. The slices were again washed with running water and soaked in eosin solution (G1001, Servicebio) for 3 min. After programmed dehydration and transparency, the slices were fixed with neutral resin and photographed under an inverted microscope (Olympus, Japan).

Transcriptome sequencing analysis

The total RNA of skin tissue was extracted by the Trizol method, and the transcriptome was sequenced in Illumina sequencer by oebiotech (Shanghai, China). High-throughput sequencing data of the skin tissue was performed on the Ubuntu 20.04 LTS (Focal Fossa) system. R software (version 4.0.5) and its R package were used for data analysis. After initial data cleaning by tidyverse and reshape2, small quantities of low expression values were screened out. The PCA (Principal Component Analysis) package is used for quality control analysis, and differentially expressed genes (DEGs) were analyzed by DESeq2 package and presented as volcano plots. GO (Gene Ontology) and KEGG (Kyoto Encylopaedia of Genes and Genomes) enrichment analysis were performed based on the reference transcriptome database of Cavia porcellus in NCBI (National Center for Biotechnology Information). After the genes were sorted from large to small according to Log2Foldchange, the GSEA-GO (Gene Set Enrichment Analysis-Gene Ontology) and GSEA-KEGG (Gene Set Enrichment Analysis-Kyoto Encylopaedia of Genes and Genomes) enrichment analysis was performed. ssGSEA (single sample Gene Set Enrichment Analysis) analysis of functional modules was carried out by GSVA (Gene Set Variation Analysis) package, and the results are analyzed by the difference of functional items and presented in the form of heatmap. PPI (Protein-Protein Interaction Networks) analysis between protein coding genes is carried out on STRING: functional protein association networks (Version: 11.5).

Quantification and statistical analysis

Statistical analysis was conducted using R software. p-value <0.05 was considered as statistically significant.

Acknowledgments

Thanks to all the partners. Thanks to Natural Science Foundation of Hunan Province (2022JJ30880) and the Fundamental Research Funds for Central Universities of the Central South University (No. 2023ZZTS0027) for their support.

Author contributions

Q.Z. designed the subject. J.C. was responsible for funding acquisition, methodology and resources. The experiments and data analyses were performed by H.G., X.Z., C.F., L.J., and Y.H. J.H., H.L., F.Z., and L.Z. are responsible for visualization of images and data.

Declaration of interests

The authors declare no competing interests.

Inclusion and diversity

We support inclusive, diverse, and equitable conduct of research. We worked to ensure diversity in experimental samples through the selection of the genomic datasets.

Published: August 9, 2023

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2023.107559.

Supplemental information

Document S1. Figures S1 and S2

mmc1.pdf^{(535.6KB, pdf)}

Data and code availability

The data of RNA-seq have been deposited at NCBI (National Center for Biotechnology Information) and are publicly available. The data link is https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE234691. This paper does not report original code. Any additional information reported in this paper will be shared by the lead contact upon request.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1 and S2

mmc1.pdf^{(535.6KB, pdf)}

Data Availability Statement

[bib1] 1.Mu Y.Z., Jiang L., Yang H. The efficacy of fractional ablative carbon dioxide laser combined with other therapies in acne scars. Dermatol. Ther. 2019;32 doi: 10.1111/dth.13084. [DOI] [PubMed] [Google Scholar]

[bib2] 2.Verma N., Yumeen S., Raggio B.S. StatPearls. 2022. [PubMed] [Google Scholar]

[bib3] 3.Lecocq C., Pirard D., del Marmol V., Berlingin E. [The use of lasers in dermatology] Rev. Med. Brux. 2013;34:12–19. [PubMed] [Google Scholar]

[bib4] 4.Borges J., Manela-Azulay M., Cuzzi T. Photoaging and the clinical utility of fractional laser. Clin. Cosmet. Investig. Dermatol. 2016;9:107–114. doi: 10.2147/CCID.S77996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5.Majidian M., Kolli H., Moy R.L. Management of skin thinning and aging: review of therapies for neocollagenesis; hormones and energy devices. Int. J. Dermatol. 2021;60:1481–1487. doi: 10.1111/ijd.15541. [DOI] [PubMed] [Google Scholar]

[bib6] 6.Agrawal N., Smith G., Heffelfinger R. Ablative skin resurfacing. Facial Plast. Surg. 2014;30:55–61. doi: 10.1055/s-0033-1364223. [DOI] [PubMed] [Google Scholar]

[bib7] 7.Tierney E.P., Hanke C.W. Treatment of CO2 laser induced hypopigmentation with ablative fractionated laser resurfacing: case report and review of the literature. J. Drugs Dermatol. 2010;9:1420–1426. [PubMed] [Google Scholar]

[bib8] 8.Grunewald S., Bodendorf M., Illes M., Kendler M., Simon J.C., Paasch U. In vivo wound healing and dermal matrix remodelling in response to fractional CO(2) laser intervention: clinicopathological correlation in non-facial skin. Int. J. Hyperthermia. 2011;27:811–818. doi: 10.3109/02656736.2011.595380. [DOI] [PubMed] [Google Scholar]

[bib9] 9.Feily A., Firoozifard A., Sokhandani T., Elosegui-Rodriguez P., Perez-Rivera E., Lange C.S., Hosseinpoor M., Ramirez-Fort M.K. Follicular Transplantation, Microneedling, and Adjuvant Narrow-band Ultraviolet-B Irradiation as Cost-Effective Regimens for Palmar-Plantar Vitiligo: A Pilot Study. Cureus. 2020;12 doi: 10.7759/cureus.7878. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10.Dayan S.H., Vartanian A.J., Menaker G., Mobley S.R., Dayan A.N. Nonablative laser resurfacing using the long-pulse (1064-nm) Nd:YAG laser. Arch. Facial Plast. Surg. 2003;5:310–315. doi: 10.1001/archfaci.5.4.310. [DOI] [PubMed] [Google Scholar]

[bib11] 11.Manstein D., Herron G.S., Sink R.K., Tanner H., Anderson R.R. Fractional photothermolysis: a new concept for cutaneous remodeling using microscopic patterns of thermal injury. Lasers Surg. Med. 2004;34:426–438. doi: 10.1002/lsm.20048. [DOI] [PubMed] [Google Scholar]

[bib12] 12.Sarnoff D., Gotkin R., Doerfler L., Gustafson C., Hanke C.W. The Safety of Laser Skin Resurfacing With the Microablative Carbon Dioxide Laser and Review of the Literature. J. Drugs Dermatol. 2018;17:1157–1162. [PubMed] [Google Scholar]

[bib13] 13.Esmat S., Shokeir H.A., Samy N.A., Mahmoud S.B., Sayed S.S.E.D., Shaker E., Hilal R.F. Automated Microneedling Versus Fractional CO2 Laser in Treatment of Traumatic Scars: A Clinical and Histochemical Study. Dermatol. Surg. 2021;47:1480–1485. doi: 10.1097/DSS.0000000000003227. [DOI] [PubMed] [Google Scholar]

[bib14] 14.Shin M.K., Choi J.H., Ahn S.B., Lee M.H. Histologic comparison of microscopic treatment zones induced by fractional lasers and radiofrequency. J. Cosmet. Laser Ther. 2014;16:317–323. doi: 10.3109/14764172.2014.957216. [DOI] [PubMed] [Google Scholar]

[bib15] 15.Paasch U., Haedersdal M. Laser systems for ablative fractional resurfacing. Expert Rev. Med. Devices. 2011;8:67–83. doi: 10.1586/erd.10.74. [DOI] [PubMed] [Google Scholar]

[bib16] 16.Laubach H.J., Tannous Z., Anderson R.R., Manstein D. Skin responses to fractional photothermolysis. Lasers Surg. Med. 2006;38:142–149. doi: 10.1002/lsm.20254. [DOI] [PubMed] [Google Scholar]

[bib17] 17.Friedman P., Chapas A., Kilmer S., Rahman Z., Roberts W., Ross E., Waibel J., Dover J. Optimizing Outcomes With the 1550/1927 nm Dual Wavelength Non-Ablative Fractional Laser: Experienced User Recommendations. J. Drugs Dermatol. 2021;20:1150–1157. doi: 10.36849/jdd.6181. [DOI] [PubMed] [Google Scholar]

[bib18] 18.Agamia N.F., Sobhy N., Abd-Elraouf A., Tawfik A. Fractional CO2 Laser for Transcutaneous Drug Delivery of Onabotulinum Toxin in Palmar Hyperhidrosis. Dermatol. Surg. 2021;47:678–683. doi: 10.1097/DSS.0000000000002901. [DOI] [PubMed] [Google Scholar]

[bib19] 19.Chen S.X., Cheng J., Watchmaker J., Dover J.S., Chung H.J. Review of Lasers and Energy-Based Devices for Skin Rejuvenation and Scar Treatment With Histologic Correlations. Dermatol. Surg. 2022;48:441–448. doi: 10.1097/DSS.0000000000003397. [DOI] [PubMed] [Google Scholar]

[bib20] 20.Jensen J.M., Proksch E. The skin's barrier. G. Ital. Dermatol. Venereol. 2009;144:689–700. [PubMed] [Google Scholar]

[bib21] 21.Bausch D., Fritz S., Bolm L., Wellner U.F., Fernandez-Del-Castillo C., Warshaw A.L., Thayer S.P., Liss A.S. Hedgehog signaling promotes angiogenesis directly and indirectly in pancreatic cancer. Angiogenesis. 2020;23:479–492. doi: 10.1007/s10456-020-09725-x. [DOI] [PubMed] [Google Scholar]

[bib22] 22.Omi T., Numano K. The Role of the CO2 Laser and Fractional CO2 Laser in Dermatology. Laser Ther. 2014;23:49–60. doi: 10.5978/islsm.14-RE-01. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib24] 24.Mohammadi S., Amiri R., Khalili M., Iranmanesh B., Aflatoonian M. Treatment protocols and efficacy of combined laser with medical treatment modalities in vitiligo. J. Cosmet. Dermatol. 2022;21:3272–3291. doi: 10.1111/jocd.14602. [DOI] [PubMed] [Google Scholar]

[bib25] 25.Nassar A., Atef H., Eldeeb F., Alakad R. Comparison of fractional laser-assisted drug delivery and intralesional injection of triamcinolone acetonide in nail psoriasis. J Dtsch Dermatol Ges. 2022;20:788–796. doi: 10.1111/ddg.14731. [DOI] [PubMed] [Google Scholar]

[bib26] 26.Banzhaf C.A., Thaysen-Petersen D., Bay C., Philipsen P.A., Mogensen M., Prow T., Haedersdal M. Fractional laser-assisted drug uptake: Impact of time-related topical application to achieve enhanced delivery. Lasers Surg. Med. 2017;49:348–354. doi: 10.1002/lsm.22610. [DOI] [PubMed] [Google Scholar]

[bib27] 27.Nestor M.S. Prophylaxis for and treatment of uncomplicated skin and skin structure infections in laser and cosmetic surgery. J. Drugs Dermatol. 2005;4:s20–s25. [PubMed] [Google Scholar]

[bib28] 28.Azzam O.A., Bassiouny D.A., El-Hawary M.S., El Maadawi Z.M., Sobhi R.M., El-Mesidy M.S. Treatment of hypertrophic scars and keloids by fractional carbon dioxide laser: a clinical, histological, and immunohistochemical study. Lasers Med. Sci. 2016;31:9–18. doi: 10.1007/s10103-015-1824-4. [DOI] [PubMed] [Google Scholar]

[bib29] 29.Chen W., Shen Z., Cai S., Chen L., Wang D. FGF21 promotes wound healing of rat brain microvascular endothelial cells through facilitating TNF-alpha-mediated VEGFA and ERK1/2 signaling pathway. Adv. Clin. Exp. Med. 2021;30:711–772. doi: 10.17219/acem/133494. [DOI] [PubMed] [Google Scholar]

[bib30] 30.Wang J., Ding X. IL-17 signaling in skin repair: safeguarding metabolic adaptation of wound epithelial cells. Signal Transduct. Target. Ther. 2022;7:359. doi: 10.1038/s41392-022-01202-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib32] 32.Jensen P.J., Wheelock M.J. The relationships among adhesion, stratification and differentiation in keratinocytes. Cell Death Differ. 1996;3:357–371. [PubMed] [Google Scholar]

[bib33] 33.Liu H.Z., Lyu K.Y. [Research advances on the mechanism of fractional laser in treating scars] Zhonghua Shaoshang Zazhi. 2021;37:386–390. doi: 10.3760/cma.j.cn501120-20200315-00166. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib35] 35.Orringer J.S., Rittié L., Baker D., Voorhees J.J., Fisher G. Molecular mechanisms of nonablative fractionated laser resurfacing. Br. J. Dermatol. 2010;163:757–768. doi: 10.1111/j.1365-2133.2010.09998.x. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Dynamic panoramic presentation of skin function after fractional CO2 laser treatment

Haoran Guo

Xiangyu Zhang

Hui Li

Chuhan Fu

Ling Jiang

Yibo Hu

Jinhua Huang

Jing Chen

Qinghai Zeng

Summary

Graphical abstract

Highlights

Introduction

Results

Dynamic changes of gene expression profile after fractional CO2 laser treatment

Figure 1.

Dynamic changes of functions and signaling pathways in skin tissue after fractional CO2 laser treatment

Figure 2.

The potential effect of fractional CO2 laser on skin barrier regulation

Figure 3.

Fractional CO2 laser normalizes collagen arrangement

Figure 4.

Fractional CO2 laser promotes drug metabolism

Figure 5.

Fractional CO2 laser affects the skin defense system

Figure 6.