Number of sessions of applications of laser photobiomodulation therapy interferes with the viability of skin flap: an experimental study in rats

Number of sessions of applications of laser photobiomodulation therapy interferes with the viability of skin flap: an experimental study in rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Number of sessions of applications of laser photobiomodulation therapy interferes with the viability of skin flap: an experimental study in rats Bruno Batista, Cintia Cristina Santi Martignago, Homero Garcia-Motta, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7014658/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Dec, 2025 Read the published version in Lasers in Medical Science → Version 1 posted 13 You are reading this latest preprint version Abstract Purpose: Several parameters influence the effectiveness of photobiomodulation therapy (PBMT) in improving skin flap viability, yet the role of the number of treatment sessions remains underexplored. This study aimed to evaluate the effect of different numbers of laser PBMT sessions on skin flap viability. Methods: Thirty-two Wistar rats were randomly divided into four groups: G1 (PBMT simulation), G2 (PBMT for 2 consecutive days), G3 (PBMT for 5 days), and G4 (PBMT for 7 consecutive days). Treatment began immediately after surgery with the following parameters: 90 J/cm² fluence, 40 mW output power, 90 seconds application time, and 3.6 J of energy per point, with irradiation at three points and 24-hour intervals between sessions, according to each group’s protocol. On the 7th postoperative day, tissue was collected from the irradiated area for analysis of necrotic area, vessel and mast cell morphometry, and immunohistochemistry for angiogenesis markers. Results: G2 showed the smallest necrotic area, and a higher percentage of VEGF- and HIF-1α-positive cells compared to G1. Conclusion: These results suggest that the number of PBMT sessions influences skin flap viability, with two applications providing the most beneficial outcome. Photobiomodulation Therapy Angiogenesis Plastic Surgery Skin Flap Figures Figure 1 Figure 2 INTRODUCTION The skin flap is a surgical technique that involves lifting, detaching, and repositioning cutaneous tissue over a target area while preserving its vascular connection to the original site [ 1 ]. Although widely used in reconstructive plastic surgery, complications can compromise the success of the procedure, with tissue necrosis being one of the most concerning outcomes [ 2 ]. Several studies have shown that laser photobiomodulation therapy (PBMT) can enhance skin flap viability [ 3 – 10 ], as it modulates key cellular events such as ATP synthesis, reduction of oxidative stress [ 11 ], neovascularization [ 12 ], improved cellular metabolism [ 13 ], regulation of the inflammatory response, and stimulation of the healing process [ 14 ]. The effectiveness of PBMT depends on several parameters, including power, irradiance, fluence, wavelength, and total energy delivered [ 6 , 9 , 15 ]. However, other critical aspects related to dose—such as the interval between sessions and the number of applications—have received limited attention [ 11 ]. To date, no studies have specifically evaluated the influence of the number of PBMT applications on skin flap viability, despite this being a key factor in defining the optimal therapeutic dose [ 11 , 16 ]. Most studies reporting positive outcomes used PBMT for five consecutive days [ 3 , 4 , 7 , 8 ], while a few have shown good results with shorter protocols of three days [ 17 , 18 ]. In this context, the present study aims to investigate the influence of the number of laser PBMT sessions on skin flap viability. To assess this, we analyzed the necrotic area, morphometry of blood vessels and mast cells, and the percentage of cells expressing fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), glycoprotein CD34, and hypoxia-inducible factor 1-alpha (HIF-1α) in the treated region. MATERIAL AND METHODS Thirty-two male Wistar rats ( Rattus norvegicus , var. Albinus , order Rodentia , class Mammalia ), aged ten weeks and weighing 283.7 ± 29.84 g, were obtained from the Central Animal Facility of the Federal University of São Carlos. The animals were randomly allocated into four experimental groups (n = 8 per group) as follows: G1 : received sham PBMT (placebo laser application); G2 : received two consecutive PBMT sessions; G3 : received five consecutive PBMT sessions; G4 : received seven consecutive PBMT sessions. All animals were housed individually in standard polypropylene cages under controlled environmental conditions: a 12/12-hour light/dark cycle, a temperature of 23 ± 1°C, and a sanitized environment. They received commercial feed (Premium®) and water ad libitum , along with daily analgesic administration throughout the experimental period. The study protocol was approved by the Animal Use Ethics Committee (CEUA) under protocol number 3805060617. Surgical procedure A cranially based random-pattern skin flap was created following the experimental model proposed by McFarlane, DeYoung, and Henry (1965) [ 19 ]. Animals were anesthetized with an intraperitoneal injection of ketamine (95 mg/kg) and xylazine (12 mg/kg). After induction, each animal was positioned on a flat surface for flap demarcation using a custom mold (4 cm wide × 10 cm long), aligned with the lower angle of the scapula. The flap was elevated from the dorsal region, including the skin, panniculus carnosus, and superficial musculature, and detached from the underlying deep muscle layer. A plastic film of the same dimensions was inserted between the flap and the donor bed to prevent revascularization and promote necrosis induction. The flap was then repositioned and secured with simple interrupted sutures using 4 − 0 nylon, with 1 cm spacing between stitches. Laser photobiomodulation therapy PBMT was performed using a class 3B AsGaAlIP (Gallium–Aluminum–Indium–Phosphide Arsenide) laser device (MMO Twin, MM Optics Ltda®), with a wavelength of 660 nm, output power of 40 mW, beam area of 0.04 cm², and continuous emission mode. The device was previously calibrated to ensure accuracy. Each irradiation point received 90 seconds of exposure, corresponding to a fluence of 90 J/cm² and a total energy delivery of 3.6 J per point (Table 1 ). Laser application began immediately after the surgical procedure and was repeated every 24 hours according to the total number of sessions designated for each group: 2, 5, or 7. Table 1 Parameters used in the treatment with photobiomodulation. Parameter G1 G2 G3 G4 Irradiance [W/cm²] Simulation 1 1 1 Exposition time [s] 90 90 90 90 Fluence [J/cm²] Simulation 90 90 90 Energy per point [J] Simulation 3.6 3.6 3.6 Number of points 3 3 3 3 Number of applications Simulation 2 5 7 Total energy [J] Simulation 10.8 10.8 10.8 Irradiated area [cm²] Simulation 0.12 0.12 0.12 Energy at the end of treatment [J] 0 21.6 54 75.6 Application technique Point contact Point contact Point contact Point contact Laser photobiomodulation was applied using the point-contact technique, with the laser probe positioned at a 90° angle in direct contact with the animal tissue throughout the exposure time. Irradiation was performed at three standardized points located at the cranial base of the skin flap, using a template to ensure consistency in application (Fig. 1 ). Sample collection and euthanasia All animals were euthanized on the seventh postoperative day by anesthetic overdose using ketamine (285 mg/kg) and xylazine (36 mg/kg). Tissue samples were collected from the irradiated region of the skin flap. Necrosis area calculation To assess the extent of necrosis, animals were photographed on the fifth and seventh days of the experiment. To ensure consistent imaging distance, a 20 cm fixed-base stand was used to position the digital camera (Sony DSC-W830). The necrotic area was quantified using ImageJ® software, based on macroscopic differences between viable and necrotic tissues—considering parameters such as hair regrowth, tissue color, and texture. The percentage of necrosis was calculated using the following formula [ 20 ]: $$\:Necrosis\:area\:\%\:=\:\frac{Pixels\:from\:area\:of\:necrosis}{Pixels\:from\:area\:of\:skin\:flap}\times\:100$$ Histological processing After collection, tissue samples were fixed in 10% formaldehyde solution for 24 hours. They were then rinsed under running water for another 24 hours. Dehydration was performed through a graded series of ethanol solutions at 70%, 90%, and 100%. Samples were immersed for 1 hour each in the 70% and 90% ethanol baths, followed by six consecutive 1-hour immersions in 100% ethanol. After dehydration, the samples underwent clearing in a 1:1 alcohol/xylene solution for 1 hour, followed by two 1-hour baths in pure xylene. Finally, the tissues were embedded in paraffin and sectioned using a rotary microtome (Spencer − 820). Morphometry of blood vessels and mast cells Slides were stained with Hematoxylin and Eosin for blood vessel counting and with toluidine blue for mast cell visualization. Images were captured using an optical microscope (OLYMPUS BX53) at 400x magnification across 10 interleaved fields. The first field was standardized at the left side of the slide, just above the fleshy panicle of each histological section. Blood vessels and mast cells were quantified independently by two observers; their inter-observer agreement was assessed, and the mean count per slide was used for statistical analysis. Immunohistochemistry of VEGF, FGF, CD34, and HIF-1α markers Paraffin-embedded tissue sections were deparaffinized through sequential baths of xylene and graded ethanol. Antigen retrieval was performed by incubating the sections in a steamer with 0.01 M citrate buffer (pH 6.0) for three cycles of 5 minutes each. Endogenous peroxidase activity was blocked by incubating the samples with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) for 5 minutes, followed by blocking with 5% serum in PBS for 10 minutes. Sections were then incubated with primary antibodies against VEGF, FGF, CD34, and HIF-1α (Santa Cruz Biotechnology, California, USA) for 2 hours at room temperature. Subsequently, a secondary antibody from the ABC kit (PK-6200, Vector Laboratories, Burlingame, CA, USA) was applied at a 1:5 dilution for 30 minutes. Colorimetric detection was carried out using diaminobenzidine (DAB) substrate (SK-4100, Vector Laboratories) and counterstained with hematoxylin. Quantitative analysis was performed on photomicrographs captured with an optical microscope (OLYMPUS BX53) at 200x magnification. Images were taken from six interleaved fields per slide, with the first field standardized adjacent to the fleshy panicle of each histological section. ImageJ® software, using the Color Deconvolution METHYL GREEN-DAB plugin, was employed to quantify the immunostained areas. The ratio of stained area to total field area was calculated to determine the relative expression of VEGF, FGF, CD34, and HIF-1α markers. Statistical analysis Data normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was checked with Levene’s test. Group comparisons were conducted using two-way ANOVA when significant differences between variables were detected (α < 0.05), followed by Tukey’s post hoc test. For variables assessed by two independent observers (mast cells and blood vessels), inter-rater reliability was evaluated using the intraclass correlation coefficient (ICC). RESULTS Necrosis area The evaluation of necrosis area is an important parameter commonly analyzed to assess tissue condition at different time points. In this study, the necrosis area was measured and compared across groups and times. Results showed no significant interaction between groups and times (p = 0.54). However, a difference between groups was observed (p = 0.034), specifically between G1 and G2 at both 5 days (p = 0.047) and 7 days (p = 0.032), as illustrated in Fig. 2 . Vessel morphometry The quantification of blood vessels was performed to evaluate vascular characteristics across the groups. The analysis indicated no statistically significant differences in vessel counts among the groups (p = 0.504). The mean vessel values for each group are presented in Table 2 . Table 2 Average number of vessels analyzed by morphometry. Groups Averages SD Confidence intervals Low High G1 2.55 0.7 1.96 3.13 G2 3.32 1.35 2.06 4.57 G3 2.78 0.94 1.99 3.57 G4 2.93 0.86 2.21 3.66 SD: Standard deviation. Confidence interval = 95% Mast cell morphometry Mast cell counts were assessed to determine their distribution in the tissue samples. No statistically significant differences were found between the groups (p = 0.422). The average mast cell counts can be found in Table 3 . Table 3 Average number of mast cells analyzed by morphometry. Groups Averages SD Confidence interval Low High G1 5.11 1.02 4.09 6.13 G2 4.33 0.89 3.44 5.21 G3 4.46 0.75 3.79 5.12 G4 4.16 1.43 2.88 5.44 SD: Standard deviation. Confidence interval = 95% Immunohistochemistry Immunohistochemical staining was conducted for VEGF, FGF, CD34, and HIF-1α markers to quantify their presence across groups. Statistically significant differences were found between groups G1 and G2 for VEGF (p = 0.013) and HIF-1α (p = 0.048). No significant differences were observed for FGF (p = 0.428) and CD34 (p = 0.860). Quantitative data for these markers are detailed in Table 4 . Table 4 Averages obtained in immunohistochemistry. Maker G1 G2 G3 G4 VEGF 22.21 ± 6.52 26.15 ± 2.84* 26.00 ± 5.31 22.51 ± 3.95 HIF-1α 18.91 ± 2.74 26.00 ± 2.83* 21.04 ± 3.67 20.09 ± 4.86 FGF 48.94 ± 4.41 45.79 ± 4.77 44.82 ± 4.11 45.00 ± 4.53 CD34 42.55 ± 4.58 42.24 ± 10.07 45.61 ± 10.07 45.31 ± 7.43 (*) indicates a statistically significant difference with G1. DISCUSSION This study aimed to evaluate whether the number of laser PBMT sessions influences skin flap viability over treatment durations of two, five, and seven days. Analysis of necrosis area revealed that Group G2 exhibited a lower percentage of necrotic tissue compared to Group G1, suggesting that the number of PBMT applications played a role in treatment effectiveness. Although the energy delivered per session was constant across groups, the cumulative energy over the entire treatment period varied, potentially contributing to the superior performance observed in G2, which received the lowest total energy dose. This outcome can be interpreted in light of the known dose-response relationship of PBMT, wherein treatment efficacy is strongly dependent on the dosage parameters [ 11 , 21 ]. PBMT dose refers to the total energy delivered to the tissue over a given period, and it is influenced by factors such as total energy, fluence, exposure time, and the number of sessions [ 11 ]. In this study, all PBMT parameters were standardized, with the number of applications being the only variable. Accordingly, the total energy delivered to groups G2, G3, and G4 was 21.6 J, 54 J, and 75.6 J, respectively. The biphasic dose-response curve of PBMT, frequently reported in the literature, describes an “optical window” in which the treatment exerts its most beneficial effects [ 21 ]. When this optimal range is exceeded, additional energy may lead to diminished or even inhibitory biological responses. Several studies have supported this biphasic model [ 22 – 27 ]. Thus, the energy dose administered to G2 appears to have fallen within this therapeutic window, promoting favorable tissue responses, whereas the higher cumulative doses in other groups may have surpassed the optimal threshold, potentially limiting the stimulation of cellular processes involved in tissue repair. Elevation of the skin flap triggers a series of physiological alterations, most notably a reduction in blood flow due to partial transection of the vascular supply. This interruption compromises the delivery of oxygen and nutrients to the affected region, which may result in tissue ischemia and necrosis, particularly in the distal portion of the flap [ 28 ]. Although vessel morphometry did not reveal statistically significant differences among the experimental groups, a higher mean number of vessels was observed in Group G2. This finding aligns with the well-established association between angiogenesis and tissue viability [ 9 , 29 ], as G2 also presented a larger area of viable tissue when compared to the other groups. Immunohistochemical analysis further supported these observations. The angiogenic markers VEGF and HIF-1α were found in greater quantities in G2, reinforcing the hypothesis of enhanced vascular response in this group. The elevated expression of these markers, coupled with the higher average number of blood vessels and the reduced necrotic area, suggests improved tissue perfusion. This increased vascularization likely contributed to a more effective delivery of nutrients and regulatory factors essential for restoring tissue homeostasis and limiting the progression of necrosis [ 29 ]. It has been shown that FGF production can be stimulated by laser PBMT, acting on tissue regeneration by the production of collagen and other extracellular matrix components [ 18 , 30 , 31 ]. However, it was not possible to detect differences in the presence of FGF between the treatments. The CD34 neoangiogenic marker showed no difference between the analyzed groups. Neves 2017 showed a positive result in the presence of this immunomarker compared to its control group, indicating that PBMT was effective in the appearance of new blood vessels. Tissue collection in the study was performed 24 hours after the end of treatment, while in this study, the collection was performed seven days after the surgical procedure, which may have caused the lack of difference between the groups. Mast cells are cells responsible for mediating different physiological events, including stimulating the growth, migration, and proliferation of essential elements for skin repair [ 32 , 33 ]. Regarding the number of mast cells, no difference was found between the groups, unlike that found by Nishioka et al. [ 34 ], where they found a correlation between the increase in mast cell number and PBMT. A plausible explanation for this disagreement is that the number of mast cells is higher near the necrosis region [ 34 ]. In our study, the material was collected at the application site, directly at the pedicle, and far enough away from the necrosis site. It would be interesting to have performed the same immunomarker analyses directly in the necrosis region to detect more accurately the effects of the treatments. The lower energy used in G2 demonstrated an improvement in tissue healing observed by the smaller area of necrosis compared to the other groups, as well as by the greater presence of VEGF, HIF-1α, corroborating with the found by other authors [ 28 , 30 ]. CONCLUSION The periodicity of laser PBMT applications may interfere with the flap viability, being two applications are more efficient in increasing skin flap viability, and a greater presence in the number of angiogenic markers VEGF and FGF compared to the five and seven applications in the proposed PBMT. STATEMENTS AND DECLARATIONS Funding: We would like to acknowledge the contributions of the Brazilian funding agency FAPESP project#2017/14998-4 for the financial support of the present research. Conflict of Interest: The authors declare that they have no competing interests. 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Cite Share Download PDF Status: Published Journal Publication published 29 Dec, 2025 Read the published version in Lasers in Medical Science → Version 1 posted Editorial decision: Revision requested 11 Aug, 2025 Reviews received at journal 11 Aug, 2025 Reviews received at journal 11 Aug, 2025 Reviews received at journal 11 Aug, 2025 Reviewers agreed at journal 10 Aug, 2025 Reviewers agreed at journal 09 Aug, 2025 Reviewers agreed at journal 09 Aug, 2025 Reviews received at journal 31 Jul, 2025 Reviewers agreed at journal 26 Jul, 2025 Reviewers invited by journal 24 Jul, 2025 Editor assigned by journal 24 Jul, 2025 Submission checks completed at journal 08 Jul, 2025 First submitted to journal 30 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7014658","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":491320713,"identity":"079cfe17-d950-4b99-a50f-b11d531a8f71","order_by":0,"name":"Bruno Batista","email":"","orcid":"","institution":"Federal University of São Carlos","correspondingAuthor":false,"prefix":"","firstName":"Bruno","middleName":"","lastName":"Batista","suffix":""},{"id":491320714,"identity":"f5a62fe7-190c-4048-bee4-9a2cdc4f6756","order_by":1,"name":"Cintia Cristina Santi Martignago","email":"","orcid":"","institution":"Federal University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Cintia","middleName":"Cristina Santi","lastName":"Martignago","suffix":""},{"id":491320715,"identity":"f9ea72aa-175b-4341-8f34-802751c97e30","order_by":2,"name":"Homero Garcia-Motta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYBACA1RuBRCzMzAwNhCnhRmo8AwDAw8zSVoY24jQYs7enfi4ooLBbm17//EHP+fZJO5nZj74cAaDnZwuDn2WPWc3G545w5C87cxhxsbebWmJPcxsyYYbGJKNzQ7gcNiN3G2SjW0MyWY3khkbeLcdBmrhMZN8wHAgcRtuLdt/wrQ0/p1DnJZtjEAtdiAtzbwNUC0b8Gk5c3azZMMZiQSzM4cNZ8scSzPuOQz0ywwDPH453rvxY0OFjb3Z8cYHH9/U2Mi2tzcffNhTYSeHSwsUSCQ2oBmFVzkY2BNWMgpGwSgYBSMWAAB8x2CgK3O7RwAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of São Paulo","correspondingAuthor":true,"prefix":"","firstName":"Homero","middleName":"","lastName":"Garcia-Motta","suffix":""},{"id":491320716,"identity":"fa52b1d5-5d24-4374-a660-31192be69729","order_by":3,"name":"Bruna Nascimento","email":"","orcid":"","institution":"Federal University of São Carlos","correspondingAuthor":false,"prefix":"","firstName":"Bruna","middleName":"","lastName":"Nascimento","suffix":""},{"id":491320717,"identity":"1dfbaaa7-5163-4497-a5f7-47d57d2fd5b2","order_by":4,"name":"Carla Roberta Tim","email":"","orcid":"","institution":"Universidade Brasil","correspondingAuthor":false,"prefix":"","firstName":"Carla","middleName":"Roberta","lastName":"Tim","suffix":""},{"id":491320718,"identity":"a4698e46-c06c-4a43-a6c7-5e637edd02f9","order_by":5,"name":"Livia Assis","email":"","orcid":"","institution":"Federal University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Livia","middleName":"","lastName":"Assis","suffix":""},{"id":491320719,"identity":"fe9f4436-28fd-48bc-9f18-1d7dd62bca7f","order_by":6,"name":"Richard Eloin Liebano","email":"","orcid":"","institution":"Federal University of São Carlos","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"Eloin","lastName":"Liebano","suffix":""},{"id":491320720,"identity":"be0833c6-8d5d-4b56-97ca-a8821438d988","order_by":7,"name":"Nivaldo Antonio Parizotto","email":"","orcid":"","institution":"Federal University of São Carlos","correspondingAuthor":false,"prefix":"","firstName":"Nivaldo","middleName":"Antonio","lastName":"Parizotto","suffix":""}],"badges":[],"createdAt":"2025-07-01 00:23:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7014658/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7014658/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10103-025-04791-3","type":"published","date":"2025-12-29T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87830410,"identity":"95e3de92-855f-4a0a-8386-8cd4739db777","added_by":"auto","created_at":"2025-07-29 12:23:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85944,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the template used to standardize laser photobiomodulation application sites. The black dots indicate the specific irradiation points. The template was positioned using the scapula as an anatomical reference to ensure consistent placement.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7014658/v1/e99a86cf2d46c494b831a118.png"},{"id":87830403,"identity":"e0c5b298-8892-4810-9d7f-03c80d3cbecd","added_by":"auto","created_at":"2025-07-29 12:23:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83122,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of necrosis area in both evaluations. (*) indicates a statistically significant difference between G1 and G2.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7014658/v1/d1032fa6d6f0917b12305f9c.png"},{"id":99545173,"identity":"10e6f840-e29d-4b57-a3d1-86b139165b95","added_by":"auto","created_at":"2026-01-05 16:00:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":837312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7014658/v1/f073ac00-5661-400b-9e55-5dbc905a6a36.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eNumber of sessions of applications of laser photobiomodulation therapy interferes with the viability of skin flap: an experimental study in rats\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe skin flap is a surgical technique that involves lifting, detaching, and repositioning cutaneous tissue over a target area while preserving its vascular connection to the original site [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although widely used in reconstructive plastic surgery, complications can compromise the success of the procedure, with tissue necrosis being one of the most concerning outcomes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSeveral studies have shown that laser photobiomodulation therapy (PBMT) can enhance skin flap viability [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8 CR9\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], as it modulates key cellular events such as ATP synthesis, reduction of oxidative stress [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], neovascularization [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], improved cellular metabolism [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], regulation of the inflammatory response, and stimulation of the healing process [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe effectiveness of PBMT depends on several parameters, including power, irradiance, fluence, wavelength, and total energy delivered [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, other critical aspects related to dose\u0026mdash;such as the interval between sessions and the number of applications\u0026mdash;have received limited attention [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To date, no studies have specifically evaluated the influence of the number of PBMT applications on skin flap viability, despite this being a key factor in defining the optimal therapeutic dose [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMost studies reporting positive outcomes used PBMT for five consecutive days [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], while a few have shown good results with shorter protocols of three days [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this context, the present study aims to investigate the influence of the number of laser PBMT sessions on skin flap viability. To assess this, we analyzed the necrotic area, morphometry of blood vessels and mast cells, and the percentage of cells expressing fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), glycoprotein CD34, and hypoxia-inducible factor 1-alpha (HIF-1α) in the treated region.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003eThirty-two male Wistar rats (\u003cem\u003eRattus norvegicus\u003c/em\u003e, var. \u003cem\u003eAlbinus\u003c/em\u003e, order \u003cem\u003eRodentia\u003c/em\u003e, class \u003cem\u003eMammalia\u003c/em\u003e), aged ten weeks and weighing 283.7\u0026thinsp;\u0026plusmn;\u0026thinsp;29.84 g, were obtained from the Central Animal Facility of the Federal University of S\u0026atilde;o Carlos. The animals were randomly allocated into four experimental groups (n\u0026thinsp;=\u0026thinsp;8 per group) as follows:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eG1\u003c/b\u003e: received sham PBMT (placebo laser application);\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eG2\u003c/b\u003e: received two consecutive PBMT sessions;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eG3\u003c/b\u003e: received five consecutive PBMT sessions;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eG4\u003c/b\u003e: received seven consecutive PBMT sessions.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eAll animals were housed individually in standard polypropylene cages under controlled environmental conditions: a 12/12-hour light/dark cycle, a temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, and a sanitized environment. They received commercial feed (Premium\u0026reg;) and water \u003cem\u003ead libitum\u003c/em\u003e, along with daily analgesic administration throughout the experimental period. The study protocol was approved by the Animal Use Ethics Committee (CEUA) under protocol number 3805060617.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSurgical procedure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA cranially based random-pattern skin flap was created following the experimental model proposed by McFarlane, DeYoung, and Henry (1965) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Animals were anesthetized with an intraperitoneal injection of ketamine (95 mg/kg) and xylazine (12 mg/kg). After induction, each animal was positioned on a flat surface for flap demarcation using a custom mold (4 cm wide \u0026times; 10 cm long), aligned with the lower angle of the scapula. The flap was elevated from the dorsal region, including the skin, panniculus carnosus, and superficial musculature, and detached from the underlying deep muscle layer. A plastic film of the same dimensions was inserted between the flap and the donor bed to prevent revascularization and promote necrosis induction. The flap was then repositioned and secured with simple interrupted sutures using 4\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon, with 1 cm spacing between stitches.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLaser photobiomodulation therapy\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePBMT was performed using a class 3B AsGaAlIP (Gallium\u0026ndash;Aluminum\u0026ndash;Indium\u0026ndash;Phosphide Arsenide) laser device (MMO Twin, MM Optics Ltda\u0026reg;), with a wavelength of 660 nm, output power of 40 mW, beam area of 0.04 cm\u0026sup2;, and continuous emission mode. The device was previously calibrated to ensure accuracy. Each irradiation point received 90 seconds of exposure, corresponding to a fluence of 90 J/cm\u0026sup2; and a total energy delivery of 3.6 J per point (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Laser application began immediately after the surgical procedure and was repeated every 24 hours according to the total number of sessions designated for each group: 2, 5, or 7.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eParameters used in the treatment with photobiomodulation.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eG1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eG2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eG3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eG4\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIrradiance [W/cm\u0026sup2;]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSimulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExposition time [s]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFluence [J/cm\u0026sup2;]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSimulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnergy per point [J]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSimulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of points\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of applications\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSimulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal energy [J]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSimulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIrradiated area [cm\u0026sup2;]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSimulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnergy at the end of\u003c/p\u003e\u003cp\u003etreatment [J]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e75.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eApplication technique\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePoint contact\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePoint contact\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePoint contact\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePoint contact\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eLaser photobiomodulation was applied using the point-contact technique, with the laser probe positioned at a 90\u0026deg; angle in direct contact with the animal tissue throughout the exposure time. Irradiation was performed at three standardized points located at the cranial base of the skin flap, using a template to ensure consistency in application (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSample collection and euthanasia\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll animals were euthanized on the seventh postoperative day by anesthetic overdose using ketamine (285 mg/kg) and xylazine (36 mg/kg). Tissue samples were collected from the irradiated region of the skin flap.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNecrosis area calculation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the extent of necrosis, animals were photographed on the fifth and seventh days of the experiment. To ensure consistent imaging distance, a 20 cm fixed-base stand was used to position the digital camera (Sony DSC-W830). The necrotic area was quantified using ImageJ\u0026reg; software, based on macroscopic differences between viable and necrotic tissues\u0026mdash;considering parameters such as hair regrowth, tissue color, and texture. The percentage of necrosis was calculated using the following formula [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Necrosis\\:area\\:\\%\\:=\\:\\frac{Pixels\\:from\\:area\\:of\\:necrosis}{Pixels\\:from\\:area\\:of\\:skin\\:flap}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistological processing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter collection, tissue samples were fixed in 10% formaldehyde solution for 24 hours. They were then rinsed under running water for another 24 hours. Dehydration was performed through a graded series of ethanol solutions at 70%, 90%, and 100%. Samples were immersed for 1 hour each in the 70% and 90% ethanol baths, followed by six consecutive 1-hour immersions in 100% ethanol. After dehydration, the samples underwent clearing in a 1:1 alcohol/xylene solution for 1 hour, followed by two 1-hour baths in pure xylene. Finally, the tissues were embedded in paraffin and sectioned using a rotary microtome (Spencer \u0026minus;\u0026thinsp;820).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMorphometry of blood vessels and mast cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSlides were stained with Hematoxylin and Eosin for blood vessel counting and with toluidine blue for mast cell visualization. Images were captured using an optical microscope (OLYMPUS BX53) at 400x magnification across 10 interleaved fields. The first field was standardized at the left side of the slide, just above the fleshy panicle of each histological section. Blood vessels and mast cells were quantified independently by two observers; their inter-observer agreement was assessed, and the mean count per slide was used for statistical analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemistry of VEGF, FGF, CD34, and HIF-1α markers\u003c/b\u003e\u003c/p\u003e\u003cp\u003eParaffin-embedded tissue sections were deparaffinized through sequential baths of xylene and graded ethanol. Antigen retrieval was performed by incubating the sections in a steamer with 0.01 M citrate buffer (pH 6.0) for three cycles of 5 minutes each. Endogenous peroxidase activity was blocked by incubating the samples with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) for 5 minutes, followed by blocking with 5% serum in PBS for 10 minutes. Sections were then incubated with primary antibodies against VEGF, FGF, CD34, and HIF-1α (Santa Cruz Biotechnology, California, USA) for 2 hours at room temperature. Subsequently, a secondary antibody from the ABC kit (PK-6200, Vector Laboratories, Burlingame, CA, USA) was applied at a 1:5 dilution for 30 minutes. Colorimetric detection was carried out using diaminobenzidine (DAB) substrate (SK-4100, Vector Laboratories) and counterstained with hematoxylin.\u003c/p\u003e\u003cp\u003eQuantitative analysis was performed on photomicrographs captured with an optical microscope (OLYMPUS BX53) at 200x magnification. Images were taken from six interleaved fields per slide, with the first field standardized adjacent to the fleshy panicle of each histological section. ImageJ\u0026reg; software, using the Color Deconvolution METHYL GREEN-DAB plugin, was employed to quantify the immunostained areas. The ratio of stained area to total field area was calculated to determine the relative expression of VEGF, FGF, CD34, and HIF-1α markers.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was checked with Levene\u0026rsquo;s test. Group comparisons were conducted using two-way ANOVA when significant differences between variables were detected (α\u0026thinsp;\u0026lt;\u0026thinsp;0.05), followed by Tukey\u0026rsquo;s post hoc test. For variables assessed by two independent observers (mast cells and blood vessels), inter-rater reliability was evaluated using the intraclass correlation coefficient (ICC).\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eNecrosis area\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe evaluation of necrosis area is an important parameter commonly analyzed to assess tissue condition at different time points. In this study, the necrosis area was measured and compared across groups and times. Results showed no significant interaction between groups and times (p\u0026thinsp;=\u0026thinsp;0.54). However, a difference between groups was observed (p\u0026thinsp;=\u0026thinsp;0.034), specifically between G1 and G2 at both 5 days (p\u0026thinsp;=\u0026thinsp;0.047) and 7 days (p\u0026thinsp;=\u0026thinsp;0.032), as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVessel morphometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe quantification of blood vessels was performed to evaluate vascular characteristics across the groups. The analysis indicated no statistically significant differences in vessel counts among the groups (p\u0026thinsp;=\u0026thinsp;0.504). The mean vessel values for each group are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAverage number of vessels analyzed by morphometry.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAverages\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eConfidence intervals\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eSD: Standard deviation. Confidence interval\u0026thinsp;=\u0026thinsp;95%\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eMast cell morphometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMast cell counts were assessed to determine their distribution in the tissue samples. No statistically significant differences were found between the groups (p\u0026thinsp;=\u0026thinsp;0.422). The average mast cell counts can be found in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAverage number of mast cells analyzed by morphometry.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAverages\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eConfidence interval\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eSD: Standard deviation. Confidence interval\u0026thinsp;=\u0026thinsp;95%\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining was conducted for VEGF, FGF, CD34, and HIF-1\u0026alpha; markers to quantify their presence across groups. Statistically significant differences were found between groups G1 and G2 for VEGF (p\u0026thinsp;=\u0026thinsp;0.013) and HIF-1\u0026alpha; (p\u0026thinsp;=\u0026thinsp;0.048). No significant differences were observed for FGF (p\u0026thinsp;=\u0026thinsp;0.428) and CD34 (p\u0026thinsp;=\u0026thinsp;0.860). Quantitative data for these markers are detailed in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAverages obtained in immunohistochemistry.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMaker\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eG4\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVEGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.21\u0026thinsp;\u0026plusmn;\u0026thinsp;6.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.51\u0026thinsp;\u0026plusmn;\u0026thinsp;3.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHIF-1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.04\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.09\u0026thinsp;\u0026plusmn;\u0026thinsp;4.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48.94\u0026thinsp;\u0026plusmn;\u0026thinsp;4.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.79\u0026thinsp;\u0026plusmn;\u0026thinsp;4.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e42.55\u0026thinsp;\u0026plusmn;\u0026thinsp;4.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e42.24\u0026thinsp;\u0026plusmn;\u0026thinsp;10.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.61\u0026thinsp;\u0026plusmn;\u0026thinsp;10.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.31\u0026thinsp;\u0026plusmn;\u0026thinsp;7.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e(*) indicates a statistically significant difference with G1.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study aimed to evaluate whether the number of laser PBMT sessions influences skin flap viability over treatment durations of two, five, and seven days. Analysis of necrosis area revealed that Group G2 exhibited a lower percentage of necrotic tissue compared to Group G1, suggesting that the number of PBMT applications played a role in treatment effectiveness. Although the energy delivered per session was constant across groups, the cumulative energy over the entire treatment period varied, potentially contributing to the superior performance observed in G2, which received the lowest total energy dose.\u003c/p\u003e\u003cp\u003eThis outcome can be interpreted in light of the known dose-response relationship of PBMT, wherein treatment efficacy is strongly dependent on the dosage parameters [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. PBMT dose refers to the total energy delivered to the tissue over a given period, and it is influenced by factors such as total energy, fluence, exposure time, and the number of sessions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this study, all PBMT parameters were standardized, with the number of applications being the only variable. Accordingly, the total energy delivered to groups G2, G3, and G4 was 21.6 J, 54 J, and 75.6 J, respectively.\u003c/p\u003e\u003cp\u003eThe biphasic dose-response curve of PBMT, frequently reported in the literature, describes an \u0026ldquo;optical window\u0026rdquo; in which the treatment exerts its most beneficial effects [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. When this optimal range is exceeded, additional energy may lead to diminished or even inhibitory biological responses. Several studies have supported this biphasic model [\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Thus, the energy dose administered to G2 appears to have fallen within this therapeutic window, promoting favorable tissue responses, whereas the higher cumulative doses in other groups may have surpassed the optimal threshold, potentially limiting the stimulation of cellular processes involved in tissue repair.\u003c/p\u003e\u003cp\u003eElevation of the skin flap triggers a series of physiological alterations, most notably a reduction in blood flow due to partial transection of the vascular supply. This interruption compromises the delivery of oxygen and nutrients to the affected region, which may result in tissue ischemia and necrosis, particularly in the distal portion of the flap [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough vessel morphometry did not reveal statistically significant differences among the experimental groups, a higher mean number of vessels was observed in Group G2. This finding aligns with the well-established association between angiogenesis and tissue viability [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], as G2 also presented a larger area of viable tissue when compared to the other groups.\u003c/p\u003e\u003cp\u003eImmunohistochemical analysis further supported these observations. The angiogenic markers VEGF and HIF-1α were found in greater quantities in G2, reinforcing the hypothesis of enhanced vascular response in this group. The elevated expression of these markers, coupled with the higher average number of blood vessels and the reduced necrotic area, suggests improved tissue perfusion. This increased vascularization likely contributed to a more effective delivery of nutrients and regulatory factors essential for restoring tissue homeostasis and limiting the progression of necrosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt has been shown that FGF production can be stimulated by laser PBMT, acting on tissue regeneration by the production of collagen and other extracellular matrix components [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, it was not possible to detect differences in the presence of FGF between the treatments.\u003c/p\u003e\u003cp\u003eThe CD34 neoangiogenic marker showed no difference between the analyzed groups. Neves 2017 showed a positive result in the presence of this immunomarker compared to its control group, indicating that PBMT was effective in the appearance of new blood vessels. Tissue collection in the study was performed 24 hours after the end of treatment, while in this study, the collection was performed seven days after the surgical procedure, which may have caused the lack of difference between the groups.\u003c/p\u003e\u003cp\u003eMast cells are cells responsible for mediating different physiological events, including stimulating the growth, migration, and proliferation of essential elements for skin repair [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Regarding the number of mast cells, no difference was found between the groups, unlike that found by Nishioka et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], where they found a correlation between the increase in mast cell number and PBMT. A plausible explanation for this disagreement is that the number of mast cells is higher near the necrosis region [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In our study, the material was collected at the application site, directly at the pedicle, and far enough away from the necrosis site. It would be interesting to have performed the same immunomarker analyses directly in the necrosis region to detect more accurately the effects of the treatments.\u003c/p\u003e\u003cp\u003eThe lower energy used in G2 demonstrated an improvement in tissue healing observed by the smaller area of necrosis compared to the other groups, as well as by the greater presence of VEGF, HIF-1α, corroborating with the found by other authors [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe periodicity of laser PBMT applications may interfere with the flap viability, being two applications are more efficient in increasing skin flap viability, and a greater presence in the number of angiogenic markers VEGF and FGF compared to the five and seven applications in the proposed PBMT.\u003c/p\u003e"},{"header":" STATEMENTS AND DECLARATIONS","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eWe would like to acknowledge the contributions of the Brazilian funding agency FAPESP project#2017/14998-4 for the financial support of the present research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eThis study was approved by the Ethics Committee on the Use of Animals under number 8577280716 and conducted according to the international norms of ethics in animal experimentation (NATIONAL RESEARCH COUNCIL, 1996).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFerreira L. Manual de cirurgia pl\u0026aacute;stica. In: Manual de cirurgia pl\u0026aacute;stica. 1995.\u003c/li\u003e\n\u003cli\u003eEsteves Junior I, Ferreira L M, Liebano R E. Pept\u0026iacute;deo relacionado ao gene da calcitonina por iontoforese na viabilidade de retalho cut\u0026acirc;neo rand\u0026ocirc;mico em ratos. Acta Cir\u0026uacute;rgica Brasileira, 2004. https://doi.org/10.1590/S0102-86502004000600008\u003c/li\u003e\n\u003cli\u003eKami T, Yoshimura Y, Nakajima T, Ohshiro T, Fujino T. Effects of low-power diode lasers on flap survival. Ann Plast Surg, v. 14, n. 3, p. 278-283, 1985. https://doi.org/10.1097/00000637-198503000-00013\u003c/li\u003e\n\u003cli\u003eAmir A, Solomon A S, Giler S, Cordoba M, Hauben D J The influence of helium-neon laser irradiation on the viability of skin flaps in the rat. Br J Plast Surg, v. 53, n. 1, p. 58-62, 2000. https://doi.org/10.1054/bjps.1999.3185\u003c/li\u003e\n\u003cli\u003eKubota J. Effects of diode laser therapy on blood flow in axial pattern flaps in the rat model. Lasers Med Sci, v. 17, n. 3, p. 146-153, 2002. https://doi.org/10.1007/s101030200024\u003c/li\u003e\n\u003cli\u003ePrado R P, Pinfildi C E, Liebano R E, Hochman B S, Ferreira L M. Diode laser in viability of random skin flap in rats. Photomed Laser Surg, New York, v. 23, n. 1, p. 155, Feb. 2005. Abstract.\u003c/li\u003e\n\u003cli\u003ePinfildi C E, Liebano R E, Hochman B S, Ferreira L M. Helium\u0026ndash;neon laser in viability of random skin flap in rats. Laser Surg Med, v. 37, n. 1, p. 74-77, 2005. https://doi.org/ 10.1002/lsm.20190\u003c/li\u003e\n\u003cli\u003eAssis L R, Marcolino A, Pinfildi C E, Prado R P. Comparison of the 904 nm and 670 nm diode laser in the viability of random skin flap in rats. Photomed Laser Surg, New York, v. 23, n. 1, p. 118-119, Feb. 2005. Abstract.\u003c/li\u003e\n\u003cli\u003eBossini P S. Laser de baixa intensidade (670nm) na viabilidade do retalho cut\u0026acirc;neo rand\u0026ocirc;mico em ratos. 2007. Tese de Doutorado. Universidade de S\u0026atilde;o Paulo.\u003c/li\u003e\n\u003cli\u003eBaldan C S, Masson I F B, Esteves J\u0026uacute;nior I, Baldan A M S, Machado A F P, Casaroto R A, Liebano R E. Inhibitory effects of low-level laser therapy on skin-flap survival in rat model. Past Surg (Oaky), v. 23, p. 35-39, 2015.\u003c/li\u003e\n\u003cli\u003eHuang Y Y, Chen A C-H, Carroll J D, Hamblin M R. Biphasic dose response in low level light therapy. Dose-Response, v. 7, n. 4, p. dose-response.09-027, 2009.\u003c/li\u003e\n\u003cli\u003eBotusan I R, Sunkari V G, Savu O, Catrina A I, Gr\u0026uuml;nler J, Lindberg S, Pereira T, Yl\u0026auml;-Herttuala S, Poellinger L, Brismar K, Catrina S B. Stabilization of HIF-1\u0026alpha; is critical to improve wound healing in diabetic mice. Proc Natl Acad Sci, v. 105, n. 49, p. 19426-19431, 2008. https://doi.org/ 10.1073/pnas.0805230105\u003c/li\u003e\n\u003cli\u003eAl-Watban F A H, Andres B L. Laser photons and pharmacological treatments in wound healing. Laser Therapy, v. 12, n. 1, p. 3-11, 2000. https://doi.org/10.5978/islsm.12.3\u003c/li\u003e\n\u003cli\u003eMedrado A R A P, Pugliese L S, Reis S R A, Andrade Z A. Influence of low level laser therapy on wound healing and its biological action upon myofibroblasts. Laser Surg Med, v. 32, n. 3, p. 239-244, 2003. https://doi.org/ 10.1002/lsm.10126\u003c/li\u003e\n\u003cli\u003eCury V. Laser vermelho e infravermelho em diferentes flu\u0026ecirc;ncias na viabilidade do retalho cut\u0026acirc;neo rand\u0026ocirc;mico em ratos. 2010.\u003c/li\u003e\n\u003cli\u003eEnwemeka C S. Standard parameters in laser phototherapy. Photomed Laser Surg, v. 26, n. 5, p. 41, 2008. https://doi.org/10.1089/pho.2008.9770\u003c/li\u003e\n\u003cli\u003eBaldan C S, Marques A P, Schiavinato A M, Casarotto R A. The effects of different doses of 670 nm diode laser on skin flap survival in rats. Acta Cirurgica Brasileira, v. 27, n. 2, p. 155-161, 2012. https://doi.org/ https://doi.org/10.1590/S0102-86502012000200010\u003c/li\u003e\n\u003cli\u003eDas Neves L M S, Leite G P M F, Marcolino A M, Pinfildi C E, Garcia S B, Ara\u0026uacute;jo J E, Guirro E C de O. Laser photobiomodulation (830 and 660 nm) in mast cells, VEGF, FGF, and CD34 of the musculocutaneous flap in rats submitted to nicotine. Lasers Med Sci, v. 32, n. 2, p. 335-341, 2017. https://doi.org/10.1007/s10103-016-2118-1\u003c/li\u003e\n\u003cli\u003eMcfarlane R M, Deyoung G, Henry R A. The design of a pedicle flap in the rat to study necrosis and its prevention. 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J Photochem Photobiol B Biol, v. 125, p. 164-170, 2013. https://doi.org/ DOI: 10.1016/j.jphotobiol.2013.06.004\u003c/li\u003e\n\u003cli\u003eHoureld N N, Ayuk S M, Abrahamse H. Expression of genes in normal fibroblast cells (WS1) in response to irradiation at 660 nm. J Photochem Photobiol B Biol, v. 130, p. 146-152, 2014. https://doi.org/ 10.1016/j.jphotobiol.2013.11.018\u003c/li\u003e\n\u003cli\u003eMartignago C C S, Oliveira R F, Pires-Oliveira D A A, Oliveira P D, Soares C P, Monzani P S, Poli-Frederico R C. Effect of low-level laser therapy on the gene expression of collagen and vascular endothelial growth factor in a culture of fibroblast cells in mice. Lasers Med Sci, v. 30, n. 1, p. 203-208, 2015. https://doi.org/10.1007/s10103-014-1644-y\u003c/li\u003e\n\u003cli\u003eNoli C, Miolo A. The mast cell in wound healing. 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Lasers Med Sci, v. 27, n. 5, p. 1045-1050, 2012. https://doi.org/10.1007/s10103-011-1042-7\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"lasers-in-medical-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lims","sideBox":"Learn more about [Lasers in Medical Science](https://link.springer.com/journal/10103)","snPcode":"10103","submissionUrl":"https://submission.springernature.com/new-submission/10103/3","title":"Lasers in Medical Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Photobiomodulation Therapy, Angiogenesis, Plastic Surgery, Skin Flap","lastPublishedDoi":"10.21203/rs.3.rs-7014658/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7014658/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePurpose: Several parameters influence the effectiveness of photobiomodulation therapy (PBMT) in improving skin flap viability, yet the role of the number of treatment sessions remains underexplored. This study aimed to evaluate the effect of different numbers of laser PBMT sessions on skin flap viability.\u003c/p\u003e\n\u003cp\u003eMethods: Thirty-two Wistar rats were randomly divided into four groups: G1 (PBMT simulation), G2 (PBMT for 2 consecutive days), G3 (PBMT for 5 days), and G4 (PBMT for 7 consecutive days). Treatment began immediately after surgery with the following parameters: 90 J/cm² fluence, 40 mW output power, 90 seconds application time, and 3.6 J of energy per point, with irradiation at three points and 24-hour intervals between sessions, according to each group’s protocol. On the 7th postoperative day, tissue was collected from the irradiated area for analysis of necrotic area, vessel and mast cell morphometry, and immunohistochemistry for angiogenesis markers.\u003c/p\u003e\n\u003cp\u003eResults: G2 showed the smallest necrotic area, and a higher percentage of VEGF- and HIF-1α-positive cells compared to G1.\u003c/p\u003e\n\u003cp\u003eConclusion: These results suggest that the number of PBMT sessions influences skin flap viability, with two applications providing the most beneficial outcome.\u003c/p\u003e","manuscriptTitle":"Number of sessions of applications of laser photobiomodulation therapy interferes with the viability of skin flap: an experimental study in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-29 12:22:56","doi":"10.21203/rs.3.rs-7014658/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-11T19:11:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T19:04:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T11:22:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T10:35:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338937522259259059367827263777832494445","date":"2025-08-10T20:07:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225274604426290816229968005103952126120","date":"2025-08-09T15:17:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183277979209494341192094940191421266394","date":"2025-08-09T04:30:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-31T05:49:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110750311205702096808804985009893405328","date":"2025-07-26T17:51:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-24T21:33:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-24T15:23:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-08T08:03:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Lasers in Medical Science","date":"2025-07-01T00:09:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"lasers-in-medical-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lims","sideBox":"Learn more about [Lasers in Medical Science](https://link.springer.com/journal/10103)","snPcode":"10103","submissionUrl":"https://submission.springernature.com/new-submission/10103/3","title":"Lasers in Medical Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"35570d00-fa4a-424e-a446-7f0dfa5bfd27","owner":[],"postedDate":"July 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T15:58:54+00:00","versionOfRecord":{"articleIdentity":"rs-7014658","link":"https://doi.org/10.1007/s10103-025-04791-3","journal":{"identity":"lasers-in-medical-science","isVorOnly":false,"title":"Lasers in Medical Science"},"publishedOn":"2025-12-29 15:56:58","publishedOnDateReadable":"December 29th, 2025"},"versionCreatedAt":"2025-07-29 12:22:56","video":"","vorDoi":"10.1007/s10103-025-04791-3","vorDoiUrl":"https://doi.org/10.1007/s10103-025-04791-3","workflowStages":[]},"version":"v1","identity":"rs-7014658","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7014658","identity":"rs-7014658","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}