Lasers in Medical Science

, Volume 31, Issue 7, pp 1363–1370 | Cite as

Laser phototherapy improves early stage of cutaneous wound healing of rats under hyperlipidic diet

  • Virgínia Dias Uzêda-e-Silva
  • Tania Tavares Rodriguez
  • Isadora Almeida Rios Rocha
  • Flávia Calo Aquino Xavier
  • Jean Nunes dos Santos
  • Patrícia Ramos Cury
  • Luciana Maria Pedreira Ramalho
Original Article

Abstract

The aim of this study was to evaluate the influence of laser photobiomodulation in cutaneous healing of rats under a hyperlipidic diet. Forty-eight Wistar Albinus rats, weaned, received standard diet (SD) or hyperlipidic diet (HD) for 20 weeks. The groups were divided into SD rats and HD rats, SD-irradiated rats (LSD), and HD-irradiated rats (LHD). Standard cutaneous wound (1 cm2) was created on the dorsum of each rat. The irradiation started immediately after surgery and every 48 h for 7 or 14 days (λ660 nm, 40 mW, 6 J/cm2, ϕ 0,04 cm2, CW), when they were killed under deep anesthesia. The specimens were removed, routinely processed, stained with hematoxylin/eosin (H/E), and evaluated by light microscopy. Rats fed with hyperlipidic diet had greater intensity in the inflammatory process and prolonged hyperemia. At day 7, the intensity of inflammation was reduced in LSD and LHD groups when compared to their control groups, SD (p = 0.002) and HD (p = 0.02). There was an increase in fibroblast proliferation and collagen deposition, especially in the LHD group. At day 14, the HD group presented more intensive hyperemia than the SD group. It can be concluded that the hyperlipidic diet modified the inflammation pattern in wound healing and that laser light has a positive biomodulative effect on the healing process only in early stages.

Keywords

Wound healing Photobiomodulation Laser phototherapy Hyperlipidic diet 

Introduction

Obesity is a metabolic disorder, which reflects an unhealthy accumulation of fat mass. The increased prevalence of obesity and obesity-associated complications is one of the most public health challenges of the twenty-first century [1]. The current definition of obesity is based on the body mass index (BMI) ≥30 kg/m2, together with other anthropometrics such as the waist circumference and an assessment of the presence and severity of obesity-related complications [2].

High-fat diet is one of the main causes of obesity development. Interaction between microbiota and high-fat diet causes excessive release of adipocytokines that favor fat deposition [3]. The use of hypercaloric or hyperlipidic diets has been used as a model of obesity induction in animals due to its similarity to the genesis and metabolic responses caused by obesity in humans [4]. Inadequate diet and a sedentary lifestyle are the most common contributing factors to the development of obesity in humans.

Studies have shown that the excess of adipose tissue, mainly in the abdomen, is a closely related risk of cardiovascular complications, such as coronary artery disease and hypertension, and metabolic abnormalities such as dyslipidemia, insulin resistance, and diabetes mellitus type II [3, 4]. In addition to these classical comorbidities, obesity has been identified as a risk factor for infectious and wound complications after a wide variety of surgical procedures which lead the increase of morbidity and prolonged hospitalization [5]. This increased risk has been attributed to immune impairment, ischemia along suture lines, larger wound areas, deficiencies in collagen synthesis, and technical difficulties [6, 7, 8]. At any rate, the decrease of tissue oxygen tension in relatively avascular adipose tissue might play a central role. Therefore, the healing process is a target for the care of this population, and new therapies are needed to facilitate tissue repair in this group.

Wound healing is a dynamic and complex process that presents three phases which have been well described: an inflammatory phase, a proliferative phase, and a remodeling phase. This process starts hours after the injury, when blood components flood the site of injury and inflammatory cells spread out inside the tissue in order to perform the critical task of phagocytosis, which removes foreign materials, bacteria, and damaged tissue. Afterward, wound reepithelialization occurs through migration and proliferation of epithelial cells originated from both margins of the lesion and epidermal appendices on the center of the lesion [9]. Incisional injuries are usually epithelized within 24–48 h after the initial injury; however, excisional wounds can take much longer to re-epithelialize [10]. Low-level laser therapy (LLLT) has been used to treat injuries and may accelerate tissue repair in its different phases. Photobiomodulation can improve both the speed of healing and the quality of scar tissue [11, 12].

The wound healing impairment observed in obese individuals can be attributed, at least in part, to tissue ischemia [7, 8]. In addition, the proinflammatory cytokines expressed by white adipose tissue contribute to the increase of circulating levels of inflammatory molecules [13, 14]. This condition favors the persistence of the inflammatory process, which may disrupt the timing in the phases of the repair process, leading to a delayed wound healing [15].

LLLT has been postulated to have photochemical, photophysical, and photobiological beneficial effects to repair. Different studies using several models of wound healing have been demonstrating that the LLLT increases mitochondrial ATP production, lymphocyte and mast cell activation, and also the proliferation of fibroblasts and other cells. Likewise, LLLT promotes analgesia and anti-inflammatory effects [16, 17, 18].

The hypothesis of this study is that the biomodulatory effects postulated for LLLT on tissue repair could compensate for the shortcomings associated with obesity. Thus, this study aimed to evaluate the effect of laser phototherapy during wound healing in rats subjected to a hyperlipidic diet.

Materials and methods

This study was approved by the Ethics Committee on Animal Use of the Dental School of the Federal University of Bahia under Protocol 02/12.

Forty-eight male Wistar rats weaning at 21 days of age and weighing an average 50 g were included in the study. The rats were housed at the Experimentation Laboratory of the Health Sciences Institute of the Federal University of Bahia in cages containing three rats each. The cages were lined with wood shavings and kept at ambient temperature (23 ± 1 °C) in a 12:12-h day/night light cycle. Water was available ad libitum. The animals were randomly assigned to one of the following groups: standard diet (SD) and hyperlipidic diet (HD). The standard diet group (n = 24) was fed with commercial feed (Nuvital, Nuvilab®, Colombo, PR, Brazil), while the hyperlipidic diet group (n = 24) was fed with a hyperlipidic diet [14]. Rats were divided into two subgroups of 12 animals each: SD—non-irradiated, LSD—laser, HD—non-irradiated, and LHD—laser (Table 1). The body weight was measured with an electronic scale (Filizola®) with a maximum capacity of 5 kg and accuracy of 5 g.
Table 1

Distribution of the experimental groups according to the diet, irradiation protocol, and time of death

Groups

Diet

Irradiation protocol

Time of death

Number

SD

Standard diet

Non-irradiated

7 and 14 days

12

LSD

Laser standard diet

GaAlAs laser λ660 nm—24 J/cm2, 40 mW, CW

7 and 14 days

12

HD

Hyperlipidic diet

Non-irradiated

7 and 14 days

12

LHD

Laser hyperlipidic diet

GaAlAs Laser λ660nm – 24 J/cm2, 40 mW, CW

7 and 14 days

12

The hyperlipidic diet consisted of commercial rat feed, roasted peanuts, milk chocolate, and biscuit cornstarch in a 3:2:2:1 proportion [19]. All components were grounded, mixed, and pelletized. This diet was composed of 17 % protein, 23 % fat, and 46 % carbohydrates. The standard diet contained 22 % protein, 4 % fat, and 57 % carbohydrates. The caloric density was about 4.5 kcal/g for the high-fat diet and 3.5 kcal/g for the standard diet.

After 20 weeks of receiving the diet, the rats were anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (14 mg/kg). The animals had their dorsal region shaved and, subsequently, antisepsis of the surgical area with 2 % chlorhexidine gluconate (Merthiolate®, Hypermarcas SA, Barueri, São Paulo, Brazil) was performed. A standardized subcutaneous excisional wound (1 × 1 cm) was made in the median dorsal region of each rat with a scalpel (Fig. 1). Hemostasis was obtained by dry gauze compression and the wound was allowed to heal without suturing or dressing. The first laser irradiation was performed immediately after surgery and repeated every 48 h during the trial period of days 7 or 14. The irradiation was performed through contact with the probe positioned perpendicularly to the irradiated tissue without pressure. Spatial average energy fluence (SAEF) from the illuminated area (1 cm2) per session was 24 J/cm2 for the groups irradiated with laser light. The wounds were irradiated with the laser device applied to the wound area, corresponding to the four point wound angles (GaAlAs, λ660 nm, 40 mW, continuous wave (CW), 6 J/cm2 per point, 150 s per point, Φ = 0.04 cm2, Twin Flex Evolution, MMoptics®, São Carlos, SP, Brazil).
Fig. 1

Standardized excisional wound created with a scalpel in the medial dorsal region. Modified surgical scalpel used (a) and clinical aspects of final wound (b)

After the experimental period (7 or 14 days), the animals were killed under general anesthetic overdose, and the specimens were removed for analysis. The retroperitoneal fat was removed as a triangular section extending from a vertex in the inguinal region up to the midline and across the power pole of the kidney, extending laterally as far as fat was visible. After its removal, it was weighed.

After sample collection, the specimens were fixed for 24 h in a 10 % formaldehyde solution and processed for routine histological technique with hematoxylin and eosin (H&E). Histological sections were analyzed under light microscopy (AxionStar®, Zeiss, Germany) by a single experienced pathologist. Descriptive and semiquantitative analyses were performed according to the table below (Table 2).
Table 2

Criteria used in the histological analysis

Parameters

Score criterion

Discreet

Moderate

Intense

Reepithelialization

Absent

Covering <50 % of the wound

Covering >50 % of the wound

Inflammatory Infiltrate

Presence of up to <25 % in the area of inflammatory cells

Presence of 25 to 50 % in the area of inflammatory cells

Presence of more than 50 % in the area of inflammatory cells

Hyperemia

Amount of blood vessels dilated and congested lower than the adjacent normal tissue

Amount of blood vessels dilated and congested similar to the adjacent normal tissue

Amount of blood vessels dilated and congested greater than adjacent normal tissue proliferation

Fibroblastic proliferation

Presence of up to 25 % of fibroblasts relative to other cell types of the tissue

Presence of 25 to 50 % of fibroblasts compared to other cell types of the tissue

Presence of more than 50 % of fibroblasts compared to other cell types of the tissue

Collagen deposition

Presence of up to 25 % deposition of collagen fibers in the area

Presence of 25 to 50 % of collagen deposition in the area

Presence of more than 50 % collagen fiber deposition in the area

The body weight and retroperitoneal fat were assessed by Student’s t test. The histological data were analyzed by Fisher’s exact nonparametric test. A 5 % significance level was defined by statistical analysis.

Results

The average final body weight of the groups showed no statistically significant difference between them (p > 0.05). However, as demonstrated in Fig. 2, the retroperitoneal fat deposit was higher in the HD group than in the SD group, demonstrating an increase of about 60 % (Student’s t test, p = 0.000).
Fig. 2

Retroperitoneal fat mass after 20 weeks of diet. Data are presented as mean ± SD. Comparison between SD and HD groups (* p = 0.000) (Student’s t test)

Seven days after the injury, a mild presence of inflammatory cells was observed in the LSD group compared to the SD group, which showed intense inflammation (p = 0.002) (Fig. 3a). When the hyperlipidic diet groups were compared, the group submitted to laser therapy showed a lower degree of intensity in the inflammatory process than the non-irradiated group did (p = 0.02) (Figs. 3b and 5a, b)
Fig. 3

Effects of diet and phototherapy on wound healing at 7 days. a Analysis of inflammatory infiltrate of SD and LSD groups (**p = 0.002). b Effects of laser therapy in the inflammatory infiltrate of the hyperlipidic groups (*p = 0.02). c Effect of laser therapy in the collagen deposition of the hyperlipidic groups (*p = 0.048). d Effect of hyperlipidic diet in the deposition of collagen of LSD and LHD groups (*p = 0.001)

At 7 days evaluation, the deposition of collagen fibers in the LHD group was significantly higher than that in the HD group (p = 0.048) (Figs. 3c and 5e, f). When the irradiated groups were compared, it was observed that the collagen deposition was significantly higher in the LHD group than in the LSD group (p = 0.001) (Fig. 3d).

Fibroblast proliferation was less intense in the LSD group than in the LHD group (p = 0.016) (Figs. 4 and 5c, d).
Fig. 4

Effects of hyperlipidic diet and phototherapy on wound healing at 7 days. Analysis of fibroblast proliferation of LSD and LHD groups (*p = 0.016)

Fig. 5

Histopathological aspects of the healing. At 7 days, a, b moderate inflammatory infiltrate in the LHD group and intense in the HD group, respectively. c, d Moderate fibroblast proliferation in the LSD group and intense in the LHD group, respectively. e, f Discreet collagen deposition in the HD group and moderate in the LHD group, respectively. At 14 days, g, h discreet hyperemia in the SD group and moderate in the HD group, respectively

Additionally, the re-epithelization and hyperemia were not different between the groups (p > 0.05).

Fourteen days after surgery, when hyperemia was analyzed, there was a statistically significant difference between the SD and HD groups. The HD group showed more intense hyperemia than did the SD group (p = 0.01) (Fig. 5g, h). Although the other variables did not show statistically significant differences, the proliferation of fibroblasts was higher in the LHD group compared with LSD group (p > 0.05). The collagen fibers showed less deposition in HD in comparison to the LHD (p > 0.05).

Discussion

In our study, not all rats exposed to HD became overweight. On the other hand, they had significantly increased retroperitoneal fat mass, which is considered positively correlated with increased overall morbidity and mortality linked to obesity. The risk of developing obesity-associated diseases seems to be more associated with the distribution of fat into specific regional depots than to the total adipose tissue mass [20]. It has been shown that obesity can lead to a reduction in the amount of food consumed in response to the high levels of leptin in obese rats [21]. The leptin, a hormone synthesized and secreted by adipose tissue, is primarily involved in appetite regulation and energy metabolism, in turn indicating to the brain that an adequate intake supply has been reached [22, 23].

A wide variety of studies have demonstrated the benefits of LLLT in various pathological conditions [16, 24, 25, 26, 27]; however, very few studies involve obesity. The choice of the wavelength used in this study was based on the fact that 660 nm has a good superficial absorption [16, 26, 28]. This characteristic is indicated for lesions that affect superficial excisional wounds, since the laser cellular effects are dependent on the wavelength and the doses used, which may influence the healing process, as already well reported in the literature [29, 30, 31].

In this study, we observed that the inflammatory infiltrate was more long-term and intense in the control SD group and HD group compared to their respective irradiated control groups at day 7. During the early stages of a wound, inflammatory cells are important for the debridement of the area and the release of cytokines and growth factors. However, when persistent, these events also increase the production of oxidants, which may lead to oxidative damage to lipids and/or proteins, and consequently, to cellular necrosis, impairing the tissue recovery [29, 30, 31].

Laser light stimulates cells with a functional deficit to proliferate after being irradiated [17, 25]. Thus, in a functional tissue, strong cells grow in an orderly and regular manner, and there is no stimulus for laser action to take place, so the effect on tissue is weak or no therapeutic effect is observed at all. On the other hand, if the tissue is altered, the laser light will act with greater effect on it through cellular function restitution. This could help to explain some results in this study, such as the intensity of inflammation, fibroblast proliferation, and collagen deposition, where better results were found in the irradiated HD group when compared with the irradiated SD groups [32, 33].

When fibroblast proliferation was evaluated in this study, it was observed that increased fibroblast activity was detected within 7 days in the LHD group compared to the LSD group. The laser proliferative action on fibroblasts has been demonstrated previously in the literature [29, 34, 35], and it has been observed that an adequate protocol of the laser can stimulate cellular responses in fibroblasts and promote cell migration and proliferation without causing stress or destruction of the cells [33].

The findings of this study demonstrated that there was no difference in the deposition and distribution of collagen fibers between SD and HD groups at day 7, which is in agreement with results found previously [36]. However, the evaluation of the LHD and HD groups in the same experimental period showed that the laser improved collagen fiber deposition in the LHD group. In this particular study, our results are similar to in vitro [18] and in vivo [37] studies that showed greater promotion of collagen maturation by LLLT during wound healing, which is essential for completion of tissue repair.

The presence of higher intensity and congested vessels in the HD group, compared to the SD group at day 14, was characterized by persistent hyperemia in this group, leading to prolonging of the vascular phase of inflammation. Neovascularization at the beginning of the repair process is crucial to carry oxygen and essential nutrients for tissue formation, with subsequent regression of these vessels in the remodeling stage [5]. The newly formed vessels are responsible for the recruitment of inflammatory cells that allow continuous release of proinflammatory cytokines, which are responsible for the continuation of inflammation. Thus, as seen in previous studies [15, 38], the HD contributed to the accumulation of fat mass and also modified the characteristics of the repair process, extending its inflammatory stage.

It is consistent in the literature that fibroblasts and adipocytes belong to the same cell lineage, as well as chondrocytes, smooth muscle cells, and osteocytes, which are not only correlated but are generally convertible to each other. However, although differentiation of these cell types is frequently irreversible, the transformation of fibroblasts into adipocytes is considered a one-way phenomenon [39]. The evidence of the reverse possibility was presented by De Andrade et al. [40]. Their hypothesis was based on the study periods, which initially showed large adipocytes with multiple fat granules and later on changed into elongated cells, in which the fat granules tended to decrease in size and number as the rough endoplasmic reticulum became increasingly prominent. This fact considered the idea that adipocytes can be transformed into fibroblasts.

The above data could explain this unusual finding in the current study, poorly reported in the literature [37, 41], where the presence of numerous adipocytes in the most superficial portion of the granulation tissue after 7 days of irradiation was found. Numerous works have been using stem cells derived from adipose tissue for wound healing, and due to their pluripotency and ability to differentiate into other cell types, they have been successful [42, 43]. Further studies are needed to clarify this matter.

The effect of LLLT on the proliferation and transdifferentiation of stem cells derived from adipose tissue in neuronal cells has been observed, and although the results have shown that laser therapy did not induce cell proliferation, it accelerated the differentiation of stem cells derived from adipose tissue into neuronal cells [44]. Shen et al. [44] believed that with the adjustment of the dose and time of the laser application and, consequently, achieving optimal parameters, better results could be obtained; hence, new studies in animal models may contribute to the development of cell therapy, which might benefit patients who suffer from stroke.

Since wound healing is a complex process that involves a series of articulated biological events in order to repair tissue, any interference in the organized process can lead to prolongation of tissue repair and thus to numerous health hazards. Research on the use of laser phototherapy LLLT should be encouraged so its parameters could increase standardization and its benefits might expand to various health problems that may interfere with meticulous repair process.

Within the limits of this study, it can be concluded that HD prolonged inflammatory response during wound healing and LLLT was able to improve healing during early phase, especially considering the intensity of inflammation. Moreover, LLLT promoted reestablishment of cellular activities and better tissue recovery when it was applied in tissues of rats fed with HD. Further studies are needed to develop appropriate laser therapy protocols as an auxiliary method of wound repair in obesity models.

Notes

Acknowledgments

The authors gratefully acknowledge the CAPES for the financial support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

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Copyright information

© Springer-Verlag London 2016

Authors and Affiliations

  • Virgínia Dias Uzêda-e-Silva
    • 1
  • Tania Tavares Rodriguez
    • 2
  • Isadora Almeida Rios Rocha
    • 1
  • Flávia Calo Aquino Xavier
    • 1
  • Jean Nunes dos Santos
    • 1
  • Patrícia Ramos Cury
    • 1
  • Luciana Maria Pedreira Ramalho
    • 1
  1. 1.School of DentistryFederal University of BahiaSalvadorBrazil
  2. 2.Department of Physiology, Health Sciences CenterFederal University of BahiaSalvadorBrazil

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