Lasers Med Sci DOI 10.1007/s10103-015-1745-2
ORIGINAL ARTICLE
Phototherapy improves wound healing in rats subjected to high-fat diet Saulo Nani Leite 1,2 & Marcel Nani Leite 2 & Guilherme Ferreira Caetano 2 & Paula Payão Ovidio 3 & Alceu Afonso Jordão Júnior 3 & Marco Andrey C. Frade 1,2
Received: 25 July 2014 / Accepted: 18 March 2015 # Springer-Verlag London 2015
Abstract This study aimed to compare the phototherapy effects on wound healing in rats submitted to normal and highfat diets. Thirty-six rats received normal lipidic diet (NL) and 36 high lipidic (HL) diet for 45 days. The nutritional status was measured by body mass, blood glucose, total cholesterol, and triglycerides levels. Four experimental groups were performed according light (L) therapy applied Bon^ or Boff^ (660 nm, 100 mW, 70 J/cm2, 2 J) on 1.5-mm-punched dorsum skin wounds as NLL+, NLL−, HLL+, and HLL−. The wound healing rate (WHR) and oxidative stress markers were analyzed on 2nd, 7th, and 14th days. Despite no difference among body mass, the HL rats presented higher blood glucose, total cholesterol, and triglycerides levels than NL rats. Respectively, on the 2nd and 14th days, the HLL+ group presented the highest WHRs (0.38±0.16/0.97±0.02) among all groups, while the HLL− (−0.002±0.12/0.81±12.1) the lowest WHRs. Hydroxyproline level was lower in HLL− (6.41±1.09 μg/mg) than HLL+ (7.71 ± 0.61 μg/mg) and also NLL+ (9.33 ± 0.84 μg/mg). HLL+ presented oxidative stress markers similar to normal control group (NLL−) during follow up and highest
* Marco Andrey C. Frade [email protected] 1
Program Interunities in Bioengineering at Engineering School of São Carlos, Institute of Chemistry of São Carlos and Ribeirão Preto Medical School, University of São Paulo (USP), Bandeirantes Avenue, 3900, Ribeirão Preto, São Paulo 14049-900, Brazil
2
Dermatology Division, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo (USP), Bandeirantes Avenue, 3900, Ribeirão Preto, São Paulo 14049-900, Brazil
3
Clinical Nutrition Division, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo (USP), Bandeirantes Avenue, 3900, Ribeirão Preto, São Paulo 14049-900, Brazil
antioxidant defense on 7th day. The results showed phototherapy accelerated the cutaneous wound healing by modulating oxidative stress in rats with metabolic disorders under a highfat diet.
Keywords Wound healing . Diet . High fat . Oxidative stress . Phototherapy
Introduction Cutaneous wound healing is characterized as a complex, dynamic, and high metabolic demand used to restore damaged tissue [1–3]. During this process, inflammatory cells (such as neutrophils), and reactions occurring in the mitochondrial respiratory chain release reactive oxygen species (ROS) in the form of free radical (O−, OH−, O−2) and non-free radical molecules such as H2O2 [4–6]. These low concentrations of ROS participate in cellular signaling during the repair process, especially in the inflammatory phase [6–8]. However, when released in high concentrations, ROS can cause cell damage through lipid peroxidation of membranes, and during the generation of hydroxyl radicals, and in the presence of iron or copper ions (Fenton reaction) modification of proteins and DNA. The antioxidant system performs the process of detoxification of ROS by means of various antioxidants low in endogenous and exogenous (catalases, peroxidases, glutathione) molecular weight. If detoxification is insufficient or ROS is released in bulk, it promotes an environment of oxidative stress [7–10]. Systemic disorders such as infections, malnutrition, metabolic diseases (diabetes mellitus, metabolic syndrome, and obesity) among others, increase the concentrations of ROS, and consequently, alter the process of wound healing [11–14]. In these situations, the inflammatory phase suffers prolonged
Lasers Med Sci
disturbances in the process of angiogenesis, a reduction of granulation tissue, and a delay in epithelialization. In addition, it alters the cellular redox environment; this causes oxidative stress which leads to important cellular changes [11, 15]. The development of metabolic diseases may be triggered by a high-calorie diet. Many studies use a high-fat diet in animals to mimic these metabolic changes in humans [15–17]. There are several treatments for healing ulcers, and in recent decades, phototherapy has been described as a valuable tool in stimulating tissue healing. That the absorbed energy (photon) is transformed into ATP [18–20] and used by the cell to power needed metabolic activities, such as cell proliferation [21, 22], collagen synthesis [22, 23], and overall acceleration of tissue repair in experimental animal studies and humans with chronic wounds [22–25]. Additionally, some phototherapy studies in vitro and in vivo have suggested that oxidative stress (such as diabetes, metabolic syndrome, obesity, malnutrition) promotes better tissue outcomes than was found in metabolically normal subjects [19, 26–28]. Despite the beneficial effects mentioned, few pathological experimental models (diabetes, obesity, malnutrition) to mimic the interaction of these systemic disorders utilize phototherapy-induced tissue healing. The aim of this study was to evaluate the effects of phototherapy on oxidative stress in the healing of skin ulcers in rats, as well as to observe the effects of oxidative stress in rats with a high-fat diet in order to induce metabolic oxidative stress Bstatus.^
Material and methods Animals Seventy-two Wistar adult male rats (Rattus norvegicus) weighing 180–200 g were obtained from the Central Animal Facility of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil. The animals were housed in individual polyethylene cages with stainless steel lids. The environmental temperature was maintained with an automatic air exhaust at a constant temperature of 22 °C, and with a relative humidity of 60 %. Artificial light was provided in an alternating cycle of darkness over a 12-h (6–18 h) period. The animals received water ad libitum. This project is in accordance with the Ethics Committee on Animal Experimentation (CETEA), at FMRP-USP, Case Number 210/2011. Induction and confirmation of the experimental model Initially, 36 rats randomly received a control diet (AIN-93), and the remaining 36 rats received a high-fat diet with a 50 % saturation of swine fat [29]. The two groups were monitored for 45 days. On day 0 and on day 45, body mass was measured in order to determine the nutritional status of the animals.
Furthermore, on the 45th day after 8 h of fasting, a tail blood sample was collected for analysis. Blood glucose levels were ascertained using a calibrated glucose meter OptiumTM XceedTM (MediSense®—Victoria, Australia; USA). The total cholesterol, HDL, and triglycerides were obtained from blood collected from animals sacrificed on the second postoperative day. Additionally, after the sacrificed of the rats, the livers were macroscopically and microscopically analyzed to confirm the hepatic steatosis status. Surgical procedure and standardization groups After 45 days, the animals were anesthetized with 2.5 % tribromoethanol intraperitoneally (100 μL/10 g bodyweight; Sigma, USA). The dorsum of all rats was shaved, and after asepsis with 70 % ethanol, two 15-mm-diameter circular cutaneous wounds were punched on the back of the rats. The wounds were maintained without any dressing, and the animals were housed individually in cages just after their recovering from anesthesia. The animals were randomly divided into four groups of NLL− (normolipidic laser off), NLL+ (normolipidic laser on), HLL− (hyperlipidic laser off), and HLL+ (hyperlipidic laser on). Irradiation protocol A solid semiconductor laser—at a wavelength of 660 nm and a power of 100 mW—was used in the application of the phototherapy Flash Laser III (DMC Equipment, São Carlos, SP, Brazil). To avoid contamination, each ulcer was covered with a 100 % transparent plastic film before applying the laser perpendicular to the ulcer in a stationary mode. The ulcers were irradiated with a total energy of 2 J, an energy density of 70 J/cm2, and a time of 25 s. The laser was applied punctually on each wound bed, immediately after the surgery and then three times per week for 14 days. Evaluation of re-epithelialization of wounds Wounds (n=12 wounds/time/treatment) were photographed using a Sony digital camera DSC-P41 (Sony Corporation, Tokyo, Japan) set on a standardized support for acquiring perpendicular wounds images from a distance of 30 cm on days 0, 2, 7, and 14 of the treatment. The images were analyzed using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). The images were used to draw the wounded areas and subsequently to calculate the wound healing rates (WHRs) with the formula = WHR (initial area −final area/initial area), where WHR=0 is the unchanged area, WHR>0 is the decreased area, WHR
ORIGINAL ARTICLE
Phototherapy improves wound healing in rats subjected to high-fat diet Saulo Nani Leite 1,2 & Marcel Nani Leite 2 & Guilherme Ferreira Caetano 2 & Paula Payão Ovidio 3 & Alceu Afonso Jordão Júnior 3 & Marco Andrey C. Frade 1,2
Received: 25 July 2014 / Accepted: 18 March 2015 # Springer-Verlag London 2015
Abstract This study aimed to compare the phototherapy effects on wound healing in rats submitted to normal and highfat diets. Thirty-six rats received normal lipidic diet (NL) and 36 high lipidic (HL) diet for 45 days. The nutritional status was measured by body mass, blood glucose, total cholesterol, and triglycerides levels. Four experimental groups were performed according light (L) therapy applied Bon^ or Boff^ (660 nm, 100 mW, 70 J/cm2, 2 J) on 1.5-mm-punched dorsum skin wounds as NLL+, NLL−, HLL+, and HLL−. The wound healing rate (WHR) and oxidative stress markers were analyzed on 2nd, 7th, and 14th days. Despite no difference among body mass, the HL rats presented higher blood glucose, total cholesterol, and triglycerides levels than NL rats. Respectively, on the 2nd and 14th days, the HLL+ group presented the highest WHRs (0.38±0.16/0.97±0.02) among all groups, while the HLL− (−0.002±0.12/0.81±12.1) the lowest WHRs. Hydroxyproline level was lower in HLL− (6.41±1.09 μg/mg) than HLL+ (7.71 ± 0.61 μg/mg) and also NLL+ (9.33 ± 0.84 μg/mg). HLL+ presented oxidative stress markers similar to normal control group (NLL−) during follow up and highest
* Marco Andrey C. Frade [email protected] 1
Program Interunities in Bioengineering at Engineering School of São Carlos, Institute of Chemistry of São Carlos and Ribeirão Preto Medical School, University of São Paulo (USP), Bandeirantes Avenue, 3900, Ribeirão Preto, São Paulo 14049-900, Brazil
2
Dermatology Division, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo (USP), Bandeirantes Avenue, 3900, Ribeirão Preto, São Paulo 14049-900, Brazil
3
Clinical Nutrition Division, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo (USP), Bandeirantes Avenue, 3900, Ribeirão Preto, São Paulo 14049-900, Brazil
antioxidant defense on 7th day. The results showed phototherapy accelerated the cutaneous wound healing by modulating oxidative stress in rats with metabolic disorders under a highfat diet.
Keywords Wound healing . Diet . High fat . Oxidative stress . Phototherapy
Introduction Cutaneous wound healing is characterized as a complex, dynamic, and high metabolic demand used to restore damaged tissue [1–3]. During this process, inflammatory cells (such as neutrophils), and reactions occurring in the mitochondrial respiratory chain release reactive oxygen species (ROS) in the form of free radical (O−, OH−, O−2) and non-free radical molecules such as H2O2 [4–6]. These low concentrations of ROS participate in cellular signaling during the repair process, especially in the inflammatory phase [6–8]. However, when released in high concentrations, ROS can cause cell damage through lipid peroxidation of membranes, and during the generation of hydroxyl radicals, and in the presence of iron or copper ions (Fenton reaction) modification of proteins and DNA. The antioxidant system performs the process of detoxification of ROS by means of various antioxidants low in endogenous and exogenous (catalases, peroxidases, glutathione) molecular weight. If detoxification is insufficient or ROS is released in bulk, it promotes an environment of oxidative stress [7–10]. Systemic disorders such as infections, malnutrition, metabolic diseases (diabetes mellitus, metabolic syndrome, and obesity) among others, increase the concentrations of ROS, and consequently, alter the process of wound healing [11–14]. In these situations, the inflammatory phase suffers prolonged
Lasers Med Sci
disturbances in the process of angiogenesis, a reduction of granulation tissue, and a delay in epithelialization. In addition, it alters the cellular redox environment; this causes oxidative stress which leads to important cellular changes [11, 15]. The development of metabolic diseases may be triggered by a high-calorie diet. Many studies use a high-fat diet in animals to mimic these metabolic changes in humans [15–17]. There are several treatments for healing ulcers, and in recent decades, phototherapy has been described as a valuable tool in stimulating tissue healing. That the absorbed energy (photon) is transformed into ATP [18–20] and used by the cell to power needed metabolic activities, such as cell proliferation [21, 22], collagen synthesis [22, 23], and overall acceleration of tissue repair in experimental animal studies and humans with chronic wounds [22–25]. Additionally, some phototherapy studies in vitro and in vivo have suggested that oxidative stress (such as diabetes, metabolic syndrome, obesity, malnutrition) promotes better tissue outcomes than was found in metabolically normal subjects [19, 26–28]. Despite the beneficial effects mentioned, few pathological experimental models (diabetes, obesity, malnutrition) to mimic the interaction of these systemic disorders utilize phototherapy-induced tissue healing. The aim of this study was to evaluate the effects of phototherapy on oxidative stress in the healing of skin ulcers in rats, as well as to observe the effects of oxidative stress in rats with a high-fat diet in order to induce metabolic oxidative stress Bstatus.^
Material and methods Animals Seventy-two Wistar adult male rats (Rattus norvegicus) weighing 180–200 g were obtained from the Central Animal Facility of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil. The animals were housed in individual polyethylene cages with stainless steel lids. The environmental temperature was maintained with an automatic air exhaust at a constant temperature of 22 °C, and with a relative humidity of 60 %. Artificial light was provided in an alternating cycle of darkness over a 12-h (6–18 h) period. The animals received water ad libitum. This project is in accordance with the Ethics Committee on Animal Experimentation (CETEA), at FMRP-USP, Case Number 210/2011. Induction and confirmation of the experimental model Initially, 36 rats randomly received a control diet (AIN-93), and the remaining 36 rats received a high-fat diet with a 50 % saturation of swine fat [29]. The two groups were monitored for 45 days. On day 0 and on day 45, body mass was measured in order to determine the nutritional status of the animals.
Furthermore, on the 45th day after 8 h of fasting, a tail blood sample was collected for analysis. Blood glucose levels were ascertained using a calibrated glucose meter OptiumTM XceedTM (MediSense®—Victoria, Australia; USA). The total cholesterol, HDL, and triglycerides were obtained from blood collected from animals sacrificed on the second postoperative day. Additionally, after the sacrificed of the rats, the livers were macroscopically and microscopically analyzed to confirm the hepatic steatosis status. Surgical procedure and standardization groups After 45 days, the animals were anesthetized with 2.5 % tribromoethanol intraperitoneally (100 μL/10 g bodyweight; Sigma, USA). The dorsum of all rats was shaved, and after asepsis with 70 % ethanol, two 15-mm-diameter circular cutaneous wounds were punched on the back of the rats. The wounds were maintained without any dressing, and the animals were housed individually in cages just after their recovering from anesthesia. The animals were randomly divided into four groups of NLL− (normolipidic laser off), NLL+ (normolipidic laser on), HLL− (hyperlipidic laser off), and HLL+ (hyperlipidic laser on). Irradiation protocol A solid semiconductor laser—at a wavelength of 660 nm and a power of 100 mW—was used in the application of the phototherapy Flash Laser III (DMC Equipment, São Carlos, SP, Brazil). To avoid contamination, each ulcer was covered with a 100 % transparent plastic film before applying the laser perpendicular to the ulcer in a stationary mode. The ulcers were irradiated with a total energy of 2 J, an energy density of 70 J/cm2, and a time of 25 s. The laser was applied punctually on each wound bed, immediately after the surgery and then three times per week for 14 days. Evaluation of re-epithelialization of wounds Wounds (n=12 wounds/time/treatment) were photographed using a Sony digital camera DSC-P41 (Sony Corporation, Tokyo, Japan) set on a standardized support for acquiring perpendicular wounds images from a distance of 30 cm on days 0, 2, 7, and 14 of the treatment. The images were analyzed using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). The images were used to draw the wounded areas and subsequently to calculate the wound healing rates (WHRs) with the formula = WHR (initial area −final area/initial area), where WHR=0 is the unchanged area, WHR>0 is the decreased area, WHR
