|Year : 2015 | Volume
| Issue : 1 | Page : 23-30
Effect of low-level laser therapy on gene expression of vascular endothelial growth factor and interleukin-1 ß in scalpel-induced and laser-induced oral wounds in rats
Inas S.M. Sayed1, Ali Saafan2, Fagr Kh Abdel-Gawad3, Tarek A Harhash2, Mostapha A Abdel-Rahman1
1 Department of Orodental Genetics, National Research Centre, El Buhouth St., Dokki, Giza, Egypt
2 Department of Medical Laser Application, National Institute of Laser Enhanced Sciences, Cairo University, Gamaa St., Giza, Egypt
3 Department of Water Pollution Research, Centre of Excellence for Advanced Science, National Research Centre, El Buhouth St., Dokki, Giza, Egypt
|Date of Web Publication||22-May-2015|
Dr. Tarek A Harhash
Department of Medical Laser Application, National Institute of Laser Enhanced Sciences, Cairo University, Gamaa St. 12613, Giza
Source of Support: None, Conflict of Interest: None
Introduction: The effect of low-level laser therapy (LLLT) on wound healing has been evaluated in several studies; however, little is known about the molecular mechanisms that underlies the biostimulatory effect of this treatment modality. Aim: to evaluate the effect of LLLT on gene expression of vascular endothelial growth factor (VEGF) and interleukin-1beta (IL-1 b) during healing of wounds created by surgical blade and by surgical laser in order to have a better image of their interactive role influenced by the laser irradiation. Materials and Methods: 40 male Wister rats were randomly assigned into four groups (A, B, C and D). In groups A and C, an incision was made in the gingival and mucosal tissues using a surgical scalpel. A similar incision was made in groups B and D using 980 nm diode laser. Group A and B were subjected twice to low level 870 nm diode laser 3 h and 24 h after the incision while group C and D served as controls. 30 min after the second irradiation, the rats belonging to all groups were euthanized, and the wound area was excised. Quantitative reverse-transcriptasepolymerase chain reaction was used to measure the gene expression of VEGF and IL-1b. Results and Discussion: LLLT caused an increase in VEGF gene expression in scalpel-induced wounds and a corresponding decrease in laser-induced wounds but without significant differences. LLLT inhibited the gene expression of IL-1b in both types of wounds, but this inhibition was only significant in scalpel wounds. Conclusion: Biostimulatory effect of LLLT could be mediated through modulation of the immune response by the inhibition of IL-1b.
Keywords: Interleukin-1 b, laser biostimulation, low-level laser therapy, vascular endothelial growth factor
|How to cite this article:|
Sayed IS, Saafan A, Abdel-Gawad FK, Harhash TA, Abdel-Rahman MA. Effect of low-level laser therapy on gene expression of vascular endothelial growth factor and interleukin-1 ß in scalpel-induced and laser-induced oral wounds in rats. J Dent Lasers 2015;9:23-30
|How to cite this URL:|
Sayed IS, Saafan A, Abdel-Gawad FK, Harhash TA, Abdel-Rahman MA. Effect of low-level laser therapy on gene expression of vascular endothelial growth factor and interleukin-1 ß in scalpel-induced and laser-induced oral wounds in rats. J Dent Lasers [serial online] 2015 [cited 2019 Apr 25];9:23-30. Available from: http://www.jdentlasers.org/text.asp?2015/9/1/23/157595
| Introduction|| |
Wound healing involves a complex series of consecutive, but overlapping, stages. Simplified, the process of wound healing is often subdivided into three phases: Inflammation, granulation formation, and matrix formation and remodeling. 
Cytokines and other growth factors flood the wound site after injury to mediate the inflammatory response, promote new cell growth, and decrease wound contraction and scarring.  Growth factors and cytokines are the signaling peptides involved in the regulation of various physiological responses. Numerous cell types including macrophages, neutrophils, lymphocytes and fibroblasts produce an excess of growth factors and cytokines. In return, these molecules control in part, their survival, proliferation and migration.  An upregulation of growth-factor expression after injury is frequently observed, suggesting a need for high growth factor levels during the repair process. There are many examples where abnormal growth-factor expression is associated with impaired wound healing or excessive scarring, indicating that a correct temporal and spatial expression of these genes is essential for normal repair. 
Vascular endothelial growth factor (VEGF), a critically important mediator of vasculogenesis, is a homodimeric 34-42 kD heparin-binding glycoprotein.  VEGF is the only specific mitogen for endothelial cells. It is produced by the endothelial cells, fibroblasts, smooth muscle cells, and the majority of inflammatory cells (macrophages, lymphocytes, neutrophils and eosinophils).  VEGF gene and protein are highly expressed in the early stage of skin injury with strong expression 1 day after injury.  VEGF stimulates wound healing via multiple mechanisms including collagen deposition, angiogenesis and epithelization. 
Interleukin 1 b (IL-1 b) is a pro-inflammatory cytokine known to be upregulated during the early phases of inflammation and the highest levels of this cytokine have been observed as early as 12-24 h after cutaneous injury. It has been reported to be predominantly expressed in neutrophils during the early phases of wound healing, followed by macrophages in the later stages of wound repair. IL-1 b can also stimulate the expression of growth factors including transforming growth factor-b, VEGF, platelet-derived growth factor and keratinocyte growth factor.  Secretion of IL-1 b within the wound bed can stimulate the production of secondary inflammatory mediators such as interleukin 6 (IL-6), IL-8, and prostaglandin E2. These mediators are part of a well-integrated inflammatory response that coordinates subsequent activity in the wound bed. Although inflammation is an integral component of wound healing, excessive inflammation has been linked to abnormal wound healing outcomes, specifically fibrosis and scar formation. On the other hand, diminished inflammatory reaction to injury, as in the case of fetal wound healing, is associated with a regenerative wound healing process that lacks scar formation and fibrosis.  IL-1 b levels are elevated in chronic wounds. In addition, infection that is common in chronic wounds further contributes to prolonged inflammation. 
The type of incisional instrument used to create a surgical wound can influence the rate of wound healing and overall wound strength.  The ability of lasers to cut, cauterize, and destroy tissue is well-known throughout the medical world. However, its role at lower power is less well appreciated. The ability of laser to nonthermally and nondestructively change cell function is known as laser biostimulation and is the basis for the current use of lasers in a number of medical fields. 
Laser enhanced biostimulation has been established to induce intracellular metabolic changes, resulting in faster cell division, rapid matrix production, and cell movement. The reported effects of low-level laser therapy (LLLT) included acceleration of wound healing, enhanced remodeling and repair of bone, pain suppression, anti-inflammatory functions, stimulation of endorphin release, modulation of immune system, a faster regeneration of severed nerves, and an increase in the formation of new capillaries through the release of growth factors. 
Since cytokines and growth factors have important roles in the regulation of different stages of repair process, the purpose of this study was to evaluate the effect of low-level diode laser on gene expression of VEGF and IL-1 b, during healing of wounds created by surgical blade and by surgical laser. This can improve our understanding of their interactive role influenced by the laser irradiation in wound healing.
| Materials and Methods|| |
Animal model and experimental groups
This study was conducted in compliance with medical research ethical committee, National Research Centre, Egypt (Ethics Registration Number 09/107).
Totally, 40 healthy male Wistar rats with weight ranging from 400 to 450 g were obtained from the Animal House, National Research Centre. The rats were randomly assigned into four groups; group A, B, C, and D (n = 10 for each group). Group A, and B are the case groups while group C, and D are the controls. The rats were anesthetized with diethyl ether. An incision was made on the gingival and mucosal tissues of the labial surface of mandibular incisors in each rat, 2 mm deep and 5 mm in length. The incision was created using No. 15 surgical blade in groups A and C. while in groups B, and D, it was made using 980 nm diode surgical laser [Figure 1] and [Figure 2]. Three hours after incision, the rats in group A and B were sedated again, and the wounds were exposed to low power diode laser irradiation [Figure 3]. The rats of the control group C and D did not receive low power laser irradiation. Twenty-four hours after primary incision, the rats in group A and B were irradiated for the second time with the same procedure. Thirty minutes after completion of the laser therapy, the rats belonging to all groups were then euthanized with the appropriate dose of diethyl ether. Excisional biopsy was taken from the site of the incision, and the excised tissues were snap frozen in liquid nitrogen and stored at −80°C.
|Figure 1: Incision on the gingival and mucosal tissues of the labial surface of mandibular incisors using #15 surgical scalpel|
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|Figure 2: Incision on the gingival and mucosal tissues of the labial surface of mandibular incisors using surgical diode laser|
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Laser devices and parameters
The incisions in group B and D were made using diode laser (Quanta C, Italy) with wavelength of 980 nm. The laser was set in a continuous wave (CW) mode at a power output of 2.5 W with a 320 mm diameter fiber tip, and it was operated in contact mode. Low-power laser irradiation in groups A, and B were performed using a diode laser (Soft-Laser SL-202, Petrolaser, Russia) with wavelength of 870 nm in CW mode. The total irradiation time was 120 s with power output of 80 mW with energy density of 9.6 J/cm 2 , delivered through K30 tip type, which has 0.03 cm 2 irradiance surface in noncontact mode at a distance 0.5 cm.
Measurement of cytokines and growth factors
RNA extraction and cDNA synthesis
RNA extraction was carried out using Qiagen RNeasy ® mini kit following the manufacturer's protocol. This was, followed by determination of quality and quantity of RNA using NanoDrop 2000 Spectrophotometer (NanoDrop Technologies, USA). Synthesis of single-stranded cDNA was carried out using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). One microgram of total RNA was reverse transcribed into 20 ml cDNA.
Quantitative reverse-transcriptase polymerase chain reaction
Real-time polymerase chain reaction (PCR) reactions were performed in StepOne Real-Time PCR System (Applied Biosystems, USA).
The sequence of Forward (F) and reverse (R) primers purchased from Invitrogen are listed in [Table 1]. b-actin was used as a constitutively expressed gene product for comparison of cytokines and growth factors' mRNA abundance between samples.
Thermocycling was done in a final volume of 25 ml (8 ml DEPC H 2 O, 2.5 ml cDNA, 1 ml of 10 pM Forward and reverse primers, and 12.5 ml of The QuantiTect SYBR ® Green Master Mix) using the following PCR conditions: For b-actin, and VEGF: The thermocycler was run for 1 cycle (95°C for 10 min), 45 cycles (95°C for 15 s, 60°C for 1 min) and 1 cycle (95°C for 15 s, 60°C for 1 min). For Il-1 b: 1 cycle (95°C for 10 min), 45 cycles (95°C for 15 s, 55°C for 1 min) and 1 cycle (95°C for 15 s, 60°C for 1 min).
The values of mean, standard deviation, standard error of the mean were calculated using the SPSS 18 software (The manufacturer details of SPSS 18 Software is (SPSS Inc., Chicago, IL, USA). The obtained data were tested for normality using Kolmogorov-Smirnov test of normality. The one way ANOVA (parametric test) was used when the data were normally distributed, while, the Kruskal-Wallis test (nonparametric tests) was used for the comparisons of means between groups when data were not normally distributed. Whenever the result of the ANOVA or Kruskal-Wallis test was significant it was, followed by a Tukey HSD tests for pair-wise comparisons among groups. Differences were considered statistically significant if the P < 0.05 [Figure 3].
| Results|| |
To assess the differences in cytokine mRNA expression between different groups, b-actin was used as a reference house-keeping gene and cytokine mRNA levels were normalized to b-actin. Kruskal-Wallis test was used for the comparisons of means of VEGF between groups as the data were not normally distributed whereas one-way ANOVA was used for IL-1 b (normally distributed data) [Table 2]. The comparison among the IL-1 b groups turned out to be highly significant with P = 0.000. The results of the pair-wise comparisons revealed that the cause of significance was (group A) [Table 3].
Effect of low-level laser therapy on gene expression of vascular endothelial growth factor
Laser irradiation of incisions created with a scalpel caused an increase in gene expression of VEGF as (group A) showed higher expression of VEGF than (group C). However, this increase was nonsignificant. There was a nonsignificant decrease in (group B) which received LLLT when compared with the control group (D) which did not receive LLLT. When comparing (group B [laser-incision and LLLT]) with (group C [Scalpel incision with no LLLT]), there was a decrease in VEGF expression. This decrease was statistically nonsignificant [Figure 4].
|Figure 4: Expression of the vascular endothelial growth factor mRNA normalized to β-actin mRNA (means ± standard error of mean) (*P < 0.05)|
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Effect of low-level laser therapy on gene expression of IL-1 b
Laser phototherapy caused marked inhibition of IL-1 b expression and the decrease in mRNA level in (group A) was highly significant in comparison with (group C). Although there was a decrease in mRNA levels of IL-1 b in laser-induced wounds submitted to LLLT (group B) in relation to (group D [laser incision without LLLT]), this decrease was not statistically significant. Gene expression of IL-1 b in (group B [laser incision and LLLT]) was slightly lower than that of (group C [scalpel incision and no LLLT]) but without significant difference [Figure 5].
|Figure 5: Expression of the interleukin-1 β mRNA normalized to β-actin mRNA (means ± standard error of mean) (*P < 0.05)|
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| Discussion|| |
The 980 nm wavelength used in this study for surgical incision has many advantages in oral and maxillofacial surgeries. It provides precise cutting similar to CO2 laser and coagulation properties comparable to Nd: YAG laser.  Both red and infrared (IR) wavelength have been used extensively in laser biostimulation. IR laser was employed in our study, which according to some studies showed more favorable effect on wound healing. ,
Whereas the laser therapy plays an important role during tissue repair, the biological mechanisms involved to this phenomenon are not completely elucidated. Because VEGF and IL-1 b are important mediators in wound healing, the study of changes in their expressions provides a valuable mean to study the effect of LLLT on the healing process. As surgical laser wounds involve a different healing pattern due to thermal damage, presence of necrotic zone, greater inflammatory reaction and delayed epithelialization, ,, we extend our research of biostimulatory effect of LLLT to include both scalpel and laser induced surgical wounds.
Several studies reported stimulatory effect of LLLT on VEGF expression. Enhanced expression of VEGF in cultured gingival fibroblast, when subjected to LLLT were found by Basso et al.  and Hakki and Bozkurt.  Similarly, irradiation of cultured endothelial cells with low-level laser caused upregulation of VEGF expression.  Lee et al.  examined the effect of the drug alendronate on oral keratinocytes and the effect of LLLT on alendronate-treated keratinocytes. Alendronate was found to inhibit the keratinocyte viability and suppress its expression of VEGF resulting in defective wound healing. Irradiation of alendronate-treated keratinocytes with LLLT increased their expression of VEGF. They suggested that LLLT could be used to partially overcome the inhibitory effects of this drug on mucosal wound healing.
Pereira et al.  and Cury et al.  reported that experimental surgical wound in rats exposed to LLLT exhibited more VEGF expression. Immunohistochemical analysis of Rats' masseter muscles subjected to LLLT of different settings revealed more intense staining for VEGF when compared to controls. 
On the other hand, the inhibitory effect of LLLT on VEGF expression was reported by Silva et al.  who compared the gene expression of VEGF in three groups of rats; the first group: Nonoperated animals, the second group: Operated animals in which a surgical wound was induced in the tongue and the third group operated animals subjected to 780 nm diode laser (70 mW, 35 J/cm 2 ). One day after surgery, the trauma-induced by the surgical wound in the second group caused an elevation in VEGF expression when compared to the nonoperated first group. Exposure of the surgical wounds in the third group to LLLT reduced the level of VEGF when compared to the second groups and returned it to levels comparable to the first nonoperated group. These authors used relatively high energy density (35 J/cm 2 ) in contrast to 9.6 J/cm 2 energy density used in our study, which could explain the contradictory results. This assumption is supported by the finding reported by Kipshidze et al.  that high energies of low-level laser were cytotoxic to vascular cells. In the current study, analysis of VEGF expression did not reveal statistically significant difference among different groups.
When we measured the gene expression of pro-inflammatory cytokine IL-1 b, we found that LLLT markedly inhibited its expression in scalpel-induced wounds. Our results are in accordance with several studied which showed inhibitory effect of LLLT on IL-1 b expression. Reduction in IL-1 b expression was noted in cultures of smooth muscle cells,  gingival fibroblasts  , as well as normal, wounded and diabetic skin fibroblasts.  Safavi et al.  and Lima et al.  reported that laser irradiation inhibited IL-1 b expression in experimental surgical wounds in rats. Viegas et al.  compared the anti-inflammatory effect of two types of laser (685 and 830 nm) on the IL-1 b mRNA levels in rats' surgical wounds with the anti-inflammatory drug Meloxicam. The Meloxicam treated group significantly inhibited IL-1 b expression. While the irradiated animals did not show results similar to those of the group that received meloxicam, the levels of IL-1 b mRNA expression detected in the irradiated animals was lower than that of the control group. de Almeida et al.  compared the anti-inflammatory effect of sodium diclofenac (topical application), cryotherapy, and LLLT on pro-inflammatory cytokine levels after a controlled model of muscle injury. Only LLLT of the three treatment modalities reduced IL-1 b expression to a significant level when compared to the controls.
Some investigators measured the levels of IL-1 b in inflammatory disorders and conditions in order to evaluate the anti-inflammatory effect of low-level laser. Yamaura et al.  found a significant decrease in both the mRNA and intracellular protein levels of IL-1 b in cultured synoviocytes isolated from rheumatoid arthritis patients when exposed to 810 nm diode laser. Experimental rats subjected to lipopolysaccharide-induced pulmonary inflammation, when exposed to LLLT showed marked decrease in IL-1 b protein concentration in bronchoalveolar lavage and lungs as well as inhibition of IL-1 b mRNA expression in trachea and lungs. ,
Wu et al.  investigated the anti-inflammatory effect of LLLT on human adipose-derived stem cells in an inflammatory environment. Lipopolysaccharide treatment of the cells strongly induced the expression of pro-inflammatory genes including IL-1 b. However, when the cells were treated with a combination of LLLT and lipopolysaccharide, the mRNA levels of IL-1 b were dramatically decreased. Alves et al.  exposed experimental rats with osteoarthritis to low power 880 nm diode laser. There was a reduction in IL-1 b mRNA expression in articular synovial lavage to values obtained in the control group (rats without osteoarthritis). LLLT has been shown to promote a decrease in mRNA expression of IL-1 b in rats' paw muscle in which inflammation was induced through the injection with carrageenan.  Yang et al.  studied the possible role of LLLT on counteracting the inflammatory effect of amyloid b-peptides on astrocytes, which is thought to mediate the progression of Alzheimer's disease. Western blot analyses of IL-1 b showed that stimulation of astrocytes by amyloid b-peptides led to a significant increase in the expressions of IL-1 b, while laser pretreatment suppressed the expressions to the control level.
We found a decrease in IL-1 b expression in laser-induced wound, but it was not statistically significant. The lack of significant effect of LLLT on IL-1 b expression in laser-induced wounds might be explained by the need for more intense inflammatory reaction in laser wounds in order to eliminate the dead and devitalized tissue and to prevent infection since the laser incisions are associated with tissue thermal damage and larger necrotic zone. ,
Under the conditions of this study, we can conclude that LLLT has a positive effect on attenuating the inflammatory response in scalpel-induced wounds. This modulation of the immune response may be responsible for the biostimulatory effect of laser therapy.
| Acknowledgment|| |
The authors would like to acknowledge the National Research Center and the National Institute of Laser Enhanced Sciences for providing access to the equipment by which the experiments were conducted. The authors thank Ms. Phoebe Magdy Abd El-Massieh for her effort in statistical data analysis.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]