|Year : 2013 | Volume
| Issue : 1 | Page : 17-21
Blood circulation changes in palatal gingiva before and after low-level laser irradiation in rats
Seda Ozturan1, Oznur Inan2, Aslihan Usumez3
1 Department of Periodontology, Istanbul, Bezmialem Vakif University, Faculty of Dentistry, Istanbul, Turkey
2 Department of Veterinary, Istanbul, Bezmialem Vakif University, Faculty of Dentistry, Istanbul, Turkey
3 Department of Prosthodontics, Istanbul, Bezmialem Vakif University, Faculty of Dentistry, Istanbul, Turkey
|Date of Web Publication||19-Sep-2013|
Department of Periodontology, Faculty of Dentistry, Bezmialem Vakif University, Fatih-34093, Istanbul
Source of Support: None, Conflict of Interest: None
Introduction: Clinical practice with low-level laser therapy (LLLT) has been investigated; however researchers and therapists have questioned the clinical benefits of laser therapy due to divergent results in the literature. Aim of the study: This research evaluated the effects of laser therapy on blood circulation following application of two diode lasers with different wavelengths with laser Doppler flowmetry (LDF). Materials and Methods: In order to evaluate the effectiveness of LLLT on circulation of blood vessels in palatal gingiva, two diode lasers were applied to the palatal gingiva at 2 mm distance to the two upper incisors of 20 rats. Ten rats were irradiated with a wavelength of 810 nm (10 J/cm 2 , Fotona) while other 10 rats were irradiated with wavelength of 660 nm (10 J/cm 2 , Helbo, Bredent) for 7 consecutive days. Rate of blood circulation in the ascending palatine artery was determined with LDF from the same gingival location before and 1 h after irradiation on each day. Data were analyzed by repeated measurements of analysis of variance (ANOVA). Results: The results indicated that blood circulation was significantly increased after 1 h following irradiation with both diode laser types (P < 0.05) and the increase was similar following each consecutive irradiation for 7 days. Conclusion: The irradiation of the palatal gingiva by LLLT enhanced the local blood circulation; however, the amount of increase was limited and similar to following identical applications on consecutive days.
Keywords: Blood circulation, diode laser, low-level laser therapy
|How to cite this article:|
Ozturan S, Inan O, Usumez A. Blood circulation changes in palatal gingiva before and after low-level laser irradiation in rats. J Dent Lasers 2013;7:17-21
|How to cite this URL:|
Ozturan S, Inan O, Usumez A. Blood circulation changes in palatal gingiva before and after low-level laser irradiation in rats. J Dent Lasers [serial online] 2013 [cited 2022 Jan 18];7:17-21. Available from: https://www.jdentlasers.org/text.asp?2013/7/1/17/118427
| Introduction|| |
The last decade has seen an explosion of research work in the application of laser technology to general dental practice. Despite the many advantages which 'hard' or 'hot' surgical lasers (such as CO 2 , neodymium:Yttrium-aluminum-garnet (Nd:YAG) and erbium:YAG (Er:YAG)) offer for both soft tissue surgical and tooth-related procedures; issues such as instrument costs and the potential for thermal injury to dental pulp from thermal changes have limited the uptake of this technology in general dental practice. At the opposite end of the equipment spectrum are the semiconductor diode lasers, which are sometimes referred to as 'cold' or 'soft' lasers. Unlike their high-powered 'hard' surgical laser counter parts, diode lasers are compact, low cost devices, which have very high electrical and optical efficiencies. In medicine and dentistry, diode lasers have been used predominantly for applications which are broadly termed low-level laser therapy (LLLT) or 'biostimulation';  however, there is controversy surrounding the effectiveness of some of these procedures. ,
The mechanisms of LLLT are complex, but essentially rely upon the absorption of particular visible red and near infrared wavelengths in photoreceptors within subcellular components, particularly the electron transport (respiratory) chain within the membranes of mitochondria. , The absorption of light by the respiratory chain components causes a short-term activation of the respiratory chain, and oxidation of the nicotinamide adenine dinucleotide pool. This stimulation of oxidative phosphorylation leads to changes in the redox status of both the mitochondria and cytoplasm of the cell. The electron transport chain is able to provide increased levels of promotive force to the cell, through increased supply of adenosine triphosphate (ATP), as well as an increase in the electrical potential of the mitochondria membrane, alkalization of the cytoplasm, and activation of nucleic acid synthesis.  Because ATP is the "energy currency" for a cell, LLLT has a potent action that results in stimulation of the normal functions of the cell.
Low-level lasers also activate ATP, ATPase, and the conversion of ATP to adenosine. Adenosine stimulates the conversion of cyclic adenosine monophosphate (cAMP) to nitric oxide (NO)  or the vascular endothelial growth factor (VEGF). , Adenosine, growth hormone (GH), FGF, and VEGF are angiogenic factors and promote new vessel growth in the same manner.  Endothelial budding is induced by angiogenic factors, which are secreted by parenchymal cells in hypoxic states, possibly as a product of anaerobic glycolysis formed at the site of these cells and diffused in all directions. , As LLLT has also been shown to cause vasodilation, with increased local blood flow, this vasoactive effect is of relevance to the treatment of inflammation, such as may occur in the temporomandibular joint (TMJ). LLLT causes the relaxation of smooth muscle associated with endothelium. This vasodilation brings in oxygen and also allows for greater traffic of immune cells into tissue. These two effects contribute to accelerated healing. ,,
Furthermore, LLLT can exert vasoactive effects by its actions on mast cells. The effects of different types of light on mast cells are well recognized.  There is direct evidence  that 660, 820, and 940 nm lights can trigger mast cell degranulation. Mast cells are distributed preferentially about the microvascular endothelium in skin, oral mucosa, and dental pulp. , Mast cells in these locations contain the proinflammatory cytokine tumor necrosis factor in their granules.  Release of this cytokine promotes leukocyte infiltration of tissues  by enhancing expression of endothelial-leukocyte adhesion molecules. In addition, mast cell proteases, such as chymase,  alter basement membranes and facilitate entry of leukocytes into tissues. Because mast cells play a pivotal role in controlling leukocyte traffic, modulation of mast cell functions by LLLT can be of considerable importance in the treatment of sites of inflammation in the oral cavity.
The blood flow changes related with vasodilation can be followed-up with laser Doppler flowmetry (LDF). The LDF technique is based on the Doppler principle. Specifically, a laser beam is emitted by an optical fiber to the tissue to be studied. The light hitting moving erythrocytes is scattered back in shifted frequency (Doppler effect) and is captured by one or more optical fibers. The light signals are then converted into electric signals and the resulting photo current is processed to provide a recording of the blood flow. Although the multiple scattering events that determine the propagation of light in tissue prevents absolute velocity measurements when used in vivo, relative blood flow measurements can be obtained. Therefore, the term used to describe blood flow is flux - a quantity proportional to the average speed of the blood cells and their concentration. This is expressed in arbitrary perfusion units (PU), which are linearly related to flux.
The aim of the current study was to find out effectiveness of different wavelengths of laser applications by measuring the blood circulation following application for making use of standardized, validated outcomes; and choosing the correct laser wavelength for true indications.
| Materials and Methods|| |
In the study, effectiveness of different wavelength laser irradiation in stimulating blood flow was investigated. Twenty adult Wistar rats with average body weights of 350-400 g were used. They were divided into two groups: One of the groups irradiated with 810 nm diode laser and the other group treated with 660 nm diode laser (n = 10). Laser applied to each rat under inhalation anesthesia. Both the lasers were applied to rats once for 7 days at the same time. The diode laser of 810 nm was applied for 20 s and 660 nm diode laser for 1 min, each day. Irradiation was done almost 2 mm to distal aspect of the right incisor for each of the rats for 7 days and small probe holders used for standardization of distance of irradiated area from the tooth. Blood flow measurements were done before the irradiation, immediately after irradiation, and 1 h after irradiation of laser every day with LDF (5010 Periflux, Perimed, Jarfalla, Sweden).
A commercially available laser Doppler flowmeter with wavelength 780 nm equipped with a standard probe (PF416 with outside diameter 1.0 mm and fiber separation 0.25 mm) was used for all measurements. The flowmeter time constant was 0.2 s, with an upper bandwidth at 20 kHz and lower bandwidth at 20 Hz. The instruments and fiberoptic probes were calibrated by means of the Perimed PF 1000 Motility Standard according to the manufacturers' specifications before each measurement. The signals were recorded in arbitrary PU and monitored using the Perisoft Software (version 2.10, Perimed AB).
Laser Doppler measures the total local microcirculatory blood perfusion including the perfusion in capillaries (nutritive flow), arterioles, venules, and shunting vessels.
Data were expressed as means ± 15 standard deviation. One-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison tests was used to compare the results. P < 0.05 were considered to be significant. Each set of experiments was repeated twice and analyzed separately, and both sets of experiments yielded comparable results.
| Results|| |
All the rats completed the study. No dropouts occurred and no adverse events were reported during the follow-up period. The effect of different wavelength of low intensity laser daily treatment for 7 days on tissue was expressed as assessment of blood flow rate (BFR) [Table 1]. The BFR was calculated as the value obtained from the device named LDF.
|Table 1: Daily Measurments of BFR before (BA), immediately after (IALA) and 1 hour after (1 H ALA) 810 nm, 660 nm diode laser application (Bonferroni's multiple comparison test)|
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Statistical comparisons showed statistically significant differences in terms of baseline on comparing with the time intervals between the groups and within the groups.
The diameter changes in BFR on the irradiated side are shown in [Table 1]. The diameters after stopping irradiation increased significantly compared to that before irradiation (P < 0.01). The changes in BFR on the irradiated side are shown in [Table 1]. A significant difference was found between before, after stopping, and 1 h after irradiation (P < 0.01). The changes in blood flow volume on the irradiated side are shown in [Table 1]. Statistical analyses revealed a significant difference between groups after stopping irradiation and the 1 h after irradiation measurements compared to that before irradiation (P < 0.01). On the second day, peak increase was higher in 810 nm laser group. In the first 5 days, there was an increase in BFR for both laser types, but more in 810 nm laser group [Table 1]. After that time the subsequent measurements on day 6 and 7, the BFR was same as for the groups.
| Discussion|| |
In the current study, blood circulation was significantly increased 1 h after following irradiation with both diode laser types and the increase was similar following each consecutive irradiation for 5 days. Both the lasers showed a significant increase on the BFR even immediately after irradiation. First 5 days, the lasers increased the BFR but the increase was the highest in 810 nm diode laser group. After the 5 th day, no significant change obtained from irradiated sites. LLLT showed significantly high BFRs between day 1 and 5 day during the treatment, a finding, which correlated with increased rates of microcirculation.
In clinical and experimental studies, the photobiological and photochemical effects of low intensity lasers have shown that low energy irradiation of injured tissues improves wound healing by accelerated epithelialization, high degree of vascularization, increased collagen synthesis, and stimulated fibroblast activity (between days 1 and 3, 7 and 13). Since the rat skin contains three layers, it is frequently used to investigate the phases of wound healing: Inflammation, proliferation, and maturation. Although it not always easy to strictly separate these three phases from each other, the inflammatory phase mostly takes part during the first 5 days, however it was seen to continue for 15 days. , The proliferatory phase generally occurs from 7 th to 15 th day and then the maturation phase takes place. , In the current study, BFR was measured during 7 consecutive days and the results showed that that both the lasers accelerates BFR for 5 days; but after that BFR decreases gradually. In 660 nm laser group, BFR was more consistent than 810 nm groups for 7 days. But in first 5 days, BFR was higher in 810 nm laser group than 660 nm laser group. When someone takes into consideration the long-term BFR effects of lasers for the treatment, it could be necessary to know how effective they are on the blood flow. Because angiogenesis is one of the major factors related to tissue repair; it is involved from the beginning of the healing process, since the vessels are responsible for reestablishing the supply of oxygen and nutrients, allowing an increase in metabolic rate and mitotic activity. Most of the recent studies on laser-induced angiogenesis have been directly or indirectly motivated by the seminal paper of Abergel et al.,  which indicated an angiogenic effect was induced by LLLT. Corazza et al., showed that LLLT with doses of 5-20 J/cm 2 demonstrated expressive results in angiogenesis on 3 rd , 7 th , and 14 th day.  In our study, the significant increase in BFRs was observed during the first 5 days. In the previous studies, blood flow was shown to play a central role in mediating the accelerated healing response.  Although the experimental model was different from the current study, their finding may help to explain the significant increase in BFRs in 5 days in our experiment.
The reasons for different results in studies may arise from differences of the study designs. Important to mention that we measured the total local microcirculatory blood perfusion including the perfusion in capillaries (nutritive flow), arterioles, venules, and shunting vessels whether or not if it is related with angiogenesis or not. In the current study, healthy model was used for searching if the laser really works on BFRs in the absence of any other effective factors. LLLT has been found to accelerate BFR possibly by stimulating oxidative phosphorylation in mitochondria and modulating inflammatory responses. By influencing the biological function of a variety of cell types, it is able to exert a range of several beneficial effects upon inflammation and healing. There is good evidence that the enhanced cell metabolic functions seen after LLLT are the result of activation of photoreceptors within the electron transport chain of mitochondria. The effect is specific for wavelength; and cannot be gained efficiently with normal, noncoherent, and nonpolarized light sources. So it is important to select true wavelength for true indications. Since we studied in healthy rats, it can be concluded that LLLT enhances the BFRs by itself without any related factors that could affect the microcirculation.
Future trials of new LLLT applications in dentistry should make use of standardized, validated outcomes; and should explore how the effectiveness of the LLLT protocol used may be influenced by wavelength, treatment duration, dosage, and the site of application [Figure 1], [Figure 2], [Figure 3].
|Figure 1: Daily BFR measurements before (BA), immediately after (IAA), and 1 h after (1HAA) 810 nm diode laser application|
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|Figure 2: Daily blood flow rate measurements before (BA), IAA, and 1 h after (1HAA) 660 nm diode laser application|
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|Figure 3: Comparison of the effectiveness of the 810 and 660 nm laser wavelength on the blood flow rate (BFR), before (BA) and 1 hour after (1HAA) application|
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| Conclusions|| |
It can be concluded that lasers significantly increase blood flow in first 5 days, but after that time no additional effect could be obtain when considering BFRs. The results indicated that LLLT enhanced microcirculation seemed to be unique in the normalization of functional features of the injured area, which could lead to occlusion of the regional blood vessels.
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[Figure 1], [Figure 2], [Figure 3]