Sri Kunarti,Devi Eka Juniarti,Yulianti Kartini Sunur,Meidi Kurnia Ariani
The different effects of low-level laser therapy before and after overinstrumentation on the expression of substance P and interleukin-10
Substance P,IL-10,Overinstrumentation,Precondition,Postcondition,Laser diode 650 nm
Background One of the causes of endodontic flare-ups is overinstrumentation during canal preparation. In general, to reduce the level of pain and swelling that is caused by endodontic flare-ups, patients it has taken analgesics and antibiotics after treatment. However, some patients have been reported to have allergic reactions to nonsteroidal anti-inflammatory drugs. Lasers have been reported to be significantly effective in reducing pain and inflammation after root canal treatment. Low-level laser therapy (LLLT) 650 nm pre- or post-conditioning is widely used as a therapy. Objective This study investigated the effect of pre- or post-conditioning with a 650 nm diode laser to reduce pain caused by overinstrumentation Methods Thirty incisor teeth from Wistar rats were overinstrumented and exposed to a 650 nm diode laser before or after overinstrumentation and then divided into six groups. Groups I and II were control groups (30 and 120 min), groups III and IV were precondition groups (30 and 120 min) and Groups V and VI were postcondition groups (30 and 120 min). Immunohistochemical analysis was conducted to check the expression of substance P and interleukin-10 (IL-10) Result The expression of substance P in the precondition LLLT groups was significantly lower than that in the control and postcondition groups. On the other hand, the expression of IL-10 in the precondition LLLT groups was significantly higher than that in the control and postcondition groups. Conclusion There was a decrease in pain after preconditioning laser diode 650 nm exposure.
The different effects of low-level laser therapy before and after overinstrumentation on the expression of substance P and interleukin-10

One of the causes of endodontic flare-ups is overinstrumentation during canal preparation. In general, to reduce the level of pain and swelling that is caused by endodontic flare-ups, patients it has taken analgesics and antibiotics after treatment. However, some patients have been reported to have allergic reactions to nonsteroidal anti-inflammatory drugs. Lasers have been reported to be significantly effective in reducing pain and inflammation after root canal treatment. Low-level laser therapy (LLLT) 650 nm pre- or post-conditioning is widely used as a therapy.

This study investigated the effect of pre- or post-conditioning with a 650 nm diode laser to reduce pain caused by overinstrumentation

Thirty incisor teeth from Wistar rats were overinstrumented and exposed to a 650 nm diode laser before or after overinstrumentation and then divided into six groups. Groups I and II were control groups (30 and 120 min), groups III and IV were precondition groups (30 and 120 min) and Groups V and VI were postcondition groups (30 and 120 min). Immunohistochemical analysis was conducted to check the expression of substance P and interleukin-10 (IL-10)

The expression of substance P in the precondition LLLT groups was significantly lower than that in the control and postcondition groups. On the other hand, the expression of IL-10 in the precondition LLLT groups was significantly higher than that in the control and postcondition groups.

There was a decrease in pain after preconditioning laser diode 650 nm exposure.

Endodontic flare-up is a pathological condition with acute symptomatic exacerbation of pulpal or periapical origin that occurs during or after root canal treatment and is characterized by pain or swelling (Sipaviciute and M. Rasmutė, 2014, Arslan et al., 2018). The prevalence of endodontic flare-up after root canal treatment is reported with an incidence of 3% – 58% (Subbiya et al., 2017, Ahangar et al., 2017). Endodontic flare-up cannot be prevented but can be minimized. Some effective ways to decrease the incidence of endodontic flare-up include complete debridement, use of potential intracanal drugs, antibiotics, analgesics, proper diagnosis, determination of the correct length of action, and selection of the right irrigation solution (Tamara., 2019). The etiology of endodontic flare-ups varies from mechanical, to chemical, to microbial. One of the causes of endodontic flare-ups is overinstrumentation during canal preparation. Overinstrumentation is a sharp object lesion that can cause injury to blood vessels and periapical sensory nerve tissue and, as a result, can cause disruption of homeostasis in the periapical area. In general, to reduce the level of pain and swelling caused by endodontic flare-ups, we can use analgesics and antibiotics. Recent studies have shown that the use of nonsteroidal anti-inflammatory drugs (NSAIDs) is effective. However, it has been reported that some patients have allergic reactions to NSAIDs. Currently, pain relief uses various methods, such as acupuncture and acupressure, with low-level laser therapy (LLLT) being widely used (Erkan, 2016). LLLT can reduce C fiber conduction in the pulp, stimulate the release of endorphins and serotonin, and increase oxygenation and lymphatic drainage, resulting in decreased pain sensation (Goyal et al., 2013). LLLT also polarizes M2 macrophages. M2 macrophages are anti-inflammatory mediators that secrete interleukin-10 (IL-10) (Carvalho et al., 2016). LLLT inhibits the synthesis of inflammatory factors and pain-related neurotransmitters. LLLT increases pain-inducing substances, including substance P and inhibits cyclooxygenase-2 (Ahangar et al., 2017). Substance P is a pro-inflammatory neuropeptide and pain biomarker. LLLT with a wavelength of 650 nm for 40 s is an effective time because it increases the proliferation of fibroblast and odontoblast-like cells (Barbosa et al., 2017). In addition, LLLT can control pain and swelling after mandibular third molar odontectomy (Subbiya et al., 2017, Carvalho et al., 2016). LLLT is also effective for pain relief in the treatment of recurrent aphthous stomatitis (Soliman and Mostafaa, 2019). It is still unclear whether LLLT should be given before or after treatment. Some literature states that endodontic flare-up therapy using LLLT is given after root canal treatment (Asnaashari et al., 2017, Nabi et al., 2018). Research on preconditioning with LLLT has not been widely reported. Research by Agrawal (2014). reported that LLLT is more effective if light is delivered to normal cells or tissues before trauma. Low-level laser therapy (LLLT) is a novel branch of medicine that employs low-power lasers in living tissues to stimulate and enhance cell function or relieve pain. The effectiveness of LLLT in reducing pain and swelling, stimulating nerve function, enhancing revascularization and improving the wound healing process has been confirmed in several studies (Eshghpour et al., 2017). The aim of this study is to determine which treatment is more effective as a pain killer on 650 nm diode laser exposure before or after root canal instrumentation. (See Table 3.1)

The research was conducted after obtaining a letter of ethical standard issued by the Ethics Commission of the Faculty of Dental Medicine of Universitas Airlangga with letter number: 216/HRECC.FODM/IV/2020. A total of 30 male white rats (Rattus norvegicus) weighing approximately 200–300 g and 12–18 weeks of age were used in this study. There were three groups as follows: control, precondition, and postcondition groups; each group was divided into two subgroups: 30 and 120 min after treatment groups. In the control groups, 30 (K1) and 120 min (K2) groups, rats were anesthetized with ketamine at 100 mg dosage (Ketalar®, Warner Lambert, Irlandia) and xylazine HCl (Rompun®, Bayer, Leverkusen, Jerman). The maxillary incisors were detached at the level of the interdental papillae using a low-speed handpiece with a fissure bur and the pulp chamber was perforated using a round bur. After the pulp chamber was exposed, C Pilot file number #6 was inserted into the root canal exceeding the apex (overinstrumentation) until the apex locater monitor indicated –3. In the precondition groups, 30 (K3) and 120 min (K4) groups, the sulci of the rats’ maxillary incisors were exposed to a 650 nm laser diode at a distance of 1 cm for 40 s before the overinstrumentation process. Meanwhile, in the post-condition groups, 30 (K5) and 120 min (K6) groups, overinstrumentation was carried out first, then exposed to a 650 nm laser diode at a distance of 1 cm for 40 s (Sampoerno et al., 2019). All rats were euthanized 30 and 120 min after treatment, and the expression of IL-10 and substance P was examined in the jawbone in the maxillary area of the incisors. Immunohistochemical (IHC) examination was conducted using a monoclonal antibody for substance P and IL-10 (Santa Cruz Biotechnology, Amerika). Histological examination was subsequently performed to count the expression of substance P and IL-10 by Meyer’s hematoxylin staining (Sigma-Aldrich). Photographs were taken using a phase contrast microscope at 1000x magnification. Data are expressed as the mean ± standard deviation. The significance of the difference was evaluated by analysis of variance (ANOVA), with Tukey HSD when comparing the difference between groups. P < 0.05 was considered statistically significant.

In this study, immunohistochemical examination was carried out to determine the periapical expression of substance P and IL-10 after exposure to 650 nm diode laser precondition and postcondition overinstrumentation. The expression of IL-10 and substance P in each group is shown in Fig. 3.1. The expression of substance P in the precondition, LLLT groups (K3 and K4) was significantly lower than that in the control (K1 and K2) and postcondition groups (K5 and K6). On the other hand, the expression of IL-10 in precondition, LLLT groups (K3 and K4) was significantly higher than that in the control (K1 and K2) and postcondition groups (K5 and K6) (See Fig. 3.2 and Fig. 3.3)

The results of this study indicate that the expression of substance P in pre-condition treatment is lower compared to postcondition. In general, substance P is pervasively present and mainly produced by nerve cells. Substance P will increase when there is a stimulus such as overinstrumentation in root canal treatment (Munoz and Covenas, 2014). Substance P is an important neuropeptide in inflammation and immunomodulation. Substance P is a pro-inflammatory mediator that can cause neurogenic inflammation, in which pain sensitivity increases and the pain threshold decreases (Chen et al., 2019). The main role of substance P is as a vascular vasodilator. Almost all pathological conditions affecting oral tissues, as well as dental procedures, can increase the production and release of substance P. Root canal preparation causes posttreatment pain (such as symptomatic apical periodontitis) because of the release of substance P in the periodontal ligament, although the amount of substance P produced will differ between procedures. An increase in substance P expression it can produce an inflammatory process in the periapical tissue (Sacerdote and Levrini, 2012). Substance P is also affected by anxiety, stress, emotion, and pain. This study used the expression of substance P and did not use the expression of calcitonin gene-related peptide (CGRP), although both are indicators of pain because CGRP is also anti-inflammatory, while substance P is purely proinflammatory (Schou et al., 2017). The results of this study also showed that the expression of IL-10 was higher in the precondition treatment group than in the postcondition treatment group. In the body, IL-10 is a ubiquitous mediator, produced by leukocytes, T cells, B cells, neutrophils, dendritic cells, resident macrophages, and monocytes. LLLT exposure stimulates the release of IL-10 from these cells (Tamara, 2019). The increase in IL-10 is important for the analgesic effect, so if it is done before a therapy that can cause pain, including root canal treatment, it is highly recommended (Cheung et al., 2013). Using a 650 nm diode laser at a dose of 40 s will reduce the sensitivity of sensory nerve endings due to a decrease in Adenosine Triphosphate (ATP). As a result, when a stimulus occurs, the amount of substance P released is not too high. In general, laser exposure will increase the production of ATP by the mitochondria. However, this effect is prioritized in the healing process and posttraumatic pain to increase oxygenation and reduce oxidative stress (Cotler et al., 2015). Until now, the anti-pain mechanism of LLLT has not been clear because it is not from thermal or temperature effects, but the effect of an appropriate dose of LLLT will decrease the membrane potential of dorsal root ganglia neurons so that ATP production is reduced. This reduction in ATP is very likely due to the presence of neural blocking. The immediate effect of nociceptor blockade is pain relief for several minutes. Inhibition of this peripheral sensitivity not only lowers the threshold for nerve activation but also decreases the release of proinflammatory neuropeptides thus, LLLT has short-, medium-, and long-term effects on pain relief that occur within minutes after application, which is the result of peripheral and sympathetic nerve blockade. It is clear that LLLT is also effective if delivered to normal cells or tissues before the actual trauma, because photons are absorbed in cytochrome (c) oxidase (unit four in the mitochondrial respiratory chain), and activation of this enzyme increases electron transport, respiration, oxygen, and ATP production in addition to the broader use of LLLT as a pre-condition to prevent pain and promote healing after surgical or medical procedures and possibly to improve athletic performance (Goldberg et al., 2014). The use of LLLT is influenced not only by the length of irradiation but also by certain parameters, including fluence, power output, wavelength, light source, and the type of laser used. Cheung, et al., (2013) evaluated the effects of LLLT on the severity of oral mucositis and inflammatory mediators (TNF-α, IL-6, IL-1β, IL-10, TGF-β, metalloproteinases, and growth factors) on salivary and blood levels of patients' hematopoietic stem cell transplantation. It has been reported that LLLT is clinically effective, and the mechanism does not appear to be entirely related to the modulation of pro- or anti-inflammatory cytokines, growth factors, or matrix metalloproteinases. The differences between the various studies are due to the different areas where the laser is applied and the laser parameters. Although root canal treatment aims to eliminate the source of inflammation, inflammation originating in the pulp tissue can extend to the periapical tissue and thereby induce inflammation. However, LLLT differs from intracanal laser application in terms of the method and location of application. Compared to intracanal application, which requires a special tip, the laser used in this study is easier to apply and can theoretically prevent pain with radiation therapy procedures before the start of the main treatment (Arslan et al., 2018). LLLT also has a positive effect on inflammation and the immune system by increasing endogenous endorphins (β-endorphins) and inducing phagocytic activity. In addition, it increases the number and diameter of lymphatic vessels while reducing vascular permeability, thereby reducing edema and the activity of C fibers and bradykinin (Chen et al., 2019).

Preconditioning with LLLT decreases pain sensitivity by increasing the pain threshold. Further research is needed in humans after being carried out on experimental animals and on diode lasers with expressions related to tissue healing.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.