Antinociceptive and anxiolytic-like effects of Lavandula angustifolia essential oil on rat models of orofacial pain

Abstract Nociceptive and inflammatory orofacial pain is highly prevalent in the population, which justifies the search for safer analgesics. There is increasing evidence of the analgesic and anxiolytic potential of Lavandula angustifolia essential oil (LAV EO), which may represent, when administered through inhalation, may represent a safer alternative for pain treatment. Objective to evaluate whether LAV EO has antinociceptive effect in the formalin test, and anti-hyperalgesic and anxiolytic-like effects in rats subjected to a model of orofacial postoperative pain. Methodology Female Wistar rats were exposed to LAV EO (5%) by inhalation for 30 minutes. After exposure, animals were injected with formalin (2.5%, 50 μL) or saline into the hind paw or upper lip and the number of flinches or facial grooming time, respectively, were evaluated. Likewise, on day 3 after intraoral mucosa incision, the animals were exposed to LAV EO and facial mechanical, and heat hyperalgesia were assessed. The influence of LAV EO inhalation on anxiety-like behavior was assessed in operated rats by testing them on the open field (OF) and elevated plus maze (EPM). Results LAV EO reduced the phase II of the paw formalin test and both phases of the orofacial formalin test. On day three post-incision, LAV EO reduced heat and mechanical hyperalgesia, from 30 minutes up to three hours, and reduced the anxiety-like behavior in operated rats without causing locomotor deficit. Conclusion LAV EO inhalation results in antinociceptive and anxiolytic-like effects in orofacial pain models, which encourages further studies on LAV EO indications and effectiveness on orofacial pain conditions.


Introduction
The orofacial region encompasses many unique structures, including the cornea, meninges, teeth, nasal and oral mucosa and temporomandibular joint, which are frequently afflicted by painful disorders. 1 Acute trigeminal pain is highly frequent even after minor oral surgical procedures, and it markedly affects patient recovery and quality of life. 2 In fact, acute trigeminal pain has been associated to emotional co-morbidities, especially anxiety, which has been considered a contributing factor for pain chronification. 3 Moreover, the prevalence of orofacial pain has been estimated twice as high in women compared with men, and female sex has also been suggested a risk factor for orofacial pain chronification. 3,4 The formalin test, applied to the orofacial region or hind paw of rats, has been widely used for the screening of compounds with analgesic potential. Regardless of the injection site, formalin induces a biphasic response, consisting of a brief acute phase, due to direct activation of peripheral nociceptors, followed by an inflammatory pain phase. 5 Inflammatory pain may also be assessed in rodents by incision surgery models, which mimic post-operative pain. In the intraoral mucosa incision model, our previous studies demonstrated that 3 days after surgery rats show spontaneous pain, heat and mechanical hyperalgesia, and anxiety-like behavior. 6 Thus, these models allow the pre-clinical investigation of antinociceptive, antihyperalgesic and anxiolytic effect of test substances.
Lavandula angustifolia has been used whole or as an essential oil (EO) for centuries for a variety of therapeutic purposes. [7][8][9][10][11] The anxiolytic effect of Lavandula angustifolia essential oil (LAV EO) has been extensively demonstrated 8,[12][13][14][15] In fact, inhalation of LAV EO has been shown to reduce peri-operative anxiety in patients undergoing oral surgery. [16][17][18][19] On the other hand, the analgesic effect of LAV EO is less studied, and most studies that addressed this topic used the oral route of administration. 20 For instance, oral administration of LAV EO reduced both phases of the paw formalin test in rats. However, the inhalation of certain EO allows a safe, simple, non-invasive and low-cost therapeutic alternative that may be useful in some clinical contexts. This route may be of special interest in orofacial pain conditions since the EO may have a rapid direct effect on peripheral trigeminal afferents, as well as a rapid transportation from the nasal mucosa to the brain through the olfactory and trigeminal nerves. 21 In light of these considerations, this study sought to investigate the antinociceptive and anxiolytic effects of LAV EO administered by inhalation in rats subjected to orofacial pain. Firstly, we assessed the effect of LAV EO by inhalation in the paw and orofacial formalin test. The first served as a control and allowed the comparison between LAVOE effect in a trigeminal versus a non-trigeminal innervated region. Then, we explored the effect of LAV EO in facial heat and mechanical hyperalgesia and anxiety-like behavior associated with facial post-operative pain. Exposure to LAV EO Exposure to LAV EO or vehicle (avocado oil 5% -VEH) was done individually in a cage box (41x32x16,5 cm), with a special device (as shown in Figure 1) for standardized volatilization, allowing the animals to inhale the EO for 30 minutes, following previous studies. 8,12 The animals were kept in separate rooms during the experiments so that the control group was not exposed to LAV EO odor. Thus, the interference of the essential oil on the control group was avoided.

Formalin Test
The orofacial formalin test was conducted as previously described by our group. 24 The formalin test was also conducted via injection in the plantar surface of one hind paw as described previously by Abbott, Franklin and Westbrook 25 (1995). Rats were placed individually in cages for an acclimatization period in order to minimize any stress-related behavioral changes for about 15 minutes. The animals received a subcutaneous (s.c.) injection of formalin (2.5%, 50 μL) or vehicle (50 μL of saline) into the upper lip or hind paw. The facial grooming (i.e. time spent rubbing the injected area with its forepaws) time was evaluated for 30 minutes, and the flinches number (i.e. shaking of the injured paw) was counted for 60 minutes, respectively. The first and second phases of the orofacial formalin test were considered 0 to 3 minutes and 12 to 30 minutes after injection, respectively, whereas after formalin injection in the hind paw, the first phase was considered 0 to 5 minutes, and the second phase 15 to 60 minutes. An incision was made in the intraoral mucosa, always on the right side (depth, 2 mm; length, 10 mm). One suture was placed in the middle of the incision and the rats were accompanied until their recover from anesthesia. The sham group was subjected to the same manipulation, but incision and suture were not performed. On day 3 after the procedures (i.e. sham or incision), facial heat and mechanical hyperalgesia and anxiety-like behavior was assessed, as described below.

Assessment of heat hyperalgesia
Facial heat hyperalgesia was evaluated in rats as previously reported by group. 6,26 Firstly, rats were habituated to the restraining method to avoid stress during the test. Facial heat sensory threshold was assessed by the approximation of a radiant heat source (about 50°C) 1 cm from the surface of the right

Elevated Plus Maze Test (EPM)
This test was performed following previous studies 28 and was carried out in a light-controlled room (60 lux). As index of locomotor activity, the frequency of entry in the enclosed arms was quantified.

Experimental protocols
Paw and orofacial formalin test: The animals were exposed to VEH or LAV EO 5% for 30 minutes and, immediately after the inhalation, received formalin or vehicle injection in the plantar surface of one hind paw, and the flinches were counted for 60 minutes.
An independent group of rats was subjected to the same procedures, but after inhalation they received an injection (s.c.) of formalin or saline into the upper lip and facial grooming time was evaluated for 30

minutes.
Postoperative orofacial pain model: Evaluation of mechanical and heat hyperalgesia was carried out in independent groups of rats. The baseline responses to heat and mechanical stimulus were assessed and rats were subjected to intraoral mucosa incision or sham procedure. On day 3 after the surgery, the animals were exposed to LAV EO or VEH for 30 minutes, followed by assessment of facial heat and mechanical hyperalgesia at 30 minutes after inhalation and at 1 hour-interval until the 4th hour.
EPM and OF tests: On day 3 after intraoral mucosa incision or sham procedure, the animals were exposed to LAV EO or VEH for 30 minutes, followed by the EPM test, and briefly transferred to the OF apparatus.
The tests were used in sequence as already describe by group. 30 The same animals were recorded for 5 minutes in each apparatus for posterior analysis of their behavior.

Statistical Analysis
The Kolmogorov-Smirnov normality test was applied to ensure that the data met the criteria for performing parametric tests. When the criteria were accepted, the results were expressed as mean ± standard error of the mean (SEM) of 10 animals per group. Two-way analysis of variance (ANOVA) with repeated measures (RM) was used to analyze data Antinociceptive and anxiolytic-like effects of Lavandula angustifolia essential oil on rat models of orofacial pain

LAV EO reduces formalin-induced nociceptive behavior
Two-way ANOVA with RM showed statistical difference in the treatment (F (3, 36)=22.80, P<0,05) and time (F (11, 396)=7.375, P<0.05) factors. Also, we observed an interaction of these factors (F (33, 396)=4.889, P<0.05). As can be seen in the Figure   2B, post hoc test showed that Formalin (2.5%, 50 μL) injected in the rats' hind paw induced a biphasic nociceptive response, which was significantly different from saline-injected rats (P<0.05). The treatment (LAV Post hoc test showed that previous exposure to LAV EO induced antinociceptive effect only in the phase II (P<0.05).
As shown in Figure 3B  LAV EO presented an anxiolytic effect in rats subjected to intraoral incision As shown in Figure 5, when we analyzed parameters from closed arms (panels A and B) one-way ANOVA did not reveal difference between the groups on number

Discussion
The results of this study show, for the first time  We highlight that the magnitude of the effect was similar after oral or topical application. Moreover, oral pre-treatment with LAV EO also reduced both phases of the response induced by formalin (2%) injected into the surface or rats' hind paw. Herein, only the second phase of the paw formalin test was attenuated by LAV EO, but both phases of the orofacial formalin test were significantly reduced. The percentage of inhibition caused by inhalation of LAV EO in the phase I and II of the orofacial formalin test was 81% and 76%, respectively, and 47% for the phase II of the paw formalin test. The reason for this discrepancy remains to be investigated, but it may be related to the different administration routes. To our knowledge, there are no reports about the distribution of LAV EO after inhalation in rats or humans, but according to our data, inhalation may favor a better effect in the orofacial region. There is evidence that LAV EO may have a direct influence on rats' trigeminal afferents, as well as may be rapidly transported directly to the brain from the nasal cavity along the olfactory and trigeminal nerves. 33 These mechanisms could explain the rapid analgesic effects of LAV EO (i.e., immediately after the exposure) and the apparent greater susceptibility of the orofacial region. The nose to brain pathway has attracted attention in the clinical setting and in the market for many reasons, including rapid effect drugs delivered to the brain with minor side effects, and may be of special interest for the treatment of orofacial pain conditions. In addition to the administration route (oral x inhalation), another difference between the present study and the one by Da Silva and colleagues is the sex of animals used. There is increasing evidence about sex differences in the effect of analgesics drugs, 34 and that would be a very interesting question to be explored in the effect of LAV EO.
Several reports of benefits of LAV EO inhalation in postoperative pain are seen in the clinical setting. [16][17][18] In spite of that, it is difficult to draw a definitive conclusion, since the protocols, conditions, and endpoints vary enormously and many studies lack methodological quality. Thus, we highly recommend the development of high quality pre-clinical and clinical standardized studies that allow the inclusion of suggest that LAV EO may be useful in treating pain and associated anxiety, which agrees with some clinical reports. 16,42 Moreover, studies conducted in humans do not clarify whether the anxiolytic effects of LAV EO are the result of action on the central nervous system (through the olfactory and limbic system) or whether the action is mediated peripherally. 10 We point out that the olfactory nerve conducts information directly to the limbic system, a fundamental region in the processing of both pain and emotion. However, the precise mechanisms underlying LAV EO analgesic and anxiolytic effects, as well as the peripheral and central structures involved, remain to be elucidated.

Conclusion
Our data suggest that LAV EO presents antinociceptive effects in orofacial pain models, corroborating clinical and non-clinical evidence. In