*Methods for Evaluating the* In Vivo *Analgesic and Anti-Inflammatory Activity of Essential Oils DOI: http://dx.doi.org/10.5772/intechopen.113832*

response of the two hind legs to be differentiated, and the threshold values are stable over time, allowing repeated measurements [ 50 , 64 ]. The animal's withdrawal threshold is measured in relation to the force exerted by the filament. Mechanical sensitivity test [ 45 , 55 , 64 ]. The von Frey bristles are nylon monofilaments, or von Frey bristles are nylon monofilaments or stiff metal bristles that exert precise levels of force when pressed against the skin. They can be used to measure mechanical stimulation.

#### *6.1.3.2 Paw pression*

 The Randall and Selitto test [ 65 ] is based on determining the pain threshold induced by the application of pressure. Using a mechanical stimulator, constant or, more often, increasing pressure is applied to the animal's hind limb. The animal's behaviour is assessed: it freezes, withdraws its limb and emits cries. Electromyographic recordings of nociceptive reflexes can alike be made [ 49 ].

 From all these tests, the tail withdrawal and hot plate tests remain the most commonly used. On the other hand, it should be noted that the rate of publications concerning the tail withdrawal, hot plate and torsion tests stabilised in the 1990s. In contrast, the number of articles based on the formalin test and various tests involving paw withdrawal from mechanical stimuli was noted to have increased [ 49 ].

#### *6.1.4 Facial coding scales*

 A facial expression is one or more movements of the muscles or skin of the face. These movements express the emotional state of the individual to an observer. Therefore, the facial expression of pain can be used as an interesting indicator. If we can estimate these expressions quantitatively using facial coding criteria, this will enable the assessment of pain [ 66 ], and recent evidence suggests that facial expressions of pain could alike be used in rodents [ 67 , 68 ]. Similarly, it is difficult to assess the internal emotional states of rodents by analysing their facial expression. Nevertheless, it has already been used, for example, to assess taste/disgust [ 69 ]. In the case of pain expression, the 'grimace scale' defined for mice [ 67 ] and rats [ 68 ] consists of noting orbital constriction. This grimace scale depends on five facial features: orbital narrowing, nose bulge, cheek bulge, ear position and moustache change. These facial action units have values of 0 (no pain), 1 (mild pain or likely pain) and 2 (severe pain or definitely present) ( **Figure 4** )[ 68 , 70 ]. This new approach to pain assessment in rodents could be facilitated by partial automation.

#### **Figure 4.**

 *Examples of visible pain expression on a rat's face, framed from the database and labelled with the rat grimace scale [ 48 , 68 ] (example adapted from the Facial Pain Expression Table for Rats: Sotocinal et al., 2011).* 

#### **6.2 Methods used to study the anti-inflammatory activity** *in vivo*

The inflammatory response is a physiological process of defence of the body against an attack, which leads to tissue damage. The primary function of the inflammatory response is to eliminate or isolate the attacking agent (bacteria, virus, parasite, damaged tissue) from the rest of the body and to allow, as quickly as possible, tissue repair. The inflammatory response is a physiological process of defence of the body against an attack, which leads to tissue damage. The inflammatory reaction allows the elimination of the aggressors and ensures the repair of the lesions. It stops when the attacks disappear [71] and the inflammatory mediators constitute all the molecules involved in the regulation of the inflammatory process and which activate and sensitize the nociceptive system. In order to relieve inflammatory pain, the body often relies on compounds that stimulate the immune response or on inflammatory mediators themselves [72]. As a consequence, among the tests that are considered a model of short-term inflammatory pain model is the formalin test.

Several methods have been used to assess the anti-inflammatory activity of medicinal plant extracts. In virtue of which, we will illustrate some of the methods used to assess anti-inflammatory activity *in vivo*:

#### *6.2.1 Carrageenan-induced paw oedema*

Definitely**,** Carrageenan-induced paw oedema is certainly one of the most popular tests [72]. Therefore, it is a very sensitive and reproducible test and has been relied upon since ancient times as a model for studying new drugs effective against inflammatory pain [73]. Likewise, carrageenan-induced inflammation enables us to detect orally active acute anti-inflammatory agents. For this reason, it has great predictive value for anti-inflammatory agents that act *via* mediators of acute inflammation [74]. This inflammatory response includes three distinct phases: a first phase involving histamine and 5-hydroxytryptamine, which promote vasodilation, plasma transudation and oedema (0–1 hour), a second phase (1.5–3 hours), which uses kinins as mediators, increases vascular permeability [75] and prostaglandin biosynthesis occurs beyond the third hour (third phase) [76]. A positive effect is explained by the inhibition of the actions or synthesis of pro-inflammatory substances.

Indeed, inflammation occurs when carrageenan is injected. In rodents, intra-plantar injection of carrageenan causes hypersensitivity, which is assessed by mechanical or thermal stimulation. Pain pharmacology: The oedema caused by this photogenic agent can be translated into volume and measured, making it possible to monitor the inflammatory process (**Figure 5**) [77]. Similarly, the behaviours observed can be characterised using a scoring scale [45].

#### *6.2.2 Croton oil-induced ear oedema*

The ear oedema is induced by croton oil, using the method of Manga and colleagues. The thickness of the ear is measured using a digital calliper before treatment and a few hours after induction of inflammation [78].

#### *6.2.3 Injection of formalin into the facial region*

Injection of formalin into the rat's upper lip: The time the rat spends grooming, scratching and rubbing is assessed [79]. The injection of formalin provokes a biphasic *Methods for Evaluating the* In Vivo *Analgesic and Anti-Inflammatory Activity of Essential Oils DOI: http://dx.doi.org/10.5772/intechopen.113832*

**Figure 5.** *Measuring the volume of the hind paw of the mice using the plethysmometer.*

response similar to the one observed during intra-plantar injection. Administration of analgesics reduces nociceptive behaviour. This test can be used to assess the pain behaviour associated with trigeminal pain, as well as the effects of potentially analgesic drugs [45, 80].
