**6. Methods used for assessment of the analgesic and anti-inflammatory activity** *in vivo*

The use of animals is widespread in biomedical research, and pain studies are no exception. As experiments on human subjects have to be limited to moderate stimuli that do not cause injury or disease, researchers have turned to animals to find answers to their questions [45].

Nociception and pain constitute a vast area of neuroscience and medical scientific research. As we know, the use of animals in scientific research has been controversial since ancient times [46], although these animal models have great merit in the advancement of biomedical sciences through their important contributions to our growing understanding pathological and biological processes [47]. Over time, various tests and models have been developed in rodents to provide fundamental and translational research tools on this subject; to study pain and try to reduce it, tests using thermal, mechanical and chemical stimuli, Hyperalgesia and pain measurements, and inflammatory or neuropathic pain models constitute one of the most important tools available to researchers in this area [48]. Preclinical therapeutic research should consequently combine pain models with nociceptive tests in order to be more relevant [49].

#### **6.1 Methods used to study the analgesic activity** *in vivo*

Most of the tests used to assess pain in animal experimentation involve inducing pain in animals using different agents [50]. Further, the tests are grouped around three basic types of pain: thermal nociception, chemical nociception and mechanical nociception. Nevertheless, in the following figure, we describe and critically analyse the most commonly used behavioural tests of nociception in animals (**Figure 1**) [49].

### *6.1.1 Thermal nociception*

In mammalian nociceptors, noxious heat above 40°C activates thermosensitive C fibres and heat above 52°C activates A fibres [51]. In addition, tests measuring

#### **Figure 1.**

*A. A number of original articles published between 1970 and 1999 in which researchers used one of the five most common pain sensation tests, B. The relative proportions of these categories of articles appearing during the year 1999 (based on Medline) [49].*

the nociceptive response to heat can be used experimentally in both rats and mice. Likewise, the stimulus can stop automatically when the animal responds [48, 49]. The animal is placed inside the heating plate and waited a few moments for it to react to the pain. The mouse must be recovered immediately after observing its response to the experiment to avoid any risk of burns. We can do the same experiment and repeat it several times to check the values, but this is often observed in some laboratory animals, either stress or habit of the protocol gives us different values. Repeating measurements for the same mouse may lead to different results. [49, 50]. There are also some limitations to this test, repeated measurements lead to learning phenomena, and these lead to variations in reaction latency [52].

When we touch something too hot or too cold, our senses translate this into a sensation of pain. If you put your hand in a fire, the resulting burning sensation will cause your body to move your hand away as quickly as possible. Feeling pain is actually proof that your body is working hard to keep you safe. So, losing the ability to do that means you find yourself in real trouble.

The hot plate and tail-flick tests measure an animal's ability to consciously remove a part of its body from a heat source, and they all test the ability to an animal to feel and respond to a certain degree of painful stimulation [52].

#### *6.1.1.1 Tail withdrawal test, D'Amour and Smith test or* Tail-flick *test*

It stands for a simple method that measures a spinal nociceptive reflex. The tail-flick test includes two types that are superficially similar but physically very different [53]. The first consists of immersing the animal's tail in water at a certain temperature. The second type involves applying radiant heat to a specific small area of the tail. More to the point, the surfaces stimulated can be very different. In fact of matter, it is surprising that authors generally consider these two tests to be equivalent [49]. The time taken for the animal to withdraw its tail is measured [49, 54]. Besides, to minimise the risk of tissue trauma due to exposure to heat, a time limit such as 10 seconds is set, at which point the animal is removed from the test [52].

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

## *6.1.1.2* Hot plate *test*

The hot plate method relies on measuring latency to assess skin sensitivity to pain. The response to pain usually involves licking the area to relieve the pain, shaking or immediately jumping off the hot plate [53]. Definitely, the hot plate is an open cylindrical space, the base of which is a heatable metal plate. The mouse is placed inside the plate that is preheated to a constant temperature. The animal is monitored and the response time to any type of behaviour is measured, namely paw licking and jumping. Both are considered to be integrated supraspinal responses [49, 53].

The aim was to verify the ability of the products tested to protect the animal against thermal pain. The extracts were administered either through oral or peritoneal way. The animal is placed on a heated metal plate maintained at between 52 and 55°C (**Figure 2**). The latency time for the appearance of behavioural responses is measured, with the animal licking itself, shaking its legs and/or jumping [55].
