Somatosensory evaluations

As indicated in Section 2.3.4.4 a), there are many different approaches concerning how and where the noxious stimuli should be administered.

The only generally accepted rule is that the medial aspect of the foot should be avoided, as the saphenous nerve caters for this region (DE LAHUNTA, 1977; DEVOR et al., 1979; DE KONING et al., 1986; VAREJÃO et al., 2004a).

It should also be borne in mind that by varying the intensity of stimuli applied, it is possible to trigger the subsequent reaction on two different levels: a subconscious withdrawal reflex or a conscious pain reaction (DE LAHUNTA, 1977; GAROSI, 2004). The withdrawal reflex is a true reflex which involves the central nervous system on the spinal level. The animal responds to the stimulation of nociceptors simply by withdrawing its leg. For a conscious reaction to pain, however, afferent signals have to reach the thalamus via the spinothalamic tracts and the medial lemniscus, and are further projected to the somaesthetic cortex via the internal capsule (DE LAHUNTA, 1977). Conscious pain perception occurs both on the thalamic and on the cortical level, and causes the animal to react with an accurate pain response such as licking its foot or vocalising its discomfort in addition to withdrawing its foot from the noxious stimulus (NAVARRO et al., 1994).

Presupposing that the afferents have been re-established after experimental nerve trauma, the question that now arises is whether a rat with residual sciatic dysfunction is capable of performing a recognisable withdrawal of the foot. This could be a problem if the effector muscles needed for this action are still denervated or incompletely or inappropriately reinnervated. Additionally, the common technique of wrapping the rat in a towel for immobilisation and restriction of vision during evaluation (Fig.19) (MASTERS et al., 1993; HU et al., 1997; VAREJÃO et al., 2004b) might not only hinder the rat from performing a withdrawal reflex, but might also make it more difficult for the examiner to recognise weak responses.

7.6.1.1 Implementation of somatosensory assessments

The somatosensory assessments in the present study were carried out by pinching the rats’ feet with atraumatic forceps in a number of places. These amounted to 14 spots on the dorsal aspect, 13 spots on the lateral aspect, 5 spots on the medial aspect and 24 spots on the plantar aspect (Fig.19), a greater number than in comparable studies. The noxious stimuli were delivered by holding the rats gently, but firmly, with one hand (Fig.19), whilst pinching their foot with the other at a maximum force of 0.8 N per mm².

Fig.19 Implementation of somatosensory assessments: pinching points (left), fixation of the rat (right). The inset illustrates the way in which rats are commonly wrapped in towels for somatosensory evaluations.

Only conscious pain reactions were counted as a definite reaction to a noxious stimulus. These took the form of definite vocalisation of protest at the moment of being pinched, with or without retraction of the foot. Often the rats also attacked the source of stimulation, the forceps, or licked their foot in an attempt to alleviate discomfort. Only in one single case of more than 320 examinations did a rat attempt to bite the examiner.

7.6.1.2 Results of somatosensory and supplementary assessments

The somatosensory tests revealed that all animals experienced roughly the same amount of sensory reinnervation in the foot, regardless of their experimental group (Fig.15). Morphometric and electronmicrosopic evaluations revealed, however, that the vast majority of rats in Groups B and C experienced no neural regrowth in the plantar extensions of the tibial nerve (Figs.15, 20), which physiologically is associated with innervation of the plantar and lateral aspect of the foot (GREENE,

1955; HEBEL & STROMBERG, 1976). This contrasted strongly with the morphometric assessments of the same neural segments of rats in Group A (Figs. 15, 20), where all rats except one experienced neural regeneration.

With the help of retrograde tracing studies (Figs 15, 20) it could be proven that it was primarily the saphenous nerve and maybe also a very proximal branch of the sciatic nerve, the musculocutaneous nerve (PUIGDELLÍVOL-SÁNCHEZ et al., 2000, 2005), which were providing sensory reinnervation to the lateral metatarsus of toe 5.

Fig.20 Results of the supplementary assessments: histological images of the plantar extensions of the tibial nerve in a rat in Group A (left) and a rat in Group C (middle). Note that there are no myelinated fibres in the neural segment of the rat in Group C. The picture on the right shows a dorsal root ganglion of L2 exhibiting fluorescence for both the tracers Fluorogold and Fast Blue. These had been injected at the medial metatarsus of toe 1 (Fluorogold) and the lateral metatarsus of toe 5 (Fast Blue).

7.6.1.3 Anatomical considerations

Physiologically, the saphenous nerve is responsible for the sensory innervation of the medial aspect of the foot, toes 1 and 2 and only the proximal phalanx of toe 3 (GREENE, 1955; PUIGDELLÍVOL-SÁNCHEZ et al., 2000). According to the literature, the peroneal nerve provides sensory innervation to the dorsum of the foot, the medial aspect of toe 1 and the adjacent aspects of toes 2, 3, 4 and 5; the tibial nerve provides sensory innervation to the plantar side of the foot and toe 1, to the lateral side of toe 5 and to the adjacent sides of toes 1 to 5 (GREENE, 1955; HEBEL & STROMBERG, 1976). In short, under physiological circumstances, the distal extensions of the tibial and peroneal nerves are responsible for innervation of the dorsal, plantar and lateral sides and all the toes of the hind paw in the rat, while the saphenous nerve only innervates the medial side of the hind paw and has

variable contributions to toes 1, 2 and 3. The musculocutaneous branch of the sciatic nerve, which has only recently been discovered and described, seems to contribute to all the toes; the exact extent and locations have unfortunately not as yet been clarified, however (PUIGDELLÍVOL-SÁNCHEZ et al., 2000). The musculocutaneous branch leaves the sciatic nerve shortly after the caudal gluteal nerve branches off. It then crosses the very proximal aspect of the thigh, running across the adductor magnus muscle, under the caudofemoralis muscle, and across the semimembranosus muscle towards the biceps femoris muscle and the popliteal fossa (PUIGDELLÍVOL-SÁNCHEZ et al., 2000).

7.6.1.4 Implications for both existing and future somatosensory

assessments

(A) The observations described in Paper 2 mean that somatosensory assessments, even if they are conducted at the most lateral aspect of the foot, can produce unreliable results. Results obtained in both withdrawal reflex tests and pain response tests should, consequently, be treated with caution. The areas tested, and the likelihood that collateral sprouting of the saphenous nerve could have taken place must be taken into account.

The musculocutaneous nerve should also be kept under observation. This nerve runs in areas of the thigh which are not commonly subjected to injury in the average sciatic nerve regeneration study, and physiologically provides sensory innervation of the foot. Under pathological circumstances the musculocutaneous nerve is also capable of sprouting (PUIDELLÍVOL-SÁNCHEZ et al., 2005). Unfortunately, however, as noted above, the territory of this nerve not even under physiological circumstances has as yet been clarified.

This means that when conducting somatosensory evaluations, care should be taken to stimulate the rats only in areas where no sprouting of neighbouring nerves has so far been observed, i.e. toe 5.

(B) Sensory assessments based on pain responses are feasible to perform and produce reliable results.

Depending on how much the rat is handicapped in performing a recognisable withdrawal reflex (towel, required muscles denervated), pain response testing might produce more reliable results than withdrawal reflex testing.

(C) The results once again show the Lewis strain to be the most suitable rats for sciatic nerve regeneration studies. Due to their docile and friendly nature (STRASBERG et al., 1999) they allow themselves be examined single-handedly

even in a painful situation and do not respond to pain with adverse reactions against the examiner.