Analysis of footprints

The evaluation of footprints is one of the most popular methods for evaluating the functional aspect of sciatic regeneration, since footprint analysis has traditionally been regarded as an overall assessment of sciatic nerve regeneration consisting of sensory input, cortical integration and motor output (DELLON & MACKINNON, 1989; KANAYA et al., 1996; BERVAR, 2000; VAREJÃO et al., 2001b; SCHIAVETO DE SOUZA et al., 2004). Analysis of the 100 most recently published papers on sciatic nerve regeneration revealed that close to 40% of the studies used footprint analysis to determine the extent of functional regeneration (Table 3). It is not easy to obtain well recognisable, characteristic and distinct footprints, as described in Section 2.3.4.3. Usually, prints from walking rats are acquired by coating the soles of their feet with (finger) paint or ink and inducing the rats to traverse a narrow corridor lined with strips of paper and leading to a darkened cage (Fig.21) (VAREJÃO et al., 2001b, 2004a). In the original version of this method developed by de Medinaceli and colleagues, the corridor was lined out with x-ray films and the rats’ feet were dipped in x-ray developer (DE MEDINACELI et al., 1982). Various authors suggested using photographic paper and film developer instead. The high costs, the slippery surface and the potentially caustic effects of the developer made it necessary to search for alternatives, however (VAREJÃO et al., 2001b).

These days, either ink or paint and white paper are used, or in a more sophisticated version, the rats traverse a perspex corridor, and are filmed from underneath with a high-speed camera. If a mirror is placed at an angle of 45º below the runway, both the side view and the plantar view can be obtained from the animals in one film session. The footprints can then be assessed in stills created from the film (VAREJÃO et al., 2001a,b, 2004a).

However, as mentioned in Section 2.3.4.3, it has repeatedly been suggested that footprint analysis primarily measures motor function (WALKER et al., 1994; YU et al., 2001) and for an overall evaluation it needs to be supplemented by sensory assessments (HADLOCK et al., 1999).

7.6.2.1 Unpublished observations and implementations of footprint

gathering

During preliminary tests with other rodents it could be observed that, whilst traversing the corridor, the animals would lean against the wall on their affected side (Fig.21). None or only very sketchy footprints of the affected hind limb could be acquired because the exorotated foot would hit the wall during the swing phase and then often be placed with the dorsum on the ground or not at all.

In view of this, the prints (SFI) in the present study were collected on a wide elevated beam, since all rats had learnt to traverse this beam for the ambulatory assessments. A fitting strip of paper was clipped onto the 8 cm wide beam and the rats were placed on this paper with the plantar aspects of their hind limbs covered from the tips of their toes to the heel with non-toxic children’s paint (Fig.21). As a result of having been trained and examined on this beam many times, the rats made their way across the beam in an unhesitant and confident manner.

Fig.21 Footprint gathering: rat leaning against the wall of a corridor (Varejão et al., 2001b) (left), covering the rats’ feet with children’s paint (top right), rat traversing the 8 cm wide beam (bottom right).

An important factor was that there was no wall, which meant that the rats had nothing to lean on, and there was no chance of exorotated foot swings hitting the wall and there being no footprints. Furthermore, the rats subconsciously trusted their unoperated hind limb more and kept toward the unoperated side, enabling clear footprints to be obtained on the operated side.

For static prints (SSI) the rats were held upright, with one leg and the tail tucked under. The rat was then lowered onto a plain sheet of paper with the extended leg anticipating the ground and the print was taken much like stamping a piece of paper.

7.6.2.2 Results achieved in footprint analysis

The results achieved in the present study and presented in Paper 3, revealed that whilst moderate differences could be noted between the different groups regarding the results achieved in the footprint analyses (SFI and SSI), only very few, if any, differences could be noted between the different surgical groups in the overall ambulatory assessments (see also Section 7.5.2). Furthermore, the differences in regeneration between the groups showed up more clearly in the SSI evaluations than in the SFI results.

7.6.2.3 Implications for both existing and future footprint assessments

(A) The acquisition of footprints whilst getting rats to traverse an elevated beam of suitable width is a feasible procedure and produces more usable footprints than the traditional method of letting the rat walk up a corridor. No additional training is required for conditioning the animals.

(B) A comparison of the different surgical groups indicated that the SSI assessments are slightly more sensitive to minimal changes than the SFI assessments.

(C) This procedure confirmed moreover the proposition frequently met in the literature, that footprint assessments are primarily only concerned with motor function (WALKER et al., 1994; HADLOCK et al., 1999; YU et al., 2001) and cannot be regarded as an overall assessment of integrated motor function. For overall assessments other methods of evaluation concerned with gait, such as the ambulatory assessments carried out in the present study, or additional somatosensory tests have to be applied.