Additional considerations

Considering the fact that human patients would be treated in a stress-free environment and would receive every chance for physiotherapy and positive motivation after surgical intervention following injury to a peripheral nerve, it was decided to expose the rats to the same conditions. Otherwise it could be argued that the results obtained are distorted and falsely negative.

7.3.1 Physiotherapy and training

Physiotherapy was supplied on both a mandatory and a voluntary basis. The twice- weekly (mandatory) ambulatory assessments trained the rats’ motor skills, while voluntary physiotherapy was provided by placing the rats daily for a few hours in a large motor enriched cage. Here they could participate in motor activities according to their individual ability and motivation.

Since all of the methods applied to assess the extent of reacquired function involved extensive handling of the rats, which potentially could result in chronic stress, the rats were familiarized with being handled twice daily for five days as from nine days before the operation (D-9 to D-5) (Fig.13). On these days the rats were also trained and conditioned to cross the elevated beams and parallel bars until they could perform this task in a confident and unhesitant manner and exhibited zero mistakes. Before commencement of the training the rats were allowed an additional four days (D-13 to D-10) to acclimatise and get their bearings in their new surroundings.

This procedure made it possible to ensure that all the reference values obtained two days before the operation (D-2) were derived from data collected in healthy and relaxed rats. A further reason for allowing the rats to acclimatise and for letting them gradually grow accustomed to the handling was to avoid the introduction of chronic stress, which would have resulted in increased plasma corticoid levels. Such changes to the adrenocortical axis have been shown to have a detrimental effect on axonal sprouting and elongation (VAN MEETEREN et al., 1997b; AMAKO & NEMOTO, 1998; VAN MEETEREN et al., 1998) and it was vital to avoid this effect at all costs.

Fig.13 Handling (top left) and housing of the rats in the motor enriched cage (right and bottom left).

7.3.2 Housing and voluntary physiotherapy

All rats were housed in groups of four on soft bedding in a temperature-controlled room with 12:12 hour light cycles, and had free access to standard rat food and water. The rats were kept in groups for two reasons. Firstly, rats are social animals which prefer living in groups (Fig.13). Therefore, being kept in groups will have a positive effect on their general well-being. Depriving rats of social contact with other rats will lead to physiological and behavioural abnormalities (MOON et al., 2006). Secondly, it has been shown that rats kept in groups are more active than those housed alone and will therefore exercise more on their own initiative (MEEK et al., 2004; MOON et al., 2006). Neurotrophin levels and their receptors, for example, are activity-dependent and can influence sprouting in the rat spinal cord after injury (BALLERMANN & FOUAD, 2006). The same has been proven for the proliferation and differentiation of oligodendrocyte progenitors and the maturation of oligodendrocytes. Consequently, this also renders myelination of the CNS highly activity-dependent (MC DONALD & BELEGU, 1996). Furthermore, it has been

shown that impoverished environments can even change gait characteristics in intact rats (MOON et al., 2006), and this had to be avoided at all costs.

For voluntary physiotherapy the rats were allowed 4 to 6 hours of ‘playtime’ daily on weekdays in a 45 cm x 55 cm x 120 cm motor enriched cage in groups of eight to twelve rats. The cage was customised to provide continuous physiotherapy by incorporating four different levels connected by oblique mesh ramps (Fig.13). A hammock-like construction was added, which provided the rats’ feet with additional stimulus due to its unsteady nature. Furthermore, the sides of the cage could be and were frequently used as climbing opportunities. Beams, foreign objects and healthy treats (pieces of carrot, cucumber or capsicum) were placed in different parts of the cage at frequent intervals to provide the rats with constant stimuli to explore, climb and balance.

7.3.3 Surgical procedures and postoperative care

In 20 male adult Lewis rats (Charles River Laboratories, Sulzfeld, Germany; 320 to 340 g) the left sciatic nerve was exposed under general anaesthesia [2 mg/kg midazolam (Ratiopharm, Germany), 150 µg/kg medetomidine (Pfizer, Germany) and 5 µg/kg fentanyl (Deltaselect, Germany) i.p.] by separating the biceps femoris muscle from the gluteus superficialis muscle in its aponeurosis and also from its lateral insertion at the tibial crest, making it possible to fold the unimpaired biceps femoris muscle towards caudal. A 14 mm segment was then excised from the sciatic nerve and the resulting gap was subsequently bridged by autograft (Group A; n=8), empty collagen tubes (Group B; n=6) or collagen tubes filled with denatured autologous muscle (Group C; n= 6) (Meek et al., 1999a) (Fig.14). All lesions were set at the same place, with the distal end located 4 mm proximal to the submersion of the tibial branch of the sciatic nerve into the gastrocnemius muscle.

Following the implantation of the interponate, the biceps femoris muscle was carefully sutured back into place and the anaesthesia was reversed [0.75 mg/kg atipamezole (Pfizer, Germany), 200 µg/kg flumazenile (Inresa, Germany) and 120

µg/kg naloxone (Deltaselect, Germany) s.c.].

For postoperative analgesia, the rats received metamizol (200 mg/kg p.o.; corresponds to 3 drops of Novalgin®; Ratiopharm, Germany) upon waking up and

buprenorphine (50 µg/kg s.c.; Temgesic®; Essex Pharma, Germany) every 12 hours for three days.

Fig.14 Surgical images (clockwise from top left): extraction of a 14 mm segment of the sciatic nerve, repair by autograft, empty collagen tube, collagen tube sutured into place.

7.4 Conclusions drawn after applying various methods of