A wide variety of factors need to be considered when planning a peripheral nerve regeneration study. As outlined in Sections 2.2.2 and 2.2.3, rats are the preferred animal models in peripheral nerve regeneration studies. The choice of strain is important and will depend on the properties required in the rat since certain parameters, e.g. size, temperament, susceptibility to autotomy or even immunodeficiency, are strain-related (see Section 2.2.4.2).
Once the strain has been selected, a decision will have to be reached as regards age, weight/size and gender.
7.1.1 Anatomical investigations (Paper 1)
In the preliminary anatomical investigations the four rat strains most commonly featured in peripheral nerve regeneration studies (Wistar Han, Sprague Dawley, Lewis and Fischer 344), and a strain of nude t-cell deficient rats (amounting to a total of 210 adult rats) were examined regarding their anatomical suitability for peripheral nerve regeneration studies featuring the sciatic nerve (Fig.11). Anatomical suitability was defined as the amount of accessible undivided sciatic nerve, i.e. the length of the sciatic nerve before it branches into its two successors, the tibial and peroneal nerves. Additionally, the length of accessible sciatic nerve after it has divided into its two successors, but with both still contained in the same epineurium, was ascertained. These two measured sections of the sciatic nerve were not only expressed as percentages of the approximate femur length, but also as absolute lengths in millimetres.
Fig.11 Anatomical overview: skinned right hind limb of a rat with the biceps femoris muscle (bf) still in place, anatomical landmarks assessed in Paper 1 (middle), demonstration of some of the landmarks after the biceps femoris (bf) muscle has been removed (right). Note that P2 cannot be clearly pinpointed from this angle.
7.1.1.1 Population examined
All rats examined were randomly donated as carcasses from various other studies. It can therefore be assumed that the assessments covered a representative cross- section of the adult laboratory rat population. By far the greater proportion of the rats were males (150 males, 60 females) as these feature more widely in research laboratories (personal communications with Charles River Laboratories, US) due to the fact that they are not affected by hormonal fluctuations, which can cause inconsistencies in a study otherwise based on standardised parameters. Additionally, they do not have any obvious negative traits, such as being strikingly more aggressive than females, are easy to keep in groups, and are slightly less expensive to acquire (see catalogue: www.criver.com). The reason why they are a little lower in cost is that rats are generally ordered by weight (in grammes) and as males grow faster than females they reach a suitable weight at a younger age. Another reason why female rats are more expensive is that they are the limiting factor in breeding programmes.
7.1.1.2 Results
The results revealed that the lengths of available sciatic nerve – where this was indeed accessible (see below) – were fairly constant in all strains of rats (Fig.12). It amounted to approximately one third of the length of the femur for stretches of undivided sciatic nerve, and up to nearly half of the femur length for stretches where the tibial and peroneal nerves were already present, but still enclosed by the
same epineurium. The average male adult Wistar Han and Sprague Dawley rats were the two largest strains with the longest femur lengths and the longest feet. Nevertheless it was ascertained that male adult Lewis rats had the greatest relative and absolute amount of sciatic nerve suitable for nerve regeneration studies (both undivided and divided), but only very marginally.
It should, however, be noted that in more than 68% of the hind limbs of the Fischer 344 rats the point at which the sciatic nerve divided into its two successors lay so far proximal that no sciatic nerve per se could be observed when the rat was dissected (Fig.12). This undesirable trait also featured, but to a lesser extent, in the other strains but least of all in the hind limbs of the Lewis rats (3.3%).
The same trends were observed in the female counterparts.
Fig.12 Division and branching of the sciatic nerve in nude, Wistar Han, Lewis, Sprague Dawley and Fischer 344 rats (from left to right).
7.1.1.3 Conclusions and practical implications
The above findings mean that from the anatomical point of view, Lewis rats are slightly more advantageous for peripheral nerve regeneration studies featuring the sciatic nerve, than the other strains examined (Wistar Han, Sprague Dawley, nude rats). The use of Fischer 344 rats is not advisable, for the reason given above. A further advantage of the Lewis strain is that these rats do not exhibit the slightest inclination towards autotomy (PANERAI et al., 1987; CARR et al., 1992; STRASBERG et al., 1999), which means that they do not subject their denervated hind paws to automutilation (see 2.2.4.2). It should therefore be possible to perform footprint assessments when evaluating the extent of regeneration. The placid and trusting nature of this strain (STRASBERG et al., 1999), which represents a further
great advantage when carrying out evaluations, is the final reason why this strain of rats was chosen for the present regeneration study.
Male rats were selected for the reasons given under Section 7.1.1.1. On the basis of the results of Paper 1 it was calculated that the size or rather the weight of rats to be ordered should be around 300 g, as male Lewis rats of this size should normally have a mean femur length of 33.72 mm. This would mean that the length of undivided sciatic nerve should therefore amount to approximately 12.34 mm; the amount of undivided sciatic nerve plus divided sciatic nerve (with the successors still surrounded by the same epineurial sheath) should amount to approximately 16.37 mm. Considering the animals were to undergo extraction of a 14 mm segment of the sciatic nerve, a weight of 300 g was considered to be sufficient, particularly as the rats were to experience ten days of preoperative acclimatisation and training in which they would also grow and increase their weight by approximately 20 to 30 g.