Animal models for peripheral nerve injury and reconstruction

Annually, the Federal Ministry of Food and Agriculture publishes statistical data on the use of experimental animals. In 2017 approximately 2.8 million animals were used for scientific

Introduction

purposes. Rodents constituted 80%, while fishes accounted for 12%, rabbits for 5%, and birds for 2% of all used experimental animals. This means that large animal models, like monkeys, dogs, cats, pigs, and sheep, were only used for approximately 1% of all animal experiments in 2017 (Federal Ministry of Food and Agriculture, 2018). As emerging from the following sections these numbers are also reflected with regard to in vivo experiments on peripheral nerve regeneration.

1.7.1. Large animal models

Peripheral nerve trauma often reveals injuries with defect sizes larger than 3 cm in human patients. Although critical defect sizes of up to 6 and 7 cm were successfully repaired by means of ANGs and allografts in rats (Saheb-Al-Zamani et al., 2013) and rabbits (Koller et al., 1997), the method of choice for studying larger defect sizes is represented by larger animal models, which are thought to be more translational to human medicine. However, no standard animal model for large peripheral nerve surgery has been established yet. Up to now researchers have referred to different species, e.g. sheep, pigs, dogs, cats, and primates. While the ulnar nerve is used in primates (Mackinnon and Dellon, 1990; Auba et al., 2006) and pigs (Atchabahian et al., 1998; Brenner et al., 2004), the peroneal nerve is reconstructed in dog models (Ide et al., 1998;

Matsumoto et al., 2000). Experimental studies in sheep report either the transection and repair of the median, tibial or the peroneal nerve (Matsuyama et al., 2000; Forden et al., 2011; Radtke et al., 2011). Cats as experimental animals are commonly used for studies in the field of ear, nose and throat surgery (Kretzmer et al., 2004). However, the sciatic nerve of cats may also be used to study biomaterials in terms of peripheral nerve repair (Suzuki et al., 1999; Sufan et al., 2001).

1.7.2. Small animal models

Small animal models are not compulsorily suitable to generate large sized peripheral nerve defects. Besides, different dynamics of nerve regeneration due to the big size differences between humans and especially rodent models are under debate (Kirk, 2003; Hoke, 2006; Kaplan et al., 2015). Nevertheless, small animal models, e.g. rabbits and rodents, are widely used to study peripheral nerve regeneration (Rochkind and Nevo, 2014; Georgiou et al., 2015; Stößel et al., 2017; Dietzmeyer et al., 2019b). Among the small animals, the rat is the most popular in vivo model (Ronchi et al., 2019). High availability, as well as moderate housing costs and simple

Introduction

handling, display advantages, making the model attractive for scientific purposes (Fitzgerald, 1983; Ronchi et al., 2019). The following sections will expand on two types of rat peripheral nerve injury and repair models, which were used for both animal studies of my PhD project, later described.

1.7.2.1. The rat sciatic nerve model

The rat sciatic nerve model is the most commonly used model for peripheral nerve reconstruction (Tos et al., 2009; Angius et al., 2012; Gordon and Borschel, 2016). The mixed sciatic nerve contains sensory as well as motor fibers and consists of mainly 3 branches: the tibial nerve, the common peroneal nerve, and the caudal sural cutaneous nerve (Swett et al., 1986; Swett et al., 1991). Via dorsolateral operation route the sciatic nerve is easily accessible over a relatively short distance, so that nerve defects, exceeding 4 cm, can be created (Saheb-Al-Zamani et al., 2013;

Kornfeld et al., 2019). As the sciatic nerve is the biggest nerve of the body, surgery can be facilitated (Ronchi et al., 2019). In this well established model, reinnervation of target muscles as signs of motor recovery can be analyzed by electrodiagnostic measurements of compound muscle action potentials (CMAPs). Other functional tests to evaluate recovery of motor function are estimation of the static sciatic index (SSI), calculated by measured distances between the toes, which are altered after denervation, and estimation of the static functional index, mainly based on the same principle as the SSI. Sensory recovery is assessed by mechanical or thermal algesimetry tests (Varejao et al., 2001; Cobianchi et al., 2013; Navarro, 2016). But the use of the rat sciatic nerve model also goes along with downsides. The artificially created injury leads to paralysis of the hind limb, which may be followed by autotomy behavior, as well as joint contractures (Carr et al., 1992; Navarro et al., 1994; Ronchi et al., 2019). These circumstances can result in an exclusion of the respective animals from functional testing, decreasing the power of the study (Ronchi et al., 2019). Therefore, the rat median nerve forelimb model has gained more interest in the recent years, as described below.

1.7.2.2. The rat median nerve model

The rat median nerve fulfills only motor function in contrast to the human median nerve. The smaller sized nerve only allows to create nerve defects of smaller gap lengths up to 10 mm. When

Introduction

compared to the rat sciatic nerve, microsurgical transection and reparation procedures are more complicated in the rat median nerve model, due to issues of scale. With the help of a battery of functional tests, the onset, progression and completeness of motor recovery can be precisely determined, allowing comprehensive regeneration analysis (Stößel et al., 2017). Similar to the sciatic nerve model, electrodiagnostic measurements of evoked CMAPs in the target muscles can be recorded (Korte et al., 2011; Navarro, 2016; Stößel et al., 2017). The staircase test, in which the animals grab sugar pellets, displays recovery of fine motor skills (Montoya et al., 1991;

Galtrey and Fawcett, 2007; Stößel et al., 2017), while the grasping test represents recovery of gross motor function (Papalia et al., 2003; Stößel et al., 2017). Complete transection of the median nerve leads to partial but not complete impairment of the upper limb function. Full sensory and part of the motor function is still preserved due to the intact radial and ulnar nerve (Sinis et al., 2006). The absence of joint contractures, and only rare occurrence of ulcers or autotomy behavior are further advantages of the median injury model (Ronchi et al., 2019). A decision on the appropriate animal model always requires considerations of translatability. The fine finger movement between rodents and humans are similar, so that the rat median nerve injury model might be comparable to human digital nerve lesions (Whishaw et al., 1992).

Furthermore, the median nerve is located in a highly mobile region just like the joint-crossing human digital nerves.