The Median Nerve Injury Model in Pre-clinical Research – A Critical Review on Benefits and Limitations

The successful introduction of innovative treatment strategies into clinical practise strongly depends on the availability of effective experimental models and their reliable pre-clinical assessment. Considering pre-clinical research for peripheral nerve repair and reconstruction, the far most used nerve regeneration model in the last decades is the sciatic nerve injury and repair model. More recently, the use of the median nerve injury and repair model has gained increasing attention due to some significant advantages it provides compared to sciatic nerve injury. Outstanding advantages are the availability of reliable behavioural tests for assessing posttraumatic voluntary motor recovery and a much lower impact on the animal wellbeing. In this article, the potential application of the median nerve injury and repair model in pre-clinical research is reviewed. In addition, we provide a synthetic overview of a variety of methods that can be applied in this model for nerve regeneration assessment. This article is aimed at helping researchers in adequately adopting this in vivo model for pre-clinical evaluation of peripheral nerve reconstruction as well as for interpreting the results in a translational perspective.


INTRODUCTION
Peripheral nerve injuries are commonly caused by motor vehicle, domestic, work or sport accidents or during surgeries (iatrogenic nerve injuries) . Nerve injuries can lead to motor and sensory deficits that may result in disabilities permanently compromising the patients' quality of life.
The general ability of peripheral nerves to regenerate has been recognised more than a century ago, but until today functional recovery outcome after severe nerve injury and reconstructive surgery is often still poor in many patients. Nowadays, the "gold standard" reconstructive technique for bridging a nerve gap is autologous nerve grafting. This technique, however, is accompanied by important drawbacks such as the donor site morbidity, the need of additional surgery and the limited availability of graft material for extended repair (Konofaos and Ver Halen, 2013;Faroni et al., 2015).
Over the past decades, substantial effort has been made to identify new strategies to improve peripheral nerve regeneration after grafting and to substitute the autologous nerve graft. Advancements in biomedical methods, the tissue-engineered technology, gene therapy approaches, nanotechnology, biology, and microsurgical skills have opened new research fields in the nerve reconstruction area. Indeed, there is an exponential increase in the number of publications dealing with experimental nerve regeneration research over the years: a literature search with the PubMed search string ("Nerve-Regeneration" [Mesh] OR nerve-regenerat * OR nerve-repair * ) AND (rat * OR mouse OR mice OR rabbit * OR sheep), delivered 26 results in 1970 and 479 in 2018. In the context of pre-clinical peripheral nerve regeneration research, the choice of the experimental animal model is of fundamental importance. When a researcher moves on to test a novel attempt in vivo, the animal model should be chosen according to the study aims [e.g., for studying the involvement of a specific molecule in the biological process of nerve regeneration, the most appropriate choice will most likely be different to the model chosen for evaluating the effectiveness of a nerve conduit for long gap (>50 mm) repair]. Obviously, the pros and cons of the different available options must also be taken into careful consideration.
The choice of the appropriate experimental nerve injury model is usually guided by several factors. For nerve repair studies, in particular, the size (diameter and length) of the model nerve is certainly one of the main aspects considered. Indeed, most nerve repair studies are conducted on the sciatic nerve especially because of its big dimension that facilitates experimental microsurgery (Varejao et al., 2004;Bozkurt et al., 2011;Sinis et al., 2011a). The sciatic nerve is the biggest nerve in the body and the choice among mouse, rat, rabbit, dog, or sheep already provides variability in nerve gap lengths to be applied (Angius et al., 2012). Different downsides resulting from experimental injury to the sciatic nerve have, however, led to increasing interest in the median nerve as alternative model nerve (Bertelli et al., 2004). At first, injury to the sciatic nerve results in a paralysis of the hind limb and, often, in automutilation behaviour, such as biting and self-amputation of denervated toes and paw areas by the subjected animal. Longer lasting paralysis (>4 weeks in the rat) often leads to joint contractures and stiffness. Automutilation behaviour and joint contractures reduce the reliability of functional tests, such as estimation of the sciatic function index or, in severe cases, will lead to exclusion of the respective animal from a study for ethical and animal welfare reasons. Furthermore, possibilities for evaluation functional recovery of motor skills after sciatic nerve lesion in the awaken animal are rather limited or need considerable efforts to be realised (Navarro, 2016).
In the recent years employment of the median nerve injury and repair model in the experimental research has increased (Papalia et al., 2003b;Ronchi et al., 2009) because of several advantages. Transection injury of the rodent median nerve, results in only partial impairment of the upper limb function . Incidence of automutilation is significantly lower in comparison to the sciatic nerve model, ulcerations are fewer and no joint contractures can be seen. This milder phenotype results from the fact that after median nerve injury, the ulnar and radial nerves still preserve sensitivity and motor function in the forearm (Sinis et al., 2006). An additional advantage of the rodent median nerve injury and repair model is that positively evaluated attempts are more likely to be translated into clinical practise, since surgical interventions for the repair of a damaged human nerve are very often performed at the upper limb level. In addition, the hand functions require a fine finger movement that is quite similar between rodents and humans (Whishaw et al., 1992). From this perspective, the possibility to apply specific and precise functional tests for motor recovery evaluation following median nerve reconstruction is a further pro of this model in preclinical research.
This review provides an overview on the use of the median nerve injury and repair experimental model in pre-clinical research. The different animal species (not only mouse and rat but also larger animals such as rabbit, sheep, and monkey) are taken into account as are the different options these species provide with regard to comprehensive analysis of the regeneration outcome.

THE PRE-CLINICAL MEDIAN NERVE INJURY AND REPAIR MODEL IN DIFFERENT ANIMAL SPECIES
The use of the median nerve injury and repair model as preclinical model has progressively increased in the last years, but the sciatic nerve injury and repair model is still more often employed [in PubMed a research on (median-nerv * ) AND (regenerat * OR repair) yielded 1002 papers, while (sciatic-nerv * ) yielded 6612 papers].
Information from our literature search is presented in Tables 1-5. The tables summarise peripheral nerve regeneration studies after median nerve injury and repair in the different animal models. In addition to the specific reference, the tables list animal strain and sex, type of injury/gap, follow up periods and type of analyses conducted. We took our best efforts to include all articles available until end of 2018; nevertheless, inadvertently, we could have missed some papers and apologise in advance with their authors.
In the following paragraph specificities of the different models listed in the tables will be reviewed in more detail.

Mouse Model
Since most of the available transgenic animal models are mice, they are often used for studying the role of specific genes in the peripheral nerve regeneration process. For this purpose, genes of interest are knocked-out, mutated or over-expressed. Moreover, mice -especially wild type strains -are economical in their keeping, simple to handle and to care for and can therefore be studied in large groups. Mice nerves can be subjected to different types of injury and repair and analysed with all kinds of functional, morphological and biomolecular assays. The most simple crush injury can be easily used and standardised. On the other hand, peripheral nerves in mice are rather small and experimental in vivo work employing more complex surgeries on them, such as end-to-end repair, requires advanced microsurgical skills. These skills are provided by many clinical researchers from disciplines routinely performing nerve surgeries but are less prevalent in the basic researcher community. Furthermore, when it comes to studies evaluating new developments for bio-artificial nerve guides, the much smaller diameter of mouse peripheral nerves is not fitting the larger one of nerve guides that are primarily commonly designed to fit human digital nerves.

Rat Model
Among rodents, rats are the most commonly used in vivo model in peripheral nerve regeneration research. This relates mainly to the dimension of their nerves. Surgery on rat peripheral nerves requires less microsurgical skills than needed for mouse models and rat limbs and nerves are long enough to allow 1.5-2 cm gap repair to be studied at least in the sciatic nerve. Moreover, rats can be investigated in large groups because their keeping is economical, and they are mostly simple to handle and to care for. Transgenic rat models are, however, less available than mouse transgenic models and also the availability of rat specific antibodies for molecular and histological examination is rather limited. But due to their prevalent employment, functional tests for evaluating motor or sensory recovery in rats are more standardised (Navarro, 2016) and comparable among different research groups. Rat nerves can be subjected to different types of injury and repair and analysed in most comprehensive functional, morphological and biomolecular assays Navarro, 2016;Ronchi et al., 2016). Different rat strains can be utilised, for which varying willingness to enrol in specific functional tests is End-to-end repair of the median nerve distal to the elbow (n = 6); End-to-end repair of the median and ulnar nerves proximal to the elbow (n = 6); Repair of the median and ulnar nerves with a 7-mm polyurethane tube (gap 5 mm) proximal to the elbow (n = 9) Up to 13 weeks Evaluation of volitional forelimb strength Ronchi et al., 2017 Wistar F Immediate, 3 and 6 months delayed nerve repair with a cross suture between the degenerated median nerve distal stump and the freshly axotomised ulnar proximal stump (n = 7/group); Controls: healthy nerve (n = 5); 9-month degenerated nerve (n = 5); 3-month regenerated end-to-end-repaired median nerves (n = 5)     reported (Nikkhah et al., 1998;Galtrey and Fawcett, 2007), but so no comparative studies investigating differences in their ability to regenerate have been published. When using rodents (mice and rats) as animal models for peripheral nerve regeneration, researchers must finally be aware of the following immanent differences to mankind: (1) gaps that can be produced are shorter than those commonly found in human nerve lesions; (2) axonal regeneration rate is faster than in humans; (3) recovery is often complete, while in humans it is often incomplete (Kaplan et al., 2015).

Rabbit Model
In peripheral nerve regeneration research, rabbits offer the possibility to study regeneration across gap lengths of up to 6 cm. Rabbits are, however, expensive to purchase and maintain, and difficult to care for. Rabbits are more delicate and less resilient than rats and mice, and their occurrence as pet animals probably creates ethical problems for animal care takers and researchers. Also, there are almost no valid functional assays that can be applied in rabbit models, besides electrodiagnostic evaluation. Finally, very few specific antibodies are available to be used on rabbit tissue samples, so that conclusions on the regeneration outcome can mainly only be based on nerve morphometry studies.

Sheep Model
The sheep as an animal model is useful when nerve regeneration across very long gaps should be evaluated. An ethical advantage of this animal is provided by the fact that a median nerve transection injury does not result in serious impairment of the limb usage ability and functional read-outs have been described in the recent years. To establish the model and to provide adequate housing conditions is, however, a considerable challenge, and research in this model will often only be realised through collaborative work.

Monkey Model
Although non-human primates could be useful to test safety and efficacy of synthetic nerve conduits -because of the similarity of non-human primates with human beings -their use is considerably limited for ethical reasons. Monkeys are considered as animal models mainly to study neuronal plasticity occurring in the brain following peripheral nerve injury and repair. Again, studies in this model will mainly be subject of collaborative work and not appropriate for early stage pre-clinical research.

Other Animals
Other large animals can be used as models to study median nerve injury and regeneration, such as pigs (Ochoa and Marotte, 1973;Marotte, 1974), dogs (Lee et al., 1999), and cats (Murray et al., 1997), but their use is limited for three main reasons: (1) animal care for large species is considerably expensive; (2) for some species the possibilities for functional testing are limited or require complex training, therefore, nerve regeneration may only be assessed with nerve morphometry; (3) dogs and cats are domestic animals, and their use in research is more restricted for ethical reasons.

METHODOLOGICAL CONSIDERATIONS -APPROACHES FOR THE TREATMENT OF NERVE INJURIES
The following criteria should be considered for selecting the most suitable animal model for pre-clinical research on peripheral nerve regeneration: (1) costs to purchase and house the animals; (2) ease of handling, such as tolerance to captivity; (3) tolerance for surgery and eventual repetitive anaesthesia, as well as resistance to infection; (4) compliance with national policies and with ethical principles; (5) inter-animal uniformity, life span of the species, biological information and available tools; (6) overall experimental plan (Angius et al., 2012). With regard to the latter, especially when several time-points are to be analysed, rodents represent the best choice. On the contrary, rodent life span is short, therefore for long experiments (>1-1.5 years), it becomes necessary to use lager animals as model organism.
In the last years, several approaches have been developed and tested in pre-clinical animal models to improve peripheral nerve regeneration. All these approaches can be applied on different nerves and are not median-nerve specific. In this paragraph we will present an overview of different techniques, including the use of different conduits to guide regenerating axons, the use of stem cell transplantation, the application of physical therapies and optogenetics, all of which have been demonstrating variable positive effects on nerve regeneration irrespectively of the model they were investigated in.

Nerve Guidance Conduits
The major disadvantage of the autologous nerve graft technique is the remarkable sensitivity loss (mainly sural nerve grafts are harvested for this) and the limited availability of donor tissue (Ray and Mackinnon, 2010). Reconstruction of a nerve with artificial or non-artificial nerve conduit grafts is particularly indicated in case of extensive nerve tissue loss. Nerve guidance conduits made of several materials, artificial or of natural origin, are available; most common and FDA approved for clinical use Japanese white rabbits -Study 1: the median nerve was elongated for 10 days (n = 6), for 15 days (n = 7) and for 20 days (n = 6). Study 2: Right arm: the proximal segment of an injured median nerve was elongated for 10 days (n = 10) and for 15 days (n = 10). Left arm: a 10-for 15-mm segment of the median nerve was removed, and a 10-for 15-mm segment, respectively, of the tibial nerve was grafted in its place.     Grabb, 1968 Rhesus monkeys F Nerves were cut and sutured primarily (4 h after nerve injury, n = 30), or secondarily (about 3 weeks after injury, n = 30).

months Electromyographic examination
are conduits made of collagen, chitosan, or poly (DL-lactideε-caprolactone) (Kornfeld et al., 2018). All devices currently on the market and FDA-approved have proven good support for the promotion of peripheral nerve regeneration in pre-clinical models. Good clinical results, however, are obtained only for lesions with a substance loss inferior to 3 cm in length, while severe and enlarged injuries remain a critical condition (Kaplan et al., 2015). For this reason, research in the field of novel nerve conduit functionalisation strategies is still highly vivid.

Application of Stem Cells and Their Secretome
In regenerative medicine and tissue engineering, the use of Stem Cells and their secretome is fast expanding with the aim to develop innovative therapeutic strategies for the treatment of peripheral nerve injuries (Caplan, 2015;Caseiro et al., 2016;Busuttil et al., 2017;Sayad-Fathi et al., 2019). In particular, Mesenchymal Stem Cells (MSCs) present relevant key features: they can be easily expanded, they can differentiate into different cell types, they are immune-privileged and immune-modulatory, they show preferential homing to injured sites (Frausin et al., 2015;Sullivan et al., 2016;Jiang et al., 2017). Moreover, the MSC secretome contains trophic mediators (Meirelles Lda et al., 2009;Fu et al., 2017), modulating the function of several tissues, including the skeletal muscle  and the peripheral nervous system (Lopatina et al., 2011;Gartner et al., 2012Gartner et al., , 2014. The most widely source of MSCs for therapeutic purposes is the bone marrow; as good alternative other sources are: the umbilical cord blood, the stromal tissue of the umbilical cord, the dental pulp, the adipose tissue (Jin et al., 2013).

Transgenic Models to Promote Peripheral Nerve Regeneration
To study the biology of peripheral nerve regeneration, different transgenic models can be used (Magill et al., 2008). Most of the available transgenic animals are mice and they represent a powerful tool to study the influence of over-expression or depletion or mutation of a specific gene in a specific cell type, using inducible systems, but it must be kept in mind that mice are difficult subjects for microsurgical models due to the small size of their nerves, as discussed above.
To investigate the function of specific genes in nerve regeneration discriminating between motor and sensitive neurons, transgenic mice over-expressing the gene of interest in postnatal motoneurons or dorsal root ganglion neurons can be obtained using Thy1 or NSE (neuron specific enolase) promoters (Michailov et al., 2004;Gomez-Sanchez et al., 2009;Velanac et al., 2012).
To obtain tissue specific expression or depletion of specific proteins, the inducible cre-lox system can be applied: transgenic mice driving motor neuron specific expression of cre recombinase with the promoter of Mnx1 (motor neuron and pancreas homeobox 1) gene can be used to specifically in/activate the expression of floxed genes in motor neurons, while specific in/activation in Schwann cells can be obtained using the promoter of Mpz (myelin protein zero) gene (La Marca et al., 2011).
Transgenic animals can also be developed as advanced experimental models to study genetic diseases giving rise to peripheral neuropathies (Hoke, 2012;Juneja et al., 2018), and they can be studied to investigate their ability to regenerate injured peripheral nerves.
Finally, the expression of neurotrophic factors or other potentially therapeutic proteins in Schwann cells or in neurons can be obtained through the use of different viral vectors (Tannemaat et al., 2008).

Physical Therapies
The efficacy of brief Electric Stimulation (ES) of the proximal stump of an injured nerve in promoting nerve regeneration in animal models has been verified in several independent studies reviewed by Gordon (2016) and Gordon and English (2016). In particular, a 14 days period of ES was chosen (Al-Majed et al., 2000) to accelerate the regenerative process and the effect was dramatic: preferential motor reinnervation of motor pathways was evident at 21 days rather than at 42 days, and, importantly, all of the motoneurons had regenerated into the motor nerve branch.
Another interesting study was aiming at evaluating the value of electromagnetic stimulation for the neural regenerative process of the rat median nerve after transection and end-to-end repair (Beck-Broichsitter et al., 2014b). From the 1st day after surgery a pulsed magnetic therapy was daily applied in the experimental group. Magnetic stimulation was positively influencing the functional regeneration in terms of grasping force and reduced muscular atrophy.

Optogenetics
Axonal regeneration and functional recovery are enhanced by activity-related therapies, such as exercise and electrical stimulation (Gordon, 2016). Unlike electrical stimulation, optogenetics allows to selectively activate or inactivate specific neurons: for example, selective expression of the light-sensitive cation channel channelrhodopsin-2 (ChR2), that is maximally activated by blue light, can be used to depolarise neurons, thereby driving action potentials. Conversely, selective expression of the light-sensitive inward chloride pump halorhodopsin (Halo), that is maximally activated by amber light, can be used to obtain neuron hyperpolarisation, thereby inhibiting action potentials (Montgomery et al., 2016). Optically induced neuronal activity has been shown to be sufficient to promote functional motor axon regeneration in vivo (Ward et al., 2016). Moreover, through the selective expression of opsins in sensory neurons or motoneurons, it was possible to investigate the effect of system-specific neuronal activation on axonal regeneration, thus demonstrating that acute activation is sufficient to enhance regeneration of both motor and sensory axons (Ward et al., 2018).
Until now, in the peripheral nervous system, optogenetics has been applied mainly to sciatic nerves in transgenic mice expressing different opsins, but given its therapeutic potential it will be certainly applied also to median nerves and in other animal models, by injection of optogenetic constructs to transduce opsin expression in peripheral nerves in the future.

Immunomodulation
The early inflammatory reactions undergoing in the course of Wallerian Degeneration of the distal nerve, comprise the activation of the complement system, arachidonic acid metabolites, and inflammatory mediators involved in myelin fragmentation and activation of repair Schwann cells. Finetuned upregulation of the cytokine/chemokine network by repair Schwann cells activates resident and hematogenous macrophages to complete the clearance of axonal and myelin debris and stimulate regrowth of axonal sprouts (Yona and Jung, 2010;Cortez-Retamozo et al., 2012;Dubovy et al., 2013;Jessen and Mirsky, 2016). An innovative approach in the field of peripheral nerve regeneration is exploiting the endogenous capacity of the body to repair itself through immune cells. In very promising studies, starting from the known different pro-inflammatory and pro-regenerative macrophage phenotypes, they were modulated through their response to different IFN-γ or IL-4 cytokines and studied in their ability to influence nerve regeneration in a critically sized, 15 mm rat sciatic nerve gap. The results of this research have shown that the administration of IL-4 at the injury site increased the pro-regenerative effect and therefore that the regenerative outcomes appeared to be influenced not only by the macrophage presence, but by their specific phenotype at the site of injury (Mokarram et al., 2012). A similar approach was conducted later by the same authors, through early stage administration of fractalkine, a chemokine able to control the phenotype in monocyte recruitment and to increase the regenerative potential. The pharmaceutical approach was evaluated from a morphological and functional point of view (Mokarram et al., 2017).

METHODOLOGICAL CONSIDERATIONS -TECHNIQUES TO INVESTIGATE PERIPHERAL NERVE REGENERATION
A number of different techniques have been developed to investigate the degree and the accuracy of nerve regeneration. While functional tests must be nerve-specific, all other methods can be applied to all types of peripheral nerves. In this paragraph we describe in detail different functional tests that are used to study the functional recovery of the median nerve and, in addition, we present an overview of other methods used for the investigation of nerve regeneration, including morphological and morphometrical analysis, gene expression analysis and fluorescent transgenic animal models.

Functional Evaluation
Several functional tests are available for rodents, both rats and mice (Galtrey and Fawcett, 2007). Some have been designated ad hoc to evaluate functional recovery following median nerve repair (e.g., grasping test), others have been adapted from tests normally used following other lesion types (injuries to the spinal cord or sciatic nerves).
The tests described below refer to the rat, but most of them are adaptable also to mice.
• The grasping test is a simple method to assess the flexor function, first introduced by . The modified method (Papalia et al., 2003a) consists in presenting a small tower with only three bars forming a triangle on its top instead of a grid for grasping. With this modification the tendency to walk on the grid is avoided and the presence of a band put just below the three bars avoids that the rat employs the wrist flexion to hold the bars. This device is connected to a precision dynamometer. The animal is hold by its tail and allowed to grasp the grid. Then, the animal is pulled upward until it loses its grip. The balance records the maximum weight that the animal managed to hold before losing the grip. • The staircase test is a functional test which assesses skilled forepaw reaching and grasping (Montoya et al., 1991). Two/three food pellets are placed on each step of two staircases located one on either side of a central platform. Both stairs are composed of seven steps. The animal is placed in a box and can only reach the pellets from the left staircase with its left paw and those from the right staircase with its right paw. The rat can grasp, lift, and retrieve food pellets from the steps of the staircase. The number of pellets completely removed from the staircase box provides a quantifiable measure of the distance and efficiency of fine motor reaching skills. Rats need to be pre-trained in the staircase test before surgery, put on restrictive diet before testing and need to be accomodated again to the test conditions for some days before the following evaluation. Therefore, this test is more complex to be applied than the grasping test described above. • The walking track analysis is used to evaluate forelimb motor recovery (Ozmen et al., 2002;Galtrey and Fawcett, 2007). The rat forepaw is dipped in an ink solution and the animal is allowed to walk down the track upon a strip of white or graph paper. The prints by the ink are left to dry and then analysed. Different parameters can be analysed (longest length and widest width of the paw impression, widest width between the second and third fingers, distance between homologous points of sequential paw impressions on a given side, perpendicular distance between the central portion of the paw impression and the direction of movement). Moreover, walking track analysis can be performed by 2D digital video motion analysis, which allows also to quantify the movement of the wrist and the metacarpophalangeal joint (Wang et al., 2008). • The Von Frey filament test is used to evaluate mechanical allodynia (Galtrey and Fawcett, 2007). The animal is placed in a box on a wire mesh floor. Von Frey filaments of different bending forces are used to examine the mechanical threshold of the rat forepaws. The test starts with the smallest bending force and continues in increasing order. Each filament is inserted through the mesh and applied in the medial surface of the forepaw.
To perform the test, the rat must be stationary and standing on the four paws. The first filament in the series that evoked withdrawal three times is regarded as the paw withdrawal threshold. • The Irvine, Beattie, and Bresnahan (IBB) scale test (Irvine et al., 2014) is used for the assessment of fine control of the forelimb and digit movements. Sphericaland donut-shaped pieces of cereal are given to the rat. The forelimb behaviour (joint position, object support, wrist and digit movement, and grasping method) used while eating both cereal shapes is analysed. An IBB score is assigned using the 10-point (0-9) ordinal scale for each shape, and the highest score reflects the greatest amount of forelimb recovery. • The ladder rung walking test is used to assess forelimb strength, stepping, placing, and co-ordination during skilled locomotion (Metz and Whishaw, 2002;Galtrey and Fawcett, 2007). The apparatus consists of two side walls with rungs inserted into the walls to create a ladder. The ladder is elevated and can also be inclined. The animal is conditioned to run on the ladder on several training sessions. Performance is scored (successful steps/total steps). • The Randall-Selitto test is used to assess the nociceptive withdrawal threshold (Galtrey and Fawcett, 2007). The test consists in the application of an increasing mechanical force with the tip of an algesimeter on the medial portion of the forepaw until a withdrawal response results. • For the cold sensory test an ice probe is made by freezing water in a 1.5-ml tube (Lindsey et al., 2000;Galtrey and Fawcett, 2007). When the rat is drinking from the water bottle, the ice probe is applied to the glabrous skin of the forepaw. The withdrawal latency is measured. At least 1 min between trials is needed to allow the skin to return to body temperature and prevent sensitisation. If the rat does not withdraw the paw after 10 s, the probe is removed. • The cold and hot plate test is used to assess temperature sensation. The rat is placed in a Plexiglas chamber where the metal base temperature is 25 • C. For cold plate testing, the temperature is rapidly lowered and the animal behaviour is observed for signs of painlike behaviour (avoiding contact with the cold plate, suspension of the affected forelimb, licking of the paw, lack of grooming and exploration vocalisation, or freezing behaviour). Once pain-like behaviour is observed, the temperature is increased to a higher temperature. For hot plate testing, the temperature is raised and the behaviour is assessed as described above (Shaikh et al., 2016). • The CatWalk automated quantitative gait analysis is a computer-assisted method that can simultaneously measure dynamic as well as static gait parameters, including duration of different phases of the step cycle and pressure applied during locomotion (Bozkurt et al., 2008). The animal is placed in the CatWalk walkway, which is comprised of a glass plate with two Plexiglas walls, a high-speed colour camera, and recording and analysis software. The animal walks voluntarily from one side of the glass plate to the other. Its footprints are captured. The intensity of the signal depends on the degree of paw floor contact and increases with pressure applied. The more pressure is exerted, the larger the total area of skin-floor contact and thus the brighter the pixel. An appropriate software visualises the prints and calculates statistics related to print dimensions and the time and distance relationships between footfalls (Chen et al., 2012a,b). • Electrophysiology is often used in rat, while in the mouse model it is less used probably for the small size. The maximum amplitude and latency of evoked compound muscle action potentials recorded from the thenar muscles are usually evaluated ).
In conclusion, the list provided above clearly demonstrates that a large variety of tests can be used to evaluate the functional recovery after median nerve transection and repair in rodents.
From a translational point of view, tests should be selected in way to model as closely as possible the course of functional recovery as it is observed in human patients. Recently, the combination of electrodiagnostic evaluation, with the commonly used grasping test (reflex-based gross motor function) and the staircase test (skilled forelimb reaching) has been described to produce results with high translatability (Stossel et al., 2017).
A final comment needs to be put on the fact that different rat strains have been described to demonstrate different motivation degrees to participate in more complex tasks, like, e.g., the staircase test. Especially, Lewis rats have been described to be less motivated and also to eventually be less capable of learning how to perform more complex motor tasks (Nikkhah et al., 1998;Galtrey and Fawcett, 2007).
In the other animals, the functional recovery is assessed mainly by electrophysiology. Indeed, while in rats and mice the fingers do a fine movement quite similar to humans and their functionality can be assessed by different suitable and specific tests, the other animals can do gross movements only.

Morphology and Morphometry (Stereology)
Regardless of the animal model used, nerve regeneration assessment must necessarily have an accurate morphological and morphometrical evaluation (Geuna and Herrera-Rincon, 2015). Among the techniques that allow this type of analysis, immunohistochemistry offers the possibility to specifically identify the different structures of the regenerating nerve, such as Schwann cells, motor or sensory axons, blood vessels, and other cell types, including macrophages, fibroblast-like cells, perineurial cells, endothelial cells (Carriel et al., 2014b). Moreover, immunofluorescence or immunohistochemical techniques allow to accurately quantify the fraction area and the intensity of the expression of specific proteins which are correlated with regenerative processes. For example, the expression of markers such as Neurofilament in neurons or S-100 in Schwann cells are indicative of an excellent regenerative process, when these levels reach the values of the control nerves (Carriel et al., 2014a).
To quantify the number of sensitive and motor neurons which were able to regenerate axons across a nerve gap, the retrogradelabelling technique can be applied. A dye will be applied into the distal nerve and taken up by regenerated axons and retrogradely transported into the neuronal soma of sensory neurons in the dorsal root ganglia or motor-neurons in the ventral horn of the spinal cord (Hayashi et al., 2007;Kemp et al., 2017).
Together with functional assessment, quantitative estimation of regenerated nerve fibres is a key investigation tool in nerve regeneration research (Kanaya et al., 1996;Geuna et al., 2004). The toluidine blue staining of resin-embedded semithin sections allows to clearly identify most of the myelinated axons and their myelin organisation thanks to the postfixation with OsO 4 . Usually, morphoquantitative analysis is performed on one randomly selected toluidine blue semi-thin transverse nerve section. The total cross-sectional area of the nerve is measured. Then, an adequate number of fields of interest (according to the size of the nerve) is randomly selected following a systematic random protocol and analysed ). The parameters used as nerve regeneration indicators are myelinated fibre number and density, fibre and axon diameter, myelin thickness and g-ratio (axon-diameter/fibre-diameter) (Geuna, 2000(Geuna, , 2005.
In order to compare results obtained by different research groups, different potential sources of bias should be considered.
First of all, the strain, the gender and the age of experimental animals, that can affect the quantification outcome.
The second aspect that can influence the results of a stereological analysis is the level at which it is conducted and, obviously, the different investigation time points. The analysed parameters can vary significantly depending on the distance from the lesion point, especially in the early time points after injury, considering that a nerve can grow approximately 1 mm/day (Santos et al., 2007). Therefore, only quantitative data taken at the same location along the nerves can actually be compared (e.g., 5 mm distal to the lesion site). Obviously, also the time point analysed gives different results and should always be considered (with the same lesion type, a 3-month regenerated nerve will be different from a 6-month regenerated nerve). Finally, it must possibly be considered to analyse all branches of a nerve. The portion of the median nerve that is usually injured and repaired in experimental studies goes from the axillary region to the elbow. In this tract, the median nerve is unifascicular, but for more distal investigation sites the anatomy of the nerve needs to be considered. The median nerve gives off three palmar digital branches more distally, at the level of the carpal bones, that in turn bifurcate between the 1st, 2nd, and 3rd digit . Other nerves (i.e., the sciatic nerve) release branches in the tract that is commonly investigated. Therefore, if the morphological/morphometrical analysis requires the nerve cross section (for example to estimate the total number of nerve fibres), all branches must be analysed.
The third aspect is represented by the chosen method for measuring the selected size parameters (computerised or manual analysis). It is important to note how computers can certainly make quantitative morphology easier and faster (Williams and Rakic, 1988;Dolapchieva et al., 2000), but a comparison of the performance of automated cell detection revealed that a manual approach is still the most appropriate method for stereological counting (Schmitz et al., 2014).

Gene Expression Analysis
Injured median nerve gene expression analysis can be carried out both at mRNA and protein level. To reduce the number of animals and comply with the 3R' Principle (Replace, Reduce, and Refine) (Tannenbaum and Bennett, 2015) a good strategy is to extract from the same nerve sample both total RNA and proteins, using commercially available kits.
The first point that should be considered when analysing a nerve sample is that protein extraction involves all nerve components, neuronal axons and peripheral cells (Schwann cells, fibroblasts, macrophages, and so on). RNA extraction mostly encloses peripheral cells, because neuronal RNA is mainly localised in the cell bodies of sensory neurons (in the dorsal root ganglia) or motor neurons (in the ventral horn of the spinal cord), with only few mRNAs locally translated in the axon after nerve injury (Terenzio et al., 2018).
The second point that should be carefully considered is the portion of the injured nerve to be analysed and the time window for the analysis. Indeed, in the 1st days after injury, regenerating axons start to colonise the proximal portion of the repaired nerve, while in the distal portion axons are still undergoing Wallerian degeneration (Girouard et al., 2018). In the following days, regeneration occurs also in the distal stump. Therefore, gene expression analysis will give information about regeneration or Wallerian degeneration taking place according to the region and the time point analysed: for gene expression analysis, like previously discussed for morphology and morphometry, the region and the time point analysed must be the same for all samples and for comparison with other studies.
For mRNA analysis, quantitative real time PCR can be carried under paying attention to the housekeeping genes used for normalisation: indeed, it is really important to normalise data to genes whose expression is not affected by nerve injury (Vandesompele et al., 2002). To this aim, stable housekeeping genes suitable for gene expression analysis were identified (Gambarotta et al., 2014;Wang et al., 2017). To avoid amplification of contaminant genomic DNA, a good strategy is to design primers on different exons, possibly separated by introns larger than 1,000 bp, or straddling two contiguous exons. For protein analysis, western blots can be carried out using the stain-free technology, which is a good strategy, because protein expression is normalised to the total protein content, bypassing the problem of the choice of suitable housekeeping genes (Gurtler et al., 2013).

Transgenic Models to Evaluate Peripheral Nerve Regeneration
A transgenic model that can be used for evaluating and monitoring peripheral nerve regeneration in mice and rats is Thy1-GFP, in which Thy1 promoter drives neuron specific expression of GFP, allowing imaging of nerve regeneration following nerve injuries (Porrero et al., 2010;Moore et al., 2012;Kemp et al., 2013).

CONCLUSION
In this review we emphasised the use of the median nerve as pre-clinical experimental model to study nerve regeneration in vivo. Accordingly, in the last years the median nerve model is increasingly used due to different reasons, including a better animal well-being, and availability of different functional tests which, specifically in rodents, allow testing the digital fine movement, making the results obtained with this model more translatable into the clinic.
With regard to the animal choice, the rat definitely represents the easiest and more translatable model, especially for studies evaluating regeneration across short nerve gaps, while for large nerve gaps the use of other, larger, animals would be recommended. In case large animal facilities are not available, an alternative and very interesting approach might be the cross-chest median nerve transfer in the rat animal model (Sinis et al., 2006). Indeed, this method would allow the use of rodents, which are less expensive and easier to handle compared to large animals, but at the same time would allow studying nerve regeneration across long gaps (up to 40 mm).

AUTHOR CONTRIBUTIONS
GR, GG, SG, and KH-T organised the manuscript. GR, GG, and MM prepared all the tables. GR, GG, MM, FF, SG, and KH-T wrote different sections of the manuscript. All authors contributed to manuscript revision, and read and approved the submitted version.